Introduction
Zinc (Zn) malnutrition is widespread in resource-poor
populations of the world. It weakens the immune function, increases the vulnerability to infection, and
affects pregnancy in women as well as the physical growth of children (Roohani et
al. 2013; Millward 2017). Young children, pregnant and lactating women are
the most affected due to Zn malnutrition (Hess 2017). Zn malnutrition can be overcome
by taking supplements and dietary intervention. Zn supplementation is
convenient especially for effected populations; however, it is not
cost-effective intervention (Meenakshi et al. 2007). Dietary
intervention is a sustainable long-term intervention
intended for the intake of diverse diets including greater consumption of
animal-source foods, commercial fortification and
biofortification (Bouis and Saltzman 2017). Grain Zn biofortification can enhance Zn status of cereals consumed by rural poor
people and can be carried
out through plant breeding, transgenic approaches, chemical fertilizers, and
with plant growth-promoting rhizobacteria (PGPR) inoculation (White and Broadley 2005; Rana et
al. 2012; Velu et al. 2014; Garg et al. 2018; Farooq et al. 2018; Rehman et al.
2018a; Younas et al. 2020). Application of PGPR is a novel
biotechnological approach through which cereals can be fortified by enhancing
the nutrient bioavailability and uptake (Rana et al. 2012; Hussain et al.
2018; Mumtaz et al. 2018; Rehman et al. 2018b; Ullah et al. 2020a).
Bioavailability of Zn for plant uptake and accumulation
in dietary foods is dependent on the Zn concentration. An increase in soil pH
decreases the Zn solubility and its availability to plants. Zn fertilizers are applied to fulfill the Zn
deficiency in plant, however, their greater fraction can become unavailable to
plants due to various edaphic factors and these can be transformed into
available forms using efficient ZSB strains (Cakmak
2008; Alloway 2009;Rehman et al.
2018c). The PGPR having the
ability to solubilize insoluble Zn are called Zn solubilizing bacteria (ZSB) (Saravanan et al. 2007; Mumtaz et al. 2017). ZSB secrete organic acids which chelate the bounded Zn and make it available to
crop plants (Fasim et al.
2002; Saravanan et al. 2007; Vidyashree et al. 2018; Mumtaz et
al. 2019). Numerous ZSB species of genera viz., Acinetobacter, Bacillus,
Cyanobacteria, Gluconacetobacter, Pseudomonas, and Serratia have been reported for their ability to solubilize non-labile-Zn in
soil (Saravanan et al. 2007; Mumtaz et al. 2017; Rehman et al. 2018b; Vidyashree et al. 2018; ;
Ullah et al. 2020b). Among these bacterial genera, Bacillus
spp. was the most dominant to solubilize Zn and to promote crop growth, yield
and improving nutrients accumulation in grains.
Zinc solubilizing Bacillus
strains are gram-positive plant-associated
bacteria having the ability to solubilize
non-labile minerals and secrete growth-promoting metabolites that enhance plant growth, nutrient availability and
suppress soil-borne plant pathogens (Chen
et al. 2006; Meena et al. 2016; Mumtaz et al. 2017, 2019).
Such strains can survive in extremely adverse environments because of their
prospective endospore formation and variable fatty acid configurations
(Diomande et al. 2015). These efficient strains can enhance the Zn availability to fulfill the
requirement of the plant and thus, helpful in cereal
grains biofortification (Sharma
et al. 2012). Previously, various Zn solubilizing Bacillus strains viz. Bacillus spp. (Shakeel et al. 2015; Mumtaz et al. 2017, 2018), B. aryabhattai (Ramesh et al. 2014; Mumtaz et al. 2017), B. thuringiensis (Khande et al.
2017), B. cereus (Khande et
al. 2017), B. firmus, B. amyloliquefaciens (Sharma et al.
