The present invention relates to a novel bacterium belonging to Bacillus family,
designated as Bacillus subtilis ssp. shriramensis exhibiting anti-microbial and/or antifungal
activity, isolation and identification of extract of the novel microbe exhibiting
antimicrobial and/or antifungal, proteolytic, amylolytic activities, composition
comprising the novel bacterium and/or extract, method of inhibiting the growth of
pathogenic microbes and/or fungi by contacting the pathogenic microbes and/or fungi
with an effective amount of the novel bacterium and/or an antimicrobial and/or antifungal
composition and/or agent and use thereof .
BACKGROUND OF THE INVENTION
The Earth’s atmosphere is known to team with airborne microorganisms, though the high
light intensities, extreme temperature variations, low concentrations of organic matter and
scarcity of water, make the environment unsuitable for microbial growth. Biological
material may contribute about 20%, 22% and 10% to the total airborne particulate matter
by volume in remote continental, populated continental and remote maritime
environments, respectively. Most of them originate from natural sources such as soil,
lakes, animals and humans. Moreover, agricultural practices, health care units and
industrial operations such as sewage treatment, animal rearing, fermentation processes,
and food processing plants also emit viable microorganisms into the environment.
Bacteria form a large domain of single-celled, prokaryotic microorganisms. Typically a
few micrometres in length, bacteria have a wide range of shapes, ranging from cocci to
rods and spirals. Bacteria are ubiquitous on Earth, growing in soil, acidic hot springs,
radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and
the live bodies of plants and animals. The bacilli are rod-shaped, gram-positive,
sporulating, aerobic or facultative anaerobic bacteria. Most bacilli are saprophytes. Each
bacterium creates only one spore, which is resistant to heat, cold, radiation, desiccation,
and disinfectants. The bacilli exhibit an array of physiological abilities that allow them to
live in a wide range of habitats, including many extreme habitats such as the desert sands,
hot springs, and Arctic soils. Bacillus species can be thermophilic, psychrophilic,
acidophilus, alkaliphilic, halotolerant, or halophilic and are capable of growing at various
pH values, temperatures, and salt concentrations.
‐ 3 ‐
Production of antimicrobial agents seems to be a general phenomenon for most bacteria.
These bacteria produce an admirable array of microbial defence systems, including
broad-spectrum classical antibiotics, metabolic by-products such as organic acids, and
lytic agents such as lysozyme. In addition, several types of protein exotoxins, and
bacteriocins, which are biologically active peptide moieties with bactericidal mode of
action, are also produced. The biological arsenal from microbes is remarkable in its
diversity and natural abundance.
The search for new antimicrobial agents is a field of utmost importance. The development
of resistance to antimicrobial agents is increasing at an alarming rate. Current solutions
involve development of a more rational approach to antibiotic use and discovery of new
antimicrobials.
Highly relevant patents
1. Novel bacterial strains and methods of controlling fungal pathogens
(WO/2000/015761).
OBJECTIVES OF THE INVENTION
The objective of the present invention is to provide a novel bacterium exhibiting
antimicrobial and/or antifungal activity.
The objective of the present invention is to isolate & identify an extract of the novel
bacterium, wherein the extract displays antimicrobial and/or antifungal activity.
The objective of the present invention is also to provide an antimicrobial and/or
antifungal composition or agent wherein the composition or the agent comprises the
novel bacterium and/or the extract of the novel bacterium.
Another objective of the present invention is to provide a method of inhibiting the growth
of pathogenic microbes and/or fungi by contacting the pathogenic microbes and/or fungi
with an effective amount of the novel bacterium and/or an antimicrobial and/or antifungal
composition and/or agent wherein the composition or the agent comprises the novel
bacterium and/or the extract of the novel bacterium and/or a mixture of the novel
‐ 4 ‐
bacterium and its extract.
The other objective of the present invention is to provide use of the novel bacterium, an
antimicrobial and/or antifungal composition or agent wherein the composition or the
agent comprises the novel bacterium and/or the extract of the novel bacterium and/or a
mixture of the novel bacterium and its extract, for inhibiting the growth of pathogenic
microbes and/or fungi.
SUMMARY OF THE INVENTION
An aspect of the present invention is to provide an isolated, novel bacterium which is
useful in producing antimicrobial and/or antifungal metabolites or agents.
One aspect of the present invention is to provide a novel form of bacterium belonging to
Bacillus species which is designated as Bacillus subtilis ssp. shriramensis having
accession number (MTCC-5674). In particular, the novel bacterium disclosed in the
present work is capable of exhibiting distinct antimicrobial and/or antifungal property.
Another aspect of the present invention is to provide a process for the production of an
antimicrobial and/or antifungal composition or agent wherein the composition or the
agent comprises Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or the extract of
the Bacillus subtilis ssp. shriramensis (MTCC-5674).
There is provided a composition comprising Bacillus subtilis ssp. shriramensis (MTCC-
5674). The composition may further comprise pharmaceutically acceptable excipients,
diluents and/or carriers.
