European Journal of Soil Biology 50 (2012) 118e126

Contents lists available at SciVerse ScienceDirect

European Journal of Soil Biology

journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Biofortification of wheat through inoculation of plant growth promoting

rhizobacteria and cyanobacteria
Anuj Rana a, Monica Joshi a, Radha Prasanna a, Yashbir Singh Shivay b, Lata Nain a, *
a
Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110 012, India
b
Division of Agronomy, Indian Agricultural Research Institute, New Delhi, India

a r t i c l e i n f o a b s t r a c t

Article history: Biofortification is a novel approach which can lead to the development of micronutrient dense staple
Received 3 February 2011 crops. However, the role of microorganisms in improving the nutritional status of plants has been less
Received in revised form investigated. In our study, one bacterial (Providencia sp. PW5) and three cyanobacterial strains CW1, CW2
5 January 2012
and CW3 (Anabaena sp., Calothrix sp. and Anabaena sp. respectively) were evaluated in a field experi-
Accepted 16 January 2012
Available online 2 February 2012
ment, for their role in improving the nutritional quality of wheat grains, in terms of protein content and
Handling editor: Kristina Lindström important micronutrients (Fe, Cu, Zn and Mn). An enhancement of 18.6% in protein content was recorded
with PW5 þ N60P60K60 as compared to fertilizer control (N60P60K60). Comparative analysis with fertilizer
Keywords:
control (N60P60K60) revealed that inoculation of Providencia sp. PW5 þ N60P60K60 recorded statically at
Biofortification par values for Zn accumulation (41.73 mg kg1) and resulted in an increase of 105.3, 36.7 and 150.0% in
Malnutrition Fe, Mn and Cu respectively. Our study clearly emphasizes the need for the inclusion of PGPR to
Micronutrients complement the existing biofortification strategies for reducing malnutrition in developing countries.
PGPR Ó 2012 Elsevier Masson SAS. All rights reserved.
Wheat

1. Introduction Group on International Agricultural Research (CGIAR; http://www.

Plant growth promoting rhizobacteria (PGPR) include beneficial
bacteria that colonize plant roots and enhance plant growth by
a wide variety of mechanisms [1]. The use of PGPR is steadily
increasing in agriculture, as it offers an attractive way to reduce the
use of chemical fertilizers, pesticides and related agrochemicals.
Micronutrient malnutrition is known to affect more than half of
the world’s population and considered to be among the most
serious global challenges to humankind (Copenhagen consensus
2004; http://www.copenhagenconsensus.com). Modern plant
breeding has been historically oriented towards achieving high
agronomic yields rather than nutritional quality and other efforts
related to alleviating this problem have been primarily through
industrial fortification or pharmaceutical supplementation. Micro-
nutrient malnutrition or the hidden hunger is very common among
women and preschool children caused mainly by low dietary intake
of micronutrients, especially Zn and Fe [2]. Biofortification has been
defined as the process of increasing the bioavailable concentrations
of essential elements in edible portions of crop plants through
agronomic intervention or genetic selection. The Consultative
* Corresponding author. Tel.: þ91 11 25847649; fax: þ91 11 25846420.
E-mail address: latarajat@yahoo.co.in (L. Nain).
cgiar.org) has been investigating the genetic potential to increase
bioavailable Fe and Zn in staple food crops such as rice, wheat,
maize, common beans and cassava [3], however, information on
interventions using PGPR or other biological agents is limited [4].
Secretion of phytosiderophores by microorganisms and plants in
restricted spatial and temporal windows represent an efficient
strategy for uptake of iron and other micronutrients by plants from
the rhizosphere. Analysis of the complex interactions between
soils, plants and microbes in relation with micronutrient dynamics
represents a unique opportunity to enhance our knowledge of the
rhizosphere ecology. Such progress can provide information and
tools enabling us to develop strategies to improve plant nutrition
and health with decrease in the application of chemical inputs.
Microorganisms are known to differ significantly in competing
with higher plants for micronutrients [5] and application of FYM
(farmyard manure) or other organic manures also play a critical role
in the kinetics of mineral uptake in plants [6]. Among bacteria, a lot
of attention has been dedicated to the siderophore-mediated iron
uptake by fluorescent pseudomonads. A number of other mecha-
nisms are also involved in the sequestration and transformation by
microorganisms in soil such as production of acids, alkalis etc. PGPR
constitute a significant part of the protective flora that benefit
plants by enhancing root function, suppressing disease and accel-
erating growth and development [1]. Steven [5] pointed out that

