Performance of low and high Fe accumulator wheat genotypes grown on soils with low or high available Fe and endophyte inoculation

Abstract

One of the important limiting factors to realising the benefits of modern high- yielding crop varieties is the availability of iron (Fe) in the soil, which often leads to Fe deficiency in food grains. The main objective of this study was to evaluate the role of two siderophore-producing endophytes (Arthrobacter sulfonivorans DS-68 and Enterococcus hirae DS-163) in the biofortification of grains with Fe and enhance yield in four genotypes of wheat (Triticum aestivum L.) in soils with low and high available Fe content. Endophyte inoculation increased the surface area, volume, length of roots and number of root tips by 78.27, 75, 71 and 44%, respectively, relative to the uninoculated control (recommended dose of fertilizers; RDF), across genotypes and soil types. In the low available-Fe soil, inoculation with endophytes increased grain yield twofold relative to the control (RDF), whereas in the high available-Fe soil, the increase was only 1.2-fold across genotypes. In general, endophyte inoculation caused an increase of 1.5-fold and 2.2-fold in iron concentration in grains over the RDF + FeSO4 treatment and uninoculated control (RDF), respectively, across all the genotypes and both soil types. Such siderophore-producing endophytes can be recommended as bioinoculants to mitigate iron deficiencies in the soil and enhance crop productivity.

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Acta Physiologiae Plantarum (2020) 42:24
https://doi.org/10.1007/s11738-019-2997-4
ORIGINAL ARTICLE
Performance oflow andhigh Fe accumulator wheat genotypes grown
onsoils withlow orhigh available Fe andendophyte inoculation
DevendraSingh1· NeelamGeat2· MahendraVikramSinghRajawat3· RadhaPrasanna4· AnilKumarSaxena3
Received: 19 February 2019 / Revised: 17 December 2019 / Accepted: 26 December 2019 / Published online: 14 January 2020
© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2020
Abstract
One of the important limiting factors to realising the benefits of modern high- yielding crop varieties is the availability of
iron (Fe) in the soil, which often leads to Fe deficiency in food grains. The main objective of this study was to evaluate the
role of two siderophore-producing endophytes (Arthrobacter sulfonivorans DS-68 and Enterococcus hirae DS-163) in the
biofortification of grains with Fe and enhance yield in four genotypes of wheat (Triticum aestivum L.) in soils with low and
high available Fe content. Endophyte inoculation increased the surface area, volume, length of roots and number of root tips
by 78.27, 75, 71 and 44%, respectively, relative to the uninoculated control (recommended dose of fertilizers; RDF), across
genotypes and soil types. In the low available-Fe soil, inoculation with endophytes increased grain yield twofold relative to
the control (RDF), whereas in the high available-Fe soil, the increase was only 1.2-fold across genotypes. In general, endo-
phyte inoculation caused an increase of 1.5-fold and 2.2-fold in iron concentration in grains over the RDF + FeSO4 treatment
and uninoculated control (RDF), respectively, across all the genotypes and both soil types. Such siderophore-producing
endophytes can be recommended as bioinoculants to mitigate iron deficiencies in the soil and enhance crop productivity.
Keywords Endophyte· Fe-biofortification· Root morphology· Siderophore· Grain yield
Introduction
The significance of iron in human health is exemplified by
its role in a wide variety of metabolic processes, includ-
ing synthesis of deoxyribonucleic acid (DNA) and several
enzymes (Abbaspour etal. 2014). The recommended die-
tary allowances (RDA) for Fe for adults in the age group
of 19–50years for males and females is 8 and 18mgday–1
respectively, but is 27mgday–1 for females during preg-
nancy period (Food and Nutrition Board 2001). Iron defi-
ciency linked-anemia was one of the major causes of sev-
eral deaths and implicated in the loss of several million
disability-adjusted life-years (DALYs) annually (4.5% of
all risk-attributable to DALYs), as reported in a Global
Burden of Disease study, undertaken in 2013 (Forouzanfar
etal. 2015). In India, more than 50% women and 74% chil-
dren have been found to be anaemic (IIPS 2000); this can
be attributed largely to the insufficient intake of iron-rich
foods and poor bioavailability of iron. Stevens etal. (2013)
showed that anaemia prevalence was highest in the pre-
school age children, pregnant and non-pregnant women in
Western Pacific, Southeast Asia and Africa. In a nationwide
study in India, Chellan and Paul (2010) found a moderate to
severe Fe-deficiency anaemia in 47.9% pre-school children
(below 6years), in 74.8% adolescent girls (10–19years)
and in 41.5% pregnant women (15–44years). The access
to quality food, which can provide optimal energy is of
major concern, however, the deficiency of micronutrients is
Communicated by P. Wojtaszek.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1173 8-019-2997-4) contains
supplementary material, which is available to authorized users.
* Anil Kumar Saxena
saxena461@yahoo.com
1 Department ofMicrobiology, College ofBasic Sciences
& Humanities, Dr. Rajendra Prasad Central Agricultural
University, Pusa, Samastipur, Bihar848125, India
2 Agricultural Research Station, Mandore, Agriculture
University, Jodhpur342304, India
3 ICAR-National Bureau ofAgriculturally Important
Microorganisms, Kushmaur, MauNathBhanjan,
UttarPradesh275103, India
4 Division ofMicrobiology, ICAR-Indian Agricultural
Research Institute, NewDelhi110012, India

Acta Physiologiae Plantarum (2020) 42:24
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equally important (Stewart etal. 2010), and biofortification
of grains using breeding or fertilization approaches has been
the choice of researchers (Bouis 2003; Cakmak 2008; White
and Broadley 2009).