There is provided a composition containing an extract of Bacillus subtilis ssp.
shriramensis (MTCC-5674). There is also provided a composition comprising an aqueous
extract of Bacillus subtilis ssp. shriramensis (MTCC-5674). The composition may
further comprise pharmaceutically acceptable excipients, diluents and/or carriers.
There is provided a method for inhibiting the growth of pathogenic microbes and/or fungi
by contacting the pathogenic microbes and/or fungi with an effective amount of Bacillus
subtilis ssp. shriramensis (MTCC-5674) or the extract of Bacillus subtilis ssp.
‐ 5 ‐
shriramensis (MTCC-5674). The Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or
the extract of Bacillus subtilis ssp. shriramensis (MTCC-5674) may optionally contain
one or more additional antimicrobial and/or antifungal agents.
There is provided in the present invention the use of Bacillus subtilis ssp. shriramensis
(MTCC-5674) and/or the extract of the Bacillus subtilis ssp. shriramensis (MTCC-5674)
in the formulation of an antimicrobial and/or antifungal composition or agent for
inhibiting the growth of pathogenic microbes and/or fungi.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel microbe belonging to Bacillus family designated
as Bacillus subtilis ssp. shriramensis and having an accession number (MTCC-5674) and
a method of producing an antimicrobial and/or antifungal composition or agent wherein
the composition or the agent comprises Bacillus subtilis ssp. shriramensis (MTCC-5674)
and/or the extract of the Bacillus subtilis ssp. shriramensis (MTCC-5674).
The present invention also provides a method of inhibiting the pathogenic microbes
and/or fungi by contacting the microbes and/or fungi with an effective amount of the
novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or the
composition comprising the novel bacterium or its extract.
The present invention also provides use of Bacillus subtilis ssp. shriramensis (MTCC-
5674), and/or an antimicrobial and/or antifungal composition or agent comprising the
novel bacterium Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or the extract of
the Bacillus subtilis ssp. shriramensis (MTCC-5674) for inhibiting the pathogenic
microbes and/or fungi.
The novel Bacillus subtilis ssp. shriramensis (MTCC-5674) may be utilized for the mass
production of antimicrobial and/or antifungal composition/preparation/agent by culturing
Bacillus subtilis ssp. shriramensis in the suitable growth medium under favourable
conditions.
Through deep and careful researches, the inventors have surprisingly found, isolated and
cultured a novel bacterium, which can produce a novel agent. Through detailed
‐ 6 ‐
experimental researches, the inventors have also invented a method of producing the said
novel agent from the said novel microorganism.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Fig. 1 – Plate showing isolation and purification of Bacillus subtilis ssp. shriramensis
(MTCC-5674).
Fig. 2 - Clone of one of the purified colonies of Bacillus subtilis ssp. shriramensis
(MTCC-5674) showing inhibition of growth of Fusarium oxysporum mycelium.
Fig. 3 - Microscopic picture of vegetative bacterial cells of Bacillus subtilis ssp.
shriramensis (MTCC-5674) along with spores.
Fig. 4 – Plate showing actively growing colonies of Bacillus subtilis ssp. shriramensis
(MTCC-5674).
Fig. 5 – Rod shaped Bacillus subtilis ssp. shriramensis (MTCC-5674) under light
microscope .
Fig. 6 – Picture showing results of catalase test.
Fig. 7 – Plate showing amylolytic activity of Bacillus subtilis ssp. shriramensis (MTCC-
5674) culture filtrate.
Fig. 8 – Picture showing results of O/F (Oxidation-Fermentation) test.
Fig. 9 – Picture showing results of Hydrogen sulphide production test.
Fig. 10 – Picture showing results of SDS-PAGE of the concentrated culture filtrate of
Bacillus subtilis ssp. shriramensis (MTCC 5674).
Fig. 11 – Culture plates showing antimicrobial and/or antifungal activity displayed by
Bacillus subtilis ssp. shriramensis (MTCC-5674) colony and culture filtrate.
Fig. 12 – Picture showing results of MIC assay of antimicrobial and/or antifungal
compound by the tube dilution method.
Fig. 13 – Picture showing results of MIC assay of antimicrobial and/or antifungal agent
by agar diffusion method.
Fig. 14 – Picture showing effect of antimicrobial and/or antifungal agent on spores of
Aspergillus niger.
Fig. 15 – Plate showing antimicrobial and/or antifungal activity of cell lysate against
Fusarium oxysporum; (1) Well containing only lysozyme (to check the effect of
lysozyme on fungus Fusarium oxysporum ) and (2) Well containing cell lysate of Bacillus
subtilis ssp. shriramensis (MTCC-5674).
‐ 7 ‐
Fig. 16 – Plate showing assay of antimicrobial and/or antifungal activity of Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells/extract against diverse types of plant
pathogenic fungal and bacterial species.
Fig. 17 – Plate showing results of antimicrobial and/or antifungal activity of Bacillus
subtilis ssp. shriramensis (MTCC-5674) extract on germination of rice seeds in presence
of Fusarium oxysporum
Fig. 18 – Plate showing results of experiments to show absence of pathogenecity of
Bacillus subtilis ssp. shriramensis (MTCC-5674) on various plant species.
