WJPR 4588

www.wjpr.net Vol 4, Issue 10, 2015.
Lad et al. World Journal of Pharmaceutical Research
“EMBEDDING POTENTIAL BIOINOCULANT STRAIN FOR
EFFECTIVE SOIL CONDITIONER PRODUCTION”
Sumant D. Lad*, Arti Shanware and Minal Trivedi
Rajiv Gandhi Biotechnology Centre, L.I.T Premises, Rashtrasant Tukadoji Maharaj Nagpur
University, Nagpur - 440033 (M.S.)
ABSTRACT
Biofertilizer is the resource and producer of plant nutrients and
promotes growth by increasing supply of essential nutrients and
growth induced to the target crop when applied to plant surfaces, seed
and soil. Biofertilizers are environmental friendly fertilizers that aid in
Nitrogen fixation, Phosphate solubilization and Potassium
mobilization. But studies regarding organisms solubilizing secondary
nutrients like sulphur and magnesium are yet to be carried out
optimally. Sulphur and Magnesium are secondary nutrients required in
small quantities. In the present investigation Sulphur oxidizing
bacteria(SOB) and Magnesium oxidizing bacteria (MOB) were
isolated from Sugarcane(Ladewadgaon, Dist.Beed, Maharashtra) &
Citrus rhizosphere (Rajiv Gandhi Biotechnology Centre, RTMNU,
Nagpur). The isolates were characterized by using Morphological and
biochemical, cultural tests and screened by PCR using specific
oligonucliotide primers. Isolates were sequenced by 16S r RNA sequencing for molecular
characterization and identification. Bacillus sp. & Serratia marcescens sp. were found to be
competent isolates for sulphur & magnesium oxidation respectively. Further green house
studies were conducted to see their impact of plant growth.
KEYWORDS: Biofertilizers, Sulphur, Magnesium, Secondary, PCR, 16s r RNA.
INTRODUCTION
Biofertilizer is one of the approaches to increase the production in sustainable agriculture.
Soil conditioner is the resource and producer of plant nutrients and promotes growth by
increasing supply of essential nutrients and growth induced to the target crop when applied to
Article Received on
3 Aug 2015,
Revised on 23 Aug 2015,
Accepted on 12 Sep 2015
*Correspondence for
Author
Sumant D. Lad
Rajiv Gandhi
Biotechnology Centre,
L.I.T Premises,
Rashtrasant Tukadoji
Maharaj Nagpur
University, Nagpur -
440033 (M.S.)
ladsumant90@gmail.com
World Journal of Pharmaceutical Research
SJIF Impact Factor 5.990
Volume 4, Issue 10, XXX-XXX. Research Article ISSN 2277– 7105

plant surfaces, seed and soil. The plant is greatly influenced by a wide range of these
nutrients. Soil is the reservoir of nutrients required by plants. But it cannot sustain the
demand by crops continuously, season after season and hence farmers have resorted to
application of fertilizers. Even here farmers apply major nutrients like Nitrogen, Phosphorus
and Potassium often and seldom the secondary and micronutrients. Magnesium and Sulphur
are secondary nutrients required in substantial quantities by crops but applied rarely.[1]
Sulphur is considered as the fourth major plant nutrient after N, P and K, and is one of the
sixteen nutrient elements which are essential for the growth and development of plants.
Sulphur is one of the essential plant nutrients and it contributes to yield and quality of crops.
Sulphur oxidizers are involved in oxidation of elemental sulphur to plant available sulphate.
Sulphur is the key element for higher pulse production and plays an important role in the
formation of proteins, vitamins and enzymes.[2]
Thiobacilli sp. play a significant role in
sulphur oxidation in soil. Sulphur oxidation is the mainly important step of sulphur cycle,
which improves soil productiveness. Subsequently, the sulphide can be oxidized by sulphur
oxidising bacteria to produce sulphate.[3]
Next secondary nutrient Magnesium, plays an
essential role in stepping up the growth and quantitative as well as qualitative features of the
plant. In reclaimed sandy soils, foliar applications of macro and micro nutrients are widely
used and preferable and show the way to significant increase in growth and productivity of
some crops.[4]
Magnesium is absorbed as the Mg2+ ion and is mobile in plants, moving from
the older to the younger leaves. To correct Magnesium deficiency in soil, use Dolomitic lime
when lime is needed; use soluble sources of Magnesium when lime is not needed.
Magnesium supplementation may be needed for crop production.[5]
Magnesium and Sulphur are secondary nutrients required in sustainable quantities by crops
but applied rarely. These secondary nutrients are taken up in smaller quantities but are
essential for plant growth.[6]
In this study we have isolated bacteria that have ability to
oxidize sulphur & magnesium from the rhizophere of sugarcane and orange. Further
Biofertilizers were made with the potential isolates and green house studies were conducted
to validate its growth enhancement property.
MATERIALS AND METHODS
The studies were conducted at Rajiv Gandhi Biotechnology Centre RTM Nagpur University,
Nagpur during the year 2014-15.