Plants require only small amounts of micronutrients
for their growth and development, but their importance is
equivalent to macronutrients in achieving optimum produc-
tion and yield of crops. Rice (Oryza sativa L.), wheat (Triti-
cum aestivum L.) and barley (Hordeum vulgare L.) account
for a major share of the foods and food products, around
the world including India; however, plants exhibit Cu, Fe,
Mn, Zn deficiency (Cakmak etal. 2010; Bouis etal. 2011).
Micronutrient supplementation with chemical fertilizers
has been an effective measure employed by farmers to gain
maximize crop yield, but its use efficiency is as low as only
2–5% of the total applied dose is utilized (Tian etal. 2008).
On the other hand, application of higher doses of chemical
fertilizers has several negative implications on ecological
aspects such as soil fertility, nutrient dynamics besides envi-
ronmental concerns to health of human beings (Lockhart
etal. 2013; Wu and Ma 2015). Biological fertilization is
an effective method to supply plant nutrients, because it is
economically and environmentally feasible and sustainable
(Miransari 2013).
Wheat (Triticum aestivum L.) is the most important crop
among cereals that provides dietary calories and protein and
ranks second in food grain production. Globally, India ranks
second in cultivated area (13.2%) and production (12.05%)
of wheat. However, India’s mean yield is lower as compared
to world’s average yield (Directorate of Economics and Sta-
tistics 2016). Deficiencies of macronutrients and micronutri-
ents, available soil water and other soil-physical constraints
are major causes of low yield in India (Mahajan and Gupta
2009; Das and Avasthe 2018). Mhoro etal. (2015) reported
that low yield of wheat in Tanzania was associated with defi-
ciency of both macronutrients and micronutrients in soils. In
general, chlorophyll concentration in leaves of wheat plant
progressively dropped with decreasing Fe supply in the soil
solution and exhibited leaf chlorosis (Celletti etal. 2016;
Zamboni etal. 2017). Astolfi etal. (2018) reported that Fe
deficiency in soil induced a significant reduction in weight
of grains and yield of wheat as compared to control (Fe
fertilized soil). White and Broadley (2009) suggested that
micronutrient deficiency in soil not only declined the crop
yield but also contributed to low crop quality, leading to
dietary micronutrient deficiencies in human beings.
Micronutrients are essential for metabolic functions and
proper development of the plant (Römheld and Marschner
1991; Welch and Shuman 1995; Hänsch and Mendel 2009).
The Fe sufficiency range in wheat was recorded between
28.8 and 56.5mgkg−1 dry weight (Graham etal. 1999),
with critical content of Fe in wheat grains being reported as
45mgkg−1 dry weight (Frossard etal. 2000). The critical
value of iron in the soil that denotes iron deficiency is
4.5mgkg−1 soil (Sillanpaa 1982; Alloway 2009). Most of
the soils throughout India have been exhibiting Fe deficiency
for quite some time (Malewar and Ismail 1995). The avail-
ability of micronutrients, particularly iron, has declined in
13% of the Indian soils (Shukla etal. 2014). This is mainly
attributed to the excessive use of chemical fertilizers, pro-
duction of high yielding crops that extract more nutrients, or
to soil erosion or the nature of soils being calcareous, water
logged, peat, alkaline, sandy or saline. Soils with excess
amounts of organic matter or phosphorus or belonging to
arid environments, also limit Fe mobilization (Alloway
2008).
Interestingly, iron deficiency in soils and human beings
has shown a close geographical relationship, indicating the
necessity to enhance the amount of Fe in crop products.
Keeping these facts and the finite land resources in view,
fulfilling demand for nutritious food for the burgeoning
population is an enormous challenge (Clark etal. 2016).
In this context, the use of microbial inoculants is a promis-
ing approach to enhance the availability of iron in the soil.
Microorganisms are known to sequester or make available
micronutrients, besides improving their uptake and trans-
location in cereal crops, leading to an enhancement of
their availability in food grains (Adak etal. 2016; Prasanna
etal. 2015; Rana etal. 2012; Saha etal. 2012). Arbuscu-
lar mycorrhizae are known to enhance iron uptake through
an increased exploration of soil volume by the root system
(Kothari etal. 1990). Root morphology, differs among
various species of plants and increased root length with an
enhanced surface area of root can affect the uptake efficiency
of micronutrients especially iron (Rengel and Graham 1995).
Colonization of endophytic microorganisms such as mycor-
rhizal fungi, in roots influence the nutrient-uptake efficiency
of iron, zinc, phosphorus and copper (Marschner and Dell
1994; Saxena etal. 2015). Inoculation with microorganisms
was found to improve the exploration of the soil resources
by the root system, as compared with uninoculated control
by enhancing the root characteristics, including length, total
volume and area, besides number of crown root, root tips and
root hairs (Rose etal. 2011).
Biofortification of grains using breeding or fertilization
approaches has been the choice of researchers to alleviate
micronutrient-deficiency in human diet. However, micro-
nutrient use efficiency is limited to only 2–5% (Tian etal.
2008) and biofortified crop varieties may not improve in
low Fe available soils over the years. Several researchers
observed that there was significant genetic variability in
terms of Fe and Zn among wheat genotypes, particularly
grain Fe content ranging from 9 to 50.8mgkg−1; while
other researchers recorded 41.4 to 67.7mgkg−1, and 20 to
60mgkg−1 (Badakhshan etal. 2013; Chatzav etal. 2010;
Goudia and Hash 2015; Morgounov etal. 2007; Oury etal.

Acta Physiologiae Plantarum (2020) 42:24
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Page 3 of 13 24
2006; Velu etal. 2011; Zhao etal. 2009). Therefore, the use
of microbiological options, such as rhizosphere microflora
or endophytes, can be a promising option to enhance the
mobilization of Fe from soil to different plant parts, includ-
ing grains in low and high accumulator genotypes, charac-
terised based on our earlier studies (Singh 2016). Hence, for
the present study, the following objectives were formulated:
(1) to analyse the influence of siderophore- producing endo-
phytes in biofortification of wheat grains with Fe; (2) to
evaluate how different wheat genotypes respond to inocula-
tion with endophytes and their performance in soils with low
and high available Fe content, in relation to Fe biofortifica-
tion of wheat grains with Fe. The main focus of the present
study was on plant attributes such as root morphology, iron
uptake, translocation of iron and yield of wheat in two dif-
ferent types of soil (sandy clay loam, with high available
Fe content soil and calcareous soil with low available Fe
content) in pot experiments.
