10.1007@s00344-016-9663-5

J Plant Growth Regul 

DOI 10.1007/s00344-016-9663-5 

Co-inoculation with Enterobacter and Rhizobacteria on Yield 

and Nutrient Uptake by Wheat (Triticum aestivum L.) 

in the Alluvial Soil Under Indo-Gangetic Plain of India 

Ashok Kumar 1,2 · B. R. Maurya 1 · R. Raghuwanshi 2 · Vijay Singh Meena 1,3 · 

M. Tofazzal Islam 4 

Received: 2 August 2016 / Accepted: 9 November 2016 

© Springer Science+Business Media New York 2017 

Abstract The aim of this work was to evaluate the effects 

of co-inoculation with phosphate-solubilizing and nitrogenfixing 

rhizobacteria on growth promotion, yield, and nutrient 

uptake by wheat. Out of twenty-five bacteria isolated 

from the rhizosphere soils of cereal, vegetable, and agroforestry 

plants in eastern Uttar Pradesh, three superior most 

plant growth-promoting (PGP) isolates were characterized 

as Serratia marcescens, Microbacterium arborescens, and 

Enterobacter sp. based on their biochemical and 16S rDNA 

gene sequencing data and selected them for evaluating their 

PGP effects on growth and yield of wheat. Among them, 

Enterobacter sp. and M. arborescens fixed significantly 

higher amounts (9.32 ± 0.57 and 8.89 ± 0.58 mg Ng −1 carbon 

oxidized, respectively) of atmospheric nitrogen and 

produced higher amounts (27.06 ± 1.70 and 26.82 ± 1.63 

TP 100 µg mL −1 , respectively) of IAA in vitro compared 

to S. marcescens (8.32 ± 0.39 mg Ng −1 carbon oxidized and 

21.29 ± 0.99 TP 100 µg mL −1 ). Although both M. arborescens 

and S. marcescens solubilized remarkable amounts 

of phosphate from tricalcium phosphate likely through 

* Ashok Kumar 

ashokbhu2010@gmail.com; ashokabt@gmail.com 

* Vijay Singh Meena 

vijay.meena@icar.gov.in; vijayssac.bhu@gmail.com 

1 

2 

3 

4 

Department of Soil Science and Agricultural Chemistry, 

Institute of Agricultural Sciences, Banaras Hindu University, 

Varanasi, Uttar Pradesh 221005, India 

Department of Botany, MMV, Banaras Hindu University, 

Varanasi, Uttar Pradesh 221005, India 

ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan, 

Almora, Uttarakhand 263601, India 

Department of Biotechnology, Bangabandhu Sheikh Mujibur 

Rahman Agricultural University, Gazipur 1706, Bangladesh 

production of organic acids, however, Enterobacter sp. was 

inactive. The effects of these three rhizobacteria were evaluated 

on wheat in alluvial soils of the Indo-Gangetic Plain 

by inoculation of plants with bacterial isolates either alone 

or in combinations in both pot and field conditions for two 

successive years. Rhizobacterial inoculation either alone 

or in consortium of varying combinations significantly 

(P ≤ 0.05) increased growth and yield of wheat compared 

to mock inoculated controls. A consortium of two or three 

rhizobacterial isolates also significantly increased plant 

height, straw yield, grain yield, and test weight of wheat in 

both pot and field trials compared to single application of 

any of these isolates. Among the rhizobacterial treatment, 

co-inoculation of three rhizobacteria (Enterobacter, M. 

arborescens and S. marcescens) performed best in promotion 

of growth, yield, and nutrient (N, P, Cu, Zn, Mn, and 

Fe) uptake by wheat. Taken together, our results suggest 

that co-inoculation of Enterobacter with S. marcescens and 

M. arborescens could be used for preparation of an effective 

formulation of PGP consortium for eco-friendly and 

sustainable production of wheat. 

Keywords Plant health · PGPR · P-solubilization · PGP 

activities · Crop productivity 

Introduction 

During the green revolution, high-yielding varieties with 

application of synthetic fertilizers and pesticides were 

introduced for increasing yields of crops. Indiscriminate 

use of hazardous synthetic fertilizers and pesticides 

caused environmental pollution and deteriorated soil health 

(Elkoca and others 2010). Moreover, production of crops 

largely depending on synthetic chemicals also decreased 

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the nutritional quality of the produce that ultimately affects 

human health. Therefore, it is a great challenge to search 

for an alternative to hazardous synthetic chemicals for ecofriendly 

and sustainable crop production with improvement 

of soil health for ensuring food and nutritional security 

of the growing population of the world. Application of 

organic fertilizers and pesticides such as composts, green 

manures, biofertilizers, and biopesticides has been suggested 

for ensuring sustainable crop production in agriculture 

(Ahmad and others 2016; Bahadur and others 2016; 