Fig. 19 – Picture showing results of experiments to show action of Bacillus subtilis ssp.
shriramensis (MTCC-5674) as bio-control agent.
Fig. 20 – Plates showing (1) Penicillium oxalicum (NFCCI-1997) fungal colonies and (2)
Bacillus subtilis ssp. shriramensis (MTCC-5674) pure colonies.
Fig. 21 – Plate showing corn seeds coated with various formulations of antimicrobial/
antifungal agent Bacillus subtilis ssp. shriramensis (MTCC-5674).
Fig. 22 – Plate showing results of bio-control activity after 2 weeks.
Fig. 23 - Plate showing results of bio-control activity after 4 weeks.
Fig.24 - Assay of Antifungal and/or antimicrobial activity against a variety of human
pathogenic fungal species.
Isolation and identification of the novel bacterium
The inventors collected air samples from 18 different locations in Hyderabad and
Patancheru (Telangana, India) while conducting a study on air flora. Disposable petri
plates containing media (T3 Medium, Travers, et al., 1987) were prepared in the
laboratory and exposed to air at different locations. The exposed plates were sealed and
incubated at 30o C in lab incubator. In one of the plates exposed to air in Patancheru area,
a bacterial colony surrounded by fungal mycelium was observed (Fig. 1A). Despite
continued incubation, the clearance zone was maintained and growth of fungal mycelium
remained restricted to the periphery of clearance zone. The microorganisms from this
colony were subjected to purification by using standard methods of microbiology (Fig.
1B). The individual colonies were tested against a common fungus Fusarium oxysporum
(Fig. 2).
‐ 8 ‐
One of the colonies showed inhibition of fungal growth and a clearance zone was
observed (Fig. 2). Microscopical examination of the bacteria from the colony revealed a
rod shaped motile bacterium (Fig. 5). After six days of incubation in the culture medium
the bacteria produced spores. The colonies of the bacteria were mucoid, raised, circular,
smooth, and creamy to off-white in color (Fig. 4), and cells showed variable gram
staining (Fig. 5).
A range of biochemical tests including carbohydrate fermentation, catalase activity,
oxidation-fermentation test, starch hydrolysis, hydrogen sulphide production test, oxidase
activity test, desoxycholate agar test were carried out. The results of these tests confirmed
that the bacteria is catalase positive, posses amylase activity, strongly aerobic, does not
produce hydrogen sulfide, oxidase positive and gram variable.
For identification of bacteria 16S DNA sequencing and FAME analysis was carried out.
The results of both studies showed that the bacteria is showing 0.37% difference in 16S
DNA sequence and FAME similarity index of 0.827; with Bacillus subtilis ssp. subtilis
and 0.84% difference in 16S DNA sequence and FAME similarity index of 0.749 with
Bacillus atrophaeus. Thus, the results suggest that this bacterium is related to Bacillus
subtilis and Bacillus atrophaeus, but not identical to any of the catalogued bacterial
species in ATCC collection.
Results of 16S DNA sequence comparison
Match % Difference Length Library Entry Name
1 0.19 535 Bacillus subtilis ssp. subtilis
2 0.84 535 Bacillus atrophaeus
3 1.03 535 Bacillus amyloliquifaciens
Results of FAME analysis comparison
S. No. Similarity Index Library Entry Name
‐ 9 ‐
1 0.827 Bacillus subtilis
2 0.749 Bacillus atrophaeus
The isolated bacterium is a new member of sub-species of the genus Bacillus. According
to bacterial nomenclature convention, the novel bacterial species was named as Bacillus
subtilis ssp. shriramensis. The bacterium is deposited in the Microbial Type Culture
Collection (MTCC) at IMTECH, Chandigarh, India. The deposition number of this novel
species is (MTCC-5674).
Characteristic features of the novel microbe having accession/deposition number
(MTCC-5674) provided by the present invention
The bacterium is a rod shaped measuring 2.45x0.88 μm, motile, spore forming, gram
variable; colonies are smooth, mucoid, off-white to creamish in early stages but turn
wrinkled on prolonged incubation. The bacterium transforms into spore as the nutrients in
the medium deplete, normally the process of sporulation takes place in 4 days of
incubation in 10 ml medium containing 100 μl of 5x108cells inoculum in a 25x150 mm
culture tube at 30oC and shaking at 200 rpm.
The novel bacterium, Bacillus subtilis ssp. shriramensis having accession number
(MTCC-5674) exhibits antimicrobial and/or antifungal activity. The extract of the novel
bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674)
exhibits antimicrobial and/or antifungal activity. The range of potential applications and
uses of the bacterium are extensive.
The present invention provides a method of producing the antimicrobial and/or antifungal
extract from the novel bacterium, Bacillus subtilis ssp. shriramensis having accession
number (MTCC-5674).