Materials andmethods
Soil collection andanalysis ofsoil properties
Soil samples of two different types of soils were collected
from farm field, ICAR-Indian Agricultural Research Insti-
tute (IARI soil), New Delhi, India situated at (28° 35′ N,
77° 12′ E) and a farm of Balsamand, near KVK Sadalpur,
Haryana Agricultural University (HAU), Hisar (Supplemen-
tary Table1). Samples collected were pooled for analyses of
different physico-chemical, pH and electrical conductivity
(EC) (Singh etal. 1999), and nutrient-related properties such
as organic carbon content (Walkley and Black 1934), avail-
able N, using the alkaline permanganate method (Subbiah
and Asija 1956), available P (Olsen 1954) and available K
(Standfold and English 1949). Available iron was analyzed
by diethylene triamine penta acetic acid (DTPA) extraction
method (Lindsay and Norvell 1978).
Selection ofendophytes, growth conditions
andinoculum preparation
Two endophytes Arthrobacter sulfonivorans DS-68 and
Enterococcus hirae DS-163, previously reported (Singh
2016) to be efficient for Fe fortification in the grains of
one low Fe accumulator wheat genotype (CIM-412) were
used. Characterisation of these isolates had shown that they
exhibited IAA, siderophore production, phosphorus solu-
bilization, and ammonia production ability (Singh etal.
2018). These bacterial isolates were grown in nutrient broth
(50mL) whose constituents (g/L) were: 5.0g peptone, 3.0g
beef extract, and 5.0g sodium chloride. The pH of medium
was adjusted to 7.2 ± 0.2, by adding either 1M NaOH or
1N HCl. Bacterial endophytes were inoculated in sterilized
nutrient broth and incubated for 18h at 30°C, while being
shaken at 150rpm.
Wheat genotypes
Four wheat genotypes, classified based on preliminary
experimentation (Singh 2016) as 4HPYT-414 (low Fe accu-
mulator in high available Fe content soil/sandy-clay-loam
soil), 4HPYT-433 (high Fe accumulator in high available Fe
content soil/sandy-clay-loam soil), CIM-412 (low Fe accu-
mulator in low available Fe content soil/calcareous soil) and
GW-07-112 (high Fe accumulator in low available Fe con-
tent soil/ calcareous soil) were used.
Pot experiments
Two pot experiments were conducted in a glasshouse of the
Division of Microbiology, ICAR-IARI, New Delhi, using
IARI soil (sandy-clay-loam soil) and Hisar soil (calcare-
ous soil) in 10kg earthen pots filled with 8kg of sterilized
soil. The first pot experiment was carried out with IARI
soil (sandy-clay-loam soil/ high available Fe content soil),
using two wheat genotypes, i.e., 4HPYT-414 (low Fe accu-
mulator in high available Fe content soil) and 4HPYT-433
(high Fe accumulator in high available Fe content soil) and
two endophytes i.e., Arthrobacter sulfonivorans DS-68 and
Enterococcus hirae DS-163 (siderophore-producing endo-
phytes). Chemical fertilizer used was FeSO4. The details of
different treatments are: T1 (4HPYT-414-only RDF; unin-
oculated control), T2 (4HPYT-414 + FeSO4), T3 (4HPYT-
414 + DS-68), T4 (4HPYT-414 + DS-163), T5 (4HPYT-433-
only RDF; uninoculated control), T6 (4HPYT-433 + FeSO4),
T7 (4HPYT-433 + DS-68) and T8 (HPYT-433 + DS-163).
Each treatment was applied in triplicate. The FeSO4 dose in
the treatments was 25kg/ha.
The second pot experiment was carried out with Hisar
soil (low available Fe content soil/calcareous soil), in which
two wheat genotypes, i.e., CIM-412 (low Fe accumulator in
Hisar soil/low available Fe content soil/calcareous soil) and
GW-07-112 (high Fe accumulator in Hisar soil/low avail-
able Fe content soil/calcareous soil), were used. Endophyte
inoculants and chemical fertilizer (FeSO4) were the same as
used in the first pot experiment. Thus, eight treatments for
biofortification with iron in Hisar soil were same as those in
IARI soil; only wheat genotypes were different.
The recommended doses of fertilizers (RDF)—90kg
N/ha, 60kg P/ha and 40kg K/ha, were applied to wheat
through urea, diammonium phosphate (DAP) and muriate of
potash (MOP), respectively, in both experiments. The treat-
ment details are also provided in the tables. Mature plants
were harvested after 110days of sowing.

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Determination ofroot morphological parameters
After 60days of sowing, the plants from three replicates
were uprooted. The roots were rinsed with water to remove
soil particles adhering to its surface. Root morphology was
investigated using a root scanner controlled by WIN RHIZO
Programme V. 2002C software (Regent Instruments Inc. Ltd.
Quebec, Canada), and attributes such as length, surface area,
volume, as well as the average number of root tips and root
diameter were recorded.
Estimation ofiron inplants andyield
Iron accumulation in harvested plant parts (root, shoot and
grains) of each treatment was analyzed after grinding the
plant samples separately using mortar and pestle, followed
by the digestion of samples, using di-acid (nitric acid and
perchloric acid in 4:1 ratio) mixture. Digestion of samples
was undertaken using a hot plate at 300°C, followed by
measurement of iron concentration using AAS (atomic
absorption spectrophotometer), as outlined by Lindsay and
Norvell (1978). Different concentrations of iron (1.0, 2.0,
3.0, 5.0 and 10.0mg Fe L−1) were used as standard.