Verma and others 2015b; Yadav and Sidhu 2016; Yasin 

and others 2016; Zahedi 2016). The plant rhizosphere is 

considered as a hot spot as well as a battle field of diverse 

microorganisms, and most of them play a crucial role in 

plant nutrition through fixation of nitrogen and/or solubilization 

of soil minerals and production of plant growth-promoting 

substances. Discovery of the elite strains of plant 

growth-promoting bacteria from the plant rhizosphere and 

applying them as biofertilizers is a new trend of research 

in eco-friendly and sustainable crop production systems 

worldwide (Hayat and others 2010; Kaymak 2011). The 

plant growth-promoting rhizobacteria (PGPR) exert a 

positive effect on plant growth either by direct mechanisms 

such as solubilization of soil nutrients, fixation of 

nitrogen, and production of growth regulators (Kumar and 

others 2014) and/or indirect mechanisms such as stimulation 

of mycorrhizal development, competitive exclusion 

of pathogens, induction of systemic resistance in plants, 

or remediation of phytotoxic substances (Bashan and de- 

Bashan 2010). Biofertilizer refers to the product, carrierbased 

solid or liquid containing agriculturally useful living 

microorganisms when applied in soil or seeds to increase 

the fertility of the soil and consequently improve crop productivity. 

The PGPR genera such as Arthrobacter, Azotobacter, 

Azospirillum, Bacillus, Enterobacter, Caulobacter, 

Erwinia, Chromobacterium, Flavobacterium, Microbacterium, 

Pseudomonas, and Serratia have been used for 

formulation of biofertilizers to enhance plant growth and 

yield (Bahadur and others 2014; Das and Pradhan 2016; 

Dominguez-Nunez and others 2016; Dotaniya and others 

2016; Velazquez and others 2016; Verma and others 

2015a). Serratia marcescens is one of the PGPR which 

promotes plant growth by enhanced P-solubilization in the 

rhizosphere and production of phytohormones (Tripura 

and others 2007). Nitrogen (N) is one of the major essential 

plant nutrients involved in improving plant growth and 

yield (Jaiswal and others 2016; Kumar and others 2015, 

2016a; Sindhu and others 2016; Teotia and others 2016). 

The rhizobacteria that can fix atmospheric nitrogen are 

also known as diazotrophic bacteria. They are capable of 

transforming atmospheric nitrogen into fixed nitrogen, that 

is, inorganic compounds usable by plants. Many efficient 

strains of diazotrophic bacteria have been formulated as 

biofertilizers. The effective biofertilizers developed from 

elite strains of diazotrophic bacteria play crucial roles in 

reducing dependency of synthetic fertilizers in eco-friendly 

sustainable agriculture (Ashrafuzzaman and others 2009; 

Meena and others 2016c). A large body of literature is 

available on plant growth promotion by PGPR and their 

mode of actions (Mukerji and others 2006; Kumar and others 

2016b). However, discovery of novel and elite strains of 

bacteria having nitrogen-fixing and phosphate-solubilizing 

abilities and/or with other plant growth-promoting traits 

from the native crop plants is important for development of 

effective biofertilizers for a particular crop. 

Plant microbe associations that improve soil fertility and 

contribute to increased overall plant growth and health are 

receiving increased attention for use as microbial inoculants 

in agriculture (Lugtenberg and Kamilova 2009; Islam 

and Hossain 2013). Most of the micronutrients such as Fe, 

Zn, Cu, and Mn are essential for plants, humans, and animals 

and play important roles in improving human nutrition 

on a global scale (Masood and Bano 2016; Saha and 

others 2016b; Sharma and others 2016; Shrivastava and 

others 2016). Several lines of evidence suggest that application 

of PGPR significantly enhance growth promotion 

and yield of crop plants, such as wheat and maize (Shankar 

and others 2013), chickpea (Akhtar and Siddiqui 2009), 

chilli (Turan and others 2012), soybean (Fernandez and 

others 2007), and decrease the use of synthetic chemicals 

in crop production (Meena and others 2016c). However, the 

application of PGPR as a consortium of compatible strains 

has been more effective than their single application in the 

practical field (Meena and others 2016c). This study aimed 

to isolate some indigenous nitrogen-fixing and P-solubilizing 

rhizobacteria and evaluate their effects either singly 

and/or combined application to seeds on growth, yield, and 

nutrient uptake by wheat in alluvium soils of IGP of eastern 

Uttar Pradesh, India. 

