Journal of Plant Nutrition

ISSN: 0190-4167 (Print) 1532-4087 (Online) Journal homepage: https://www.tandfonline.com/loi/lpla20

Effects of plant growth promoting microorganisms

inoculums on mineral nutrition, growth and
productivity of rice (Oryza sativa L.)

Esmaeil Bakhshandeh, Hemmatollah Pirdashti, Khadejeh Shahsavarpour

Lendeh, Zahra Gilani, Mohammad Yaghoubi Khanghahi & Carmine Crecchio

To cite this article: Esmaeil Bakhshandeh, Hemmatollah Pirdashti, Khadejeh Shahsavarpour

Lendeh, Zahra Gilani, Mohammad Yaghoubi Khanghahi & Carmine Crecchio (2020): Effects of
plant growth promoting microorganisms inoculums on mineral nutrition, growth and productivity of
rice (Oryza�sativa L.), Journal of Plant Nutrition, DOI: 10.1080/01904167.2020.1739297

To link to this article: https://doi.org/10.1080/01904167.2020.1739297

Published online: 12 Mar 2020.

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JOURNAL OF PLANT NUTRITION
https://doi.org/10.1080/01904167.2020.1739297

Effects of plant growth promoting microorganisms inoculums

on mineral nutrition, growth and productivity of rice
(Oryza sativa L.)
Esmaeil Bakhshandeha, Hemmatollah Pirdashtia, Khadejeh Shahsavarpour Lendeha,
Zahra Gilania, Mohammad Yaghoubi Khanghahib, and Carmine Crecchiob
a
Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural
Resources University, Sari, Iran; bDepartment of Soil, Plant and Food Sciences, University of Bari Aldo Moro,
Bari, Italy

ABSTRACT ARTICLE HISTORY

Two field experiments were carried out separately to evaluate the ability of Received 4 November 2019
three plant growth promoting microorganisms (PGPM, Pantoea ananatis, Accepted 27 January 2020
Enterobacter sp. and Piriformospora indica) as bio-fertilizer on rice (Oryza
KEYWORDS
sativa L.) productivity under different levels of potassium sulfate fertilizer
agronomic efficiency;
(PSF). The results clearly showed that the PGPM significantly enhance the physiological efficiency;
dry weight of rice plant tissues as compared with the control. Uptake of plant growth promoting
phosphorus and potassium in rice plant tissues and also nutrient use effi- microorganism; potassium
ciency (NUE) remarkably increased especially when the co-inoculation with sulfate fertilizer; rice
P. ananatis and P. indica in experiment I and the combined (seed þ see-
dling root) inoculation method in experiment II were used. Grain yield (GY)
increased linearly with the addition of PSF up to 110 kg ha1 for the co-
inoculation treatment (23% lower than the control), then remained con-
stant at its maximum (5348 kg ha1, 32% higher than the control) up to
180 kg ha1 in experiment I. Similarly, the maximum value of GY (5330 kg
ha1, 20% more than the control) was obtained at 100 kg ha1 PSF in
experiment II. Consequently, both co-inoculation and combined inoculation
methods could be selected as the best ways for improving NUE and plant
growth of rice in sustainable production systems.

Abbreviations: AE: agronomic efficiency; APE: agro-physiological efficiency;

ARE: apparent recovery efficiency; AS: anthesis stage; DW: dry weight; GLAS:
dry weight of green leaves at AS; GLPM: dry weight of green leaves at PM;
GrainPM: grain yield at PM; HI: harvest index; K: potassium; KSB: potassium
solubilizing bacteria; N: nitrogen; NUE: nutrient use efficiency; P: phos-
phorus; PE: physiological efficiency; PGPM: plant growth promoting micro-
organism; PM: physiological maturity; PSB: phosphate solubilizing bacteria;
PSF: potassium sulfate fertilizer; StAS: dry weight of stem at AS; StPM: dry
weight of stem at PM; StrAS: dry weight of straw at AS; StrPM: dry weight of
straw at PM; YLPM: dry weight of yellow leaves at PM

Introduction
Rice (Oryza sativa L.) is known as an important cereal crop for 50% of the world’s population,
particularly in developing counties where it is eaten as a staple food (Bakhshandeh, Pirdashti, and
Shahsavarpour Lendeh 2017). It has been cultivated in an area of 163 million ha1 around the

CONTACT Carmine Crecchio carmine.crecchio@uniba.it Department of Soil, Plant and Food Sciences, University of Bari
Aldo Moro, Via Amendola 165/a, 70125 Bari, Italy.
ß 2020 Taylor & Francis Group, LLC
2 E. BAKHSHANDEH ET AL.

world with an annual grain production of 749 million tonnes (Mt) in 2016 (FAO 2016).
However, rice production should increase 65% by the year 2020 as a result of rapid growth of
global population for meeting food security in the world (Fageria 2015).
Therefore, to achieve higher yields in crop production (e.g., rice), farmers usually use large
amounts of chemical fertilizers and pesticides which can lead to serious problems in the quality
of soil and environment (Bakhshandeh, Pirdashti, and Shahsavarpour Lendeh 2017).
Among essential plant nutrients, nitrogen (N), phosphorus (P) and potassium (K) are known
as three important micronutrients which have a great influence on the growth and development
of plants because they are involved in many physiological and biochemical processes. For
example, N is required for the production of chlorophyll, proteins, amino acids and nucleic acids
(Ohyama and Sueyoshi 2010), P is needed for cell division, energy metabolism and signal trans-
duction cascades (Bakhshandeh et al. 2015), K is required for the activity of enzymes, plant resist-
ance to stresses and also is a metabolic pathways regulator (Bakhshandeh, Pirdashti, and
Shahsavarpour Lendeh 2017).
FAO (2016) reported that the world demand for N, P and K fertilizer increased from 113.1,
42.7 and 31.0 Mt in 2014 to 119.4, 46.6 and 34.4 Mt in 2018 (estimated), respectively, with an
annual growth of 1.4, 2.2, and 2.6% in the world and 2.1, 6.3 and 4.0% in West Asia, including
Iran, respectively. In addition, 40–70% N, 80–90% P and 50–70% K of the total applied fer-
tilizers are unavailable for plant uptake and will be lost in the environment, as a result of the
strong binding force between these elements and other minerals, run-off, soil erosion and leach-
ing which is related to soil dynamics (Fageria, Gheyi, and Carvalho 2014; Yaghoubi Khanghahi
et al. 2019a; Zeraatpisheh et al. 2020). This level of loss in agrochemical inputs (i.e., fertilizers
and pesticides) not only declines crop productivity but also is harmful for the sustainable agricul-
ture (Meena et al. 2017). Therefore, many researchers have made efforts to introduce appropriate
ways for reducing chemical fertilizers, improving nutrient use efficiency (NUE) along with mini-
mizing negative impacts of pollutants in today’s crop production systems (Bakhshandeh et al.
2015; Delshadi, Ebrahimi, and Shirmohammadi 2017).
Application of plant growth promoting microorganisms (PGPM) as biofertilizer is introduced
as an effective way to reduce agrochemical fertilizers, improve soil quality and support an envir-
onment-friendly crop productivity in the last decade (Bakhshandeh et al. 2015; Bakhshandeh,
Pirdashti, and Shahsavarpour Lendeh 2017). The most important PGPM belong to the
Azospirillium, Azotobacter, Bacillus, Burkholderia, Enterobacter, Erwinia, Pantoea, Pseudomonas,
Rhizobium and Serratia genera for bacteria and Aspergillus and Glomus mosseae for fungi which
are living immediately around the rhizosphere (affected by root exudates) and can nicely influ-
ence plant growth and health interacting at macro and micro level (Huang et al. 2017; Lopez-
Gomez et al. 2016; Bakhshandeh et al. 2015, 2020).
Microbial associations (i.e., PGPM) are vital components of soil in sustainable agricultural eco-
systems, play a key role in soil quality and nutrient cycle and can be useful for plant productivity
(Bakhshandeh, Francaviglia, and Renzi 2019). Generally, PGPM use several direct and indirect
mechanisms to regulate the nutrient flow in soil and to promote plant growth (Meena et al.
2016). Some mechanisms are phytohormone production (e.g., auxin, cytokinin, gibberellin and
kinetin), N fixation, solubilization of organic and inorganic minerals (e.g., P, K, Zn, Fe and etc)
and synthesis of bio-control agents like siderophore, hydrogen cyanide, antibiotics and enzymes
(Bakhshandeh, Pirdashti, and Shahsavarpour Lendeh 2017; Meena et al. 2017).
Numerous studies clearly documented positive effects of PGPM on plant growth and nutrient
uptake to date. For example, rice seed inoculations with phosphate solubilizing bacteria (PSB) sig-
nificantly increased plant height (10.8-15.1%), biomass (27.4-65.3%) and K uptake by leaves
(35.5-76.9%), stem (17.6-52.9%) and root (25.0-75.0%), depending on the PSB strain compared to
the control (Bakhshandeh, Pirdashti, and Shahsavarpour Lendeh 2017). It has also been reported
that application of potassium solubilizing bacteria (KSB) significantly increased K uptake by grain
 JOURNAL OF PLANT NUTRITION 3