2012) and B. subtilis (Mumtaz et al. 2017, 2018) were reported as
potential candidates for Zn biofortification in cereals. These ZSB
strains biofortified the cereals grains using different mechanisms including
biological nitrogen fixation from the atmosphere,
solubilization of non-labile mineral compounds, production of phytohormones,
1-aminocyclopropane-1-carboxylate deaminase activity, production of
siderophores and antifungal activities which promote yield and grain quality of cereals (Meena et al. 2016; Mumtaz et al. 2017;
Dinesha et al. 2018). These strains can be used as biofertilizers that synergistically promote
nutrient absorption and accumulation in grains (Vaid et al. 2014; Mumtaz et
al. 2017).
Biofortification of cereals through inoculation with ZSB
strains is an emerging biotechnological approach that can promote grain quality
and human health. It is well understood that the ZSB strain can increase the Zn
solubility in soil and its accumulation in grains but its role under Zn
deficient soils is poorly understood. Considerable research is needed in this
area to recognize novel strains as well
as their role for biofortification of cereals under Zn deficient soil
conditions. The current experiment describes
the potential of selected Zn solubilizing Bacillus strains possessing
multiple plant growth-promoting characteristics for improving growth, grain yield, and biofortification of maize under
native soil Zn conditions.
Materials and Methods
Collection
of bacterial strains and preparation of inoculum
Four Zn
solubilizing Bacillus strains viz., Bacillus
spp. ZM20, B. aryabhattai ZM31, B.
subtilis ZM63 and B. aryabhattai S10 (Genbank
accession numbers KX086260, KX788860, KX788861 and KX788862, respectively) were
obtained from the gene bank of Soil Microbiology and Biotechnology Laboratory,
Department of Soil Science, the Islamia University of Bahawalpur. The strains
were grown in Dworkin and Foster (DF) minimal media modified with ZnO (0.1% of
Zn w/v) as described by Mumtaz et al.
(2017) and incubated at 30 ± 1°C under shaking (100 rpm) conditions (Model
SI9R-2, Shellab, USA) for 48 h. After incubation, the
bacterial cells were harvested by centrifugation at 9000 rpm and 22°C for 20
min (Model: UNIVERSAL 320R, Hettich, Germany). The
supernatant was discarded, and the pellets were re-suspended in sterilized
distilled water. This washing procedure was repeated, and the pellets were
dissolved in sterilized distilled water to get uniform cell density (OD = 0.45;
cell count 108 CFU mL-1). The final cultures with a
uniform population were taken in a sterile
flask and used for inoculation.
Experimental management
The
experiment was conducted in farmer field located
at Latitude: 29.46°N, Longitude: 71.70°E and 115 m elevations above the Arabian
Sea level. Before crop sowing, a composite soil sample from 0–20
cm depth was taken and analyzed for physicochemical
characteristics by following standard procedures reported by Ryan et al. (2007). Available Zn
concentration in the soil before crop sowing was estimated by following
diethylene triamine penta-acetic acid (DTPA) extraction method (Lindsay and
Norvell 1978). The Zn concentration in extractant was determined in an Atomic
Absorption Spectrophotometer (Agilent
Technologies, Australia) using Zn lamp. For determination of total Zn
concentration in soil, 0.5 g of soil was digested with hydrofluoric acid (HF)
and perchloric acid (HCIO4) and analyzed through Atomic Absorption
Spectrophotometer (Yawar et al.
2010). The field soil was sandy loam and low in organic matter, nitrogen (N)
and phosphorus (P) but contains enough potassium (K). The bacterial strains
along with their co-inoculation combinations were inoculated on maize seeds of
cultivar Pioneer-30Y87 (Pioneer Seed Ltd., Pakistan) by
preparing slurry with peat, inoculum (bacterial culture) and sugar solution
(10%) in the ratio of 5:4:1. For co-inoculation formulation, broth of
respective cultures was used in the ratio
of 1:1 for slurry preparation. Uninoculated control was maintained through
coating seed with peat, control broth (without culture) and sugar solution.
Maize seeds from each treatment were sown on four ridges of 75 cm apart and
thinning was performed after 15 days of emergence through pulling out the
extra/ weak plants to maintain ten plants at a distance of 20–25 cm in each
ridge.
