Zinc solubilizing Bacillus sp. ZM20 and Bacillus aryabhattai ZM31 promoted the productivity in okra (Abelmoschus esculentus L.)

Abstract

Zinc solubilizing bacteria (ZSB) improve crop productivity by increasing bioavailability of zinc (Zn). A pot experiment was conducted to evaluate the effectiveness of five promising ZSB strains on the productivity of okra. The experiment was conducted using Completely Randomized Design (CRD) with four replications. Data regarding physiological, growth, and yield parameters were collected and statistically analyzed. Results showed that inoculation of ZSB strains significantly increased these attributes of okra. Inoculation of strain Bacillus sp. ZM20 followed by Bacillus aryabhattai ZM31 was significantly more effective among the tested ZSB strains. Strain Bacillus sp. ZM20 improved relative water contents up to 17%, chlorophyll a and b up to 67 and 71%, respectively, plant height up to 30%, shoot fresh weight up to 19%, shoot dry weight up to 31%, root length up to 79%, root fresh weight up to 58%, root dry weight up to 66%, number of fruits plant-1 up to 89%, fruit fresh weight up to 79%, fruit dry weight up to 78%, concentration of N up to 20%, P up to 65%, K up to 20%, and protein contents up to 20% as compared to uninoculated control. It is concluded that inoculation of ZSB strains like Bacillus sp. ZM20 and Bacillus aryabhattai ZM31 is an effective approach to improve the productivity of okra (Abelmoschus esculentus L.).

Full text

BIOLOGIA (PAKISTAN) PKISSN 0006 – 3096 (Print)
December, 2018, 64 (II), 179-185 ISSN 2313 – 206X (On-Line)
Author’s Contribution: I.F. Performed all experiments, analyzed data and wrote first draft of manuscript; M.J., & M.A. Developed the idea
of research, supervised research & edited manuscript.; M.L. Helped for the analysis of data; M.Z.M., S.H., A.H. Contributed to writing the
manuscript, S.R.K Helped for the preparation of revision of manuscript suggested by reviewers
Zinc solubilizing Bacillus sp. ZM20 and Bacillus aryabhattai ZM31 promoted the
productivity in Okra (Abelmoschus esculentus L.)
ISHRAT FATIMA1, MOAZZAM JAMIL1, AZHAR HUSSAIN1*, MUHAMMAD ZAHID MUMTAZ2,
MUHAMMAD LUQMAN1, SAJID HUSSAIN3, SAIF UR REHMAN KASHIF4 & MAQSHOOF AHMAD1
1Department of Soil Science, University College of Agriculture & Environmental Sciences, The Islamia University of
Bahawalpur, Pakistan
2Institue of Molecular Biology and Biotechnology, The University of Lahore, Defense Road Campus, Lahore Pakistan
3Stat Key Laboratory of Rice Biology, China National Rice Research Institute Hangzhou, Zhejiang Province, P.R. China
4Department of Environmental Sciences, University of Veterinary & Animal Sciences, Lahore, Pakistan
ARTICLE INFORMAION
ABSTRACT
Received: 11-07-2018
Received in revised form:
04-09-2018
Accepted: 07-09-2018
Zinc solubilizing bacteria (ZSB) improve crop productivity by increasing
bioavailability of zinc (Zn). A pot experiment was conducted to evaluate the
effectiveness of five promising ZSB strains on the productivity of okra. The
experiment was conducted using Completely Randomized Design (CRD) with four
replications. Data regarding physiological, growth, and yield parameters were
collected and statistically analyzed. Results showed that inoculation of ZSB
strains significantly increased these attributes of okra. Inoculation of strain
Bacillus sp. ZM20 followed by Bacillus aryabhattai ZM31 was significantly more
effective among the tested ZSB strains. Strain Bacillus sp. ZM20 improved
relative water contents up to 17%, chlorophyll a and b up to 67 and 71%,
respectively, plant height up to 30%, shoot fresh weight up to 19%, shoot dry
weight up to 31%, root length up to 79%, root fresh weight up to 58%, root dry
weight up to 66%, number of fruits plant-1 up to 89%, fruit fresh weight up to 79%,
fruit dry weight up to 78%, concentration of N up to 20%, P up to 65%, K up to
20%, and protein contents up to 20% as compared to uninoculated control. It is
concluded that inoculation of ZSB strains like Bacillus sp. ZM20 and Bacillus
aryabhattai ZM31 is an effective approach to improve the productivity of okra
(Abelmoschus esculentus L.).