1164-5563/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejsobi.2012.01.005
 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126
microorganisms differ in competing with higher plants for micro-
nutrients. Shivay et al. [7] observed that various species of Azoto-
bacter differed in their competitiveness with wheat plants in
extracting Fe and Zn.
Biofortification of crops through application of PGPRs can be
therefore considered as a possible supplementary measure, which
alongwith breeding varieties, can lead to increased micronutrient
concentrations in wheat crop, besides improving yield and soil
fertility [8,9]. In many micronutrient-deficient regions, wheat is the
dominant staple food making up more than 50% of the diet and
efforts towards enrichment using environment friendly options can
go a long way in translating into reduction in mortality of under-
nourished populations globally. Research efforts have shown that
there are varietal and even agronomic practices related differences
with respect to the ability to extract/absorb Fe or Zn from soil by
plants. Nitrogen nutrition is known to affect the abundance of
transporter proteins involved in the root uptake or root-to-shoot
translocation or phloem loading of Zn and other micronutrients,
such as ZIP family proteins and YSL transporters [10]. Hence in our
study, the interrelationship between major macronutrients and
micronutrients was also investigated in depth. Our study therefore
focused towards understanding the role of PGPR in enhancing the
protein and micronutrients concentration in wheat grains.
2. Materials and methods
2.1. Microorganisms
The bacterial strain PW5 was isolated from the wheat rhizo-
sphere and characterized by polyphasic approaches [11]. The strain
PW5 was identified as Providencia sp. on the basis of 16S rDNA
sequence analysis and submitted to Genbank with accession no.
FJ866760. The three cyanobacterial isolates CW1, CW2 and CW3
were also isolated from wheat/rice rhizosphere and identified using
the taxonomic keys of Desikachary [12]. All these cultures were also
screened for plant growth promoting traits and evaluated for their
plant growth promotion capability under controlled conditions in
phytotron as well as green house conditions [11e13].
2.2. Plant growth promoting traits
The production of indolic compounds by the bacterial isolates
was estimated using Salkowski reagent and quantified spectro-
photometrically by measuring the intensity of pink colour at
530 nm, using calibration curve of standard IAA stock solution
(10e100 mg ml1) prepared in 50% ethanol [14]. Quantitative esti-
mation of total soluble proteins was done spectrophotometrically
using the protocol of Herbert et al. [15] against bovine serum
albumin as standard. Qualitative assay for P/Zn solubilzation was
done in Pikovskaya medium containing tricalcium phosphate/zinc
carbonate [16]. The presence of a clear halo zone around the culture
spot, after incubation at 30  C for 48 h, indicated the P/Zn solubi-
lization capacity of the isolate. HCN production was evaluated by
the qualitative method of Kremer and Souissi [17]. Detection of
ammonia production was done by adding 1 ml of Nessler’s reagent
to 72 h old cultures grown in peptone broth and positive samples
exhibited yellowish brown colour [18].
A qualitative assay of siderophore production was conducted in
Chrome Azurole’s (CAS) agar media [19]. The development of
a yellow orange halo around the colony was considered as a posi-
tive result. The activity of 1-aminocyclopropane-1-carboxylate
(ACC) deaminase enzyme in the induced bacteria was quantified
by measuring the amount of a-ketobutyrate produced as a result of
the enzymatic cleavage of ACC [20]. The antifungal activity was
assessed against the test pathogen (Macrophomina phaseolina) by
119
dual culture technique on Potato Dextrose Agar (PDA) medium [21].
The diameter of zone of inhibition was measured after 48 h.
2.3. Field trial of promising plant growth promoting bacterial and
cyanobacterial strains
The promising bacterial (PW5) and cyanobacterial strains were
tested in a field experiment with wheat (Triticum aestivum variety
WR 544) in plots of 12 sq m. The experiment was conducted in the
Research Farm of Indian Agricultural Research Institute (IARI), New
Delhi, situated at a latitude of 28 400 N, longitude of 77120 E and
altitude of 228.6 m above the mean sea level (Arabian sea). The
climate of Delhi is of sub-tropical and semi-arid type with hot and
dry summer and cold winter and falls under the agro-climatic zone
‘Trans-Gangetic plains’. The annual mean pan evaporation is about
850 mm. Wheat was sown in the last week of November and har-
vested in the last week of March (Rabi season). The details of soil
type are given as Supplementary Table 1.
A suspension of log phase (12e14 d old) cultures of the cyano-
bacterial strains was applied at the rate of 2 g chlorophyll ha1. The
inoculum of bacterial strain PW5 contained 1011 cells ml1 and
500 ml culture ha1 was used for seed application. Bacteria and
cyanobacteria were mixed with compost as a carrier amended with
1% CMC (Carboxymethyl cellulose) prior to sowing of seeds and the
amended compost was used for seed coating at the rate of
500 g ha1. Seeds were sown at the rate of 120 kg ha1. Preliminary
evaluation undertaken on seed germination using single, dual
and multiple strain combinations revealed the promise of eight
treatments (data submitted elsewhere for publication). Further
experimentation was conducted with the eight promising treat-
ments in three replicates, as randomized block design (RBD):
Absolute control; Full dose N120P60K60; N60P60K60; N90P60K60;
N60P60K60 þ CW1; N60P60K60 þ PW5; N60P60K60 þ CW1 þ PW5;
N60P60K60 þ CW1 þ CW2 þ CW3. The recommended dose of
chemical fertilizer for wheat crop was 120 kg ha1 nitrogen (which
was applied in two split doses of urea), 60 kg ha1 phosphate in the
form of single super phosphate (SSP) and 60 kg ha1 potassium in
the form of muriate of potash (MOP) as single dose at the time of
sowing. Half of the recommended dose of N along with full dose of
P and K fertilizers was applied in the treatments receiving microbial
inoculants. Rhizospheric soil samples were collected at mid and
harvest stage and analyzed for soil microbiological parameters
(FDA hydrolysis, dehydrogenase, alkaline phosphatase, microbial
biomass C). Plant biomass, grain yield and harvest index were also
recorded. NPK and micronutrients concentration (Zn, Cu, Mn and
Fe) were also estimated in the wheat grains.
2.3.1. Soil microbiological parameters
Alkaline phosphatase activity was assayed in soil by the method
of Tabatabai and Bremner [22], and the enzymatic activity was
expressed as mg p-nitro phenol released g1 soil h1. The FDA
(Fluorescein diacetate) hydrolysis assay was carried out using
Fluorescein standard by the method outlined by Green et al. [23].
The values were represented as mg Fluorescein released g1 soil h1.
Dehydrogenase activity was assayed using the method of Casida
et al. [24]. The values were expressed as mg of Triphenyl Formazon
(TPF) g1 soil d1.
Microbial biomass C was estimated by the method of Nunan
et al. [25], using aliquots of K2SO4 extracts through dichromate
digestion. Microbial biomass carbon was measured after back