The recommended dose of N, P, and K (120: 90: 60 kg ha-1) was applied in the form of urea, diammonium phosphate (DAP) and sulfate of potash (SOP),
respectively. A full dose of P, K and
half of N was applied at sowing time. The
remaining half dose of N was given at the anthesis stage. The experiment
was conducted by applying treatment in Randomized
Complete Block Design (RCBD) with three replications. Irrigation need of each
plot was fulfilled through flooding the
field with good quality underground irrigation water (Ayers and Westcot 1985).
Thinning was done after germination to maintain plant density. All standard
agronomic practices were carried out as and when required. Growth and yield
contributing attributes were recorded on harvest at maturity. Plant and grain samples were analyzed to measure
biofortified nutrient concentrations. At physiological maturity, data about
growth and yield parameters were recorded. For determination of SPAD value, ten mature leaves (3rd from flag
leaf) from different plants were selected randomly and reading was noted by using SPAD meter model CL-01
(Hansatech Instruments Ltd., England). For the determination of N, P, K, Fe and
Zn, in maize straw and grains, samples from each treatment and replication were
oven-dried at 67°C and wet
digested following the method as described by Wolf (1982).
For the
determination of P concentration in digested plant samples, the standard procedure of Ryan et al. (2007) was followed. Flame
photometer (BWP Technologies, U.K.) was used to determine K in digested plant samples. The N,
Fe, and Zn were determined by using
commercial service of Central Hi-Tech
Laboratory, University of Agriculture Faisalabad, Punjab, Pakistan. The N concentration in plant extract was determined
following the standard procedure of the Kjeldahl
method as described by Ryan et al.
(2007). For Fe and Zn analysis, samples
were analyzed by using Atomic Absorption
Spectrophotometer (Agilent Technologies, Australia) as described by Helrich
(1990).
Statistical analysis
The statistical method was developed to evaluate the
effect of sole and co-inoculation with ZSB strains on growth, yield and quality
of maize. The group of variables were randomly split into bacterial inoculation
treatment and collected data were analyzed
by using one-way analysis of variance
technique (ANOVA) and means were compared by Least Significant Difference (LSD)
Tests at 5% level of significance (Steel et
al. 1997) through computer software Statistix v. 8.1 (Analytical Software,
Tallahassee, FL, USA).
Results
Field soil
characterization
The experimental field soil used in this study was
characterized by physicochemical properties. The result revealed that the
experimental soil was sandy loam (70% sand, 16% silt and 14% clay) alkaline in
nature (pH 8.1) having 0.28 dS m-1 electrical conductivity of soil
extract and 0.88% organic matter contents. Before sowing, soil showed total N
contents up to 0.05%, available P up to 4.6 mg kg-1, and extractable K up to 169 mg kg-1. There was 46.5 mg kg-1 total
Zn concentration in the soil while 3.2 mg kg-1 of Zn was present in
the available form (data is not given).
Growth
attributes
Maize growth
parameters, including SPAD (Soil-Plant Analyses Development) unit value, plant
height and shoot dry weight in retort to sole or co-inoculation with ZSB
strains are given in Table 1. The inoculation/co-inoculation gave a significant increase in growth attributes of
maize in terms of SPAD unit value, plant height and shoot dry weight.
Uninoculated control showed significantly lowest SPAD value of 50.7. The
increase in the SPAD unit value of maize was observed due to both inoculation
and co-inoculation, however, co-inoculation treatments were more effective to
show an increase in SPAD value except the combination of Bacillus spp. ZM20 and B. aryabhattai S10. The maximum SPAD value was
observed due to the co-inoculated
combination of B. aryabhattai ZM31 and B. aryabhattai S10 that
showed 65.3 of SPAD value. The co-inoculation combination of B. aryabhattai S10 × B. subtilis ZM63 also showed better SPAD value (61.4).