Keywords: Okra (Abelmoschus esculentus L.), Bacillus strains, Nutrient
solubilization, Plant nutrition, Sustainable
*Corresponding Author:
Azhar Hussain
azharhaseen@mail.com
Original Research Article
INTRODUCTION
Okra (Abelmoschus esculentus L.) is widely
consumed vegetable crop. It provides high
nutritional contents such as carbohydrates,
minerals, proteins, calcium, iron and vitamins to
human diet (Bawa & Badrie, 2016). In Pakistan,
13.9 thousand hectares area is under okra
cultivation with a production of 102.6 thousand tons
(Khokhar, 2014). Its production is very low in most
developing countries including Pakistan because of
its reliance on natural soil fertility.
Low solubility of Zn in soils is an important
factor for reducing crop yield and production as it
plays important role in metabolism of nucleic acid,
cell division, synthesis of proteins and synthesis of
indole acetic acid (MacDonald, 2000; Rout & Das,
2009). Zn deficiency occurs in 70% soils of
Pakistan due to calcareous nature, low organic
matter and high pH and causes crop failure (Bapiri
et al., 2012). Low Zn solubility in soils is the main
cause of Zn deficiency in crops rather than a low
total Zn contents (Cakmak, 2008; Alloway, 2009).
Its low availability decreases the yield and leads to
the inferior quality of crop products and is
responsible for its deficiency in humans (Rehman et
al., 2018).
Zinc deficiency in rhizosphere is being
corrected via use of manures and chemical
fertilizers. Chemical fertilizers enhance the fertility
status of soils and productivity of crops but these
also affect the soil chemistry negatively and are
very costly (Steinshamn et al., 2004). Application of
manure fulfills the Zn requirements but depends on
the factors like soil physico-chemical properties,
temperature, moisture, characteristics of manure,
and microbial activity in soil (Alloway, 2009).
Researcher reported the increase in crop
production and quality of food produced by use of
rhizobacteria. They colonize in rhizosphere and
increase the plant growth through number of
primary and secondary metabolites involved in
solubility of phosphorus (P), potassium (K), Zn, iron
(Fe), biological nitrogen (N) fixation, production of

2 I. FATIMA ET AL BIOLOGIA PAKISTAN
siderophores, syntheses of phytohormones and
control of plant pathogens (Freitas et al., 2007;
Lugtenberg & Kamilova, 2009; Mumtaz et al.,
2017). Most of soils contain significant Zn
concentration in unavailable forms which can be
converted in to available form by inoculation of Zn
solubilizing bacterial (ZSB) strains (Saravanan et
al., 2004; Bapiri et al., 2012; Mumtaz et al., 2017,
2018; Khanghahi et al., 2018). They dissolve the
insoluble Zn compounds via producing organic
acids like gluconic acid and 2-keto gluconic acids
(Bapiri et al., 2012). Inoculation of ZSB in
rhizosphere enhances the concentration of Zn and
decreases the dependence on synthetic fertilizers.
Unwise and indiscriminate use of perilous
agricultural chemicals can be reduced by
inoculation of ZSB which are good substitute of
chemicals for increasing the growth and yield of
plants (Vessey, 2003). Therefore, keeping in view
the above scenario, the present study was
conducted to evaluate the impact of ZSB strains on
growth, physiology and productivity of okra.
. MATERIALS AND METHODS
Collection of bacterial strains and preparation
of inoculum
Five ZSB strains viz. ZM19, ZM20, ZM27,
ZM31, and ZM50 were obtained from gene bank of
Soil Microbiology and Biotechnology Laboratory,
Department of Soil Science, The Islamia University
of Bahawalpur. These strains were previously
characterized, screened, and evaluated for plant
growth promotion by Mumtaz et al. (2017, 2018).
Among these tested strains, strains ZM20 and
ZM31 were identified as Bacillus sp. ZM20 and
Bacillus aryabhattai ZM31 (Mumtaz et al., 2017).
The bacterial cultures were grown in DF-minimal
salt broth amended with 0.1% zinc oxide (ZnO) in
shaking incubator (Model SI9R-2, Shellab-USA) for
48 h. After incubation, bacterial cultures were
maintained to uniform population (cell count of 108
cfu ml-1) and used for seed inoculation.