in rice plants, which was 2.7 times more than non-inoculated plants (Yaghoubi Khanghahi et al.
2019b). In other study, Rodrigues et al. (2008) showed that rice plants inoculated with PGPM
had higher grain dry matter accumulation (7–11%) compared to the control. Yaghoubi
Khanghahi et al. (2018) indicated that KSB inoculation significantly increased grain yield in rice
20-38% in a pot experiment and 20-52% in a field experiment.
NUE is an important ecological parameter as it integrates a variety of physiological processes
related to plants nutrients generally used for the production of biomass and grain (Ramesh et al.
2014; Suprapta, Maulina, and Khalimi 2014). NUE can be optimized by fertilizer management
practices that apply nutrients at the right rate in both conventional and organic farming. In add-
ition, a balance between optimal NUE and optimal crop productivity is necessary (Naeem,
Ansari, and Gill 2017). PGPM alternatively, can improve NUE through enhancing nutrient bio-
availability and promote nutrient acquisition by plants (Pii et al. 2015). Yaghoubi Khanghahi
et al. (2018) reported that the bacterial inoculums of rice plants enhanced agronomic efficiency
(AE), apparent recovery efficiency (ARE), physiological efficiency (PE), agro-physiological effi-
ciency (APE), internal utilization efficiency (UE), partial factor productivity (PFP) and partial
nutrient balance (PNB).
The objectives of this study, therefore, were to determine (i) the effect of three effective PGPM
including Pantoea ananatis, Enterobacter sp. and Piriformospora indica as bio-fertilizer on rice
productivity at different levels of potassium sulfate fertilizer (PSF); (ii) the amount of P and K
uptake by rice plant tissues; (iii) the NUE changes when PGPM and PSF applied together; (iv)
the amounts of PSF required for achieving the maximum yields in rice.

Materials and methods

Field experiments
Two field experiments were separately conducted in a split-plot arrangement based on a random-
ized complete block design with three replicates in Mazandaran province in 2016. The climate
was Mediterranean with a mean annual temperature 17.9  C and mean annual precipitation
789 mm. Both experiments were conducted under optimal agronomic conditions based on
local recommendations (see more details in Table 1). Plots were irrigated with a water depth of
4-5 cm from transplanting until two weeks before harvesting time in both experiments
(83 days after transplanting). Some information about the experiments is presented in Table 1.
In both experiments, different levels of PSF were used as the main plots and the various inocu-
lation methods served as the sub-plots (see more details in Table 1). In total, we had 48 plots (4
levels of PSF  4 inoculation methods  3 replications) in experiment I and 72 plots (6 levels of
PSF  4 inoculation methods  3 replications) in experiment II. Each main plot size was 10 m
long and 5 m width (50 m2) related to PSF levels and different subplots (each subplot was 5 m
long  2.5 m width ¼ 12.5 m2) related to the inoculation methods, which were defined within
each of the main plot. Rice seedlings were cultivated with a row spacing of 20 cm, included 12
rows with four and three seedlings hill in experiment I and II, respectively. The distance between
each hill on the rows was 20 cm.

Main properties of the microorganisms

In these experiments we used three microorganisms, including Enterobacter sp. (KM977992) and
P. ananatis (KM977993) which were grown in nutrient broth (NB; Scharlau, Spain; 8 g l1)
medium as followed Bakhshandeh et al. (2015) to a final density of 107 CFU ml1 as well as an
endophyte fungus P. indica which was grown in Kafer medium to a final density of 109 CFU
ml1 as in Sherameti et al. (2005). All microorganisms were provided by Genetics and
4 E. BAKHSHANDEH ET AL.

Table 1. Summary of agronomic practices and measurements in two field experiments.

Experiment I
Location Jouybar (located at 36 330 N, 53 E and
25.7 m asl)
Rice cultivar Tahrom Mahalli
Previous culture Fallow
Soil type Clay loam
Sand (24%), Clay (33%), Silt (43%)
Soil properties Organic carbon of 0.59%; pH of 7.75;
electrical conductivity of 3.15 dS
m1; total nitrogen of 0.13%;
available P of 14.2 mg kg1 and
available K of 92 mg kg1
Sowing date 6 May 2016
Agronomic practices Weeds controlled by hand (twice
during the experiment) and
chemically using butachlor
Chilo suppressalis controlled by
Trichogramma cards
Naranga aenescens controlled by
Malathion
Magnaporthe grisea controlled by
Tricyclazole
Fertilization 50 kg ha1 of triple super phosphate
150 kg ha1 of urea (100 kg ha1 at
transplanting time and 50 kg ha1 at
booting stage)
Treatments (T) four levels of PSF (0, 60, 120 and
180 kg ha1)
four levels of inoculation;
(EITI) control (non-inoculation)
(EITII) inoculation with P. ananatis
(EITIII) inoculation with P. indica
(EITIV) co-inoculation (EITII þ EITIII)
Measurements Anthesis and physiological maturity
PSF: potassium sulfate fertilizer (contained at least 44% soluble K).
Both are low-yielding rice cultivars and selected because of their high grain quality in northern Iran.
Experiment II
Babol (located at 36 460 N, 52 560 E
and 25 m asl)
Tahrom Hashemi
Fallow
Sandy loam
Sand (73%), Clay (20%), Silt (7%)
Organic carbon of 1.59%; pH of 7.76;
electrical conductivity of 0.63 dS
m1; total nitrogen of 0.16%;
available P of 39 mg kg1 and
available K of 125 mg kg1
7 May 2016
Weeds controlled by hand (twice
during the experiment) and
chemically using butachlor
Chilo suppressalis controlled by
Trichogramma cards
Naranga aenescens controlled by
Malathion
Magnaporthe grisea controlled by
Tricyclazole
50 kg ha1 of triple super phosphate
120 kg ha1 of urea (50 kg ha1 at
transplanting time, 35 kg ha1 at
three weeks after tillering stage and
35 kg ha1 at booting stage)
Six levels of PSF (0, 25, 50, 75, 100 and
125 kg ha1)
four methods of inoculation;
(EIITI) control (non-inoculation)
(EIITII) pre-germinated seed
inoculation with Enterobacter sp. in
the nursery
(EIITIII) seedling root inoculation with
Enterobacter sp. before transplanting
(EIITIV) combined method (EIITII
þ EIITIII)
Anthesis and physiological maturity

Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari, Iran. They are known as mul-
tiple plant growth promoting microorganisms (PGPM). Enterobacter sp. and P. ananatis were
able to solubilize insoluble P (Ca3(PO4)2, 254 and 172 mg ml1 after 5 days at 28  C, respectively),
K (mica, 33.6 and 38.9 mg ml1 after 25 days at 28  C, respectively) and production of indole-3-
acetic acid (IAA, 3 and 8 mg ml1 after 3 days at 28  C, respectively) (see more details in
Bakhshandeh et al. 2014, Bakhshandeh, Pirdashti, and Shahsavarpour Lendeh 2017). P. indica was
also known as an effective plant growth promoting mycorrhizal fungus whole characteristics are
fully described in Varma et al. (2012).