Uninoculated control showed the lowest plant height up to 220.5 cm and shoot
dry weight up to 187.6 g. The sole inoculation of most of the strains gave a significant
increase in maize height and shoot dry
weight, however, co-inoculation was more
effective as compared to sole inoculation. The maximum plant height of 255.9 cm
was recorded in the treatment involving the combined use of B. aryabhattai ZM31
and B. subtilis ZM63 which was statistically similar to co-inoculation
of B. aryabhattai S10 × B. subtilis ZM63 (249.8 cm). Combined
application of B. aryabhattai ZM31 and B. subtilis ZM63 also gave maximum shoot dry weight
216 g which was statistically similar to co-inoculation with Bacillus spp. ZM20 × B. subtilis ZM63.
Yield
attributes
Yield attributes in terms of cob length, cob dry weight
and 100-grains weight were enhanced when treated with sole and/or
co-inoculation (Table 2). Most of the treatments were non-significant to each
other but statistically different from uninoculated control. Co-inoculation
with B. aryabhattai ZM31 and B. subtilis ZM63 resulted in maximum
cob length of 17 cm while minimum cob length was 15.3 cm shown by uninoculated
control. Uninoculated control also showed a minimum cob dry weight of 131.4 g
and a 100-grains weight of 18.2 g. Co-inoculation with B. aryabhattai ZM31 and B. subtilis ZM63 showed maximum cob dry weight
of 158.3 g which was non-significant with sole inoculation of B. aryabhattai S10, however, these
treatments were statistically significant with uninoculated control. The
maximum 100-grains weight of 22.8 g was observed due to co-inoculation with B. aryabhattai ZM31 and B. subtilis ZM63 followed by co-inoculation
with Bacillus spp. ZM20 and B. subtilis ZM63 (22.4 g). These combinations were non-significant to each other
but significantly different from uninoculated control.
Table 1: Effect of Zn solubilizing Bacillus strains inoculation/co-inoculation on SPAD value, plant height and shoot dry weight of
maize sown in field conditions
|
Inoculation/co-inoculation* |
SPAD value |
Plant height (cm) |
Shoot dry weight
(g) |
|
Uninoculated Control |
50.7 f |
220.5 f |
187.6 i |
|
Bacillus spp. ZM20 |
55.9 cde |
242.3 cd |
201.5 f |
|
B. aryabhattai
ZM31 |
54.7 de |
236.8 de |
207.3 c |
|
B. aryabhattai S10 |
54.9 cde |
234.1 e |
192.2 h |
|
B. subtilis ZM63 |
57.9 bcd |
235.8 de |
205.3 e |
|
Bacillus spp. ZM20 × B. aryabhattai ZM31 |
57.8 bcd |
244.8 bc |
211.6 b |
|
Bacillus spp. ZM20 × B. aryabhattai S10 |
53.1 ef |
241.2 cde |
206.0 de |
|
Bacillus spp. ZM20 × B. subtilis ZM63 |
58.5 bc |
239.4 cde |
215.3 a |
|
B. aryabhattai
ZM31 × B. aryabhattai S10 |
55.6 cde |
243.5 bc |
196.3 g |
|
B. aryabhattai
ZM31 × B. subtilis ZM63 |
65.3 a |
255.9 a |
216.0 a |
|
B. aryabhattai S10
× B. subtilis ZM63 |
61.4 b |
249.8 ab |
208.8 c |
|
LSD (P ≤ 0.05) |
3.7026 |
6.6079 |
1.9428 |
*Inoculation/co-inoculation of Zn solubilizing Bacillus strains; LSD = least
significant difference; data are mean values of three replicates; Means sharing the same letter (s) do not
differ significantly according to LSD test
Table 2: Effect of Zn solubilizing Bacillus strains inoculation/co-inoculation on cob length, dry
weight and 100-grains weight of maize sown in field conditions
|
Inoculation/co-inoculation* |
Cob length (cm) |
Cob dry weight (g) |
100-grains weight (g) |
|
Uninoculated Control |
15.3 e |
131.4 e |
18.2 f |
|
Bacillus spp. ZM20 |
16.0 cd |
138.2 d |
18.6 ef |
|
B. aryabhattai