Statistical analyses
The data generated were analyzed as one-way ANOVA and
two-way ANOVA using Minitab statistical software version
17 and OPSTAT software, respectively. All the values pre-
sented here represent the mean values of three replications.
Comparisons between means were carried out via Tukey’s
test at 5% level of probability (P < 0.05).
Results
Soil characteristics
Both IARI and Hisar soils showed distinct differences in
chemical and nutritional parameters such as pH, EC, nitro-
gen, phosphorus, potassium and micronutrients, especially
zinc and iron. Hisar soil showed higher pH (8.70 ± 0.10)
and EC (0.31 ± 0.03dS m−1) than IARI soil. However, the
availability of macronutrients and micronutrients in Hisar
soil was low, with concentration of available iron in Hisar
soil and IARI soil recorded as 1.34 ± 0.11 mg kg−1 soil
and 4.75 ± 0.14mgkg−1 soil, respectively (Supplementary
Table1).
Response ofwheat genotypes tobacterial
endophyte inoculation andassociated root
morphological traits
Analyses of root parameters revealed higher values in both
the low and high iron accumulating genotypes of wheat,
when grown in high available Fe content soil than in low
available Fe content soil. In terms of root parameters, the
high Fe-accumulator wheat genotype showed significantly
higher values, as compared to low Fe accumulator wheat
genotype (Table1, Figs.1, 2, and Supplementary Table2).
Fertilization with FeSO4 was statistically significantly higher
than uninoculated control (RDF) relative to surface area of
CIM-412 genotype and root length of GW-07-112 genotype
in low available Fe content soil; whereas, in high available
Fe content soil, fertilization with FeSO4 was statistically
Table 1 Effect of FeSO4 fertilization and bacterial endophytes inoculation on root parameters of low and high Fe accumulating wheat genotypes,
grown in high available Fe content (IARI) and low available Fe content (Hisar) soils
Treatments in column, with four treatments having different letters are statistically different at 5% level of significance via Tukey’s Test, gener-
ated through analysis of variance (ANOVA)
DS-68 = Arthrobacter sulfonivorans
DS-163 = Enterococcus hirae
Soil type Treatments Root length (cm) Total root volume
(cm3)
Average root
diameter
(mm)
Root length (cm) Total root volume
(cm3)
Average root
diameter (mm)
Low avail-
able Fe
content
(Hisar
soil)
CIM-412 genotype (Low Fe accumulator) GW-07-112 genotype (High Fe accumulator)
RDF 600 ± 100b0.57 ± 0.03c0.42 ± 0.03b750 ± 50c0.65 ± 0.04b0.41 ± 0.08c
RDF + FeSO4866 ± 57ab 0.65 ± 0.07bc 0.35 ± 0.02b985 ± 79.8b0.80 ± 0.05b0.47 ± 0.04bc
RDF + DS-68 1133 ± 152a1.00 ± 0.11a0.57 ± 0.06a1300 ± 100a1.11 ± 0.09a0.60 ± 0.03ab
RDF + DS-163 1066 ± 115a0.81 ± 0.09ab 0.53 ± 0.03a1100 ± 100ab 0.99 ± 0.05a0.62 ± 0.04a
High avail-
able Fe
content
(IARI
soil)
4HPYT-414 genotype (Low Fe accumulator) 4HPYT-433 genotype (High Fe accumulator)
Only RDF 878 ± 78b1.70 ± 0.09c0.44 ± 0.03b950 ± 58d1.92 ± 0.10c0.59 ± 0.07b
RDF + FeSO4966 ± 57b1.97 ± 0.13c0.50 ± 0.02ab 1155 ± 42c2.16 ± 0.28c0.65 ± 0.01ab
RDF + DS-68 1577 ± 167a3.61 ± 0.23a0.51 ± 0.02a1744 ± 44a4.30 ± 0.52a0.71 ± 0.03a
RDF + DS-163 1366 ± 57a2.74 ± 0.36b0.50 ± 0.01ab 1523 ± 40b3.07 ± 0.15b0.67 ± 0.04ab

Acta Physiologiae Plantarum (2020) 42:24
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significant over the uninoculated control relative to root
length and number of root tips in 4HPYT-433 genotype
(Table1, Figs. 1, 2). The ANOVA for root parameters
showed that although genotype and treatments were highly
significant (1% probability), their interaction (T X G) was
significant only for the average number of root tips (Table2).
With respect to root length parameter. treatments were more
significant (1% probability) in iron deficient soil (Hisar soil)
as compared to iron sufficient soil (IARI soil).
Endophyte inoculation led to significant increases in root
length, surface area, root volume and number of root tips
over RDF (Table1, Figs.1, 2). In low available Fe content
soil, endophyte inoculation increased the root surface area
and average number of root tips by twofold and 1.6-fold,
respectively, over the control (RDF), irrespective of geno-
types. However, in high available Fe content soil, these two
attributes only increased by 1.5-fold and 1.2-fold, respec-
tively, over the control (RDF), irrespective of genotypes
(Supplementary Table2). In low available Fe content soil,
only DS-68 inoculation was statistically significant over
FeSO4 fertilization for average number of root tips and
root surface area of both wheat genotypes (CIM-412 and
GW-07-112) (Fig.2). However, in high available Fe content
soil, both DS-68 and DS-163 treatments led to significantly
higher values of root surface area, when compared with
FeSO4 fertilization, in both low and high Fe accumulator
wheat genotypes (4HPYT-414 and 4HPYT-433) (Fig.1). In
general, endophyte inoculation led to significantly higher
values over the FeSO4 fertilization treatments, for all the root
morphological parameters evaluated. Endophyte inoculation
increased the root length, surface area and total root volume
by 36, 40 and 57%, respectively, over the RDF + FeSO4,
irrespective of genotypes or soil type (Table1 and Supple-
mentary Table2).