Soil characteristics
Some physiochemical properties of the soils (at a depth of 0-30 cm) are shown in Table 1. Soil P
was determined using the method of Olsen et al. (1954) and soil K was extracted by ammonium
acetate method (Page, Miller, and Keeney 1982). Triple super phosphate (TSP) was used before
transplanting and also urea was top-dressed twice in experiments II and three times in
 JOURNAL OF PLANT NUTRITION 5

experiments I (see more details in Table 1). Furthermore, different levels of PSF were applied
before transplanting in both experiments.

Inoculation treatments
In experiment I, roots of rice seedling (35 days old with 4-5 fully leaves) were separately inocu-
lated with P. ananatis (EITII) and P. indica (EITIII), which were diluted in water to a final density
of 106 and 108 CFU ml1, respectively. A co-inoculation treatment with P. ananatis þ P. indica
(1:1) was also used as EIITIV. Rice seedling roots were dipped into the suspensions, for 12 h at
room temperature (RT) before transplanting in experiment I. Roots of rice seedling were also
treated in the same way with non-inoculated NB mediums as a control in both experiments (as
EITI and EIITI, respectively).
In experiment II, rice seeds were firstly dipped in water for 24 h and then drained off the
water and kept in warm conditions for 5 days until seed germination occurred. Pre-germinated
seeds (with a root length 3 mm) were inoculated with Enterobacter sp. suspension which was
provided in the NB medium. Briefly, 700 g pre-germinated seeds were dipped into 4 l water con-
taining 400 ml of the suspension with a density of 106 CFU ml1 for 5 h, and then the seeds were
cultivated in a nursery with a distance 3 mm from each other. Thirty days after planting, rice
seedlings which were grown in optimal agronomic conditions based on local recommendations in
the nursery, were directly used as EIITII treatment. For EIITIII treatment, the roots of rice seedling
(4-5 fully expanded leaves) were dipped into the bacterial suspension with a density of 106
CFU ml1, for 12 h at RT before transplanting in the paddy field. We used a combination
method of EIITII and EIITIII as EIITIV treatment (namely, root rice seedlings produced in EIITII
were treated again using the bacterial suspension of EIITIII treatment).

Measurements of agronomical and physiological traits

In both experiments at anthesis stage (AS) and physiological maturity (PM), we randomly
selected seven rice hills to measure dry weight (DW) of rice plant tissues (green and yellow
leaves), stems and grain, separately). For this purpose, the tissues were oven-dried at 70  C until
constant weight. Harvest index (HI) for K and P was calculated using the model suggested by
Bakhshandeh, Pirdashti, and Shahsavarpour Lendeh (2017). In order to determine K and P con-
centration into the rice plant tissues (leaves, stems and grain, separately) the method suggested by
the International Center for Agricultural Research in the Dry Areas was used (Estefan, Sommer,
and Ryan 2013). Nutrient uptake (NU) in each tissue was calculated by the following equation
[NU (kg ha1) ¼ (Nutrient concentration (%)  DW of tissue (kg ha1))/100]. We also used
5.95 as a conversion factor to convert total N content (%) to protein (%) in rice grain (Arab,
Wittler, and Schettler 2012). Furthermore, in both experiments we determined some important
parameters related to NUE, such as physiological efficiency which was calculated by the following
equation {PE (kg kg1) ¼ [(BYf – BYuf)/(NUf – NUuf)]}, agro-physiological efficiency calculated
by {APE (kg kg1) ¼ [(GYf – GYuf)/(NUf – NUuf)]}, agronomic efficiency calculated by {AE (gr
gr1)¼ [(GYf – GYuf)/Nf]} and apparent recovery efficiency calculated by {ARE (%) ¼ [(NUf –
NUuf)/Nf]  100} (Fageria, Dos Santos, and Coelho 2011; Naeem, Ansari, and Gill 2017;
Yaghoubi Khanghahi et al. 2018). In the above equations, BY, GY, NU and N indicate the bio-
logical yield, grain yield, nutrient uptake in grain plus straw and the amount of applied nutrient,
respectably, and also the f and uf indices show values at the fertilized and unfertilized plots,
respectively.
6 E. BAKHSHANDEH ET AL.

Statistical analysis
All statistical analyses were performed using the statistical analysis system (SAS) 9.4 (SAS
Institute, Cary, NC, USA) and figures were drawn by SigmaPlot 11 software. Analysis of variance
was performed by GLM procedure and the least significant difference (LSD) values were used to
compare the means of treatments. Regression analysis was also conducted to describe rice plant
response to the studied treatments.