ZM31 |
15.9 cd |
151.9 b |
19.4 e |
|
B. aryabhattai S10 |
16.1 bcd |
157.1 a |
21.6 bc |
|
B. subtilis ZM63 |
16.2 bcd |
146.8 c |
18.3 f |
|
Bacillus spp. ZM20 × B. aryabhattai ZM31 |
15.7 de |
148.7 c |
21.1 cd |
|
Bacillus spp. ZM20 × B. aryabhattai S10 |
16.6 b |
136.4 d |
18.9 ef |
|
Bacillus spp. ZM20 × B. subtilis ZM63 |
16.6 b |
153.5 b |
22.4 ab |
|
B. aryabhattai
ZM31 × B. aryabhattai S10 |
16.3 bc |
138.5 d |
20.5 d |
|
B. aryabhattai
ZM31 × B. subtilis ZM63 |
17.0 a |
158.3 a |
22.8 a |
|
B. aryabhattai S10
× B. subtilis ZM63 |
16.6 b |
153.8 b |
21.8 bc |
|
LSD (P ≤ 0.05) |
0.5265 |
2.5868 |
0.9801 |
*Inoculation/co-inoculation of Zn
solubilizing Bacillus strains; LSD =
least significant difference; data are mean values of three replicates; Means sharing the same letter (s) do not
differ significantly according to LSD test
The data regarding the effect of inoculation and
co-inoculation on stover and grain yield and harvest index are given in Table
3. Results revealed that uninoculated control showed a minimum stover yield of
17410 kg ha-1, grain yield of 8598 kg ha-1 and harvest
index of 26.6%. The maximum grain yield of 9826 kg ha-1 was obtained
due to co-inoculation with B. aryabhattai ZM31 and B. subtilis ZM63
followed by the combined application of Bacillus
spp. ZM20 × B. subtilis ZM63 that showed 9741 kg ha-1
of grain yield. These treatments were also non-significant with combined
application of B. aryabhattai S10 and B. subtilis ZM63. Co-inoculated
combination of B. aryabhattai ZM31 and B. subtilis ZM63 showed
the maximum stover yield of 19599 kg ha-1
and harvest index of 31%. This combination was statistically similar to the
co-inoculation of Bacillus spp. ZM20 and B. subtilis ZM63, however, these treatments were
significantly different from uninoculated control.
Macronutrients
concentration
Inoculation with Zn solubilizing Bacillus strains improved N, P, and K concentration in straw and
grains of maize (Table 4). Uninoculated control was lowest to show N, P, and K
concentration in maize straw up to 0.83, 0.73 and 1.25%, respectively and in
maize grains up to 1.15, 0.77 and 0.62%, respectively. The maximum N
concentration in straw and grains was 1.57 and 2.34%, respectively, shown by a
co-inoculation combination of Bacillus
spp. ZM20 × B. aryabhattai ZM31. This treatment was
non-significant with a co-inoculation combination of B. aryabhattai ZM31 × B. subtilis ZM63, however, these treatments were
significantly different from uninoculated control. Co-inoculation with B.
aryabhattai S10 and B. subtilis ZM63 reported the highest P concentration in maize straw and
grain up to 1.05% and 0.94%, respectively and was non-significant with other
sole and co-inoculation combinations, however, it was significantly different
uninoculated control. Co-inoculation with B. aryabhattai S10 and B.
subtilis ZM63 reported maximum K concentration in straw (1.83%) and grain
(0.85%) followed by co-inoculation with Bacillus
spp. ZM20 and B. subtilis ZM63. These treatments were non-significant
to each other but significantly different from uninoculated
control.
Zn and Fe
concentration in grains
Table 3: Effect of Zn solubilizing Bacillus strains inoculation/co-inoculation on stover
and grain yield and harvest index of maize sown in field conditions
|
Inoculation/co-inoculation* |
Stover
yield (kg ha-1) |
Grain
yield (kg ha-1) |
Harvest
index (%) |
|
Uninoculated Control |
17410 h |
8598 f |
26.6 f |
|
Bacillus spp.