Biofortication withFe
In soil with high available Fe content (IARI soil), endo-
phyte inoculation showed significant and enhanced uptake
of iron through roots and biofortification of Fe in grains, as
compared with uninoculated control (only RDF). Likewise,
in low available Fe content soil, endophyte inoculation also
showed significant results over the uninoculated control
(RDF) with respect to Fe concentration in root, shoot and
grains. In general, both FeSO4 fertilization and endophyte
inoculation treatments were almost equally significant over
the uninoculated control (RDF) with respect to Fe concentra-
tion in root, shoot and grains (Table3).
In high available Fe content soil, there were no differ-
ences between RDF and RDF + FeSO4 treatments with
regard to Fe translocation. Low Fe accumulator wheat geno-
type 4HPYT-414 showed almost 46 and 22% Fe transloca-
tion from root to shoot and shoot to grains, respectively, with
RDF/RDF + FeSO4 treatments. On the other hand, 59 and
20% Fe translocation from root to shoot and shoot to grains
was recorded in the high Fe accumulator wheat genotype
4HPYT-433. The average percent values of Fe translocation
Fig. 1 Influence of siderophore producing bacterial endophytes on
root surface area and number of root tips in high available Fe content
soil, using wheat genotypes with different nutrient use efficiency
Fig. 2 Influence of siderophore producing bacterial endophytes on
root surface area and number of root tips in low available Fe content
soil using wheat genotypes with different nutrient use efficiency

Acta Physiologiae Plantarum (2020) 42:24
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24 Page 6 of 13
from root to shoot and shoot to grains respectively, due to
endophyte inoculation were 54% and 31%, in low Fe accu-
mulator wheat genotype (4HPYT-414), while values of 75%
and 25%, respectively were recorded in high Fe accumulator
wheat genotype 4HPYT-433. These results clearly showed
that although the high Fe-accumulating wheat genotype
4HPYT-433 showed higher Fe translocation from root to
shoot; the low Fe-accumulating wheat genotype 4HPYT-
414 showed greater Fe translocation from shoot to grain as
a result of endophyte inoculation (Fig.3a, b).
In the low available Fe content soil, no significant
differences were observed relative to Fe translocation
between RDF and RDF + FeSO4 treatments. The low
Fe accumulator wheat genotype CIM-412 showed about
71% and 21% Fe translocation from root to shoot, and
from shoot to grains, respectively, with respect to RDF/
RDF + FeSO4 treatments. The high Fe-accumulator
wheat genotype GW-07-112 showed about 59% and 20%
Fe translocation from root to shoot, as well as shoot to
grains, respectively. Endophyte inoculation led to average
Table 2 Analysis of variance for various root parameters to FeSO4 fertilization and bacterial endophytes inoculation in high available Fe content
(IARI soil) and low available Fe content (Hisar soil) soils
Significant at *5% and **1% probability level, respectively
df degree of freedom
Soil type Source of variance Df Mean sum of squares
Root length Root surface area Total root volume Average root
diameter
Average num-
ber of root tips
Low available
Fe content
(Hisar soil)
Treatments (T) 3 324,873.0** 14,702.4** 0.23** 0.056** 654,838.4**
Genotypes (G) 1 82,837.5* 4455.4** 0.10** 0.020** 623,070.4**
Interaction (T × G) 3 5240.5 66.4 0.003 0.005 14,928.4
Error 16 9805.8 210.4 0.005 0.002 14,246.9
Total 23 53,481.3 2266.4 0.04 0.010 124,361.8
High available
Fe content
(IARI soil)
Treatments (T) 3 707,133.4** 15,342.5** 5.623** 0.011** 1,431,112.5**
Genotypes (G) 1 128,334.4** 9,126.0** 0.767** 0.165** 1,196,173.5**
Interaction (T × G) 3 3943.4 285.0 0.079 0.001 135,100.5**
Error 16 6141.9 300.5 0.074 0.001 24,885.4
Total 23 102,601.5 2644.2 0.828 0.009 273,607.8
Table 3 Effect of FeSO4 fertilization and bacterial endophytes inoculation on fortification of Fe in roots, shoots and grains, in low and high Fe
accumulating wheat genotypes, grown in high available Fe content (IARI) and low available Fe content (Hisar) soils
Treatments in column, with four treatments having different letters are statistically different at 5% level of significance via Tukey’s Test, gener-
ated through analysis of variance (ANOVA)
DS-68 = Arthrobacter sulfonivorans
DS-163 = Enterococcus hirae
Soil type Treatments Iron content (mg kg−1 dry weight)
Low accumulator wheat genotype High accumulator wheat genotype
Root Shoot Grain Root Shoot Grain
4HPYT-414 genotype 4HPYT-433 genotype
High avail-
able Fe
content
(IARI
soil)
RDF 281.83 ± 21.10b133.52 ± 17.27c29.67 ± 4.50c690.33 ± 26.27b402.87 ± 12.80c76.52 ± 9.80b
RDF + FeSO4448.33 ± 52.99a202.17 ± 25.47b46.49 ± 8.50bc 788.33 ± 34.12a473.00 ± 22.51b100.67 ± 7.63b
RDF + DS-68 437.33 ± 30.02a255.63 ± 20.20a77.11 ± 13.00a858.33 ± 25.16a641.33 ± 31.90a174.00 ± 10.97a
RDF + DS-163 396.67 ± 24.66a198.33 ± 17.55b64.67 ± 7.02ab 798.33 ± 28.86a608.67 ± 28.29a150.00 ± 10.00a
CIM-412 genotype GW-07-112 genotype
Low avail-
able Fe
content
(Hisar
soil)
RDF 213.33 ± 17.56b149.00 ± 19.00c27.80 ± 6.93c445.33 ± 40.81b259.33 ± 24.01c51.86 ± 10.00c
RDF + FeSO4316.67 ± 20.82a228.00 ± 20.00ab 47.88 ± 8.65bc 663.33 ± 32.15a397.99 ± 20.50b83.57 ± 10.0b
RDF + DS-68 343.33 ± 23.09a260.83 ± 10.82a73.93 ± 8.01a669.99 ± 20.00a509.33 ± 24.00a129.5 ± 19.4a
RDF + DS-163 306.67 ± 30.55a213.67 ± 14.43b63.67 ± 5.51ab 608.33 ± 38.19a448.67 ± 12.06b110.00 ± 11.5ab

Acta Physiologiae Plantarum (2020) 42:24
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Page 7 of 13 24
values of Fe translocation of 73% and 29% from root to
shoot and shoot to grains respectively, in low Fe accumu-
lator wheat genotype CIM-412. Likewise, in the high Fe-
accumulator wheat genotype (GW-07112), these values
were 75% and 25%, respectively (Fig.3c, d), which was
similar to the trend recorded for high available Fe content
soil. Percent increase in Fe in root and shoot was almost
similar with FeSO4 fertilization and endophytes inocula-
tion. However, a twofold increase in the Fe concentration
in grains over the control and a onefold increase over the
FeSO4 fertilization were recorded, as a result of endo-
phyte inoculation (Table3 and Supplementary Table3).