Materials and Methods 

Isolation and Purification of Diazotrophic 

Rhizobacteria 

A total of 90 composite rhizosphere soil samples were collected 

from rice, wheat, vegetables, and agro-forestry fields 

from seven districts of the IGP of eastern Uttar Pradesh, 

India (82°59′ East, 25°15′ North and 82°33′ East, 25°8′ 

North) in 2010. The nitrogen-fixing rhizobacteria (NFR) 

were isolated on nitrogen-free Ashby medium (with agar) 

by a serial dilution technique followed by purification on 

the same solid medium with a repeated plating method 

(Schmidt and Belser 1982). Twenty-five pure isolates were 

obtained and tested for their N-fixing (Bremner 1965) and 

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P-solubilizing abilities (Gaur 1990) using standard protocols. 

Among them, the three most efficient NFR strains 

were selected based on their higher N-fixing capacities. 

These superior nitrogen-fixing bacteria were identified 

through 16S rDNA gene sequencing. 

Amplification of 16S rDNA Genes by Polymerase Chain 

Reaction (PCR) 

The universal primer was used for the amplification of 16S 

rDNA genes for all three rhizobacterial isolates. Primer 

was custom synthesized by Bangalore Genei, Bangalore, 

India. The 50 μL of reaction mixture consisted of 50 ng 

of genomic DNA, 2.5 U of Taq polymerase, 5 μL of 10× 

buffer (100 mMTris-HCl, 500 mM KCl pH 8.3), 200 μMd 

NTP, 1.5mM MgCl 2 , and 10 pmoles of each primer. The 

forward primer 27F (5′-AGA GTT TGA TCC TGG CTC 

AG-3′) and reverse primer 1492R (5′-TAC GGT TAC CTT 

GTT ACG ACT T-3′) were used (Narde and others 2004). 

The amplification was performed by PCR (PCR System 

2720, Applied Biosystems, Singapore) conditions-initial 

denaturation at 94 °C for 5 min, followed by 34 cycles 

of denaturation at 94 °C for 1 min, annealing at 52 °C for 

1.5 min, extension at 72 °C for 2 min, and a final extension 

at 72 °C for 7 min. 

Amplified PCR products (5 µL) were resolved on a 1.5% 

(w/v) agarose gel at 100 V for 45 min in 1× TAE buffer 

containing ethidium bromide (EtBr) along with about 

500 bp DNA ladder (Bangalore Genei Pvt., Ltd. Bangalore, 

India). The PCR product size was approximately 1500 bp. 

The PCR product was purified using a PCR purification kit 

(Bangalore Genei, Bangalore, India) for the sequencing of 

16S rDNA genes. 

Sequencing 

Sequencing of 16S rDNA was carried out at Bangalore 

Genei Pvt. Ltd., Bangalore, India. The 16S rDNA 

sequences were analyzed with the nucleotide database 

available at the GenBank using the BLASTN tool of NCBI 

(http://www.ncbi.nlm.nih.gov) for homology search, and 

then the 16S rDNA sequences of the selected strains were 

submitted to the NCBI GenBank for obtaining accession 

numbers (Table 1). 

Seed Treatment 

Seeds of the wheat variety HUW 234 were collected from 

Banaras Hindu University, Agriculture Farm, Varanasi, 

Uttar Pradesh, India. The seeds were surface sterilized 

using 0.1% HgCl 2 for 2 min followed by rinsing five times 

with sterilized double distilled water. Enterobacter, M. 

arborescens, and S. marcescens were grown in nutrient 

broth at 120 rpm in a shaking incubator at 30 °C for 48 h. 

The healthy wheat seeds were treated with 5-day-old broth 

cultures of each of the rhizobacterial strains singly and/or 

in combinations along with 1 mL of sticker solution (2.5 g 

gum acacia + 5 g sugar in 100 mL) at a rhizobacterial population 

at a range of about 10 7 to 10 8 CFU seed −1 . Untreated 

healthy wheat seeds were used as controls. 

Pot and Field Experiments 

To study the individual and combined effects of rhizobacterial 

strains, pot and field experiments were conducted 

with the wheat variety HUW 234 during Rabi season of 

2011–12 and 2012–13. Three rhizobacterial strains namely 

Enterobacter, M. arborescens, and S. marcescens were 

inoculated in seven treatment combinations including controls 

with four replications of each treatment. For the pot 

experiment, alluvial soil collected from the agricultural 

research farm, and BHU was sieved through a 10-mesh 

sieve and filled in approximately 5 kg of earthen pots lined 

with polythene. The recommended doses of fertilizers for 

N-P-K (120-60-60) were thoroughly mixed in required 

quantity of soil. The chemical properties of pot and field 

soil were pH (7.84 and 7.80), EC 0.164 and 0.161 dS m −1 , 

organic carbon 0.50 and 0.502% (Walkley and Black 1934), 

available N (0.095 g kg − 1 and 202.45 kg ha − 1 ) (Subbiah 

and Asija 1956), P (0.01 g kg − 1 and 19.28 kg ha − 1 ) (Olsen 

Table 1 Plant growth-promoting (PGP) activity of diazotrophic isolates under in vitro conditions 