Production and Isolation of Antimicrobial and/or Antifungal Agent
Composition of culture medium for the growth of Bacillus subtilis ssp. shriramensis
having accession number (MTCC-5674) is as follows
1. Tryptone : 0.32 % (w/v)
2. Tryptose : 0.24 % (w/v)
3. Yeast Extract : 0.18 % (w/v)
‐ 10 ‐
4. NaH2PO4.H2O : 0.044 M
5. Na2HPO4 : 0.062 M
6. MnCl2 : 0.000 5% (w/v)
pH = 6.8
1. The medium was prepared as per the method given in Annexure -I (I) and 100 ml
aliquots were transferred into 500 ml conical flasks. The media was sterilised by
autoclaving at 121oC for 15 min.
2. Each flask was inoculated with a single pure colony of Bacillus subtilis ssp.
shriramensis (MTCC-5674) and incubated at 300C, 200 rpm for 60 hours.
Isolation of antimicrobial and/or antifungal agent from culture medium
Following the growth of bacteria in T3 broth for 60 hours, the culture was centrifuged at
12000 rpm for 10 min at 4oC. The supernatant was collected and filtered using 0.22 μm
disc filter (Millipore/Sartorius). The filterate was preserved under appropriate storage
conditions for detailed experiments to study antimicrobial and/or antifungal activity.
The present invention particularly provides a novel microorganism, Bacillus subtilis ssp.
shriramensis having an accession number (MTCC-5674) and a method for the production
of antimicrobial and/or antifungal composition from the novel bacterium and/or its extract
or a mixture of the novel bacterium and/or its extract.
One embodiment of the present invention provides an isolated novel bacterium belonging
to Bacillus subtilis ssp. shriramensis exhibiting antimicrobial and/or antifungal activity,
having the accession number (MTCC-5674).
In one embodiment of the present invention is provided the novel bacterium designated as
Bacillus subtilis ssp. shriramensis having the accession number (MTCC-5674).
In another embodiment of the present invention there is provided a pure culture of the
novel bacterium, Bacillus subtilis ssp. shriramensis having the accession number
(MTCC-5674).
In one embodiment of the present invention there is provided an extract of the novel
bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) wherein the extract exhibits
‐ 11 ‐
antimicrobial and/or antifungal activity.
In another embodiment of the present invention there is provided an extract of the novel
bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) wherein the extract
exhibiting antimicrobial and/or antifungal activity is an aqueous extract. In yet another
embodiment of the present invention there is provided a process for the production of the
extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) where
in the process comprises growing the novel bacterium, Bacillus subtilis ssp. shriramensis
in a nutrient medium and recovering the extract having antifungal activity by using
conventional methods.
In another embodiment of the present invention there is provided a process for the
production of the extract of the novel bacterium, Bacillus subtilis ssp. shriramensis
(MTCC-5674) wherein the process comprises growing the novel bacterium, Bacillus
subtilis ssp. shriramensis (MTCC-5674) under aerobic conditions.
In yet another embodiment of the present invention there is provided a process for the
production of the extract of the novel bacterium, Bacillus subtilis ssp. shriramensis
(MTCC-5674) wherein the process comprises growing the novel bacterium, Bacillus
subtilis ssp. shriramensis in a nutrient medium, recovering the extract having
antimicrobial and/or antifungal activity and optionally comprises concentrating the
extract using conventional methods.
In one embodiment of the present invention, there is provided a composition comprising
the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) wherein the
composition has antimicrobial and/or antifungal activity.
In another embodiment of the present invention, there is provided a composition
comprising the extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674) wherein the composition has antimicrobial and/or antifungal activity.
In another embodiment of the present invention, there is provided a composition
comprising the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) and the
extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) wherein
‐ 12 ‐
the composition has antimicrobial and/or antifungal activity.
In one embodiment of the present invention, there is provided a composition comprising
the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) and/or an extract
of the said novel bacterium, Bacillus subtilis ssp. shriramensis, or a combination thereof
that optionally comprises one or more antimicrobial and/or antifungal agents.
In another embodiment of the present invention, there is provided a composition
comprising an extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674) that optionally comprises one or more antimicrobial and/or antifungal agents.
In yet another embodiment of the present invention there is provided a composition
comprising the combination of the novel bacterium, Bacillus subtilis ssp. shriramensis
(MTCC-5674) and its extract that optionally comprises one or more antimicrobial and/or
antifungal agents.
In one embodiment of the present invention there is provided a composition comprising
the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) or an extract of the
novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) or a combination
thereof, that optionally comprises agriculturally or pharmaceutically acceptable carrier.
In another embodiment of the present invention there is provided a composition
comprising the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) that
optionally comprises agriculturally or pharmaceutically acceptable carrier.
In yet another embodiment of the present invention there is provided a composition
containing an extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674) that optionally comprises agriculturally or pharmaceutically acceptable carrier.
In still another embodiment of the present invention there is provided a composition
comprising the combination of the novel bacterium, Bacillus subtilis ssp. shriramensis
(MTCC-5674) and an extract of the said novel bacterium, Bacillus subtilis ssp.
shriramensis, which optionally comprises agriculturally acceptable carrier (See Annexure
III).