ZM20 |
18076 f |
8836 de |
28.8 d |
|
B. aryabhattai
ZM31 |
18727 d |
8723 ef |
26.8 ef |
|
B. aryabhattai S10 |
17478 h |
9229 b |
29.6 c |
|
B. subtilis ZM63 |
18431 e |
8643 ef |
27.1 ef |
|
Bacillus spp.
ZM20 × B. aryabhattai ZM31 |
19044 c |
9007 cd |
27.4 e |
|
Bacillus spp.
ZM20 × B. aryabhattai S10 |
18655 d |
8638 ef |
27.0 ef |
|
Bacillus spp.
ZM20 × B. subtilis ZM63 |
19380 b |
9741 ab |
30.5 ab |
|
B. aryabhattai
ZM31 × B. aryabhattai S10 |
17687 g |
9093 c |
29.9 bc |
|
B. aryabhattai
ZM31 × B. subtilis ZM63 |
19599 a |
9826 a |
31.0 a |
|
B. aryabhattai S10
× B. subtilis ZM63 |
18745 d |
9638 ab |
28.7 d |
|
LSD (P ≤ 0.05) |
184.55 |
233.32 |
0.7121 |
*Inoculation/co-inoculation of Zn solubilizing Bacillus strains; LSD = least
significant difference; data are mean values of three replicates; Means sharing the same letter (s) do not
differ significantly according to LSD test
Table 4: Effect of Zn solubilizing Bacillus strains inoculation/co-inoculation on N, P and K
concentration in maize straw and grains sown in field conditions
|
Inoculation/co-inoculation* |
N concentration (%) |
P concentration (%) |
K concentration (%) |
|||
|
|
Straw |
Grains |
Straw |
Grains |
Straw |
Grains |
|
Uninoculated Control |
0.83 g |
1.15 g |
0.73 d |
0.77 e |
1.25 ef |
0.62 de |
|
Bacillus spp.
ZM20 |
1.17 e |
1.22 fg |
0.97 ab |
0.91 ab |
1.67 b |
0.66 cde |
|
B. aryabhattai
ZM31 |
0.84 g |
1.93 d |
0.91 abc |
0.88 bcd |
1.29 e |
0.60 e |
|
B. aryabhattai S10 |
1.22 e |
1.50 e |
0.95 ab |
0.85 d |
1.56 c |
0.67 cde |
|
B. subtilis ZM63 |
1.53 ab |
1.25 f |
0.80 cd |
0.84 d |
1.58 c |
0.68 cde |
|
Bacillus spp.
ZM20 × B. aryabhattai ZM31 |
1.57 a |
2.34 a |
0.80 cd |
0.93 a |
1.21 f |
0.63 de |
|
Bacillus spp.
ZM20 × B. aryabhattai S10 |
0.92 f |
1.87 d |
0.96 ab |
0.86 cd |
1.28 ef |
0.75 abc |
|
Bacillus spp.
ZM20 × B. subtilis ZM63 |
1.50 b |
2.04 c |
0.96 a |
0.91 ab |
1.76 a |
0.85 a |
|
B. aryabhattai
ZM31 × B. aryabhattai S10 |
1.41 c |
1.27 f |
0.83 bcd |
0.90 abc |
1.38 d |
0.72 bcd |
|
B. aryabhattai
ZM31 × B. subtilis ZM63 |
1.54 ab |
2.26 ab |
0.99 ab |
0.91 ab |
1.60 c |
0.81 ab |
|
B. aryabhattai S10
× B. subtilis ZM63 |
1.32 d |
2.19 b |
1.05 a |
0.94 a |
1.83 a |
0.85 a |
|
LSD (P ≤ 0.05) |
0.0677 |
0.0873 |
0.1491 |
0.0436 |
0.0733 |
0.1075 |
*Inoculation/co-inoculation of Zn
solubilizing Bacillus strains; LSD =
least significant difference; data are mean values of three replicates; Means sharing the same letter (s) do not
differ significantly according to LSD test
%20691-698_files/image002.png)
Fig. 1: Effect of Zn solubilizing Bacillus strains inoculation/co-inoculation on Fe concentration in
maize grains sown in field conditions
Co-inoculation with B. aryabhattai ZM31 and B.