The ANOVA related to Fe fortification in plant tissues
revealed that genotypes and treatments were highly sig-
nificant (1% probability) for Fe levels in root, shoot and
grains. However, their interaction (T X G) was significant
only for values recorded in root and shoot, but not in
grains (Table4).
Yield andyield attributes
In both low/high available Fe content soils, low and high
iron accumulating genotypes showed an increased number
of grains per spike among treatments compared with unin-
oculated control, but no statistically significant differences
were recorded among these treatments in high iron-accu-
mulating genotypes of wheat. Low iron accumulating geno-
types of wheat showed statistically significant differences
relative to FeSO4 fertilization treatments and Arthrobacter
sulfonivorans DS-68 and Enterococcus hirae DS-163 inocu-
lation, as compared with uninoculated control (Fig.4a–d;
Supplementary Table4). However, there were no signifi-
cant differences among the two inoculated treatments. The
ANOVA showed that treatments were highly significant (1%
probability) for grain yield and genotypes were highly signif-
icant (1% probability) for 100 grain weight. However, their
interaction (T×G) was not significant for yield and yield
0
10
20
30
40
50
60
70
RDFRDF+FeSO4 RDF+DS-68RDF+DS-163
% Fe translocaon
Treatments
4HPYT-414 (Low Fe accumulator)
Root to Shoot
a
0
10
20
30
40
50
60
70
80
90
RDFRDF+FeSO4 RDF+DS-68RDF+DS-163
% Fe translocaon
Treatments
4HPYT-433 (High Fe accumulator)
Root to Shoot
Shoot to Grains
b
0
10
20
30
40
50
60
70
80
RDFRDF+FeSO4 RDF+DS-68RDF+DS-163
% Fe translocaon
Treatments
CIM-412 (Low Fe accumulator)
Root to Shoot
Shoot to Grains
c
0
10
20
30
40
50
60
70
80
RDFRDF+FeSO4 RDF+DS-68RDF+DS-163
% Fe translocaon
GW-07-112 (High Fe accumulator)
Root to Shoot
Shoot to Grains
d
Treatments
Fig. 3 Percent translocation of Fe from root to shoot and shoot to
grains, a Low Fe accumulator in high available Fe content soil;
b High Fe accumulator in high available Fe content soil; c Low Fe
accumulator in low available Fe content soil; d High Fe accumulator
in low available Fe content soil

Acta Physiologiae Plantarum (2020) 42:24
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24 Page 8 of 13
Table 4 Analysis of variance
for Fe content in roots, shoots
and grains, in response to
FeSO4 fertilization and
endophytes inoculation in high
available Fe content (IARI) and
low available Fe content (Hisar)
soils
Significant at *5% and **1% probability level, respectively
df degree of freedom
Soil type Source of variance df Mean sum of squares
Iron content (mgkg−1)
Root Shoot Grain
High available
Fe content
(IARI soil)
Treatments (T) 3 29,951.042** 37,146.033** 6392.426**
Genotypes (G) 1 925,705.236** 669,553.863** 30,086.498**
Interaction (T × G) 3 1953.588 8335.373** 870.803**
Error 16 1010.353 485.563 85.672
Total 23 45,112.34 35,381.17 2315.08
Low available
Fe content
(Hisar soil)
Treatments (T) 3 38,778.690** 34,568.671** 4009.724**
Genotypes (G) 1 546,531.468** 218,782.865** 9204.534**
Interaction (T × G) 3 3733.802* 6095.656** 367.982
Error 16 847.440 350.558 115.747
Total 23 29,896.868 15,060.20 1051.72
Fig. 4 Influence of siderophore producing endophytic bacteria on
yield and yield attributes of different wheat genotypes in low/high
available Fe content soil: a Low Fe accumulator in high available Fe
content soil; b High Fe accumulator in high available Fe content soil;
c Low Fe accumulator in low available Fe content soil; d High Fe
accumulator in low available Fe content soil

Acta Physiologiae Plantarum (2020) 42:24
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Page 9 of 13 24
attributes (Table5). As 1000 grain weight is a factor of the
weight of grains and the number of grains, further in-depth
analyses of contributing factors and conduct of trials over a
period of two or more years can throw light on this aspect.