‐ 13 ‐
In one embodiment of the present invention there is provided a method for inhibiting
growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting
the pathogenic fungi and/or bacteria with an effective amount of the novel bacterium,
Bacillus subtilis ssp. shriramensis (MTCC-5674) or a composition comprising the said
novel bacterium, or its extract or a combination thereof.
In one embodiment of the present invention there is provided a method for inhibiting
growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting
the pathogenic fungi and/or bacteria with an effective amount of the novel bacterium,
Bacillus subtilis ssp. shriramensis (MTCC-5674).
In another embodiment of the present invention there is provided a method for inhibiting
growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting
the pathogenic fungi and/or bacteria with an effective amount of a composition
comprising the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674).
In yet another embodiment of the present invention there is provided a method for
inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises
contacting the pathogenic fungi and/or bacteria with an effective amount of a composition
comprising an extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674) wherein the extract has antimicrobial and/or antifungal activity.
In yet another embodiment of the present invention there is provided a method for
inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises
contacting the pathogenic fungi and/or bacteria with an effective amount of a composition
comprising the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) and
an extract of the said novel bacterium, Bacillus subtilis ssp. shriramensis , wherein the
extract has antimicrobial and/or antifungal activity.
In one embodiment of the present invention there is provided a use of the novel
bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) or a composition comprising
the said novel bacterium or its extract or a combination thereof, for the preparation of an
antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi
‐ 14 ‐
and/or bacteria.
In another embodiment of the present invention there is provided a use of the novel
bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) for the preparation of an
antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi
and/or bacteria.
In another embodiment of the present invention there is provided a use of the composition
comprising the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-5674) for the
preparation of an antimicrobial and/or antifungal composition for inhibiting the growth of
pathogenic fungi and/or bacteria.
In another embodiment of the present invention there is provided a use of the composition
comprising an extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674) for the preparation of an antimicrobial and/or antifungal composition for inhibiting
the growth of pathogenic fungi and/or bacteria.
In another embodiment of the present invention there is provided a use of the composition
comprising the extract of the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674) and an extract of the said novel bacterium, Bacillus subtilis ssp. shriramensis, for
the preparation of an antimicrobial and/or antifungal composition for inhibiting the
growth of pathogenic fungi and/or bacteria.
In another embodiment, there is provided a pharmaceutical and agriculturally effective
composition comprising the novel bacterium, Bacillus subtilis ssp. shriramensis (MTCC-
5674).
In another embodiment, there is provided a pharmaceutical and agriculturally effective
composition comprising the extract of the novel bacterium, Bacillus subtilis ssp.
shriramensis (MTCC-5674).
In yet another embodiment of the present invention there is provided a method of
producing the said effective composition from the novel bacterium, Bacillus subtilis ssp.
shriramensis (MTCC-5674).
‐ 15 ‐
In yet another embodiment of the present invention, the steps and time required for the
production of the said composition/extract are kept at the minimum duration coupled with
the maximum recovery of the compound.
Other advantages or benefits of the present invention
The bacterium Bacillus subtilis ssp. shriramensis (MTCC-5674) along with antimicrobial
and/or antifungal agent also produces strong thermophilic protease and amylase which are
active even after exposure to high temperature i.e., 121°C for 15min.
The present invention is further explained by the following examples. However, the
present invention is not limited to these examples in any manner. The following examples
is intended to illustrate the working of disclosure and not intended to take restrictively to
apply any limitations on the scope of the present invention. Those persons skilled in the
art will understand that the equivalent substitutes to the specific substances described
herein, or the corresponding improvements are considered to be within the scope of the
invention.
Detailed Methodology is explained in the following examples:
The methods employed in the present work are well-known in microbiology with the
respective parameters varied and optimized for the present study.
Example 1
1.1 Collection and preliminary screening of air samples
Air sampling was carried out at different locations in Hyderabad and Patancheru
(Telangana, India). Disposable Petri plates containing T3 medium were prepared in
the laboratory and exposed to air at different locations. The exposed plates were
sealed and incubated at 30oC in lab incubator. In one of the plates exposed to air in
Patancheru area, a bacterial colony surrounded by fungal mycelium was observed.
1.2 Preliminary screening of air samples for antimicrobial and /or antifungal activity
Despite continued incubation the clearance zone was maintained and growth of
fungal mycelium remained restricted to the periphery of clearance zone. The
microorganisms from this colony were subjected to purification by using standard
methods of microbiology (Fig. 1B). The individual colonies were tested against a
common fungus Fusarium oxysporum (Fig. 2). One of the colonies showed inhibition
of fungal growth and a clearance zone was observed (Fig. 2).
‐ 16 ‐
1.3 Screening of novel isolate
Evaluation of the bacteria under microscope revealed that it is a rod shaped motile
bacterium (Fig. 5). After six days of incubation the bacteria produced spores.
The colonies of the bacteria were mucoid, raised, circular, smooth, and creamish to
off-white in color (Fig. 4) and the cells showed variable gram staining.