Diazotrophic strains 

Nitrogen fixation mg 

Ng −1 carbon oxidized 

IAA at TP 100 µg mL − 1 pH of broth cultures P-solubilization 

of broth μg mL − 1 

Accession number 

2 day 2 day 

Control ND ND 7.00 ± 0.09 c ND NA 

Serratia marcescens 8.32 ± 0.39 a 21.29 ± 0.99 b 5.88 ± 0.11 b 101.04 ± 1.12 b KC453982 

Enterobacter 9.32 ± 0.57 b 27.06 ± 1.70 c 7.00 ± 0.10 c ND KC453983 

Microbacterium arborescens 8.89 ± 0.58 b 26.82 ± 1.63 c 5.67 ± 0.09 a 112.27 ± 1.44 c KC453984 

IAA indole acetic acid, TP tryptophan, ND not dedicated, NA not applicable, SD standard deviation. Data are average of four replicates ±SD. 

Mean with different letters in the same column differ significantly at P ≤ 0.05 (Fisher’s protected LSD) 

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and others 1954), and K 0.118 g kg − 1 and 250.16 kg ha − 1 

(Jackson 1973), respectively. Ten rhizobacteria-treated 

seeds were sown in each pot. The uninoculated seeds were 

also sown in the pots as a control. Three plants were maintained 

after full emergence of the first leaf in each pot. 

The pots were arranged in a complete randomized design 

(CRD). During the whole experimental period, recommended 

agronomic practices were followed. A field experiment 

was conducted following a randomized block design 

(RBD) in four replications with alluvial soil at a farmer’s 

field of Bhadohi district, Uttar Pradesh, India, with plots 

size 5 m × 2 m (10 m 2 ). Each plot was fertilized with N-P-K 

(120-60-60) ha − 1 . 

The rhizobacteria-treated seed @ 150 kg ha − 1 was sown 

to each plot in line with 20 × 20 cm distance between lines. 

Seeds were sown at a depth of about 3 cm using a seed 

drill. Untreated wheat seeds were sown in control plots. 

The plots were irrigated timely, and recommended agronomical 

intercultural practices were followed. At the time 

of maturity, pot and field crops were harvested and threshed 

after optimum (~12% moisture in grain) drying. The grain 

yield in terms of g pot − 1 and q ha −1 was recorded for pot 

and field experiments, respectively. The acquisition of N 

and P by grain and straw and contents of Cu, Zn, Mn, and 

Fe in grain were determined by following the procedure as 

described by Iswaran and Marwah (1980). 

Statistical Analysis 

The data were analyzed by one-way analysis of variance 

(ANOVA). The significant differences between means were 

compared using Fisher’s protected LSD test at P ≤ 0.05. 

Statistical analysis was performed using SPSS software 

version 16.0. 

Results and Discussion 

In Vitro Plant Growth Promotion (PGP) 

by Diazotrophic Rhizobacteria 

Twenty-five diazotrophic bacteria were isolated from the 

rhizospheric soils from IGP of eastern Uttar Pradesh, India. 

Among them, the three best nitrogen-fixing isolates were 

selected for further studies and identified as Enterobacter 

sp., Microbacterium arborescens, and Serratia marcescens 

by 16 S rDNA gene sequencing (Table 1). The accession 

numbers of these rhizobacterial strains (S. marcescens, 

KC453982; Enterobacter sp., KC453983; and M. arborescens, 

KC453984) were deposited in NCBI GenBank. In vitro 

PGP activities of these three rhizobacteria were quantitatively 

assessed and presented in Table 1. Among these bacteria, 

Enterobacter sp. produced the maximum (~27 µg mL − 1 ) 

IAA followed by M. arborescens (~26 µg mL −1 ) and S. 

marcescens (22 µg mL −1 ) at 100 µg mL −1 of tryptophan. 

IAA increases surface area and root branching in plants and 

facilitates nutrient uptake by plants. Jia and others (2013) 

demonstrated that diazotrophic rhizobacteria, S. marcescens 

(~17 µg mL − 1 ), and M. trichotecenolyticum (~19 µg mL −1 ) 

produce appreciable amounts of IAA at 0.1% tryptophan. 