Results
Dry weight of rice tissues
The results of rice tissues DW affected by different levels of PSF, various PGPM and different
inoculation methods are summarized in Table 2. Our results showed that DW of rice tissues sig-
nificantly enhanced with the increasing PSF in both experiments although there was an exception
for DW of stem at AS (StAS), DW of straw at AS (StrAS), DW of green leaves at PM (GLPM) and
DW of yellow leaves at PM (YLPM) in experiment II (P > 0.05). The range of increase in DW of
green leaves at AS (GLAS), StAS, StrAS, GLPM, YLPM, DW of stem at PM (StPM), DW of straw at
PM (StrPM) and grain yield at PM (GrainPM) was 3.12-43.0, 2.11-14.7, 3.82-21.1, 16.0-83.8, 0.59-
26.8, 0.67-24.3 and 3.41-31.6% as compared to the control, respectively, with the addition of PSF
in both experiments (Table 2). On the other hand, the maximum values of these traits were
observed at 180 and 125 kg ha1 PSF in experiment I and II, respectively. The minimum values
of YLPM also obtained at 120 and 75 kg ha1 PSF in experiment I and II, respectively (14.3 and
15.3% lower than the control, respectively) and their values increased with the addition of PSF
which can be due to an increase in biomass production at higher levels of PSF (Table 2).
In addition, higher amounts of DW of rice tissues were observed in the presence of PGPM
relative to the control in experiment I. Although the effect of PGPM on the rice DW was varied
but the maximum GrainPM (4904 kg ha1, averaged for all PSF levels) was obtained when the
co-inoculation treatment with P. ananatis and P. indica (EITIV) was applied than others
(Table 2). In experiment II, the highest amount of DW at AS was obtained from the seedling
root inoculation with Enterobacter sp. before transplanting (EIITIII) which was about 8-14% more
than those in the control. Moreover, the maximum DW of rice tissues at PM belonged to the
combined inoculation method (EIITIV), although it did not have a significant difference with
other inoculation methods (Table 2).
An example of the changes in GrainPM (Figure 1a,b) and StrPM (Figure 1c,d) with PSF was
quantified using the regression analysis method suggested by Bakhshandeh, Pirdashti, and
Shahsavarpour Lendeh (2017). In each panel we compared the best treatment from each experi-
ment (EITIV and EIITIV in the experiment I and II, respectively) with the control conditions
(Figure 1a-d). According to the results, at all PSF levels, GrainPM and StrPM values were signifi-
cantly higher than the control when PGPM were applied in both experiments (Figure 1a,b). In
fact, the GrainPM increased linearly with the addition of PSF up to 110 kg ha1 for the co-inocu-
lation treatment (PSF application reduced by 23% than the control) and then remained constant
at its maximum value (5348 kg ha1, 32% higher than the control) up to 180 kg ha1 in the
experiment I. Similarly, the maximum value of GrainPM (5330 kg ha1, 20% more than the con-
trol) was obtained at 100 kg ha1 PSF in the experiment II (Figure 1a,b, Table 3). A similar trend
was observed in StrPM, as an example in the best treatments, StrPM increased linearly with the
rate of 14.3 kg for experiment I and 4.7 kg for experiment II until 134 and 80 kg ha1 PSF,
respectively, then remained constant at the maximum value in both experiments (Figure 1c,d,
Table 6). In fact, the response of rice plants to PSF in experiment I was more than experiment II
which can be resulted in a difference soil properties such as low available nutrients and high elec-
trical conductivity in the soil in experiment I (Table 1).
Table 2. Analysis of variance and means comparsion on the studied traits related to rice organs dry weight under different levels of potassium sulfate fertilizer (PSF, kg ha1) and inocula-
tion with three plant growth promoting microorganisms (PGPM) and various inoculation methods (IM).
Dry weight at anthesis Dry weight at physiological maturity
Sources Degree of Green leaves Stem Straw Green leaves Yellow leaves Stem Straw Grain
Experiments of variation freedom (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1)
Experiment I F-test
PSF 3        
PGPM 3        
PGPM  PSF 9        
CV (%) 6.11 5.64 7.27 4.21 5.52 6.73 5.40 4.21
Means comparison†
PSF (kg ha1) 0 1558 d 5352 d 6912 d 154 d 680 a 5906 c 6744 c 3790 d
60 1758 (þ12.8)c 5630 (þ5.17)c 7388 (þ6.89)c 242 (þ51.2)c 578 (15.0)b 6520 (þ10.4)b 7342 (þ8.87)b 4420 (þ16.6)c
120 2200 (þ41.4)b 6060 (þ13.2)b 8264 (þ19.6)b 266 (þ72.7)b 584 (14.3)b 7404 (þ25.4)a 8256 (þ22.4)a 4898 (þ29.2)b
180 2220 (þ43.0)a 6142 (þ14.7)a 8372 (þ21.1)a 284 (þ83.8)a 608 (10.6)b 7488 (þ26.8)a 8382 (þ24.3)a 4986 (þ31.6)a
PGPM EITI 1702 d 5370 c 7072 c 183 c 654 a 6588 b 7426 b 4058 c
EITII 1958 (þ15.1)b 5892 (þ9.72)b 7850 (þ11.0)b 261 (þ42.6)a 598 (8.41)b 6970 (þ5.79)a 7830 (þ5.45)a 4566 (þ12.5)b
EITIII 1924 (þ13.1)c 5892 (þ9.72)b 7816 (þ10.5)b 239 (þ30.8)b 590 (9.83)b 6940 (þ5.33)a 7770 (þ4.63)a 4566 (þ12.5)b
EITIV 2160 (þ27.3)a 6034 (þ13.4)a 8200 (þ15.9)a 263 (þ43.7)a 610 (6.57)b 6822 (þ3.54)a 7696 (þ3.64)a 4904 (þ20.8)a
Experiment II F-test
PSF 5  Ns Ns Ns Ns   
IM 3 Ns    Ns   
IM  PSF 15 Ns Ns Ns Ns Ns Ns Ns Ns
CV (%) 8.95 6.65 6.07 10.1 12.4 7.57 7.60 8.47
Means comparison
PSF (kg ha1) 0 820 ab 5170 a 5919 a 465 a 817 a 6536 c 7818 c 4232 c
25 876 (þ6.79)ab 5570 (þ7.73)a 6446 (þ8.90)a 542 (þ16.5)a 754 (7.72)a 6574 (þ0.59)bc 7871 (þ0.67)bc 4376 (þ3.41)c
50 846 (þ3.12)ab 5466 (þ5.72)a 6286 (þ6.20)a 539 (þ16.0)a 722 (11.6)a 6872 (þ5.15)abc 8135 (þ4.05)abc 4402 (þ4.02)bc
75 849 (þ3.57)ab 5310 (þ2.71)a 6160 (þ4.07)a 575 (þ23.6)a 691 (15.3)a 6785 (þ3.82)abc 8052 (þ2.99)abc 4604 (þ8.81)bc
100 849 (þ3.51)b 5279 (þ2.11)a 6146 (þ3.82)a 589 (þ26.7)a 771 (5.66)a 6959 (þ6.48)ab 8320 (þ6.42)ab 4782 (þ13.0)ab
125 951 (þ16.0)a 5650 (þ9.28)a 6602 (þ11.5)a 600 (þ29.0)a 804 (1.65)a 7022 (þ7.44)a 8426 (þ7.78)a 5002 (þ18.2)a
IM EIITI 836 a 5162 b 5999 b 462 c 767 a 6283 b 7513 b 4054 b
EIITII 844 (þ0.92)a 5186 (þ0.47)b 6031 (þ0.54)b 547 (þ18.3)b 744 (2.95)a 6956 (þ10.7)a 8248 (þ9.78)a 4706 (þ16.0)a
EIITIII 908 (þ8.59)a 5902 (þ14.3)a 6810 (þ13.5)a 566 (þ22.3)ab 770 (þ0.35)a 6827 (þ8.65)a 8163 (þ8.65)a 4648 (þ14.6)a
EIITIV 829 (0.91)a 5380 (þ4.23)ab 6200 (þ3.35)ab 632 (þ36.7)a 759 (1.07)a 7098 (þ12.9)a 8490 (þ13.0)a 4856 (þ19.7)a
,  and Ns represent values significant at 0.01, 0.05 probability level and non-significant based on the least significant difference test (LSD), respectively. CV is the coefficient of variation
which was related to overall data.
†
Means with the same letter are not significantly different at the probability level of 0.05. Values in parentheses indicate percent change (posetive and negative) in the respective studied
traits in comparison with the control condition. EITI, EITII, EITIII and EITIV are the control, inoculation with P. ananatis, inoculation with P. indica and co-inoculation (EITII þ EITIII) in the
experiment I, and EIITI, EIITII, EIITIII and EIITIV are the control, pre-germinated seed inoculation with Enterobacter sp. in the nursery, seedling root inoculation with Enterobacter sp. before
JOURNAL OF PLANT NUTRITION

transplanting and combined method (EIITII þ EIITIII) in the experiment II, respectively.
7
8 E. BAKHSHANDEH ET AL.

Figure 1. Effect of potassium sulfate fertilizer on rice grain yield (A and B), straw yield (C and D), phosphorus uptake in grain (E
and F) and potassium uptake in grain (G and H) in the experiment I (A, C, E and G) and the experiment II (B, D, F and H). The cul-
tivars were ‘Tahrom Mahalli’ and ‘Tahrom Hashemi’ for the experiment I and II, respectively. Data represent the mean ± standard
error of the mean (n ¼ 3). The solid lines are the fitted models to the control data and the dash lines represent regression lines
which were fitted to the best treatment in each experiment (the co-inoculation with P. ananatis and P. indica for the experiment
I (EITIV) and the combined method [pre-germinated seed inoculation with Enterobacter sp. in the nursery þ seedling root inocula-
tion with Enterobacter sp. before transplanting] for the experiment II (EIITIV)). All regression coefficients are presented in Table 6.