Example 2
2.1 Characterization and Identification of the novel microorganism
2.1.1 Characterization of the novel isolate Bacillus subtilis ssp. shriramensis
(MTCC-5674)
A range of biochemical tests, including carbohydrate fermentation, catalase
test, oxidation-fermentation test, starch hydrolysis, hydrogen sulfide
production test, oxidase activity test, were carried out. The results of these
tests confirmed that the bacterium is catalase positive, amylase positive,
oxydase positive and strongly aerobic.
2.1.1.1 Bacillus subtilis ssp. shriramensis (MTCC-5674)- Colony
Morphology
Colonies of Bacillus subtilis ssp. shriramensis (MTCC-5674) are
mucoid, raised, circular, smooth, and creamish to off-white in color
(Fig. 4).
2.1.1.2 Culture Characteristics
Bacillus subtilis ssp. shriramensis (MTCC-5674) shows optimum
growth at 300C (can grow from 15OC to 55OC). As it is an aerobic
bacterium, it requires adequate oxygen for its growth, needs
continuous shaking for culturing in broth.
2.1.1.3 Cell morphology
Bacillus subtilis ssp. shriramensis (MTCC-5674) cells are rod shaped,
diplobacilli and motile (Fig. 5).
2.1.1.3.1 Comparison of colony growth and morphology of Bacillus subtilis
ssp. shriramensis (MTCC-5674) with that of Bacillus subtilis and Bacillus
atrophaeus
‐ 17 ‐
B. subtilis ssp.
shriramensis
B. subtilis ssp,
subtilis
B. atrophaeus
Colony
Morphology
Mucoid, circular,
smooth, (rough after
prolonged
incubation), 2.0-4.0
mm in diameter.
Mucoid,circular,
entire, opaque, 2.0-
4.0 mm in diameter
Opaque,
smooth,
circular, entire
and 1.0-2.0 mm
diameter
(Nakamura
L.K. 1989).
Colony
Color
Cream to off-white Off-white to brown Dark brown to
black
2.1.1.4 Catalase test
Material
 Culture tubes of Bacillus subtilis ssp. shriramensis (MTCC-5674)
 Hydrogen Peroxide
Method
 Three tubes containing LB medium were labelled as “test”,
“positive control” and “negative control”. A loop full of Bacillus
subtilis ssp. shriramensis (MTCC-5674), Escherichia coli and
Streptococcus pneumonia were inoculated in the labelled tubes.
Following incubation at 30oC for 24 hours, few drops of hydrogen
peroxide were added in all the tubes and observed for formation of
bubbles.
Result
Gas bubbles were formed both in “test” and “positive control”
labelled tubes indicating that the Bacillus subtilis ssp. shriramensis
(MTCC-5674) is catalase positive (Fig. 6).
2.1.1.5 Starch Hydrolysis
Material
 Bacillus subtilis ssp. shriramensis (MTCC-5674) culture filtrate
 Starch agar plates
 Iodine
‐ 18 ‐
 Incubator
Method
The starch agar medium was prepared as per the method provided in
the Annexure – I (VI). Two wells were bored at equal distances in
the plate containing starch agar medium and labelled as “test” and
“negative control”. An aliquot of 500 μl each of Bacillus subtilis ssp.
shriramensis (MTCC-5674) culture filtrate and sterile distilled water
were dispensed into the wells labelled “test” and “negative control”.
The plate was incubated at 50oC for 4 hours.
Result
After 4 hours of incubation, the blue color surrounding the test well
disappeared indicating that the Bacillus subtilis ssp. shriramensis
(MTCC-5674) culture filtrate has amylolytic activity. No change in
the blue color was observed in the area surrounding control well (Fig.
7).
2.1.1.6 O/F (Oxidation-Fermentation) Test
Material
 Hugsh Leifson’s OF Basal Medium
 Test tubes
 E.coli culture
 Bacillus subtilis ssp. shriramensis (MTCC-5674)
 Incubator
Method
Three tubes containing Hugsh Leifson’s OF basal medium (OFBM)
(Annexure – I (VII)) were labelled as “negative control”, “positive
control” and “test”. A loop full of Alcaligenes faecalis, Escherichia
coli and Bacillus subtilis ssp. shriramensis (MTCC-5674) was
inoculated in the tubes labelled as “negative control”, “positive
control” and “test”, respectively. The tubes were incubated at 30°C
for 48 hours and observed for change of color.
Result
It has been concluded from the observations that the test organism
(Bacillus subtilis ssp. shriramensis (MTCC-5674) is strictly aerobic
‐ 19 ‐
as it did not ferment carbohydrate (neither gas formation nor the
color change) deep inside the medium. Due to availability of oxygen
on the surface of medium some color change was observed. Whereas
E. coli grew very well deep inside the medium and fermented the
carbohydrates (both gas formation and change in color of the
medium) indicating that it is a facultative anaerobe (Fig. 8). In the
negative control neither gas formation nor color change was
observed.