Besides IAA production, all three rhizobacteria fixed higher 

amounts of atmospheric nitrogen in vitro. Both Enterobacter 

sp. (9.32 ± 0.57 mg Ng −1 carbon utilized) and M. arborescens 

(8.89 ± 0.58 mg Ng −1 carbon utilized) fixed significantly 

higher amounts of nitrogen than S. marcescens 

(8.32 ± 0.39 mg Ng − 1 carbon utilized) (P ≤ 0.05). Fixation of 

higher amounts of atmospheric nitrogen (2–15 mg Ng − 1 carbon 

oxidized) by Bacillus, Klebsiella, Pseudomonas, and two 

rhizobacterial strains of Enterobacter has also been reported 

by Fayez (1990). Both nitrogen fixation and IAA production 

by B. megaterium, A. chlorophenolicus, and Enterobacter 

have been reported by many investigators (Kumar and others 

2014; Maurya and others 2014; Saha and others 2016a). 

To see whether selected diazotrophic rhizobacteria can 

solubilize insoluble phosphorus, we evaluated their ability 

in broth culture using TCP as a source of insoluble P. 

Although S. marcescens (101.04 ± 1.12 μg mL −1 ) and M. 

arborescens (112.27 ± 1.44 μg mL −1 ) solubilized appreciable 

amounts of P from TCP 2 days after inoculation, Enterobacter 

sp. was practically inactive (Table 1). The solubilization 

of P by both rhizobacteria was linked to a significant 

decrease in pH of the culture broth indicating the production 

of low molecular weight organic acids by the bacteria. Solubilization 

of P from TCP in broth culture by the production 

of various organic acids (citric acid, oxalic acid, succinic 

acid, and glycolic acid) by PGPR has been reported (Chen 

and others 2006; Islam and Hossain 2013; Meena and others 

2013, 2016a). P-solubilization by PGPR genera Bacillus, 

Pseudomonas, and Serratia has widely been reported 

(Hameeda and others 2008; Islam and Hossain 2013). 

Effect of Diazotrophic Strains on Growth, Yield, 

and Test Weight Under Pot Experiment 

Application of rhizobacteria alone or in various combinations 

significantly (P ≤ 0.05) increased growth and yield of 

wheat compared to untreated controls (Table 2). Co-inoculation 

of Enterobacter with S. marcescens and M. arborescens 

significantly increased (up to 14% over control) plant 

height of wheat followed by dual inoculation (up to 11% 

over control) compared to control. Single inoculation of S. 

marcescens showed somewhat more plant height as compared 

to M. arborescens and Enterobacter. Enhancement of 

plant height of wheat by the application of S. marcescens 

and Enterobacter has been reported (Zinniel and others 

2002; Kumar and others 2014). 

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Table 2 Effect of diazotrophic strains on growth, yield, and test weight under pot and field experiments 

Treatments Pot experiment Field experiment 

Plant height at 

60 DAS 

Grain yield (g 

pot −1 ) 

Straw yield (g 

pot −1 ) 

Test weight 

(g) 

Plant height at 

60 DAS 

Grain yield (q 

ha −1 ) 

Straw yield (q 

ha −1 ) 

Test weight (g) 

Control 57.50 ± 0.29 a 4.71 ± 0.31 a 6.77 ± 0.03 a 34.60 ± 0.98 a 33.50 ± 0.03 a 31.80 ± 0.35 a 47.35 ± 1.80 a 33.85 ± 0.66 a 

Enterobacter 60.50 ± 0.29 b 6.43 ± 0.17 bc 9.27 ± 0.27 c 39.50 ± 1.50 bc 41.00 ± 0.22 cd 36.50 ± 0.87 b 55.42 ± 1.46 cd 35.99 ± 0.13 ab 

S. marcescens 62.50 ± 0.29 bc 5.34 ± 0.08 b 7.53 ± 0.23 ab 38.17 ± 0.89 b 40.00 ± 0.19 c 36.33 ± 0.21 b 53.78 ± 1.76 bc 34.63 ± 0.56 a 

M. arborescens 

Enterobacter 

+ S. 

marcescens 

Enterobacter 

+ M. 

arborescens 

Enterobacter 

+ S. 

marcescens+ 

M. 

arborescens 

60.00 ± 1.45 b 5.88 ± 0.81 bc 8.73 ± 0.06 bc 39.19 ± 2.27 b 37.00 ± 0.22 b 33.65 ± 0.49 a 49.69 ± 1.03 ab 34.10 ± 1.10 a 

64.00 ± 0.88 cd 7.35 ± 0.60 cd 11.02 ± 0.58 d 40.84 ± 0.65 c 44.00 ± 0.58 de 38.36 ± 0.74 cd 58.48 ± 2.28 cd 38.65 ± 0.55 c 