Seed inoculation and experimental management
Seeds of okra variety Sabz Pari was
purchased from local seed market of Bahawalpur
and sterilized by following method of Khalid et al.
(2004) and dipped in the respective bacterial culture
for 30 mints before seed sowing. Whereas, the
control seeds were dipped in broth. Pot experiment
was performed at the wire house of Department of
Soil Science, The Islamia University of Bahawalpur,
Pakistan, located at Lat: 29.40N, Lon: 71.68E and
116 meters elevation above the sea level. Pots
were filled with 12 kg sieved loamy soil. Inoculated
seeds were sown in pots arranged in Completely
Randomized Design (CRD) having four replicates.
Recommended doses of N, P and K (50: 25: 25 kg
ha-1) were applied in the form of Urea, Diammonium
Phosphate (DAP) and Muriate of Potash (MOP),
respectively. Full P and K were applied at sowing
time while N was applied in three splits doses: first
dose at sowing and remaining at 15 days interval.
All the recommended agronomic practices were
carried out. At physiological maturity, data
regarding physiological attributes were recorded
while at harvesting; growth and yield parameters
were estimated.
Plant analysis
At flowering stage, relative water content
(RWC) of top fully developed okra leaf was
determined by using formula described by Mayak et
al. (2004). Chlorophyll a and b contents were also
determined spectrophotometically and values were
calculated by method of Arnon (1949). Okra fruits
were harvested at marketable stage and biometrical
observation like number of fruits plant-1, fresh and
dry weight of fruits were recorded.
For chemical analysis, 100 g of mix okra
shoot and leaf were dried in an oven at 65 °C to
constant weight and grounded into powder. Plant
samples were digested as described by Wolf
(1982). The N contents in plant samples were
determined by Kjeldal method while P concentration
was estimated through adopting procedure of
Jackson (1973). The K concentration was
determined through flame photometer model BWB-
XP (BWB technology Ltd. UK). Values were
compared with calibration curve of KCl standard
ranging from 0 to 100 ppm and actual concentration
was calculated.
The data of various attributes was analyzed
for analysis of variance techniques (ANOVA) in
accordance with CRD design and means were
compared by least significant difference (LSD) test
at 5% probability (Steel et al., 2007).
RESULTS
Physiological parameters
Inoculation with ZSB strains significantly
increased RWC and chlorophyll ‘a’ and ‘b’ contents
(Table I). Inoculation with strain ZM20 reported
maximum increase up to 16.8% in RWC as
compared to uninoculated control. The strain ZM31
also showed better increase up to 4.2% and was
non-significant to strains ZM19, ZM27, and ZM50

VOL. 64 (II) PROMOTION OF OKRA PRODUCTIVITY THROUGH ZN SOLUBILIZING STRAINS 3
but significantly different from uninoculated control.
The maximum chlorophyll ‘a’ and ‘b’ contents with
an increase up to 66.7 and 70.6%, respectively
were observed due to strains ZM20 followed by
strain ZM31 that gave 54.5 and 61.8% more
chlorophyll a and b contents, respectively, over
uninoculated control.
Table I: Effect of zinc solubilizing bacteria on relative water contents, chlorophyll ‘a’ and chlorophyll ‘b’
contents of okra leaves
Treatments
Relative water contents
(%)
Chlorophyll ‘a’
(µg/g)
Chlorophyll ‘b’
(µg/g)
Control
68.46 c
0.99 d
1.02 f
ZM19
69.63 bc
1.39 c
1.57 c
ZM20
79.98 a
1.65 a
1.74 a
ZM27
69.71 bc
1.32 c
1.50 d
ZM31
71.48 ab
1.53 b
1.65 b
ZM50
70.65 ab
1.05 d
1.39 e
LSD (p≤0.05)
1.5517
0.0752
0.0582
Means sharing different letters are statistically significant from each other at 5% level of probability (n = 4)
Table II: Effect of zinc solubilizing bacteria on plant height, shoot fresh and dry weight of okra
Treatments
Plant height
(cm)
Shoot fresh weight
(g plant-1)
Shoot dry weight
(g plant-1)
Control
80.19 f
125.32 f
75.74 f
ZM19
93.90 c
138.91 c
91.59 c
ZM20
104.03 a
149.26 a
99.29 a
ZM27
90.28 d
134.39 d
85.21 d
ZM31
98.66 b
145.87 b
96.93 b
ZM50
86.53 e
130.44 e
81.17 e
LSD (p≤0.05)
2.5505
2.4762
2.0172
Means sharing different letters are statistically significant from each other at 5% level of probability (n = 4)
Table III: Effect of zinc solubilizing bacteria on root length, root fresh and dry weight of okra
Treatment
Root Length
(cm)
Root fresh weight
(g plant-1)
Root dry weight
(g plant-1)
Control
44.02 f
59.75 f
29.41 f
ZM19
64.13 c
80.75 c
38.56 c
ZM20
78.77 a
94.50 a
48.83 a
ZM27
61.07 d
76.75 d
35.36 d
ZM31
72.80 b
89.25 b
42.82 b
ZM50
53.80 e
71.50 e
32.16 e
LSD (p≤0.05)
1.7231
1.8856
1.2940
Means sharing different letters are statistically significant from each other at 5% level of probability (n = 4).