2.1.1.7 Hydrogen Sulfide Production Test
Material
 SIM (Sulfide Indole Motility) medium
 Culture tubes
 E.coli culture
 Bacillus subtilis ssp. shriramensis (MTCC-5674)
 Incubator
Method
 The tubes containing SIM [Sulfide Indole Motility, {Annexure – I
(VII)}] medium were labelled as “negative control” and “test”. A
loop full of E. coli and Bacillus subtilis ssp. shriramensis (MTCC-
5674), were inoculated in the tubes labelled “test” and “negative
control”, respectively, and incubated at 30oC for 24 hours and
observed for color change.
Result
From the observations, it has been concluded that the test organism is
negative for H2S production as the medium did not turn black. The
same result was observed in the negative control (Fig. 9).
2.1.1.8 Effect of pH on the growth of Bacillus subtilis ssp. shriramensis
(MTCC-5674)
Culture tubes containing standard culture medium (LB) adjusted to
different pH values ranging from 3.4 to 11.0 (acidic to basic) were
used to grow Bacillus subtilis ssp. shriramensis (MTCC-5674) under
standard conditions. Growth of Bacillus subtilis ssp. shriramensis
‐ 20 ‐
(MTCC-5674) was observed in a pH range of 6.4 to 7.2 and the
optimum pH was found to be 7.0.
2.1.1.9 Antibiotic Sensitivity Test of Bacillus subtilis ssp. shriramensis
(MTCC-5674)
A 24 hours old Bacillus subtilis ssp. shriramensis (MTCC-5674)
culture was spread over the surface of T3 agar. Different antibiotic
discs were placed on the surface of the T3 agar plates labelled with
the respective antibiotic. The plates were incubated at 30°C for 24
hours.
Table-1 Observations on antibiotic sensitivity of Bacillus subtilis ssp. shriramensis
(MTCC-5674)
S.
No. Antibiotic Code Zone Result
Standards
RES INT SEN
1 Ampicillin A10 0 RES 13 14-16 17
2 Gentamicin G10 20 SEN 12 13-14 15
3 Tobramycin TB10 15 SEN 12 13-14 15
4 Carbenicillin CB100 15 RES 19 20-22 23
5 Vancomycin VA30 20 SEN <15 15
6 Oxacillin OX1 11 INT 10 11-12 13
7 Novobiocin NV30 24 SEN 17 18-21 22
8 Sulfisoxazole SF300 33 SEN 12 13-16 17
9 Amikacin AK30 15 INT 14 15-16 17
10 Kanamycin K10 13 RES 13 14-17 18
11 Streptomycin S10 12 INT 11 12-14 15
12 Cephalothin CH30 40 SEN 14 15-17 18
13 Chloramphenicol C30 25 SEN 12 13-17 18
14 Erythromycin E15 16 INT 13 14-22 23
15 Enrofloxacin EX10 41 RES N/A N/A N/A
16 Lincomycin L10 12 RES N/A N/A N/A
17 Amoxicillin AC30 0 RES 19 20
‐ 21 ‐
18 Clindamycin CD2 10 RES 14 15-20 21
19 Ceftriaxone CI30 28 SEN 13 14-20 21
20 Bacitracin B10 21 SEN 8 9-12 13
21 Neomycin N30 12 RES 12 13-16 17
22 Azithromycin AT15 10 RES 13 14-17 18
Result
It has been concluded from the observations that Bacillus subtilis ssp. shriramensis
(MTCC-5674) is resistant to the antibiotics - ampicillin, carbenicillin, kanamycin,
enrofloxacin, lincomycin, amoxicillin, clindamycin, neomycin, azithromycin. The
test bacterium is sensitive to gentamicin, tobramicin, vancomicin, novobiocin,
sulfisoxazole, cephalothin, chloramphenicol, ceftriaxone and bacitracin and
showed intermediate resistance to oxacillin, amikacin, streptomycin and
erythromycin.
2.1.2 Identification of the novel isolate Bacillus subtilis ssp. shriramensis (MTCC-
5674)
For identification of bacteria 16S DNA sequencing and FAME analysis were
carried out. The results of both studies showed that the bacteria is showing 0.37%
difference in 16S DNA sequence and FAME similarity index of 0.827; with
Bacillus subtilis ssp. subtilis and 0.84% difference in 16S DNA sequence and
FAME similarity index of 0.749 with Bacillus atrophaeus. Thus, the results
indicate that this bacterium is related to Bacillus subtilis and Bacillus atrophaeus,
but not identical to any of the catalogued bacterial species in ATCC collection.
2.1.2.1 Results of 16S DNA sequence comparison
Match % Difference Length Library Entry Name
1 0.19 535 Bacillus subtilis ssp. subtilis
2 0.84 535 Bacillus atrophaeus
3 1.03 535 Bacillus amyloliquifaciens
‐ 22 ‐
2.1.2.2 Results of FAME analysis comparison
S. No. Similarity Index Library Entry Name
1 0.827 Bacillus subtilis
2 0.749 Bacillus atrophaeus
The isolated bacterium is a new member of the genus Bacillus.
According to bacterial nomenclature convention, the novel bacterial
species was named as Bacillus subtilis ssp. shriramensis. The
bacterium is deposited in the Microbial Type Culture Collection
(MTCC) at IMTECH, Chandigarh, India. The deposition number of
this novel species is (MTCC-5674).