64.00 ± 0.58 cd 7.67 ± 0.58 cd 11.26 ± 0.90 d 41.65 ± 0.49 cd 43.00 ± 1.49 d 37.67 ± 0.45 bc 57.71 ± 1.12 cd 37.77 ± 1.21 bc 

65.50 ± 0.87 d 8.47 ± 0.78 d 12.09 ± 0.52 d 44.60 ± 2.14 d 45.00 ± 0.06 e 39.45 ± 0.61 d 59.77 ± 1.93 d 39.94 ± 0.61 c 

‘M.’, Microbacterium; ‘S.’, Serratia; ‘g’, grams; ‘DAS’, Days after sowing; Data are average of four replicates ± SD. Mean with different letters 

in the same column differ significantly at P ≤ 0.05 (Fisher’s protected LSD) 

Co-inoculation of Enterobacter with S. marcescens 

and M. arborescens significantly increased grain (up to 

80%) and straw yield (up to 79%) of wheat compared to 

untreated control (Table 2). Both single and dual inoculations 

of wheat with the rhizobacteria also significantly 

increased both straw and grain yield of wheat compared 

with untreated controls. Although either single or dual 

inoculation of rhizobacteria produced statistically similar 

higher grain yields of wheat over controls, dual inoculation 

of Enterobacter with S. marcescens or M. arborescens 

produced significantly higher straw yield than single inoculation 

of any bacteria. These significant enhancements of 

growth and yield of wheat by the PGPR might be linked 

with their PGP traits recorded under the in vitro experiments 

(Table 1) as well as synergistic effects due to coinoculation. 

Inoculation of winter wheat with Enterobacter 

cloacae has been shown to significantly enhance yield 

under a growth chamber (Renato de Freitas 2000). Zahir 

and others (2009) demonstrated that S. proteamaculans 

significantly increased plant height, grain yield, 100-grain 

weight, and straw yield of wheat under less than 15 dS m −1 

salinity. A significant increase in wheat grain yield by inoculation 

with various PGPR strains has also been demonstrated 

(Kumar and others 2014). In the current study, all 

treatments of PGPR either alone or in combination showed 

significant positive influences in test weights of wheat. 

Co-inoculation of Enterobacter with S. marcescens and 

M. arborescens gave significantly higher (up to 29%) test 

weight compared to the control plot. Increased test weight 

by the effects of PGPR has been reported (Khalid and others 

2004; Askary and others 2009). 

Effects of Diazotrophic Rhizobacterial Strains 

on Growth and Yield Attributes of Wheat in Field 

Conditions 

The effect of diazotrophic rhizobacterial strains either 

applied singly or in various combinations significantly 

increased plant height (10–34%) compared to control 

(Table 2). Similarly, co-inoculation of wheat with dual or 

triple combinations of Enterobacter, S. marcescens, and 

M. arborescens significantly promoted straw yield, grain 

yield, and test weight of wheat compared with untreated 

control (Table 2). Among the treatments, a combination of 

Enterobacter, S. marcescens, and M. arborescens showed 

the highest enhancement in growth (45 ± 0.06 cm), grain 

(39.45 ± 0.61 q/ha) and straw (59.77 ± 1.93 q/ha) yields 

of wheat which were more or less statistically similar to 

the application of dual inoculations of these diazotrophic 

rhizobacteria (Table 2). However, co-inoculation of Enterobacter 

with S. marcescens resulted in increased grain 

yield, straw yield, and test weight of wheat by 21, 24, and 

14% over untreated controls followed by a combination of 

Enterobacter with M. arborescens (up to 18, 22, and 11%). 

Single inoculation of Enterobacter showed slightly higher 

yield and test weight compared to S. marcescens and M. 

arborescens possibly due to its higher PGP activities as 

shown in the in vitro assays (Table 1). The plant growthpromoting 

effects of PGPR shown in this study might be 

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due to stimulated plant growth, absorption of nutrients, 

and their efficiency by the inoculated PGPR. Enhancement 

of growth, yield, and nutrient uptake by pearl millet and 

wheat by the co-inoculation of rhizobacterial inoculants, S. 

marcescens, Pseudomonas, and Enterobacter sp. has been 

reported (Hameeda and others 2006; Meena and others 

2015a). 