Phosphorus uptake by rice tissues

In experiment I, the amount of P uptake at AS (by GLAS, StAS and StrAS) and at PM (GLPM and
GrainPM) reached a maximum value at 120 kg ha1 PSF, which was about 46.5, 14.4, 22.1, 143
and 40.4% higher than those in the control, respectively (Table 3). However, the maximum P
Table 3. Analysis of variance and means comparsion on the studied traits related to the phosphorus uptake by different rice tissues under different levels of potassium sulfate fertilizer
(PSF, kg ha1) and inoculation with three plant growth promoting microorganisms (PGPM) and various inoculation methods (IM).
P uptake at anthesis P uptake at physiological maturity
Sources Degree Green leaves Stem Straw Green leaves Yellow leaves Stem Straw Grain
Experiments of variation of freedom (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1)
Experiment I F-test
PSF 3  Ns   Ns   
PGPM 3     Ns Ns  
PGPM  PSF 9 Ns Ns Ns  Ns Ns Ns Ns
CV (%) 10.9 17.6 14.5 14.7 19.4 19.6 15.8 11.3
Means comparison†
PSF (kg ha1) 0 20.39 b 64.13 a 84.52 b 0.145 b 0.628 a 4.303 b 5.017 b 9.540 b
60 23.09 (þ13.2)b 64.85 (þ1.12)a 89.98 (þ6.46)ab 0.322 (þ122.6)a 0.554 (11.7)a 4.998 (þ16.1)ab 5.906 (þ17.7)ab 12.04 (þ26.2)ab
120 29.89 (þ46.5)a 73.37 (þ14.4)a 103.3 (þ22.1)a 0.351 (þ143.0)a 0.475 (24.4)a 5.158 (þ19.8)ab 5.984 (þ19.2)ab 13.39 (þ40.4)a
180 28.77 (þ41.0)a 67.54 (þ5.32)a 96.92 (þ14.6)ab 0.325 (þ124.5)a 0.490 (21.9)a 6.616 (þ53.7)a 7.378 (þ47.0)a 13.20 (þ38.3)a
PGPM EITI 20.47 b 53.82 b 75.24 b 0.157 c 0.587 a 4.509 a 4.94 b 9.50 b
EITII 27.32 (þ33.5)a 66.77 (þ24.0)a 94.09 (þ25.0)a 0.286 (þ82.36)b 0.514 (12.3)a 5.741 (þ27.3)a 6.561 (þ32.8)a 12.41 (þ30.6)a
EITIII 25.48 (þ24.5)a 76.25 (þ41.6)a 103.3 (þ37.2)a 0.282 (þ79.92)b 0.517 (11.9)a 5.177 (þ14.8)a 5.920 (þ19.8)ab 12.08 (þ27.1)a
EITIV 28.48 (þ39.1)a 74.02 (þ37.5)a 102.5 (þ36.2)a 0.417 (þ165.7)a 0.529 (9.88)a 5.917 (þ31.2)a 6.953 (þ40.7)a 14.18 (þ49.2)a
Experiment II F-test
PSF 5    Ns Ns   
IM 3     Ns   
IM  PSF 15 Ns   Ns Ns   Ns
CV (%) 15.2 17.0 12.7 18.3 18.0 17.5 15.3 15.5
Means comparison
PSF (kg ha1) 0 13.57 b 44.87 b 58.44 b 0.774 a 1.057 a 8.012 b 9.843 b 13.54 b
25 13.58 (þ0.10)b 51.29 (þ14.3)b 64.86 (þ10.9)b 0.941 (þ21.55)a 0.883 (16.4)a 8.087 (þ0.94)b 9.910 (þ0.68)b 14.28 (þ5.46)b
50 13.73 (þ1.17)ab 51.78 (þ15.4)b 65.51 (þ12.1)b 0.875 (þ12.94)a 0.884 (16.3)a 9.023 (þ12.6)b 10.78 (þ9.54)b 13.79 (þ1.91)b
75 13.85 (þ2.06)ab 50.87 (þ13.3)b 64.72 (þ10.7)b 1.045 (þ34.92)a 0.894 (15.3)a 9.222 (þ15.1)b 11.16 (þ13.3)b 14.98 (þ10.6)ab
100 14.01 (þ3.24)ab 59.05 (þ31.6)ab 71.05 (þ21.5)ab 1.105 (þ42.68)a 1.036 (1.95)a 13.94 (þ74.0)a 16.09 (þ63.4)a 15.73 (þ16.2)ab
125 15.06 (þ10.9)a 75.18 (þ67.5)a 90.24 (þ54.4)a 1.102 (þ42.35)a 1.054 (0.25)a 15.99 (þ99.5)a 18.24 (þ85.3)a 16.51 (þ21.9)a
IM EIITI 11.82 b 35.61 c 47.42 c 0.734 c 1.008 a 7.067 c 8.77 c 12.44 c
EIITII 13.79 (þ16.6)a 50.43 (þ41.6)b 64.21 (þ35.4)b 0.940 (þ28.05)b 0.992 (1.61)a 11.55 (þ63.4)b 13.48 (þ53.7)b 15.19 (þ22.1)ab
EIITIII 14.98 (þ26.7)a 69.00 (þ93.8)a 83.98 (þ77.0)a 1.003 (þ36.63)b 0.969 (3.88)a 11.12 (þ57.3)b 13.13 (þ49.7)b 14.96 (þ20.3)b
EIITIV 13.94 (þ18.0)a 66.99 (þ88.1)a 80.94 (þ70.6)a 1.217 (þ65.82)a 0.970 (3.80)a 13.11 (þ85.5)a 15.30 (þ74.4)a 16.63 (þ33.7)a
,  and Ns represent values significant at 0.01, 0.05 probability level and non-significant based on the least significant difference test (LSD), respectively. CV is the coefficient of variation
which was related to overall data.
†
Means with the same letter are not significantly different at the probability level of 0.05. Values in parentheses indicate percent change (posetive and negative) in the respective studied
traits in comparison with the control condition. EITI, EITII, EITIII and EITIV are the control, inoculation with P. ananatis, inoculation with P. indica and co-inoculation (EITII þ EITIII) in the
experiment I, and EIITI, EIITII, EIITIII and EIITIV are the control, pre-germinated seed inoculation with Enterobacter sp. in the nursery, seedling root inoculation with Enterobacter sp. before
JOURNAL OF PLANT NUTRITION

transplanting and combined method (EIITII þ EIITIII) in the experiment II, respectively.
9
10 E. BAKHSHANDEH ET AL.

concentration in StPM and StrPM at PM belonged to the application of 180 kg ha1 PSF which
was equal to 6.6 and 7.4 kg ha1, respectively. Application of 125 kg ha1 PSF in experiment II
significantly increased GLAS, StAS, StrAS, GLPM, StPM, StrPM and GrainPM, which were nearly 10.9,
65.7, 54.4, 42.3, 99.5, 85.3 and 21.9% more than the control (Table 3). The results of the regres-
sion analysis showed that P uptake by grain remarkably enhanced with the addition of PSF in
both experiments particularly when the best treatments were used (EITIV and EIITIV in experi-
ment I and II, respectively). Generally, P uptake by grain increased linearly from zero to 127.0
and 90.0 kg ha1 PSF with the rate of 0.034 and 0.023 kg per kg increase in PSF when the best
treatment in experiment I and II were applied, respectively, then remained constant at its max-
imum value until 180 kg ha1 PSF for experiment I and 125 kg ha1 PSF for experiment II
(Figure 1e,f, Table 6). According to the model estimates, the maximum P uptake by grain was
16.6 and 17.2 kg ha1, 62 and 30% more than the control condition in experiment I and II,
respectively (Table 6).
Results also showed that the PGPM inoculations and their application methods had a signifi-
cant effect on P uptake by rice tissues in both experiments. In this regard, the co-inoculation
with P. ananatis and P. indica significantly increased GLAS, GLPM, StPM, StrPM and GrainPM com-
pared to the non-inoculated plants in experiment I, up to 39.1, 165.7, 31.2, 40.7 and 49.2%,
respectively (Table 3). However, the highest amount of StAS and StrAS were obtained from the
inoculation with P. indica (applied alone; 76.2 and 103.3 kg ha1, respectively), which had no sig-
nificant difference with other PGPM (Table 3). The maximum values of P uptake by GLAS, StAS
and StrAS at AS in experiment II were observed in the seedling root inoculation with Enterobacter
sp. before transplanting (EIITIII) treatment equivalent to 26.7, 93.8 and 77% more than the con-
trol, respectively (Table 3). Nevertheless, the combined inoculation method (EITIV) had a higher
effect on P uptake by GLPM, StPM, StrPM and GrainPM to about 65.8, 85.5, 74.4 and 33.7% more
than those in the control, respectively (Table 3).