titration with ferrous ammonium sulphate and calculated using the
equation: Biomass C ¼ 2.64  CE where CE ¼ (organic C from
fumigated soil)  (organic C from unfumigated soil). Microbial
biomass C was expressed as mg C g1 soil.
120 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126
2.3.2. Analysis of micronutrients and NPK concentration in wheat
grains
Wheat grains were ground and the digested samples were used
for analysis of micronutrients (Fe, Cu, Zn and Mn), concentration
with Atomic Absorption Spectrophotometer at the most sensitive
wavelengths for Fe (248.7 nm), Cu (324.6 nm), Zn (213.7 nm), and
Mn (279.5 nm). Potassium concentration was analyzed with Flame
photometer and compared with standard ranging from 0 to
100 ppm of KCl. P concentration was measured by the method of
Jackson [26]. The total nitrogen of grain samples was estimated by
Kjeldahl’s method and the percent nitrogen content in the samples
was recorded using the N-autoanalyzer [26].
2.4. Statistical analysis
The data of the various parameters was analyzed in triplicates
for the growth and biochemical parameters and subjected to
ANOVA (Analysis of variance) in accordance with the experimental
design (completely randomized block design) using SPSS-10
statistical package to quantify and evaluate the source of varia-
tion. The treatment means were compared at a significance level of
0.05 and the ranking of treatments denoted by alphabets. The
treatments denoted by different letters in the each column of tables
and figures represent significantly different values among the
treatments.
3. Results
The selected bacterial strain PW5 was found to be positive for all
the tested PGPR traits (ammonia production, siderophore, HCN and
indolic compound production (17.37  3.01 mg ml1), antifungal
activity, phosphorous and zinc solubilization) under in vitro condi-
tions (Supplementary Table 2). The bacterial strain exhibited ACC
deaminase activity (3.13  0.03 mm of a-ketobutyrate mg1 h1).
This strain exhibited antagonism against the pathogenic fungi
Macrophomina phaseolina. The cyanobacterial isolates (CW1, CW2
and CW3) exhibited production of ammonia and indolic compound.
These strains were also found to inhibit the pathogenic fungi (M.
phaseolina). These cultures were identified using keys of Desikach-
ary as Anabaena sp., Calothrix sp. and Anabaena sp., respectively.
3.1. Field experiment
3.1.1. Soil microbiological parameters
At mid crop stage, the inoculated treatments, especially with
PW5 þ N60P60K60 recorded highest values for FDA hydrolysis
(Fig. 1a) and along with CW1 þ PW5 þ N60P60K60 exhibited signifi-
cantly higher dehydrogenase activity (Fig. 1b) as compared with
N60P60K60 control. Microbial inoculants recorded statistically at par
values of alkaline phosphatase activity as compared with fertilizer
control N60P60K60 (Fig. 1c), while microbial biomass carbon (Fig. 1d)
was significantly higher in the treatments inoculated with
CW1 þ PW5 þ N60P60K60 and CW1 þ CW2 þ CW3 þ N60P60K60. At
harvest stage, highest values for FDA (Fig. 2a) was observed in
treatments CW1 þ CW2 þ CW3 þ N60P60K60, while alkaline phos-
phatase activity was highest in PW5 þ N60P60K60 (Fig. 2c). Treatment
receiving CW1 þ N60P60K60 recorded highest dehydrogenase
activity (Fig. 2b). Microbial biomass carbon (Fig. 2d) was signifi-
cantly higher in CW1 þ PW5 þ N60P60K60.
3.1.2. Yield parameters
Plant parameters were recorded at the harvest stage of wheat
crop (Table 1). No significant differences were recorded in terms of
thousand grain weight among all the treatments but the highest
plant biomass was recorded in full dose fertilizer control N120P60K60,
which was statistically at par with the treatments- N60P60K60,
N90P60K60, CW1 þ N60P60K60 and CW1 þ CW2 þ CW3 þ N60P60K60.
However, in terms of grain yield, treatments involving
CW1 þ PW5 þ N60P60K60 and PW5 þ N60P60K60 recorded signifi-
cantly higher values than absolute control and fertilizer control
(N60P60K60). Similarly, highest harvest index (35.97%) was recorded
in CW1 þ PW5 þ N60P60K60 which was statically at par with
PW5 þ N60P60K60.
3.1.3. Nitrogen, phosphorous and potassium (NPK) content in grains
Inoculation with the bacterial/cyanobacterial strains along with
N60P60K60 also resulted in enhanced N uptake in wheat grain. The
percent N, P and K values are given in Table 2 and the highest N
(2.45%) and protein content (15.3%) was observed in treatment
PW5 þ N60P60K60. No significant differences were recorded in
terms of K (%).
3.1.4. Micronutrients in grains
In terms of micronutrients (Fe, Zn, Mn, and Cu), the highest
values were recorded in the treatment involving inoculation of
PW5 þ N60P60K60 (Table 3). A percent enhancement of 105.3, 36.7
and 150.0 in Fe, Mn and Cu concentration, respectively, was
recorded in this treatment, as compared to fertilizer control
N60P60K60. Regression analysis indicated a positive relationship
between micronutrient concentrations and N% (Supplementary
Fig. 1).
4. Discussion
Biofortification is an agricultural strategy that aims to increase
the content of selected micronutrients, including zinc, in staple
food crops such as rice, wheat, maize, pearl millet, and others.
Mineral packed seeds can have a tremendous spin-off for
increasing farm productivity in developing countries in an envi-
ronment friendly way, as such seedlings also exhibit an increased
survival capacity and resistance to disease and other environmental
stresses [27]. Although, the concentrations of some essential
mineral elements, such as Se and S, are highest in the embryo,
others, such as Fe, Zn and Cu, are highest in the bran [3,28]. Milling
or polishing cereal seeds is known to remove large quantities of
minerals from the seeds; the extent of these losses is genotype
dependent. Most of the approaches involving biofortification have
focused towards plant breeding or transgenics. Targetted plant
breeding helps to increase the concentration of several minerals
simultaneously in edible tissues, without incurring a yield penalty
[2], while transgenic approaches rely on improving mobilization
from the soil, uptake from the rhizosphere, translocation to the
shoot and accumulation of mineral elements in bioavailable forms
in edible tissues. Our investigation was aimed towards identifying
one or more plant growth promoting microbial strains, which can
enhance the mineral uptake in wheat grains, from soil, either
through solubilization or enzymatic activity. The combination of
three cyanobacterial (CW1, CW2 and CW3) and bacterial strain
(PW5) were selected on the basis of their superior performance in
Phytotron as well as pot experiments, which revealed significant
yield enhancement due to their plant growth promoting abilities
[13]. All these strains possessed promising PGPR traits, under
in vitro conditions [11,13].
Imbalanced use of macronutrient fertilizers, decreased use of
organic manure, reduced recycling of crop residues, and bumper
harvests in the past three decades have led to secondary and
micronutrient deficiencies in the soil. In several areas with inten-
sive cropping, zinc (Zn) deficiency appeared initially and subse-
quently the deficiencies of iron (Fe), manganese (Mn), boron (B),
and molybdenum (Mo) were recorded. Wheat grain is the most
 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126 121