Effect of Diazotrophic Strains on N and P Uptake 

in the Pot Experiment 

To see whether uptake of essential plant nutrients is 

enhanced by the inoculation of PGPR alone or in combination, 

we analyzed contents of N and P in grain and straw 

of wheat. It revealed that total uptake of N and P was significantly 

increased by the rhizobacterial inoculation of 

wheat compared to untreated control (Table 3). The coinoculation 

of Enterobacter with S. marcescens and M. 

arborescens significantly increased N uptake by grain (up 

to 127%) and straw (up to 156%) of wheat over untreated 

controls. Similarly, P uptake was also increased by grain 

(up to 118%) and straw (up to 83%) of wheat over untreated 

controls due to inoculation with PGPR. However, co-inoculation 

of Enterobacter with S. marcescens caused higher 

amounts of N and P uptake than Enterobacter with M. 

arborescens which might be due to the better PGP activity 

of strains and their yield. The single inoculation of Enterobacter 

sp. gave somewhat higher N and P uptake by grain 

and straw than either S. marcescens or M. arborescens that 

can be explained on the basis of their content, yield, and 

PGP activity. The increased N content might be linked with 

the enhancement of different fractions of mineral N in soils 

and the PGP mechanisms through N 2 -fixation, which positively 

influenced plant growth and crop yields (Asghar and 

others 2004). The enhancement in available P content in 

soil shown in this study might be due to the higher solubilization 

of soil insoluble P from soils by S. marcescens 

and M. arborescens. Selvakumar and others (2008) demonstrated 

earlier that inoculation of S. marcescens and E. 

asburiae significantly enhanced N and P uptake by plants. 

Co-inoculation of plants with PGPR and enhancement of 

nutrient uptake by plants have been described in many 

reports (Abbasi and others 2011; Meena and others 2015a). 

Effect of Diazotrophic Strains on N and P Uptake 

in the Field Experiment 

Co-inoculation of Enterobacter with S. marcescens and M. 

arborescens consistently enhanced N and P uptake by grain 

and straw of wheat in the field experiment. As shown in the 

pot experiment, co-inoculation of these three bacteria gave 

the highest N and P uptake by the grain and straw of wheat 

which were significantly higher than untreated controls. 

Similarly, a combination of any two bacteria in seed inoculation 

also significantly increased N and P uptake by wheat 

compared to untreated control (Table 3). Among single 

inoculations, Enterobacter significantly increased N and P 

uptake by grain and straw of wheat over untreated control. 

Our results showed that nitrogen-fixing and P-solubilizing 

activities of the tested strains significantly influenced better 

N and P uptake by wheat which ultimately impacted on better 

growth and yield of wheat by the application of PGPR. 

Enhancement of growth, yield, and nutrient uptake in 

wheat by the application of nitrogen-fixing, P-solubilizing 

and IAA-producing bacteria has been reported (Khalid and 

others 2004). Enhancement of nutrient uptake in wheat by 

the application of S. marcescens and Enterobacter has also 

been demonstrated (Selvakumar and others 2008) (Fig. 1). 

Table 3 Effect of diazotrophic strains on N and P uptake by grain and straw under pot and field experiments 

Treatments Pot experiment Field experiment 

N uptake (g pot − 1 ) P uptake (g pot − 1 ) N uptake (kg ha − 1 ) P uptake (kg ha − 1 ) 

Grain Straw Grain Straw Grain Straw Grain Straw 

Control 6.91 ± 0.50 a 5.15 ± 0.31 a 1.31 ± 0.20 a 0.83 ± 0.01 a 45.93 ± 0.79 a 40.59 ± 1.95 a 8.32 ± 0.90 a 5.74 ± 0.22 a 

Enterobacter 11.57 ± 0.14 bc 10.51 ± 0.74 c 2.10 ± 0.05 bc 1.13 ± 0.06 bc 64.32 ± 1.86 d 51.56 ± 2.33 bc 11.26 ± 1.39 bc 6.83 ± 0.21 b 

S. marcescens 10.59 ± 1.29 b 7.80 ± 0.55 b 1.91 ± 0.27 bc 1.11 ± 0.01 bc 60.50 ± 1.20 c 49.72 ± 0.75 b 11.00 ± 0.26 b 6.52 ± 0.24 ab 

M. arborescens 8.75 ± 0.22 ab 7.23 ± 0.50 b 1.68 ± ± 0.02 ab 0.93 ± 0.03 ab 53.51 ± 1.20 b 47.67 ± 0.96 b 10.40 ± 0.08 b 6.27 ± 0.20 ab 

Enterobacter + S. 

marcescens 

Enterobacter + M. 

arborescens 

Enterobacter + S. 

marcescens + M. 

arborescens 

14.76 ± 1.45 d 11.77 ± 0.61 cd 2.30 ± 0.27 c 1.28 ± 0.16 cd 67.72 ± 0.74 de 56.28 ± 1.12 cd 12.69 ± 1.10 d 7.93 ± 0.60 cd 