Potassium uptake by rice tissues

Based on the results, the amount of K uptake at AS (by GLAS, StAS and StrAS) and PM (by GLPM
and StPM) reached their maximum values at 120 kg ha1 PSF, up to 46.2, 13.6, 21.7, 92.1 and
19.6% more than those in the control in experiment I, respectively (Table 4). On the other hand,
there was not a statistically significant difference between PSF treatments for these parameters in
experiment II. The highest PSF application rate, 180 kg ha1 in experiment I and 125 kg ha1 in
experiment II, significantly increased K uptake by StrPM and GrainPM at PM which was equal to
43.7 and 9.7 kg ha1 in experiment I and 44.2 and 7.6 kg ha1 in the experiment II, respectively
(Table 4).
Like P, K uptake by grain increased linearly from zero to 136.8 and 106.6 kg ha1 PSF with
the rate of 0.025 and 0.017 kg per kg increase in PSF when the best treatments were applied in
experiment I and II, then remained constant at their maximum values until 180 and 125 kg ha1
PSF for experiment I and II, respectively (Figure 1g,h, Table 6). According to the model esti-
mates, the maximum K uptake by grain was 10.8 and 8.30 kg ha1, 30 and 32% more than the
control in experiment I and II, respectively (Table 6). This indicates that rice plants in experiment
I were more affected by PSF, which can be due to cultivar characteristics and/or low available K
in the soil (Table 1).
Results clearly showed that the K uptake by different rice tissues was significantly influenced
by PGPM inoculations. Accordingly, K uptake by GL, St, Str in both stages and GrainPM at PM
reached the maximum value in the co-inoculation with P. ananatis and P. indica which was about
7.5-43.3% more than those in the control in experiment I (Table 4). In addition, the seedling root
inoculation with Enterobacter sp. before transplanting (EIITIII) treatment had a higher effect on K
uptake in GLAS, StAS and StrAS at AS which were 27.4, 112.4 and 139.9 kg ha1 compared to the
Table 4. Analysis of variance and means comparsion on the studied traits related to the potassium uptake by different rice tissues under different levels of potassium sulfate fertilizer (PSF,
kg ha1) and inoculation with three plant growth promoting microorganisms (PGPM) and various inoculation methods (IM).
K uptake at anthesis K uptake at physiological maturity
Sources Degree Green leaves Stem Straw Green leaves Yellow leaves Stem Straw Grain
Experiments of variation of freedom (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1) (kg ha1)
Experiment I F-test
PSF 3  Ns   Ns   
PGPM 3        
PGPM  PSF 9 Ns Ns Ns  Ns Ns Ns Ns
CV (%) 13.9 8.90 6.64 11.3 15.7 7.36 7.21 7.79
Means comparison†
PSF (kg ha1) 0 36.75 b 110.8 a 147.6 b 0.507 b 1.848 a 31.64 b 34.28 b 6.972 c
60 41.78 (þ13.6)b 116.2 (þ4.87)a 158.0 (þ7.06)b 0.837 (þ65.2)a 1.816 (1.72)a 32.44 (þ2.54)b 35.09 (þ2.37)b 8.277 (þ18.7)b
120 53.76 (þ46.2)a 126.0 (þ13.6)a 179.7 (þ21.7)a 0.974 (þ92.1)a 1.744 (5.64)a 37.84 (þ19.6)a 40.46 (þ18.0)a 8.837 (þ26.7)ab
180 51.37 (þ39.7)a 122.7 (þ10.7)a 174.1 (þ17.9)a 0.922 (þ81.9)a 1.808 (2.16)a 40.95 (þ29.4)a 43.72 (þ27.5)a 9.756 (þ39.9)a
PGPM EITI 39.23 b 107.2 c 146.5 c 0.639 c 1.997 a 34.18 b 36.41 b 7.562 c
EITII 46.16 (þ17.6)a 120.3 (þ12.2)b 166.5 (þ13.6)b 0.878 (þ37.5)ab 1.788 (10.4)ab 35.75 (þ4.60)ab 39.12 (þ7.44)a 8.376 (þ10.7)b
EITIII 47.40 (þ20.8)a 117.3 (þ9.37)b 164.7 (þ12.4)b 0.811 (þ26.9)b 1.677 (16.0)b 36.20 (þ5.91)ab 38.69 (þ6.26)ab 8.653 (þ14.4)b
EITIV 50.87 (þ29.6)a 130.9 (þ22.0)a 181.8 (þ24.1)a 0.915 (þ43.3)a 1.753 (12.1)ab 36.74 (þ7.50)a 39.41 (þ8.25)a 9.251 (þ22.3)a
Experiment II F-test
PSF 5 Ns Ns Ns Ns Ns   
IM 3 Ns    Ns   
IM  PSF 15 Ns Ns Ns Ns Ns Ns Ns Ns
CV (%) 19.9 16.7 15.7 16.8 12.1 11.4 11.1 10.1
Means comparison
PSF (kg ha1) 0 23.95 a 96.53 a 122.6 a 1.27 a 2.051 a 36.70 ab 39.51 b 6.365 c
25 25.62 (þ6.97)a 103.0 (þ6.66)a 128.6 (þ4.86)a 1.488 (þ17.1)a 1.796 (12.4)a 36.83 (þ0.35)b 40.02 (þ1.29)ab 6.493 (þ2.01)c
50 24.06 (þ0.45)a 101.7 (þ5.36)a 125.8 (þ2.56)a 1.583 (þ24.7)a 1.816 (11.4)a 37.42 (þ1.97)ab 40.82 (þ3.32)ab 6.623 (þ4.06)bc
75 26.36 (þ10.0)a 97.80 (þ1.32)a 124.2 (þ1.26)a 1.554 (þ22.3)a 1.783 (13.0)a 37.93 (þ3.34)ab 41.26 (þ4.43)ab 6.891 (þ8.27)bc
100 26.10 (þ8.94)a 98.22 (þ1.75)a 132.2 (þ7.79)a 1.629 (þ28.3)a 2.058 (þ0.34)a 39.01 (þ6.29)ab 42.69 (þ8.05)ab 7.205 (þ13.2)ab
125 27.72 (þ15.7)a 101.6 (þ5.30)a 138.4 (þ12.8)a 1.630 (þ28.3)a 2.093 (þ2.02)a 40.52 (þ10.4)a 44.24 (þ12.0)a 7.602 (þ19.4)a
IM EIITI 24.75 a 94.54 b 120.9 b 1.266 c 1.943 a 35.48 b 38.69 b 6.034 b
EIITII 26.34 (þ6.40)a 96.00 (þ1.54)b 121.3 (þ0.39)b 1.490 (þ17.6)bc 1.929 (0.72)a 38.24 (þ7.79)ab 41.66 (þ7.68)ab 7.037 (þ16.6)a
EIITIII 27.45 (þ10.8)a 112.4 (þ18.9)a 139.9 (þ15.7)a 1.560 (þ23.2)ab 1.948 (þ0.25)a 37.28 (þ5.08)b 40.79 (þ5.43)b 7.050 (þ16.8)a
EIITIV 25.34 (þ2.38)a 102.9 (þ8.88)ab 127.7 (þ5.63)ab 1.785 (þ40.9)a 1.910 (1.71)a 40.87 (þ15.1)a 44.56 (þ15.1)a 7.331 (þ21.4)a
,  and Ns represent values significant at 0.01, 0.05 probability level and non-significant based on the least significant difference test (LSD), respectively. CV is the coefficient of variation
which was related to overall data.
†
Means with the same letter are not significantly different at the probability level of 0.05. Values in parentheses indicate percent change (posetive and negative) in the respective studied
traits in comparison with the control condition. EITI, EITII, EITIII and EITIV are the control, inoculation with P. ananatis, inoculation with P. indica and co-inoculation (EITII þ EITIII) in the
experiment I, and EIITI, EIITII, EIITIII and EIITIV are the control, pre-germinated seed inoculation with Enterobacter sp. in the nursery, seedling root inoculation with Enterobacter sp. before
JOURNAL OF PLANT NUTRITION

transplanting and combined method (EIITII þ EIITIII) in the experiment II, respectively.
11
12 E. BAKHSHANDEH ET AL.

values 24.7, 94.5 and 120.9 kg ha1 obtained from the control in the experiment II, respectively.
Nevertheless, the highest K uptake in GLPM, StPM, StrPM and GrainPM at PM were achieved in
the combined inoculation method (EITIV) which was 40.9, 15.1, 15.1 and 21.4% higher than the
non-inoculated treatment, respectively (Table 4). Our finding showed that the value of K uptake
by YLPM was lower than others when EITIV and/or EIITIII were applied. However, the minimum
values of K uptake by YLPM also observed at 120 and 75 kg ha1 PSF in experiment I and II,
respectively (5.64 and 13.0% lower than the control, respectively) and their values increased with
the addition of PSF as a result of increasing biomass production at higher levels of PSF (Table 2).