Fig. 1. Effect of plant growth promoting bacteria on soil microbiological parameters at mid crop stage of wheat a. Fluorescein diacetate activity; b. Dehydrogenase, TPF represents
triphenyl formazon; c. Alkaline phosphatase, p-NP represents p-nitro phenol; d. Microbial biomass carbon. Bars marked with the same letter are not significantly different
(P < 0.05). Error bars represent standard deviation. *A basal dose of N60P60K60 was applied in all these treatments.

important source of calorie intake in a number of countries in the
developing world, but wheat is inherently too low in Zn (generally
20e30 mg of Zn/kg of grain) to meet the recommended dietary
allowance of Zn [29].
Zinc (Zn) deficiency, like iron, is known to affect billions of
people, destroying immune system, and hampering growth and
development. In many micronutrient-deficient regions, wheat is
the dominant staple food making up greater than 50% of the diet. In
our study, application of PW5 þ N60P60K60, recorded an increase in
the concentrations of Fe (105%) and Cu (150%), besides Zn, which
was statically at par with the application of chemical fertilizers
alone (control; N60P60K60). The values of Fe, Cu and Zn ranged from
129 to 272, 37-99 and 36e41 mg kg1 in the treatments receiving
microbial inputs. Plant breeding approaches exploiting the
inherent genetic variation in micronutrient concentrations in the
available germplasm, led to a similar level of enhancement in Fe
concentration (dry weight basis) in wheat grains in El Batan,
Mexico [30]. A cross between T. durum and A. tauschii was made to
develop a variety of hexaploid wheat with high concentrations of
Fe, Zn, Cu and Mn in the grain that can be crossed with modern
wheat [31]. Maize hybrids and varieties that yield grain with
25e30% more Fe and Zn than common cultivars have been devel-
oped as part of the Harvest Plus program [32].
Evidence suggests that nitrogen (N) nutritional status of plants
can have a positive impact on root-shoot translocation of nutrients.
Nitrogen status of plants is also known to exert positive effects on
root uptake of Zn and Fe and positive correlation between grain Zn
and protein concentrations are known to occur under high soil
applications of Zn and N [3]. In biological systems, proteins are
highly dependent on Zn ions to maintain their activities. Zinc is
needed for numerous proteins, having both a catalytic and a struc-
tural role. A positive, frequently close, relationship between the
grain concentrations of protein, Zn and Fe has been found in plant
species such as wheat [33,34]. There are several steps during the
uptake and transport of Zn and Fe in plants, which might be
affected by N nutrition and mediated by microorganisms. It has
been hypothesized that triggering root growth can stimulate root
mediated exudation of organic compounds [35,36].
N may also influence the mobility and root uptake of Zn and Fe
from soils. In maize, increasing N application was effective in
122 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126

Fig. 2. Effect of plant growth promoting bacteria on soil microbiological parameters at harvest stage of wheat a. Fluorescein diacetate activity; b. Dehydrogenase, TPF represents
triphenyl formazon; c. Alkaline phosphatase, p-NP represents p-nitro phenol; d. Microbial biomass carbon. Bars marked with the same letter are not significantly different
(P < 0.05). Error bars represent standard deviation. *A basal dose of N60P60K60 was applied in all these treatments.

enhancing C partitioning into roots and promoting exudation of C-
containing compounds from roots into rhizosphere [37]. The
expression level of Zn and Fe transporter proteins located on the
root cell membranes such as ZIP family transporter proteins [38]
are known to be affected by the plant N nutritional status. These
transporter proteins greatly influence uptake and accumulation of
Zn and Fe in plant cells.
To our knowledge, there is no published data concerning the
effects of PGPR on N nutrition and expression level or the amount of
transporter proteins for Zn and Fe in root cells. It is established that
following root absorption, Zn and Fe are transported into the shoot
through xylem vessels either as free ions or chelated to low
molecular-weight organic compounds. Similar to root uptake, root-
to-shoot transport of Zn and Fe is known to be facilitated by N,

Table 1
Effect of bacterial and cyanobacterial strains alone and in combination on plant yield parameters.

Treatments Thousand grains weight (g) Grain yield (t ha1) Total biomass (t ha1) Harvest index (%)
A C
Absolute control 35.74  1.51 4.18  0.13 14.15  0.10BCD 29.52  1.10CD
N120P60K60 38.11  2.70A 4.70  0.51AB 15.72  0.68A 29.85  2.58CD
N60P60K60 35.81  0.95A 4.37  0.21BC 14.54  1.36ABCD 30.23  3.34CD
N90P60K60 36.29  1.07A 4.66  0.12AB 14.54  0.68ABCD 32.07  1.52BC
*CW1 35.53  1.46A 4.15  0.31C 15.33  0.59AB 27.09  1.55D
*PW5 37.17  2.77A 4.77  0.40A 13.76  0.70CD 34.75  1.43AB
*CW1 þ PW5 36.77  1.41A 4.87  0.21A 13.56  0.61D 35.97  1.30A
*CW1 þ CW2 þ CW3 35.72  1.10A 4.66  0.31AB 14.94  0.34ABC 31.20  1.10C

Mean  Standard deviation, n ¼ 3; superscripts indicate significant differences based on the LSD 581 at 0.05 levels. *A basal dose of N60P60K60 was applied in all these
treatments.
 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126 123

Table 2
Effect of bacterial and cyanobacterial strains alone and in combination on nitrogen (N), phosphorous (P) and potassium (K) content of wheat grains.

Treatments N (%) P (%) K (%) Protein (%)

Absolute control 1.89  0.38C 0.70  0.17CD 0.35  0.02A 11.83  1.31C
N120P60K60 1.97  0.15BC 0.71  0.20CD 0.34  0.03A 12.30  0.92BC
N60P60K60 2.06  0.31ABC 0.75  0.23BCD 0.35  0.01A 12.89  1.21ABC
N90P60K60 2.16  0.28ABC 1.04  0.21AB 0.34  0.04A 13.51  1.77ABC
*CW1 2.05  0.21ABC 0.66  0.14D 0.34  0.03A 12.81  0.36ABC
*PW5 2.45  0.17A 1.10  0.16A 0.33  0.03A 15.29  1.05A
*CW1 þ PW5 2.14  0.11ABC 0.99  0.07ABC 0.31  0.03A 13.37  0.70ABC
*CW1 þ CW2 þ CW3 2.34  0.25AB 0.74  0.24CD 0.33  0.02A 14.61  1.56AB

Mean  Standard deviation, n ¼ 3; superscripts indicate significant differences based on the LSD at 0.05 levels. *A basal dose of N60P60K60 was applied in all these treatments.