Table IV: Effect of zinc solubilizing bacteria on number of fruits plant-1, fresh and dry weight of okra fruit
Treatment
Number of fruits Plant-1
Fruit fresh weight (g)
Fruit dry weight (g)
Control
8.5 e
66.09 f
11.45 e
ZM19
13.6 b
97.05 c
15.09 c
ZM20
16.0 a
118.03 a
20.49 a
ZM27
12.2 c
94.25 d
13.94 d
ZM31
14.8 b
110.11 b
16.99 b
ZM50
10.9 d
87.90 e
13.53 d
LSD (p≤0.05)
0.0264
1.4779
0.9722
Means sharing different letters are statistically significant from each other at 5% level of probability (n = 4)

4 I. FATIMA ET AL BIOLOGIA PAKISTAN
Table V: Effect of zinc solubilizing bacteria on NPK and protein % age in okra
Treatment
Nitrogen (%)
Phosphorus (%)
Potassium (%)
Protein (%)
Control
2.18 e
0.26 d
1.50 f
13.63 e
ZM19
2.42 c
0.34 c
1.76 c
15.13 c
ZM20
2.62 a
0.43 a
1.80 a
16.37 a
ZM27
2.38 c
0.33 c
1.71 d
14.91 c
ZM31
2.46 b
0.39 b
1.82 b
15.40 b
ZM50
2.31 d
0.27 d
1.63 e
14.48 d
LSD (p≤0.05)
0.0414
0.0196
0.0264
0.2577
Means sharing different letters are statistically significant from each other at 5% level of probability (n = 4)
Agronomic parameters
Results showed that inoculation of ZSB
strains was effective in improving agronomic
attributes in terms of plant height, shoot fresh and
dry weight, root length, and root fresh and dry
weight as compared to uninoculated control (Table
II). The maximum increase in plant height, shoot
fresh and dry weight was given by strain ZM20 with
increase up to 29.7, 19.1, and 31.1%, respectively,
followed by ZM30 that improved these attributes up
to 23.0, 16.4, and 28.0%, respectively, over
uninoculated control.
Significant variation in root growth in terms
of root length, root fresh and dry weight was
observed in as inoculated as compared to
uninoculated control (Table III). Inoculation of ZM20
reported maximum increase up to 78.9, 58.2, and
66.0%, in root length, root fresh and dry weight,
respectively, of okra plants as compared to
uninoculated control. Inoculation of strain ZM31
were also able to show better root length, root fresh
and dry weight with increase up to 65.4, 49.4, and
45.6%, respectively, over uninoculated control.
Yield parameters
Among yield contributing attributes of okra,
number of fruits plant-1 were significantly promoted
due to inoculation with ZSB strains (Table IV).
Uninoculated control reported minimum number of
fruits plant-1 which were 8.5. Among inoculation
treatment, strain ZM20 reported maximum number
of fruits plant-1 over uninoculated control which were
16, while strain ZM50 gave poor number of fruits
plant-1 however significantly different from
uninoculated control. Data regarding the effect of
ZSB strains on okra fruit fresh and dry weight
(Table IV) showed that fruit biomass was improved
due to inoculation. The maximum fruit fresh and dry
weights were observed due to strain ZM20 being
118.0 and 20.5 g plant-1, respectively, followed by
the inoculation with ZM31 that showed 110.1 and
16.9 g plant-1 of fruit fresh and dry weight,
respectively. Minimum fruit fresh and dry weights
were observed from uninoculated control.