subtilis ZM63 reported the highest Fe concentration of 56.5 mg kg-1
in maize grains (Fig. 1) followed by co-inoculation with B. aryabhattai S10 and B.
subtilis ZM63. Uninoculated control showed the lowest Fe concentration of
42.2 mg kg-1 in maize grains. Co-inoculation with B. aryabhattai ZM31 + B. subtilis ZM63 and B. aryabhattai S10 + B. subtilis ZM63 reported maximum Zn
concentration in grains of 52.0 mg kg-1 and 51.36 mg kg-1, respectively (Fig. 2). Zn concentration in maize grains was lowest in case of
inoculated control that showed 44.892 mg kg-1.
%20691-698_files/image004.png)
Fig. 2: Effect of Zn solubilizing Bacillus strains inoculation/co-inoculation on Zn concentration in
maize grains sown in field conditions
Discussion
Zinc is the key component of plants and required for
their growth and development. Its deficiency is most common in crops that cause
a reduction in crop yield. Application of Zn fertilizers are underutilized in
many countries including Pakistan and also not are cost-effective. Moreover,
Zn-fertilizers become converted into insoluble form soon after their
applications due to alkaline nature of the soil. Growing crops on such
Zn-deficient soil could hinder crop growth and produced staple grains that have
resulted in Zn-deficient. Inoculation with Zn solubilizing bacteria is an
effective strategy to solubilize the insoluble Zn compound that increases the nutrient
availability in soil and crop productivity (Mumtaz et al. 2018). These bacteria use various direct and indirect
mechanisms that can contribute to enrich the cereals grains with Zn. The present investigation was aimed to
biofortify the maize grains along with increasing crop productivity through the
application of Zn solubilizing Bacillus
strains (Bacillus spp. ZM20, B. aryabhattai
ZM31, B. subtilis ZM63 and B. aryabhattai S10) Previously, we have reported the
multiple growth-promoting traits of
these Zn solubilizing Bacillus
strains and their potential to increase growth, yield, and nutrient uptake in
maize (Mumtaz et al. 2017, 2018).
In the present study, sole and
co-inoculation with Zn solubilizing Bacillus
strains promoted maize growth and yield, however, co-inoculation treatments
showed better increase in maize growth and yield that might be due to better
competency of the strains in plant growth-promoting attributes e.g.,
solubilization of Zn and P minerals, production of phytohormones, siderophore,
urease, catalase activity, and ammonia and exopolysaccharides production
ability (Mumtaz et al. 2017; Dinesha et al. 2018). Microbial solubilization of P and Zn through secretion of organic acids may cause a drop in pH that played a key role in increasing their solubility and
uptake (Ramesh et al. 2014).
Co-inoculation with these strains may result in mutualistic interaction that
altered root morphology to acquire more nutrients in the plant to increase yield. These Zn solubilizing Bacillus strains were well-reported for
indole acetic acid (IAA) that plays a very
important role in plant-microbe
interactions that stimulate and facilitate plant growth. Microbial secreted IAA
interacts with plant developmental processes which may alter the endogenous pool of plant IAA and induces cell elongation and cell division (Spaepen et al. 2007). Moreover, several studies
related to Zn solubilizing bacterial strains were reported to promote plant growth
parameters (Ramesh et al. 2014;
Shakeel et al. 2015; Khande et al. 2018).
In the present, the increase in maize growth and yield
could also be due to the increase in nutrient uptake and their accumulation in
various plant parts due to co-inoculation with Zn solubilizing Bacillus strains. The N uptake was more
due to inoculation with Bacillus spp.