either through modulation of the levels of proteins contributing to
xylem loading or chelation of Zn in the xylem by nitrogenous
compounds for xylem transport by nicotianamine and mugineic
acid family phytosiderophores [39,40]. Methionine, a sulfur-
containing proteinogenic amino acid, is a critical precursor in the
biosynthesis of nicotianamine and mugineic acid family phytosi-
derophores [41]. Hence, low N supply may reduce the pool of
methionine and in turn negatively affect the biosynthesis.
Enhanced knowledge of the effects of N on the amount or expres-
sion levels of transporter proteins of Zn and Fe will contribute to
better understanding the positive association between N, Zn, and
Fe in seeds.
It is important to emphasize that increased absorption of
a micronutrient by roots does not always correlate with high grain
accumulation of this micronutrient, as recognized for Fe in rice [42].
Remobilization and retranslocation of Zn and Fe from vegetative
tissues into seeds through the phloem may be affected by the level
of N nutrition and retranslocation of micronutrients deposited in
shoot tissues plays a critical role in the grain accumulation of
micronutrients. Increasing the N supply is known to increase the
concentration of total free amino acids in leaves and stimulate the
phloem export of amino acids. The wheat genotype used in our
study was highly responsive to PGPR inoculation, which in turn
aided in mobilization of micronutrients through siderophores. In
wheat, <70% of the Zn in vegetative plant parts is remobilized [43],
whereas only 20% of Fe is remobilized in rice [42]. Genotypic
differences in grain yield may also affect grain concentrations of
micronutrients, despite a similar root uptake rate or shoot accu-
mulation of micronutrients. A high pH of phloem sap is considered
a major factor reducing solubility and translocation of Zn and Fe
into sink organs. It can be hypothesized that by affecting the pool of
amino acids and peptides, N nutrition may contribute to chelation
of Zn and Fe and promote their transport through phloem into
seeds.
Although Zinc and Fe transporter proteins located on root cell
membranes have also been identified on the plasma membranes of
phloem cells, suggesting that these transporters are possibly
involved in phloem transport of Zn and Fe into seeds, the mecha-
nism is still not well understood. Grain concentrations of Fe, Mn
and also Cu respond positively to increasing N supply, which
suggests that these micronutrients may share similar N-dependent
mechanisms with Zn for uptake/translocation or storage in the
grain. In contrast to the grain concentrations of micronutrients, the
grain K concentration did not respond to increasing N supply,
rather a decrease was recorded with increasing N supply, which
clearly shows that the effect of N on grain concentrations of
micronutrients seem to be very specific [10]. Interestingly, in our
study, both N and P positively influenced the uptake of micro-
nutrients, but K did not have any significant effect.
Zn nutritional status of wheat plants is known to influence the
grain concentrations of phosphorous (P) and phytic acid (PA). A
decrease in grain P concentration by Zn application is associated
with a corresponding decrease in grain phytate concentration and
the phytate to Zn molar ratios. Formation of insoluble phytate
complexes of Zn and Fe are suggested to be a major reason for the
high incidence of micronutrient deficiency in countries with diets
high in phytate. In our investigation, a positive correlation between
P levels and Cu was recorded (r ¼ 0.944). The correlation coefficient
between N% and Fe was higher than that with Zn or Cu or Mn. Grain
Cu and Zn, followed by Fe, correlated positively with grain yield
(p < 0.01) but to a lower extent with microbial biomass carbon.
Further in depth analysis with respect to phytate concentrations
needs to be undertaken to understand the mechanism involved.
There is no information available regarding the interrelation-
ships between microbial activity and micronutrient mobilization. It
was interesting to note that although the treatments exhibiting
micronutrient levels in grains showed a positive correlation with
plant parameters, its relationship with microbiological activity was
mixed, i.e. FDA and Dehydrogenase were either negatively corre-
lated or showed low levels of positive correlation. But alkaline
phosphatase activity and microbial biomass carbon were positively
correlated. Grain Fe and Zn also correlated significantly (p < 0.01),
with alkaline phosphatase activity (data not shown).
It should be borne in mind that compared with Zn; Fe is less
phloem mobile in cereals [36]. Positive effects of N nutrition on
uptake of Zn and Fe, a very close relationship between the
concentrations of grain Zn, Fe, protein, and co-localization of the
genes on same chromosomes affecting their concentrations are

Table 3
Effect of bacterial and cyanobacterial strains alone and in combination on micronutrient concentrations of wheat grains.

Treatments Fe (mg kg1) Zn (mg kg1) Mn (mg kg1) Cu (mg kg1)

C D E
Absolute control 67.73  16.07 31.60  1.93 22.93  3.35 33.13  6.72C
N120P60K60 107.00  10.90C 37.20  1.31BC 27.73  4.27CDE 41.07  5.67BC
N60P60K60 132.00  30.00BC 40.93  1.80A 39.07  5.94B 39.60  12.35BC
N90P60K60 109.47  7.50C 37.20  2.71BC 24.87  3.34DE 68.27  8.92AB
*CW1 136.93  30.30BC 36.27  0.62C 38.13  2.52BC 41.93  9.16BC
*PW5 271.93  23.43A 41.73  1.01A 53.40  4.38A 99.00  6.84A
*CW1 þ PW5 206.13  31.20AB 39.80  1.74AB 34.33  1.42BCD 81.60  16.85A
*CW1 þ CW2 þ CW3 129.40  12.33BC 37.60  3.79BC 33.40  1.13BCDE 37.53  9.16BC