NPK and protein contents
The improvement in NPK and Protein
concentration in straw was observed due to
inoculation treatments (Table V). The inoculation of
strain ZM20 increased N concentration up to 20.2%
as compared to uninoculated control. The maximum
increase in P, K, and protein concentration up to
65.0, 20.0, and 20.1%, respectively was also
observed by strain ZM20 as compared to
uninoculated control.
DISCUSSION
Zinc solubilizing bacteria (ZSB) can
promote crop productivity through improving soil
fertility. These microbes improved plant health
under normal as well as environmental stress
conditions and reduced the dependence on
hazardous chemicals. In the present study, ZSB
strains improved the physiological attributes like
RWC, chlorophyll ‘a’ and chlorophyll ‘b’ contents of
okra plants. Improvement in RWC might be due to
increase in root surface area that enhanced water
uptake. These results were supported by Ahmad et
al. (2011) who described that co-inoculation of
rhizobacterial and rhizobial strains improved root
length that helped water uptake from depth.
Similarly, Egamberdiyeva (2007) and Mumtaz et al.
(2018) also found that application of rhizobacteria
improved root length and root surface area that
increased water uptake from far places and resulted
in improvement of relative water content. Nayak et
al. (1986), stated that plants inoculated with PGPR
showed increase in chlorophyll contents and
photosynthetic rate which led to overall
improvement in plant health. Increase in chlorophyll
a and b was similar to findings of Sharma et al.
(2003) who reported the increased in chlorophyll ‘a’
and chlorophyll ‘b’ in rhizobacterial inoculated
plants that resulted in increased growth and yield.
The present study showed that inoculation
of ZSB strains significantly improved the growth of
okra. It could be due to the ability of bacterial
strains to create favorable conditions for vegetative

VOL. 64 (II) PROMOTION OF OKRA PRODUCTIVITY THROUGH ZN SOLUBILIZING STRAINS 5
growth and to increase shoot and root growth by
making nutrients more available to the roots
(Adesemoye & Ugoji, 2006). Han et al. (2007)
studied that soil microbes used as bio-fertilizers
play vital functions in decaying organic matter,
nutrient cycling and supporting crop growth and
health. Richardson (2001) described that
rhizobacterial inoculation efficiently increased the
root surface area and biomass due to more
production of phytohormones by bacterial strains
that facilitated more nutrient absorption. Current
study also reported the increase in fruit biomass
due to ZSB strains which was similar to the findings
of Jayapandi & Balakrishnan (1990) who reported
the increase in yield component of okra as a result
of application of rhizobacterial strains. It is well-
documented that biofertilizer enhanced plant growth
and yield through making nutrient more available
and improving soil health (Iqbal et al., 2013).
Inoculation of ZSB strains increased the
NPK and protein contents as compared to
uninoculated control under current study. The
increase in nutrient concentration in plants could be
due to their effect on initiation and development of
lateral roots and increased root weight. Ahmad et
al. (2014) reported the increase in root surface area
through root proliferation as a result of bacterial
strains inoculation which were responsible for the
availability of nutrients. The secretion of acids by
bacteria and other behavior of soil microbiota affect
the equilibrium towards more nutrient solubility and
bioavailability to plant roots for absorption
(Saravanan et al., 2004). Similarly Estrada et al.
(2013) and Abaid-Ullah et al. (2015) evaluated the
bacterial strains for secretions of organic acids in
response of insoluble nutrient like P, K, and Zn and
reported the production of gluconic, oxalic, citric,
malic acids, etc. These organic acids have the
power to acidified the soil medium and solubilize
insoluble compounds. Thus, Zn solubilizing
bacterial strains in present work improved the
productivity of okra through improving physiology,
growth, and yield and increasing the accumulation
of nutrients in okra.
CONCLUSION
Inoculation of zinc solubilizing bacterial
strains significantly improved the physiological,
growth, yield attributes and nutrients concentration
in okra. The strains Bacillus sp. ZM20 followed by
Bacillus aryabhattai ZM31 showed more promising
results. These strains are well-capable to convert
unavailable forms of nutrients into available forms
which is unconventional tool to lessen the nutrients
deficiency in plant and produce superior quality
plant products. These strains could also be better
substitute for farmers to lessen the application of
chemical fertilizers for sustainable production of
crops. ACKNOWLEDGEMENT
We are thankful to Department of Soil
Science, University College of Agriculture and
Environmental Sciences, the Islamia University of
Bahawalpur for provision of research facilities for
this study.
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