Phosphorus use efficiency

Different results were obtained from experiments I and II in terms of PUE parameters such as
PE and APE. Accordingly, the highest PHI was 120 kg ha1 PSF in experiment I which was not
statistically significant in comparison with others (Table 5 and Figure 1e, Table 6). However,
120 kg ha1 PSF increased AE, PE, APE and ARE values to about 6.5, 7.5, 5.0 and 1.7 times more
than the zero PSF treatment, respectively (Table 5). Co-inoculation with P. ananatis and P. indica
(EITIV) significantly increased AE (242.6%) and ARE (214.3%) and decreased PE (48.1%) as com-
pared to the control in experiment I. Three PGPM inoculations had no significantly effects on
PHI and APE (Table 5).
In experiment II, the maximum PHI was observed at PSF lower than 75 kg ha1 (57.4 kg ha1,
P > 0.05), and then its value declined significantly with the increasing PSF until 125 kg ha1
(Table 5). However, the maximum AE, ARE and PE were obtained from 125, 125 and 50 kg ha1
PSF, respectively. Inoculation with PGPM significantly decreased PHI, PE and APE in experiment
II which was in contrast with the results obtained in experiment I. Nevertheless, the increments
in AE and ARE were recorded by various inoculation methods, in particular, application of the
combined method (EIITIV), as compared to the non-inoculated plants (Table 5).

Potassium use efficiency

PSF application had no significant effect on KHI and ARE in both experiments and on PE and
APE in experiment II. The highest values of AE, PE and APE were recorded by application of
60 kg ha1 PSF in experiment II, equal to 29.5, 386.6 and 182.4 gr gr 1, respectively (Table 5).
The results achieved from the experiment I confirmed that the KUE was affected by PGPM
inoculation treatments, except for PE. In this regard, co-inoculation with P. ananatis and P. ind-
ica significantly increased KHI, AE, APE and ARE with the highest value to about 19, 34.2,
160.7 gr gr1 and 20.5%, respectively, which was around 1.1, 3.4, 1.5 and 1.9 times more than
those in the control. In addition, PGPM inoculations significantly increased AE and ARE com-
pared to non-inoculated treatment in experiment II. The maximum value of AE and ARE were
observed in the combined inoculation method (EIITIV) equal to 42.4 gr gr2 and 38.4%, which
were about 2.3 and 2.2 times more than those in the control, respectively. The seedling root
inoculation with Enterobacter sp. before transplanting (EIITIII) significantly increased KHI value
to about 9.3% higher than those obtained in the control (Table 5).

Discussion
According to the results of this study, PSF application had positive impacts on plant DW and
nutrients uptake as compared to the control. This result is confirmed by Bakhshandeh, Pirdashti,
and Gilani (2018) and Yaghoubi Khanghahi et al. (2019b). Similarly, Fageria (2015) reported an
increment in rice growth and DW with increasing K fertilizer. PGPM inoculations also signifi-
cantly increased the DW of different plant tissues at AS and PM in both experiments. The same
Table 5. Analysis of variance and means comparsion on the potassium and phosphorous use efficiency in rice under different levels of potassium sulfate fertilizer (PSF, kg ha1) and inocu-
lation with three plant growth promoting microorganisms (PGPM) and various inoculation methods (IM).
P use efficiency K use efficiency
PHI AE PE APE ARE KHI AE PE APE ARE
Experiments Sources of variation Degree of freedom % gr gr2 gr gr2 gr gr2 % % gr gr2 gr gr2 gr gr2 %
Experiment I F-test
PSF 3 Ns    Ns Ns    Ns
PGPM 3 Ns   Ns      
PGPM  PSF 9 Ns  Ns  Ns Ns  Ns Ns Ns
Means comparison†
PSF (kg ha1) 0 67.97 a 9.250 d 58.86 c 38.42 b 21.92 a 16.60 a 93.07 c 52.36 c
60 67.83 a 35.52 c 246.8 b 129.0 a 28.06 a 18.61 a 29.48 a 386.6 a 182.4 a 17.64 a
120 68.78 a 57.33 b 392.6 ab 165.5 a 37.97 a 18.37 a 24.96 b 314.9 ab 139.4 ab 18.70 a
180 64.26 a 60.23 a 443.8 a 191.9 a 38.09 a 18.24 a 16.96 c 226.9 b 98.87 bc 17.44 a
PGPM EITI 67.78 a 23.96 d 483.1 a 169.4 a 19.09 b 17.58 ab 10.11 d 354.6 a 106.0 b 11.04 b
EITII 65.88 a 43.25 c 316.5 b 140.7 a 31.73 a 17.17 b 22.90 c 233.8 b 97.32 b 21.10 a
EITIII 67.25 a 47.29 b 303.7 b 141.0 a 34.41 a 18.21 ab 24.88 b 253.6 ab 124.2 ab 18.92 a
EITIV 67.59 a 58.13 a 251.0 b 141.3 a 40.92 a 19.01 a 34.19 a 261.9 ab 160.7 a 20.55 a
Experiment II F-test
PSF 5    Ns  Ns  Ns Ns Ns
IM 3        Ns Ns 
IM  PSF 15 Ns Ns Ns Ns Ns Ns Ns Ns  Ns
Means comparison
PSF (kg ha1) 0 57.15 a 11.00 bc 217.2 ab 91.99 a 33.58 c 14.16 a 64.80 a 146.1 a 66.27 a 37.00 a
25 59.62 a 19.75 c 199.4 abc 106.8 a 32.94 c 14.53 a 31.81 b 260.1 a 119.8 a 34.97 a
50 56.06 a 27.13 bc 248.1 a 89.10 a 31.22 c 14.23 a 25.21 b 263.5 a 109.8 a 24.79 a
75 56.90 a 38.78 abc 212.5 ab 100.0 a 38.70 c 14.71 a 22.95 b 235.5 a 104.8 a 22.18 a
100 50.13 b 43.90 ab 182.6 bc 75.21 a 59.03 b 14.84 a 24.56 b 266.0 a 114.6 a 20.97 a
125 48.93 b 58.72 a 161.5 c 76.89 a 76.66 a 15.08 a 64.80 a 210.4 a 106.5 a 37.00 a
IM EIITI 58.78 a 22.69 b 262.8 a 109.6 a 22.71 c 13.83 b 18.38 b 203.8 a 100.4 a 17.53 b
EIITII 53.94 b 42.93 a 205.7 b 93.89 b 48.02 b 14.86 ab 34.96 a 263.3 a 107.0 a 27.79 ab
EIITIII 54.72 b 38.89 a 188.7 b 86.07 bc 45.72 b 15.12 a 35.29 a 252.9 a 129.3 a 27.83 ab
EIITIV 52.13 b 45.99 a 174.9 b 74.18 c 61.56 a 14.54 ab 42.42 a 234.4 a 96.07 a 38.40 a
,  and Ns represent values significant at 0.01, 0.05 probability level and non-significant based on the least significant difference test (LSD), respectively. CV is the coefficient of variation
which was related to overall data.
†
Means with the same letter are not significantly different at the probability level of 0.05. Values in parentheses indicate percent change (posetive and negative) in the respective studied
traits in comparison with the control condition. EITI, EITII, EITIII and EITIV are the control, inoculation with P. ananatis, inoculation with P. indica and co-inoculation (EITII þ EITIII) in the
experiment I, and EIITI, EIITII, EIITIII and EIITIV are the control, pre-germinated seed inoculation with Enterobacter sp. in the nursery, seedling root inoculation with Enterobacter sp. before
transplanting and combined method (EIITII þ EIITIII) in the experiment II, respectively.
JOURNAL OF PLANT NUTRITION
13
14 E. BAKHSHANDEH ET AL.