Collection of soil samples
Isolation was performed using soil samples collected from two field sites viz., Citrus
rhizosphere soil Orange (Citrus sinensis) Field, RGBC, RTM Nagpur University, Nagpur,
Maharashtra) & Sugarcane (Saccharum officinarum L.) rhizosphere soil Ladewadgaon,
Dist.Beed, Maharashtra.
Isolation of sulphur oxidizing bacteria
Isolation was performed by using samples collected from rhizosphere soil sample of
Sugarcane field from (Ladewadgaon, Dist.Beed, Maharashtra).
The media used for the isolation of sulphur oxidizing bacteria was thiosulphate broth
having 5.0 g Na2S2O3, 0.1 g K2HPO4, 0.2 g NaHCO3, 0.1 g NH4Cl in 1000 ml distilled
water, with pH 8.0. 0.0025 g of Bromo phenol blue (BPB) was used as an indicator.[7]
The
obtained isolates were inoculated in the growth media incubated at 32 o
C for 15 days. The
isolates were screened for their efficacy to reduce the pH 8.0.
For qualitative screening, the isolated bacteria from agar plate were further grown on the
thiosulphate agar and broth.[8]
The selected isolates were further studied for their
morphology, Gram reaction, colony characters etc.
Isolation of Magnesium Solubilizing Bacteria
Isolation was performed by using samples collected from Sweet orange field rhizosphere soil
(Sweet orange, RTM Nagpur University, Nagpur).
The solubilization of Magnesium was investigated in liquid culture and in soil incubation
studies. Basal medium (Glucose-10g; (NH4 )SO4 1.0g; KCl-0.2g; K2 HPO4 -0.1g; MgSO4 -
0.2g; DIS-1000ml; pH 7.0) was prepared and dispensed in 100ml quantities in 500ml
Erlenmeyer flasks to which talc, dolomite and Magnesium trisilicate were added separately
at 0.25% levels.[1]
The obtained isolates were inoculated in the growth media incubated at 32
o
C for 15 days. The isolates were screened for their efficacy to reduce the pH 7.0. The
selected isolates were further studied for their morphology, Gram reaction, colony characters
etc.
Morphological Biochemical and Cultural Characterization
The selected isolates were plated in Sodium Thiosulphate and Magnesium Trisilicate agar
medium to study colony morphology. The isolates were subjected to Gram staining and