ZM20 and B. aryabhattai ZM31 that might be due to the presence of nitrogenase
enzyme in these strains and having the ability
to fix atmospheric N that may facilitate its uptake (Spaepen et al. 2007). In current investigations,
co-inoculation with B. aryabhattai S10 and B. subtilis ZM63
reported the highest uptake of P, K, and
Fe in maize grains. Highest Zn biofortification in maize stover and grains was
observed from a combined use of B. aryabhattai ZM31 and B. subtilis ZM63.
Our findings are supported by the results of Rana et al. (2012), Ramesh et al.
(2014) and Abaid-Ullah et al.
(2015). Macronutrient uptake had a positive impact on micronutrient uptake
which correlated to their accumulation in grains (Cakmak et al. 2010). Translocation and mobilization
of Fe and Zn in grains depend on their concentration in vegetative tissue, N status, and
different species and cultivars. Microbial
production of the organic acid may cause a reduction
in pH and shifted the dynamic equilibrium of minerals from non-labile to
labile form and may promoted nutrient accumulation in plants (Wani et al. 2007).
The present study revealed that the co-inoculation with
Zn solubilizing Bacillus strains
enriches the maize grains with Zn and Fe that might be due to the increase in
the availability of Zn and Fe for plant uptake. The co-inoculation combination
of aryabhattai ZM31 + B. subtilis ZM63 and aryabhattai ZM31
and B. subtilis ZM63 showed the promising result to biofortify the maize
grain with Zn and Fe. These co-inoculation combinations might be more
compatible and competative to solubilize the insoluble native soil Zn contents
and improved it uptake and accumulation in maize grains as compared to other
sole and co-inoculation combination. Bacillus
strains also have the ability to produce siderophores which is important for solubilization, mobilization and phytoextraction of metals (Whiting et al. 2001). The increase in Zn and Fe
concentration in maize grains due to inoculation may also cause a reduction in antinutrients agent e.g.,
phytic acid, gluten, tannins, oxalates, lectins, leptins, and saponins which is helpful to improve the bioavailability of nutrients for human
consumption. Phytic acid in grains is not bio-available and binds to Fe and Zn
in grains and makes them unavailable to humans (Thompson 1989). Previous
findings of Ramesh et al. (2014)
reported the reduction in phytic acid accumulation in grains which could be a possible reason for the biofortification of
maize in the current study. The mechanism
for biofortification upon inoculation is unknown however, it is thought to be
due to their growth-promoting characteristics that modulate root morphology,
improve nutrient acquisition, and accumulation in grains.
Biofortification of maize through Bacillus strains has immense importance to mitigate micronutrients
malnutrition and illness in developing countries (Bouis and Welch 2010). As
people consume cereal-based diets to meet
daily nutritional requirements which contain
too low Zn concentration. Cereal grains must contain at least 45 mg kg-1
of Zn concentration for a significant impact on adult health by assuming daily
intake of 400 g of chapatti made from cereal flour (Cakmak 2008). Zn
concentration in grains due to inoculation with Bacillus strains in the current
study is relatively high for maize, even in the control, is above the minimum
level of required Zn concentration to meet daily intake. Its accumulation in
grains due to inoculation in the current
study might have large implications in terms of remediation of malnutrition in
rural population. Co-inoculation of Zn solubilizing Bacillus strains have a significant
impact on crop productivity and biofortification of maize and potentially to be
promoted as bio-inoculants to overcome the nutrient
deficiency in cereals.
Conclusion
The combined use of Bacillus
strains viz., B. aryabhattai ZM31 & S10 and B. subtilis ZM63 were found highly effective for
the biofortification of maize along with improvement in growth and yield
parameters. These inoculants would be effective in the context of increasing
food quality and reducing the use of chemical fertilizers in agriculture. The
current study clearly demonstrates that
tested Bacillus strains have the
potential to biofortify maize grains under field conditions and are recommended
to use as potential bio-inoculant for Zn biofortification under
nutrient-deficient soils.
Acknowledgments
The authors acknowledge the
financial support by the Endowment Fund Secretariat, University of Agriculture
Faisalabad. The workspace was provided by the Department of Soil Science, The Islamia University of Bahawalpur-Pakistan.
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