Table 6. Estimated parameter values based on a segmented model fitted to the data of potassium sulfate fertilizer (PSF) and
the grain yield (A and B), straw yield (C and D), phosphorus uptake in grain (E and F) and potassium uptake in grain (G and
H) in the experiment I (A, C, E and G) and the experiment II (B, D, F and H). This model was fitted to the data related to the
control conditions and the best treatment in each experiment (the co-inoculation with P. ananatis and P. indica for the experi-
ment I (EITIV) and the combined method (pre-germinated seed inoculation with Enterobacter sp. in the nursery þ seedling root
inoculation with Enterobacter sp. before transplanting) for the experiment II (EIITIV).
Experiments Fig. 1 Traits Treatments (kg ha1) a b x0 Maximum value R2
Experiment I A Grain yield Control 3606.6 6.083 144.2 4483 0.98
Co-inoculation 4028.0 12.00 110.0 5348 0.99
C Straw yield Control 6671.4 17.02 69.50 7854 0.99
Co-inoculation 6552.5 14.36 134.3 8481 0.94
E P uptake by grain Control 7.420 0.045 61.42 10.21 0.99
Co-inoculation 12.28 0.034 127.0 16.66 0.99
G K uptake by grain Control 6.950 0.009 142.8 8.292 0.99
Co-inoculation 7.310 0.025 136.8 10.83 0.99
Experiment II B Grain yield Control 3752.1 5.688 75.00 4178 0.97
Combined method 4429.2 9.010 100.0 5330 0.89
D Straw yield Control 6987.4 13.07 55.52 7713 0.88
Combined method 8109.4 4.770 80.63 8494 0.84
F P uptake by grain Control 11.21 0.026 80.85 13.32 0.92
Combined method 15.11 0.023 91.01 17.28 0.96
H K uptake by grain Control 4.820 0.023 62.25 6.301 0.95
Combined method 6.470 0.017 106.6 8.303 0.97
a, b and x0 are the initial value of trait at zero PSF (as control), the rate of the linear increase in the trait as PSF increased
and the amount of PSF when the trait reached its maximum value, respectively. R2 is the coefficient of determination. The
maximum value of traits was calculated using the model parameters for each treatment. More details are presented in
Fig. 1.

results reported by Varma et al. (2012) who showed that the inoculation of barley seedlings with
Pseudomonas putida and P. indica remarkably increased tissues DW of this plant. Bakhshandeh
et al. (2015) also indicated that the PGPM inoculations significantly increased DW of rice about
12 to 31% more than non-inoculated plants, depending on cultivars and TSP values applied.
Our results proved that the PGPM inoculations not only increased K and P uptake by different
tissues of the rice plant, but also enhanced both KUE and PUE, especially when co-inoculation
treatment with P. ananatis and P. indica were applied. These findings are in agreement with the
results obtained by Bakhshandeh, Pirdashti, and Shahsavarpour Lendeh (2017; Bakhshandeh,
Pirdashti, and Gilani 2018), Bahadur et al. (2016) and Yaghoubi Khanghahi et al. (2018) who
indicated that the bio-inoculation can enhance the nutrient uptake by rice plant. In fact, these
PGPM are known as multiple plant growth promoting microorganisms which can solubilize
insoluble form of P and K for plant and also produce some phytohormones like IAA
(Bakhshandeh et al. 2014). Previous studies have shown that the nutrient uptake by rice plant in
response to inoculations with various PGPM significantly increased through the ability of solubi-
lizing insoluble phosphate and silicate compounds minerals in the rhizosphere, production of
plant hormone such as IAA and gibberellic acid and or synthesis of antibiotics, enzymes, fungi-
cidal compounds and competition with detrimental microorganisms (Pii et al. 2015; Agrawal
et al. 2017) which can result in a better absorption of nutrients uptake by plants. Furthermore, it
has already been reported that PGPM inoculation increased plant growth by increasing leaf
photosynthesis efficiency up to 12, 12, 16 and 20% in rice (Khanghahi et al. 2019c), eggplant
(Han and Lee 2005), cucumber and pepper (Han, Supanjani, and Lee 2006), respectively, as com-
pared to the control, which could result in higher CO2 assimilation (Kramer et al. 2004) and con-
tribute to enhanced GY (Khanghahi et al. 2019c).
The studied traits values in the combined inoculation method were higher than other methods
such as the soil inoculation and or seedlings root inoculation alone with the PGPM suspension.
Therefore, it seems that the combined method could increase the transfer of the inocula toward
the roots and enhance the efficacy of the inoculation. Differences were statistically significant
among the inoculation methods as compared to the non-inoculated plants in terms of some studied
 JOURNAL OF PLANT NUTRITION 15

traits such as DW of rice plant tissues at PM and P uptake by the plant. Many studies recently indi-
cated that the amount of DW and nutrient uptake by rice plant was influenced by various incubation
methods such as rice seeds (cv. ‘Tarom’) inoculation with Enterobacter sp. (Bakhshandeh et al. 2015),
Bacillus sp. (Panhwar et al. 2013), Pseudomonas sp. (Deshwal and Kumar 2013), P. indica (Ghabooli,
Hosseini Salekdeh, and Sepehri 2015) and rice seedlings root inoculation with Pseudomonas sp.
(Ebrahimi-Chamani, Yasari, and Pirdashti 2015). In this regard, it has already been reported an incre-
ment in plant growth and GY when P. indica was applied as a root inoculation in many different
crops (Varma et al. 2012; Yaghoubian et al. 2014). It has conclusively been shown that simultaneous
inoculation with different PGPR, arbuscular mycorrhizal fungi (AMF) and P. indica can result in
increasing crops growth and yield as compared to single inoculation through improving nutrient
uptake by plant (Malusa, Sas-Paszt, and Ciesielska 2012; Mansotra, Sharma, and Sharma 2015). A
likely explanation is that these increments are a result of the efficient root colonization by P. indica,
leading to greater acquisition of water and nutrients (Vyas, Nagori, and Purohit 2008).
The results showed that both KUE and PUE were remarkably affected by different levels of
PSF, various PGPM and different inoculation methods. This is in agreement with the results
obtained by Meena et al. (2017) and Pii et al. (2015) who showed that the PGPM are able to
increase NUE and nutrient acquisition by plants. Similarly, Duarah et al. (2011) reported that
application of NPK chemical fertilizers in combination with PGPM increased NUE and decreased
the nutrient runoff or leaching in rice fields. Similar results were reported by Yaghoubi
Khanghahi et al. (2018) who stated that KSB inoculums coupled with K chemical fertilizer
increased NUE in rice (i.e., AE, ARE, PE, APE, UE, PFP and PNB).
There were some differences between two experiments in terms of NUE and K uptake by the
plant. Accordingly, the amounts of GLAS, StAS, StrAS and GLPM in experiment I significantly
increased when PSF was applied as compared to the control while their increment was not signifi-
cant in experiment II. On the other hand, the highest values of PHI, APE and PE for P belonged to
the noninoculated plants in experiment II. It seems that the effect of the PGPM on these traits was
lower in experiment II than in experiment I which can be related to higher amount of soil organic
carbon in experiment I (Table 1). These results are in contradiction with other studies such as Malik
et al. (2018) and Crecchio et al. (2007) who reported that soil organic carbon availability can increase
the whole specific microbial activity in soil, although it is not strictly relevant to the effects that
some microorganisms may have on plants. In addition, these differences may be due to other soil
conditions such as soil texture and electrical conductivity (Table 1) which can reflect significant
changes in the biological function of the soil (Chau, Bagtzoglou, and Willig 2011; Yan et al. 2015).
The results reported here appear to support the assumption that the microbial inoculants
based on these PGPM can be used as an alternative approach not only in enhancing the K and P
concentration in plant and their use efficiency, but also in reducing the use of K chemical fertil-
izer. These results agree with the findings of other studies, showing that the PGPM inoculation in
nutrient management strategies aimed at decreasing chemical fertilizer consumption (Maheshwari
2010; Meena et al. 2016). It is believed that the biofertilizer application can reduce the amount of
K chemical fertilizers up to 60% (Chandra, Ingle, and Bihari 2002) and increase the K fertilizer
efficiency (Bahadur, Meena, and Kumar 2014), especially under interactions between AMF and
PGPM (Singh and Adholeya 2003). In another study, Bakhshandeh et al. (2015) in rice reported
that the application of TSP along with PSB strains decreased the use of TSP up to 67% compared
with the control and increased NUE as well.

Conclusion
In conclusion, our results clearly showed that the three PGPM here used (P. ananatis,
Enterobacter sp. and P. indica) can represent a viable alternative to enhance DW of rice plant tis-
sues, P and K uptake, NUE, especially when a combined inoculation method (i.e., pre-germinated
16 E. BAKHSHANDEH ET AL.

seed inoculation in the nursery along with the seedlings root inoculation before transplanting)
was applied. Therefore, this information will help to select the best PGPM and/or inoculation
method for improving plant growth and NUE of rice in sustainable production systems.

Acknowledgments
The authors thank the Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT) and Sari
Agricultural Sciences and Natural Resources University, Sari, Iran for providing financial support for this study.

Conflict of interest
No conflict of interest declared.

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