Motility test. Also biochemical tests like Indole Acetic acid test and IMViC Test were
performed and the results were recorded.
Isolation of Genomic DNA From Bacteria
The genomic DNA extraction and PCR techniques are found to be the very fast and precise
methods for evaluating Sulphur oxidizing and Magnesium oxidizing bacterial isolates ability.
Total genomic DNA was isolated from SOB and MOB isolates. Genomic DNA isolation was
carried out from the isolates by using the phenol/chloroform/ isoamyl alcohol method.[9]
The
total genomic DNA isolated from SOB and MOB isolates was amplified by PCR. Polymerase
Chain Reaction was performed using the Eppendorf Master Cycler, Gradient (Eppendorf,
Germany). PCR amplification was carried out using 16S rRNA universal primer.[10]
Screened
isolates were further characterised by amplification with oligonucleotides two 16S F & 16S R
primers.
Preparation of Biofertilizer
Bioinoculant are used to prepare as carrier based inoculants containing effective
microorganisms. Among various type of Bioinoculant which includes Sulphur oxidizing
bacteria (SOB) and Magnesium oxidizing bacteria (MOB). Basically, the carrier based
inoculant of these bacteria can be prepared by a standard procedure, which is routinely being
followed for the said preparations.[11]
Green House Study
Soil was collected from agricultural field, sterilized at 1210
C for 15 min at 15 lbs pressure.
The sterile soil was cooled to room temperature and dispensed in to sterile pots and watered.
The soil sample with concentration of 25% co- inoculums of Sulphur oxidizing, Silicate
Solubilizing & Magnesium oxidizing bacteria (with charcoal) were showed in pots and were
maintained in green house.
RESULT AND DISCUSSION
Growth in Selective Media
Isolation of Sulphur oxidizing Bacteria
Qualitative screening of isolated Sulphur Solubilizing Bacteria (SOB).
The Thiosulphate medium for isolating Sulphur Oxidising Bacteria (SOB) was a simple way
to detect SOB through the formation on agar plates containing Sodium Thiosulphate as a only

„Sulphur‟ source. The isolates were facultative autotrophic in nature. Isolation of Autotrophic
sulphur oxidizers from different ecological niches. The sulphur oxidising bacterial isolates
obtained from the Chick pea soil could reduce the pH up to 4.3 to 4.2 from the initial pH 8.0
of thiosulphate broth within 11 days of incubation. The isolates were selected based on the
better pH reduction ability on the Bromophenol blue containing sulphur oxidising broth and
agar medium by changing the colour of the media purple to colourless (Fig.1). The Sulphur
oxidising bacterial isolates obtained from the Sugarcane soil initial pH 8.0 of thiosulphate
broth within 11 days of incubation. In vitro studies showed that this Bacillus can solubilize
Sulphur from the minerals. It is imperative that Sulphur oxidizing bacteria can liberate
sulphate from bearing S minerals. Thiobacillus thiooxidans cells oxidized elemental S to
SO4
2−
in growth medium.[12]
Figure 1: Screening of sulphur oxidising bacteria on thiosulphate broth supplied with
bromophenol blue as an indicator.
Isolation of Magnesium Oxidising Bacteria
Qualitative screening of isolated Magnesium Oxidising Bacteria (MOB)
Use of Magnesium Trisilicate for isolating Magnesium Oxidising Bacteria (MOB) was a
simple way to detect MOB through the formation on agar plates containing Magnesium
Trisilicate as a sole “Magnesium” source Fig (2). In vitro studies showed that this Serratia
marcescens sp. can solubilize Magnesium from the Magnesium minerals. In the current study
utilization of Magnesium by the Serratia marcescens sp. under in vitro condition was clearly
recognized. The capability to solubilise Talc, Dolomite and Magnesium trisilicate in vitro and
when added to the soil point to that this bacterium can attack other silicate mineral in soil and
liberate Magnesium.[13]

Figure 2: Isolation of Magnesium oxidizing bacteria.
Figure 3: (A-B) Inoculation of Sulphur Oxidising Bacteria (SOB) and Magnesium
oxidizing bacteria (MOB).
Morphological and Biochemical characterization of isolates
Three Bacterial isolates were characterized on the basis of morphological and biochemical
tests. The result obtained from biochemical tests like Indole Acetic acid test and IMViC Test
are depicted in Table 1.