Mean  Standard deviation, n ¼ 3; superscripts indicate significant differences based on the LSD at 0.05 levels. *A basal dose of N60P60K60 was applied in all these treatments.
124 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126
highly important and relevant for further research. Correlation
analysis showed that both Fe and Zn correlated highly significantly
(p < 0.01) with grain P and N concentrations [44]. It seems very
likely that N improves root uptake or retranslocation (remobiliza-
tion) of Zn in wheat and the positive impact of N on tissue Zn
concentration could be related to the role of N in improving the
plant growth, which in turn enhances uptake by roots and accu-
mulation of Zn in shoots, an effect that is being exploited in phy-
toremediation of metal-contaminated soils [10].
Micronutrient deficiency in plants greatly increases their
susceptibility to diseases, especially fungal root diseases of the
major food crops. It has been reported that breeding for micro-
nutrient efficiency can confer resistance to root diseases [45,46].
Effectiveness in the control of soil-borne plant diseases has been
ascribed, at least in part, to competition for limiting nutrients
between beneficial microbes and pathogenic organisms. Many
rhizobacteria and fungi release iron chelators (siderophores) with
high binding affinity and specificity for Fe (III). The excretion of
these compounds by microbes, among other mechanisms, explains
the contribution of microbial activity to Fe acquisition by plants.
Nutrients like iron and boron, which are not readily translocated
from old to young leaves under stress conditions within the plant
are called immobile, while due to the variable mobility of zinc,
copper, molybdenum, and manganese under conditions of their
deficiency, the location of their symptoms in different crops and
crop species may vary depending upon the degree of their mobility.
Iron is a ubiquitous element in soils and sediments, usually found in
the range of crustal abundance around 3.5%. Multiple interactions
between soils, plants and microorganisms drive a complex iron
cycle in the rhizosphere and their activity is promoted by the
release of root exudates. Plants and microbes have evolved active
strategies of iron uptake, among which regulated exudation of
organic ligands by plants and microorganisms is a known response
to iron limitation [36]. These iron-mediated interactions between
soils, plants and microbes impact the plant growth and health and
their analysis, together with that of the resulting iron dynamics, is
of a major agronomic interest. The iron speciation in soil solution
includes inorganic hydrolysis species and a range of inorganic and
organic complexes. Iron is the fourth most abundant element of the
earth crust. However, in cultivated soils at pH values compatible
with plant growth, the solubility of iron is controlled at extremely
low levels by stable hydroxides, oxyhydroxides and oxides. Iron is
known to enter the plant via the root from where it is distributed
inside the plant. Generally, iron is present in concentrations of
about 10e500 mg Fe g1 dry weight in plant tissues. Under Fe
deficiency, graminaceous monocots release high-affinity Fe-
chelating substances from the mugineic acid family, called phyto-
siderophores. These substances solubilize Fe3þ and the resulting
Fe3þ- phytosiderophore complexes are taken up by the root cells
via a specific plasma membrane transport system without reduc-
tion of the ferric ion. This mechanism is termed Strategy II [47] and
it might resemble the microbial siderophore strategy. In our study,
the Fe concentration in the grains in the different treatments
ranged from 67.73 in the absolute control to 271.93 and
206.13 mg kg1 in inoculated treatments, which indicates the
promise of our strains. Also, no adverse effect of the microbial
inoculants on the Cu, Mn, and Zn nutritional status of the wheat
was recorded, indicative of positive effect on plant, despite the
possibility of potential competition for nutrients between micro-
organisms and plants.
Microbial exudates can also supply additional Mn, Zn and Cu to
plants. The increased Fe availability may result in a reduced avail-
ability of other micronutrients as a result of antagonistic effects.
This phenomenon is well documented for Mn. Manganese defi-
ciency is in localized sites where rice-wheat crop rotation is
practiced in coarse-textured soils and often decreases crop yield.
However, plant species differ in their susceptibility to Mn defi-
ciency. Poaceae are often inefficient, whereas Brassicaceae seem to
be efficient in Mn uptake [48,49]. Their results indicated that
differences in Mn efficiency among the crops studied are related to
their ability to affect the solubility of Mn in the rhizosphere. The
microbial strains used in our study seem to have played an
important role in enhancing its uptake, especially Providencia sp.
(PW5) which brought about a 36.7% increase in Mn content as
compared to N60P60K60 fertilizer control. Copper deficiency is not
widespread, but deficiency based on plant analysis is higher than
soil analysis. Therefore, the critical limits used for soil copper or
plant copper need to be re-calibrated as they can become a serious
problem in the years to come [38].
Our earlier studies have shown that Providencia sp. (PW5) and
cyanobacterial strains possess the potential to inhibit pathogenic
fungi, which may be playing a role in bringing about a significant
enhancement with respect to macro (NPK) and micronutrient
concentration in wheat grains. This emphasizes the promise of
PGPRs as an easy, direct and economically favorable approach for
biofortification. The PGPR traits may help the plants to grow deeper
roots in mineral deficient soils, produce ligands/siderophores or
acids/alkali to mobilize macro/micro nutrients. In our study, all the
treatments with microbial inputs included only half of the dose of N
fertilizer which further resulted in N savings. In spite of low N
fertilizer input, the inoculation of single and combination of strains
increased N accumulation by the plant, ranging 1.89e2.34% N in
wheat grain. It was reported by Cakmak et al. [3] that N status of
plants may also exert positive effects on root uptake of Zn and Fe,
which are transported into the shoot through xylem vessels either