In high available Fe content soil, inoculation with Arthro-
bacter sulfonivorans DS-68 in a low iron-accumulating gen-
otype 4HPYT-414, showed significant differences, as com-
pared with uninoculated control (RDF) with respect to 100
grain weight. However, in high iron-accumulating genotype
4HPYT-433, there were no significant differences among
the treatments (Fig.4a, b). In low available Fe content soil,
endophyte Arthrobacter sulfonivorans DS-68 inoculation of
low iron-accumulating genotype CIM-412 and high iron-
accumulating genotype GW-07-112 showed significantly
higher values in comparison with uninoculated control for
100 grain weight (Fig.4c, d).
Discussion
The most common micronutrient deficiency in the world
is insufficiency of iron (Umbreit 2005), and despite scanty
information available regarding the extent of iron deficiency
globally, nearly two billion people are known to suffer from
anemia (World Health Organization (WHO) 2011), and the
most common cause globally is attributed roughly half to
a lack of adequate iron (Stoltzfus etal. 2004; Kassebaum
etal. 2014). The present investigation was initiated with
soil analyses, followed by growth-related response of wheat
genotypes to both soil types and endophyte inoculation, fol-
lowed by soil–plant analyses of nutrients. Sillanpaa (1982)
and Alloway (2009) reported that the critical value of Fe
in soil was 4.5mgkg−1. Based on the above-noted proper-
ties of soil, IARI soil (sandy clay loam soil) and Hisar soil
(calcareous soil) were designated as high available Fe con-
tent soil and low available Fe soil, respectively.
It is well established that root morphological parameters
such as total root length and root surface area play an impor-
tant role in nutrient uptake, particularly, under micronutri-
ent-limiting conditions (Batista etal. 2016), which supports
the results of this study. Treatments involving the inocula-
tion of siderophore-producing endophytes led to positive
effects on the root parameters, emphasizing their role as a
major contributing factor towards increased uptake of Fe.
Chen etal. (2014) reported that inoculation with endophytic
bacteria Sphingomonas SaMR12 significantly improved the
root length, root surface area, and average number of root
tips in Sedum alfredii plant, as compared with uninoculated
treatment. Hosseini etal. (2017) also reported that endo-
phytic fungus Piriformospora indica played an important
role in mitigation of drought stress because it improved root
morphology and increased the absorption sites for water
and nutrients. The hypothesis that endophyte inoculation
enhances the root volume, surface area, root length, root
diameter and average number of root tips in wheat is also
supported by previously published work (Singh etal. 2017a).
Iron fortification by endophyte inoculation might be the
result of indole acetic acid (IAA) secretion by endophytes, as
IAA has a pronounced effect on root morphological param-
eters (Pitts etal. 1998; Rahman etal. 2002; Ishida etal.
2008; Peret etal. 2009; Xu etal. 2014; Liu etal. 2015). Both
the endophytic bacteria Arthrobacter sulfonivorans DS-68
and Enterococcus hirae DS-163 produced 28 and 19µg/ml
of IAA (Singh 2016).
Although a large amount of Fe is present in the earth’s
crust, plants face stress relative to Fe availability. Insoluble
ferric oxide is the predominant form of Fe at higher soil
pH (≥ 7 pH). About 30% of the arable land of the world
Table 5 Analysis of variance
for yield and yield attributes,
as influenced by FeSO4
fertilization and bacterial
endophytes inoculation in high
available Fe content (IARI soil)
and low available Fe content
(Hisar soil) soils
Significant at *5% and **1% probability level, respectively
df degree of freedom
Soil type Source of variance df Mean sum of squares
Yield and yield attributes
Number of
grain/spike
100 grain
weight (g)
Grain yield (g/pot)
High available
Fe content
(IARI soil)
Treatments (T) 3 106.0** 0.33 3.91**
Genotypes (G) 1 28.2 1.76** 28.86**
Interaction (T × G) 3 13.5 0.27 0.48
Error 16 12.04 0.11 0.29
Total 23 25.12 0.23 2.03
Low available Fe
content (Hisar
soil)
Treatments (T) 3 187.04** 1.29** 14.50**
Genotypes (G) 1 3.37 0.20** 0.26
Interaction (T × G) 3 6.26 0.04 0.21
Error 16 22.58 0.02 0.25
Total 23 41.07 0.20 2.10

Acta Physiologiae Plantarum (2020) 42:24
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24 Page 10 of 13
is calcareous to alkaline soil (Takahashi etal. 2001) and
cereal crops such as rice, wheat and barley are more suscep-
tible to Fe deficiency than wild grassy plants. Plants exhibit
important mechanisms to grow under iron deficiency, which
includes the secretion of phenolic substances, production
of phytohormones, changes in root morphology and over-
expression of genes involved in Fe uptake (Marschner 1995;
Malinowski and Belesky 2000; Colangelo and Guerinot
2004; Dinneny etal. 2008). Morgounov etal. (2007) and
Chatzav etal. (2010) reported significant genetic variability
for Fe and Zn among wheat genotypes. From the context
of Indian wheat cultivars, Khokhar etal. (2018) recorded
6.43 ± 85.9mgkg−1 Fe in a set of 36 Indian-adapted wheat
genotypes in multiplicational trials over a period of two
years. A wide range in grain Fe content in the 286 recom-
binant inbred lines (RILs), derived from a cross between
an old Indian wheat variety WH542 and a synthetic deriva-
tive (Triticum dicoccon PI94624/Aegilops squarrosa [409]//
BCN), as reported by Krishnappa etal. (2017).
Plant–microbe interactions have crucial roles in improv-
ing the nutritional status of soil and enrichment of micro-
nutrients through metal solubilization, mobilization and
translocation to the different parts of the plant (Kothari etal.
1990; Rana etal. 2012; Chen etal. 2014). Aras etal. (2018)
also reported that the available Fe content in micronutri-
ent-deficient soil and plant tissues can be enhanced using
plant growth promoting rhizobacteria; this was mediated
mainly through increased release of organic acids and ferric
chelate reductase activity in plant and soil. Besides rhizos-
pheric microorganisms, the internal microbiome of plants
or microbial endophytes has become a fascinating research
area for the fortification of micronutrients in food grains.