13.81 ± 0.51 cd 11.25 ± 0.46 cd 2.19 ± 0.07 bc 1.20 ± 0.11 c 66.88 ± 0.61 de 55.33 ± 1.31 cd 12.41 ± 0.21 c 7.49 ± 0.28 c 

15.67 ± 1.61 d 13.20 ± 0.97 d 2.86 ± 0.13 d 1.52 ± 0.06 d 69.19 ± 1.03 e 58.08 ± 2.01 d 13.02 ± 0.75 d 8.52 ± 0.27 d 

‘M.’, Microbacterium; ‘S.’, Serratia; ‘g’, grams; Data are average of four replicates ± SD. Mean with different letters in the same column differ 

significantly at P ≤ 0.05 (Fisher’s protected LSD) 

1 3


Effect of Diazotrophic Strains on Micronutrient 

Content in Grain in the Pot and Field Experiments 

Co-inoculation of Enterobacter, S. marcescens, and 

M. arborescens significantly increased the contents of 

micronutrients, for example, Cu, Zn, Mn, and Fe, in wheat 

grain under both pot and field experiments compared to 

control (Fig. 2). Significantly increased micronutrient 

(Zn, Fe, and Cu) contents in grain of wheat due to inoculation 

with siderophore-producing PGPR strains has been 

Fig. 1 Effect of rhizobacterial strains on micronutrient contents 

in grain under pot conditions, ‘S.m.’, Serratia marcescens; ‘M.a.’, 

Microbacterium arborescens; Data are average of four replicates 

±SD. Mean with different letters in the same column differ significantly 

at P ≤ 0.05 (Fisher’s protected LSD) 

Fig. 2 Effect of rhizobacterial strains on micronutrient contents in 

grain under field conditions, ‘S. m.’, Serratia marcescens; ‘M. a.’, 

Microbacterium arborescens; Data are average of four replicates 

±SD. Mean with different letters in the same column differ significantly 

at P ≤ 0.05 (Fisher’s protected LSD) 

1 3


reported (Kumar and others 2014). The co-inoculation of 

Enterobacter with S. marcescens significantly enhanced 

Cu, Zn, Mn, and Fe contents in wheat grain by 56, 32, 52, 

and 18% in the pot and 43, 23, 48, and 16% in the field 

experiment. Co-inoculation of wheat with Enterobacter 

with M. arborescens also showed similar performances in 

micronutrient uptake by the wheat. However, single inoculation 

of rhizobacterial strains, Enterobacter and M. arborescens, 

displayed a significant effect over controls regarding 

micronutrient content in grain. The synergistic effects 

of PGPR inoculation leads to increased micronutrient content 

of wheat plants which might be due to a positive effect 

for improving the translocation of micronutrients from soils 

to plants. Micronutrient uptake from the rhizosphere soil is 

the first step in the process of accumulation in plants, prior 

to translocation to seed. These PGPR strains may be elucidated 

by their ability to enhance the nutrient availability in 

the soil system, which enhanced nutrient uptake by the crop 

(Abdel-Wahab and others 2008; Verma and others 2010). 

The micronutrient concentrations in grain for all treatments 

were slightly higher under the pot as compared to the field 

experiment, which could be due to reduced chelation, limited 

ion exchange, no leaching, and lack of weeds in the pot 

culture compared to the field conditions (Kumar and others 

2014; Meena and others 2015b). 

Conclusions 

Isolation of elite strains of PGPR and integration of a possible 

combination of synergistic bacteria in the consortium 

application to plants is crucial for the highest promotion 

of plant growth and yield. In the current study, we isolated 

and identified three potential diazotrophic bacteria 

(Enterobacter sp., S. marcescens, and M. arborescens) and 

applied them alone or in varying combinations to assess 

their effects on growth, yield, and nutrient uptake by wheat. 

Co-inoculation of these three PGPR consistently increased 

nutrient uptake, growth, and yield of wheat under both the 

pot and field experiments. Our results suggest that application 

of PGPR in a consortium with synergistic bacteria having 

multiple plant growth-promoting traits such as nitrogen 

fixation; P-solubilization and IAA production could be used 

for boosting wheat production in a sustainable manner. The 

findings of this research could be used for development of 

an eco-friendly and cost-effective technology for biofertilization 

of wheat which might reduce the application of 

chemical fertilizers and save fossil fuel consumption. 

Acknowledgements The authors are thankful to Uttar Pradesh 

Council of Agricultural Research for funding the research work. 

Thanks are also due to the Head, Department of Soil Science and 

Agricultural Chemistry, Institute of Agricultural Sciences, Banaras 

Hindu University, Varanasi, India, for providing the necessary facilities 

required for conducting the research work. 

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