a free ions or chelated to organic compounds such as siderophores.
In the present investigation, Providencia sp. (PW5) is known to
produce siderophore along with capacity for P and Zn solubilization
[11], which may have helped the wheat plant in better uptake of
macro and micronutrients.
A positive correlation was observed between the wheat grain
yield and concentrations of Nitrogen, Zn and Fe. Similar findings are
also reported by Morgounov et al. [33] in wheat triticale [50], maize
[32] and wild emmer wheat [51]. Durum wheat has often been
reported to be very sensitive to Zn deficiency [52]. This high
sensitivity of durum wheat cultivars to Zn deficiency has been
described to their low capacity to take up adequate Zn under Zn
deficient conditions and low release rate of Zn mobilizing phyto-
siderophores from roots into rhizosphere [53]. The Zn solubiliza-
tion capacity of Providencia sp. (PW5) can play an important role in
translocation and accumulation of Zn from root rhizosphere to
grain in such cultivars.
Wheat is considered as a phosphorous demanding crop and
uptake of micronutrients from soil requires a bulk of absorbed
phosphorous to provide energy in the form of ATP. In our study, this
was facilitated by the solubilization of soil phosphate by the inoc-
ulated PGPR strains. The major advantage of using PGPR is their
dual beneficial effect, resulting in nitrogen fertilizer savings as well
as biofortification of wheat, due to synergistic interactions between
bacterial and cyanobacterial strains as recorded earlier [13]. Grain
concentrations of Fe, Zn, Mn and Cu are positively affected by PGPR
inoculants, which suggest that these micronutrients may share
similar mechanisms for uptake or translocation and storage in the
grain. In our study, enhancement in the uptake of N, P and micro-
nutrients by the bacterial inoculants PW5 and cyanobacteria CW1
is supported by the PGPR traits possessed by these strains [11,13].
Our interesting findings regarding the positive involvement of
PGPR traits in enhancement of yield, micronutrient concentration
and their uptake in wheat grains is novel and constitutes a first time
report.
 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126
5. Conclusions
Plant-microbe interactions can be represented by a feed-back
loop, this being under the control of the soil physicochemical
properties, which impact the micronutrient bioavailability. Treat-
ments involving inoculation with single strain (Providencia sp.
PW5) and two-strain consortium (CW1þPW5) along with
N60P60K60 brought about 11e16% enhancement in grain yield,
harvest index and protein content, as compared to application of
chemical fertilizer (N60P60K60) alone. Our study demonstrated that
inoculation of Providencia sp. PW5 þ N60P60K60 led to an increase of
36e150% in Fe, Mn and Cu concentration, as compared to fertilizer
control N60P60K60. These observations demonstrate the merit of
deploying selected PGPR strains as a biofortification strategy for
wheat, which not only leads to 50% savings of N fertilizer, but also
does not compromise with the quality of grains. Therefore, inte-
grated use of PGPRs along with N60P60K60 can represent a major,
low-cost and sustainable option for enhancing micronutrient
concentration in wheat grains.
Acknowledgments
The authors are grateful to Indian Council of Agricultural
Research (ICAR), New Delhi for funding the AMAAS project on
Nutrient Management. We are also thankful to the Division of
Microbiology, IARI, New Delhi for providing necessary facilities for
undertaking this study.
Appendix. Supplementary material
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejsobi.2012.01.005.
References
[1] B.R. Glick, The enhancement of plant growth by free-living bacteria, Can. J.
Microbiol. 41 (1995) 109e117.
[2] H.E. Bouis, Micronutrient fortification of plants through plant breeding: can it
improve nutrition in man at low cost? Proc. Nutr. Soc. 62 (2003) 403e411.
[3] I. Cakmak, W.H. Pfeiffer, B.M. Clafferty, Biofortification of durum wheat with
zinc and iron, Cereal Chem. 87 (1) (2010) 10e20.
[4] A. de Santiago, J.M. Quintero, M. Aviles, A. Delgado, Effect of Trichoderma
asperellum strain T34 on iron, copper, manganese, and zinc uptake by wheat
grown on a calcareous medium, Plant Soil 342 (2011) 97e104.
[5] F.J. Steven, Organic matter-micronutrient reactions in soil, in: J.J. Mortvedt,
F.R. Cox, L.M. Shuman, R.M. Welch (Eds.), Micronutrient in Agriculture, Soil
Science Society of America, Madison, WI, USA, 1991, pp. 145e186.
[6] B.N. Mishra, R. Prasad, B. Gangaiah, Organic manures for increased rice
productivity and sustained supply of Fe to rice, Acta Agron. Hungarica 52
(2004) 371e397.
[7] Y.S. Shivay, R. Prasad, A. Rahal, Studies on some nutritional quality parameters
of organically or conventionally grown wheat, Cereal Res. Comm. 38 (3)
(2010) 345e352.
[8] H.E. Bouis, B.M. Chassy, O. Ochanda, Genetically modified food crops and their
contribution to human nutrition and food quality, Trends Food Sci. Tech. 14
(2003) 191e209.
[9] P.J. White, M.R. Broadley, Biofortifying crops with essential mineral elements,
Trends Plant Sci. 10 (2005) 583e586.
[10] U.B. Kutman, B. Yildiz, L. Ozturk, I. Cakmak, Biofortification of durum wheat
with zinc through soil and foliar applications of nitrogen, Cereal Chem. 87 (1)
(2010) 1e9.
[11] A. Rana, B. Saharan, M. Joshi, R. Prasanna, K. Kumar, L. Nain, Identification of
multi trait PGPR isolates and evaluating their potential as inoculants for
wheat, Ann. Microbiol. 61 (2011) 893e900.
[12] T.V. Desikachary, ICAR Monograph on Algae, Indian Council of Agricultural
Research, New Delhi, 1959, Cyanophyta.
[13] L. Nain, A. Rana, M. Joshi, S.D. Jadhav, D. Kumar, Y.S. Shivay, S. Paul,
R. Prasanna, Evaluation of synergistic effects of bacterial and cyanobacterial
strains as biofertilizers for wheat, Plant Soil 331 (2010) 217e230.
[14] A.S. Gordon, R.P. Weber, Colorimetric estimation of indole acetic acid, Plant
Physiol. 26 (1951) 192e195.
[15] D. Herbert, P.J. Phipps, R.E. Strange, Chemical analysis of microbial cells, in:
J.R. Norris, D.W. Ribbons (Eds.), Methods in Microbiology, vol. VB, Academic
Press, New York, 1971, pp. 209e344.
125
[16] R.I. Pikovskaya, Mobilization of phosphorous in soil in connection with vital
activity of some microbial species, Mikrobiologiya 17 (1948) 362e370.