This is based on their unique relationship with various plant
parts. The major mechanisms employed by microorganisms
for enrichment of micronutrients in the plant involve one or
a combination of these: (i) siderophore production (Schalk
etal. 2011; Saha etal. 2012); (ii) increased efflux of pro-
tons in the rhizosphere (Malhi etal. 1998); (iii) secretion
of organic acids that decrease the rhizospheric pH and act
as chelating agents of metal ions (Raja etal. 2006; da Silva
Lima etal. 2014); (iv) modifications in root structure, such
as enhanced root length and absorption area (Singh etal.
2017a); and (v) upregulation of genes responsible for tak-
ing up the respective micronutrient and modification of root
anatomy (Singh etal. 2017b). Endophytes also possess the
ability to enhance plant growth via a number of mechanisms
including phytohormone production, bioactive molecules,
heat stress alleviation (Waqas etal. 2015), salt stress allevia-
tion (Asaf etal. 2018), control of phytopathogens and insect
pests (Dutta etal. 2014), and nutrient uptake, even under
deficient conditions (Lugtenberg and Kamilova 2009).
Our results are also supported by previous work in which
inoculation of these siderophore-producing endophytes
brought about structural changes in the root and the expres-
sion of TaZIP3 and TaZIP7 genes was upregulated in the
roots and shoots in a hydroponic experiment. This syner-
gistically facilitated Fe translocation and the higher values
of Fe in roots and shoots (Singh etal. 2017b). Similar stud-
ies undertaken by Gopalakrishnan etal. (2016) illustrated
that inoculation with siderophore-producing bacteria led to
higher Fe concentration in grains of chickpea and pigeonpea.
In the present investigation, endophyte inoculation led to
enhanced transfer of micronutrients from not only root to
shoot, but also shoot to grains, as compared with uninocu-
lated control as micronutrients are involved in efficient elec-
tron transport system, photosynthesis, respiration, chloro-
phyll synthesis, reproduction and protein synthesis in plants.
Kabir etal. (2016) reported 100–600mgg−1 dry weight in
roots, which support our observations on the higher Fe val-
ues in the plant tissues; they also emphasized that this was
the net result of the diversity among genotypes and avail-
ability of auxin-related and antioxidant mechanisms, which
protect the plant tissues against any toxicity-related effects.
Our yield results were supported by Waller etal. (2005)
who reported that inoculation with endophytic fungus Piri-
formospora indica increased the yield of barley by 10%
over the uninoculated control, which correlated positively
with the enhanced number of ears. Ray and Valsalakumar
(2010) also reported that Piriformospora indica colonization
increased the green gram yield, number of pods per plant
and number of seeds per pod. Enhanced wheat productiv-
ity and accumulation of N, P, K+, Fe, Zn and Cu in wheat
grains was recorded as a result of mycorrhizal colonization,
in plants grown in saline soil (Talaat and Shawky 2013).
In the present investigation, treatment with endophyte
exhibited more pronounced effects on the yield as compared
with FeSO4 fertilization, in either low or high Fe-accumu-
lating wheat genotypes in low available Fe content soil.
The high available Fe content soil, FeSO4 fertilization and
endophyte inoculation gave almost similar responses which
corroborate the trend observed in our earlier study in which
endophyte inoculation led to significantly higher values, as
compared to ZnSO4 fertilization, in terms of yield and yield
parameters (Singh etal. 2017a). All these investigations
support the hypothesis that endophytes play an important
role in enhancing grain yields because of their multifarious
activities, particularly related to better nutrient mobilization
growth and yields, which in turn, are influenced to a certain
extent by genotypic differences and the soil type.
In conclusion, endophyte inoculation enhances the
translocation of Fe from root to shoot, and from shoot to
grains, as compared with uninoculated control (RDF). Per-
cent increase in Fe translocation to grains was significantly
greater because of endophyte inoculation, as compared
with FeSO4 fertilization, irrespective of soil type (low or
high Fe availability) or cultivar potential to accumulate Fe.

Acta Physiologiae Plantarum (2020) 42:24
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Page 11 of 13 24
Inoculation with siderophore-producing endophytes signifi-
cantly improved the root characteristics, including length,
surface area, root volume, root diameter and average number
of root tips which directly facilitated Fe fortification, empha-
sizing this as a safe and effective option. Therefore, future
recommendations should include the use of siderophore-
producing endophytes as bioinoculants, besides possible
substitution or supplementation with FeSO4 for enhanced
Fe fortification in grains of wheat genotypes, irrespective
of their differential micronutrient-accumulating efficiency
or type of soil. This investigation illustrates that endophytes
can be valuable, not only for Fe fortification, but also for
improving growth and yields of the modern cultivars glob-
ally and need to be integrated in the package of practices
recommended to farmers for biofortification of grains.
Author contribution statement Devendra Singh: Concep-
tualization and design of experiments, Collection of data
and data processing, Writing of first draft of the manuscript.
Neelam Geat: Collection of data. Mahendra Vikram Singh
Rajawat: Collection of data, Analysis and data processing.
Radha Prasanna: Critical editing of draft and finalisation of
manuscript for submission. Anil Kumar Saxena: Design of
the experiments; Final approval of the article, with critical
suggestions and arrangement of funds.
Acknowledgements The authors thank ICAR-Indian Agricultural
Research Institute and Indian Council of Agricultural Research
(ICAR), New Delhi for the financial support through NASF project.
The Division of Microbiology, ICAR-IARI, New Delhi is gratefully
acknowledged for the facilities provided, during the present study.
Compliance with ethical standards
Conflict of interest The authors declare no conflicts of interest.
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