Table 1: Showing Morphological and Biochemical characterization of bacterial isolates.
S.No Test SOB MOB
1 Gram reaction Positive Negative
2 Colony morphology Rods Coccobacilli
4 Indole Acetic Acid Test Negative Negative
5 Indole test Negative Positive
6 Methyl red test Positive Positive
7 Vogesproskauer test Positive Negative
8 Citrate Test Positive Negative
16s RNA SEQUENCING CHARECTERIZATION
Sulphur Oxidizing Bacteria (SOB) of 16s RNA Sequencing Characterization
16s rRNA and gene amplification of SOB isolates
The selective SOB isolates were subjected to PCR using specific primers like 16S rRNA
oligonucleotide primers. When the genomic DNA of selected SOB isolates were amplified
by PCR using primers, a major amplification band of 1500bp was observed and 16sr RNA
yielded 1623 bp band which was notified by running the PCR product on agarose gel.
The sequence information for nucleotide was obtained by comparison of the sequence and it
showed highest similarity (90 % similarity) with the sequence of Bacillus sp. Respectively
(Fig. 4 & 5) & table 2.
Table 2: Identification of bacteria.
Sl No Sample ID Identification (BLAST) Percentage similarity
1 SOB Bacillus sp 90
Figure 4: Gel electrophoresis of amplified of SOB isolates through PCR.

Figure 5: Phylogenetic analysis of obtained sequence of 16S Region with Bacillus sp.
sequences from Genbank.
Magnesium Oxidising Bacteria of 16s RNA Sequencing Characterization
16s rRNA and gene amplification of MOB isolates
The selective MOB isolates were subjected to PCR using specific primers like 16S rRNA
oligonucleotide primers. When the genomic DNA of selected MOB isolates were amplified
by PCR using primers, a major amplification band of 1500bp was observed and 16sr RNA
yielded 1477 bp band which was notified by running the PCR product on agarose gel.
The sequence information for nucleotide was obtained by comparison of the sequence and it
showed highest similarity (97% similarity) with the sequence of Serratia
marcescens respectively (Fig.6 & 7) & table 2.
Table 3: Identification of bacteria.
Sl No Sample ID Identification (BLAST) Percentage similarity
1 MOB Serratia marcescens 97
Figure 6: Gel electrophoresis of amplified of MOB isolates through PCR.

Figure 7: Phylogenetic analysis of obtained sequence of 16S Region with Serratia sp.
sequences from Genbank.
GREEN HOUSE STUDY
The results of present Green house study clearly showed that selected isolates effectively
enhance the plant growth, total Sulphur and Magnesium uptake and soil fertility.
The result of present study green house visibly showed growth enhancement ultimately and
development of plant under green house condition. The biofertilizer use 25% and 75% soil.
Field trials were conducted under green house condition to study the influence of
concentration of Sulphur Oxidizing and Magnesium Oxidizing co- inoculums. Pots were
placed in a greenhouse under a 25/18ºC day/night temperature regime with a 14-h
photoperiod. Showing shoot length Sulphur & Magnesium soil conditioner treatment
recorded maximum length of shoot and found significantly superior over rest of control
treatment (Figs.8 A&B).
Figure 8: Growth promotion of plant with Sulphur Oxidizing (A) & Magnesium
Oxidizing Bacterial (B) soil conditioner.

CONCLUSION
In current study we were able to isolate Sulphur & Magnesium oxidizing bacteria, it was
found that these can substitute the chemical fertilizer, might be used to reduce the alkalinity
of soil by neutralization phenomenon through organic acid exudation and can survive in the
soil system to retain the Sulphur and Magnesium potential for long time. Use of these SOB as
bio-inoculants can be incorporated to enhance sulphur oxidation in soil and to increase soil
available sulphate to minimize the S-fertilizer application. The present study revealed that the
isolates SOB & MOB have the potential for developing into a farmer friendly bioinoculant or
soil conditioner for enhancing the Sulphur & Magnesium nutrition, growth promotion in
several crops after testing under field conditions.
ACKNOWLEDGMENT
The authors are grateful to DBT, Govt. of India for providing financial support to carry out
this project dissertation work.
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