[17] R.J. Kremer, T. Souissi, Cyanide production by rhizobacteria and potential for
suppression of weed seedling growth, Curr. Microbiol. 43 (2001) 182e186.
[18] D.W. Dye, The inadequacy of the usual determinative tests for identification of
Xanthomonas sp, New Zealand J. Sci. 5 (1962) 393e416.
[19] B. Schwyn, J.B. Neilands, Universal chemical assay for the detection and
determination of siderophores, Anal. Biochem. 160 (1987) 47e56.
[20] D.M. Penrose, B.R. Glick, Methods for isolating and characterizing of ACC
deaminase-containing plant growth promoting rhizobacteria, Plant Physiol.
118 (2003) 10e15.
[21] C. Dennis, J. Webster, Antagonistic properties of species-groups of Tricho-
derma I. Production of non-volatile antibiotics, Trans. Br. Mycol. Soc. 57 (1971)
25e39.
[22] M.A. Tabatabai, J.M. Bremner, Use of p-Nitrophenyl phosphate for assay of soil
phosphatase activity, Soil Biol. Biochem. 1 (1969) 301e307.
[23] V.S. Green, D.E. Stott, M. Diack, Assay of fluorescein diacetate hydrolytic
activity-optimization of soil samples, Soil Biol. Biochem. 38 (2006) 693e701.
[24] L.E.J. Casida, D.A. Klein, T. Santoro, Soil dehydrogenase activity, Soil Sci. 98
(1964) 371e376.
[25] N. Nunan, M.A. Morgan, M. Herlihy, Ultraviolet absorbance (280 nm) of
compounds released from soil during Chloroform fumigation as an estimate of
the microbial biomass, Soil Biol. Biochem. 30 (12) (1998) 1599e1603.
[26] M.L. Jackson, Soil Chemical Analysis, Prentice Hall of India Pvt. Ltd., New Delhi,
1967, pp. 134 e 144.
[27] P. Nestel, H.E. Bouis, J.V. Meenakshi, W.H. Pfeiffer, Biofortification of staple
food crops, J. Nutr. 136 (2006) 1064e1067.
[28] R.M. Welch, R.D. Graham, Breeding crops for enhanced micronutrient content,
Plant Soil 245 (2002) 205e214.
[29] I. Cakmak, Enrichment of cereal grains with zinc: agronomic or genetic bio-
fortification? Plant Soil 302 (2008) 1e17.
[30] R.D. Graham, D. Senadhira, S. Beebe, C. Iglesias, I. Monasterio, Breeding for
micronutrient density in edible portions of staple food crops: conventional
approaches, Field Crops Res. 60 (1999) 57e80.
[31] D.F. Calderini, I. Ortiz-Monasterio, Grain position affects grain macronutrient
and micronutrient concentrations in wheat, Crop Sci. 43 (2003) 141e151.
[32] M. Banziger, J. Long, The potential for increasing the iron and zinc density of
maize through plant-breeding, Food Nutr. Bull. 21 (2000) 397e400.
[33] A. Morgounov, H.F. Gomez-Becerra, A. Abugalieva, M. Dzhunusova,
M. Yessimbekova, H. Muminjanov, Y. Zelenskiy, L. Ozturk, I. Cakmak, Iron and
zinc grain density in common wheat grown in Central Asia, Euphytica 155
(2007) 193e203.
[34] C.J. Peterson, V.A. Johnson, P.J. Mattern, Influence of cultivar and environment
on mineral and protein concentrations of wheat flour, bran, and grain, Cereal
Chem. 63 (1986) 118e186.
[35] E. Paterson, A. Sim, D. Standing, M. Dorward, A.J.S. McDonald, Root exudation
from Hordeum vulgare in response to localized nitrate supply, J. Exp. Bot. 57
(2006) 2413e2420.
[36] H. Marschner, Mineral Nutrition of Higher Plants, second ed. Academic Press,
London, 1995.
[37] E. Liljeroth, P. Kuikman, J.A. Vanveen, Carbon translocation to the rhizosphere
of maize and wheat and influence on the turnover of native soil organic-
matter at different soil-nitrogen levels, Plant Soil 161 (1994) 233e240.
[38] N. Grotz, M.L. Guerinot, Molecular aspects of Cu, Fe and Zn homeostasis in
plants, Biochem. Biophys. Acta 1763 (2006) 595e608.
[39] C. Curie, G. Cassin, D. Couch, F. Divol, K. Higuchi, M.L. Jean, J. Misson, A. Schikora,
P. Czernic, S. Mari, Metal movement within the plant: contribution of nic-
otianamine and yellow stripe 1-like transporters, Ann. Bot. 103 (2009) 1e11.
[40] S. Alam, S. Kamei, S. Kawai, Metal micronutrients in xylem sap of iron-
deficient barley as affected by plant-borne, microbial, and synthetic metal
chelators, Soil Sci. Plant Nutr. 47 (2001) 149e156.
[41] S. Mori, N. Nishizawa, Methionine as a dominant precursor of phytosider-
ophores in Graminaceae plants, Plant Cell Physiol. 28 (1987) 1081e1092.
[42] M.A. Grusak, J.N. Pearson, E. Marentes, The physiology of micronutrient
homeostasis in field crops, Field Crops Res. 60 (1999) 41e56.
[43] H.S. Grewal, R.D. Graham, Residual effects of subsoil zinc and oilseed rape
genotype on the grain yield and distribution of zinc in wheat, Plant Soil 207
(1999) 29e36.
[44] F.J. Zhao, Y.H. Su, S.J. Dunham, M. Rakszegi, Z. Bedo, S.P. McGrath, P.R. Shewry,
Variation in mineral micronutrient concentrations in grain of wheat lines of
diverse origin, J. Cereal Sci. 49 (2009) 290e295.
[45] D.H. Sparrow, R.D. Graham, Susceptibility of zinc deficient wheat plants to
colonization by Fusarium graminearum Schw, Group I. Plant Soil 112 (1988)
221e266.
[46] R.D. Graham, A.D. Rovira, A role for manganese in the resistance of wheat to
take all, Plant Soil 78 (1984) 441e444.
[47] V. Romheld, H. Marschner, Evidence for a specific uptake system for iron
phytosiderophores in roots of grasses, Plant Physiol. 80 (1986) 175e180.
[48] M.H. Sayyari-Zahan, U.S. Sadana, B. Steingrobe, N. Classen, Manganese effi-
ciency and manganese-uptake kinetics of raya (Brassica juncea), wheat (Tri-
ticum aestivum), and oat (Avena sativa) grown in nutrient solution, J. Plant
Nutr. Soil Sci. 172 (2009) 425e434.
[49] U.S. Sadana, D. Samal, N. Claassen, Differences in manganese efficiency of wheat
(Triticum aestivum L.) and raya (Brassica juncea L.) as related to root-shoot
relations and manganese flux, J. Plant Nutr. Soil Sci. 166 (2003) 385e389.
126 A. Rana et al. / European Journal of Soil Biology 50 (2012) 118e126

[50] B. Feil, D. Fossati, Mineral composition of triticale grains as related to grain
yield and grain protein, Crop Sci. 35 (1995) 1426e1431.
[51] Z. Peleg, Y. Saranga, M.A. Yazici, T. Fahima, L. Ozturk, I. Cakmak, Grain zinc,
iron and protein concentrations and zinc-efficiency in wild emmer wheat
under contrasting irrigation regimes, Plant Soil 306 (2008) 57e67.
[52] R.D. Graham, J.S. Ascher, S.C. Hynes, Selecting zinc efficient cereal genotypes
for soils of low zinc status, Plant Soil 146 (1992) 241e250.
[53] N. Cakmak, H. Sari, H. Marschner, H. Ekiz, M. Kalaycı, A. Yılmaz, H.J. Braun,
Phytosiderophore release in bread and durum wheat genotypes differing in
zinc efficiency, Plant Soil 180 (1996) 183e189.