Example 3
3.1 Production and screening of the antimicrobial and/or antifungal agent
3.1.1 Production of antimicrobial and/or antifungal agent
Material
 T3 broth – 1 L
 Conical Flask – 2 L capacity
 Kanamycin (30 μg/ml)
 Bacillus subtilis ssp. shriramensis (MTCC-5674) inoculum
 Shaking Incubator (set at 30oC temperature & 200 rpm shaking)
Method
The T3 broth was prepared according the method described in Annexure – I
(1). A 1 ml culture of Bacillus subtilis ssp. shriramensis (MTCC-5674) was
inoculated into the sterile T3 broth and incubated in the shaking incubator at
300C for 60 hours, while shaking at 200 rpm. Followed by the growth of
Bacillus subtilis ssp. shriramensis (MTCC-5674) in the T3 broth for 60 hours,
the culture medium was centrifuged at 12000 rpm and 4oC for 10 min. The
supernatant was collected and passed through 0.22 μm filters to separate out
any residual bacterial cells. The filtrate was maintained at 4oC.
3.1.2 Screening the antimicrobial and/or antifungal activity of the culture filtrate
collected in step 3.1.1 above
Material
‐ 23 ‐
 Culture Filtrate of Bacillus subtilis ssp. shriramensis (MTCC-5674) containing
antimicrobial and/or antifungal agent
 PDA plates
 Test fungus Fusarium oxysporum
 Incubator
Method
To test the activity of antimicrobial and/or antifungal agent in the filtrate, a well
was bored in one corner of the PDA agar plate, 500 μl of the filtrate was poured
in the well. A loop full of the fungus Fusarium oxysporum was inoculated at
the other corner in the same PDA agar plate and incubated for 5 days at room
temperature. Inhibitory activities of the filtrate against the fungus Fusarium
oxysporum were recorded as the inhibitory zone surrounding the well in
millimetres.
Result
Clear inhibitory zone of 14 mm was observed surrounding the well, suggesting
that the method used for the production of antimicrobial and/or antifungal agent
is optimum.
3.2 Characterization of antimicrobial and/or antifungal agent
The antimicrobial and/or antifungal activity associated with Bacillus subtilis ssp.
shriramensis was investigated to ascertain the nature of agent causing antimicrobial
and/or antifungal activity.
Material
 T3 broth – 1 L
 Conical Flask – 2L capacity
 Kanamycin (30 μg/ml)
 Bacillus subtilis ssp. shriramensis (MTCC-5674) inoculum
 Shaking Incubator
Method
The T3 broth was prepared according the method described in Annexure – I (1). A 1
ml aliquot of 24 hours old Bacillus subtilis ssp. shriramensis (MTCC-5674) was
inoculated into the sterile T3 broth and incubated in the shaking incubator at 300C for
60 hours, while shaking at 200 rpm. Following growth of Bacillus subtilis ssp.
shriramensis (MTCC-5674) in the T3 broth for 60 hours, the culture medium was
‐ 24 ‐
centrifuged at 12000 rpm and 4oC for 10 min. The supernatant was collected and
passed through 0.22 μm filters to remove any remaining bacterial cells.
3.2.1 Antimicrobial and/or antifungal assay with the Bacillus subtilis ssp.
shriramensis (MTCC- 5674) bacterial cells
To test the activity of antimicrobial and/or antifungal agent by the cells, a loop
full of Bacillus subtilis ssp. shriramensis (MTCC-5674) inoculated in one
corner of the T3 agar plate and loop full of the fungus Fusarium oxysporum
was inoculated at the other corner in the same T3 agar plate and incubated for 5
days at room temperature. Inhibitory activities of the bacterial cells against the
fungus Fusarium oxysporum were recorded as the inhibitory zone surrounding
the bacterial colony in millimetres.
Result
Clear inhibitory zone of 14 mm (Fig. 11A) was observed surrounding the
bacterial colony, suggesting that the active compound secreted by the bacterial
cells is getting diffused out in the culture medium resulting in clearance zone
away from bacterial colony.
3.2.2 Antimicrobial and/or antifungal assay with the Bacillus subtilis ssp.
shriramensis (MTCC-5674) culture filtrate
To test the nature of antimicrobial and/or antifungal agent in the filtrate, a well
was bored in the PDA agar plate and 500 μl of the filtrate was filled in the well.
A loop full of the fungus Fusarium oxysporum was inoculated at the diagonally
opposite end of the same PDA agar plate and incubated for 5 days at room
temperature. Inhibitory activities of the filtrate against the fungus Fusarium
oxysporum were recorded as the inhibitory zone surrounding the well in
millimetres.
Result
Clear inhibitory zone of 14 mm (Fig. 11B) was observed surrounding the well,
suggesting that the filtrate retained antimicrobial and/or antifungal activity, thus
indicating the active compound is secreted outside the bacterial cell in to the
culture medium.
3.3 Determination of MIC of Bacillus subtilis ssp. shriramensis (MTCC-5674)
antimicrobial and/or antifungal agent
‐ 25 ‐
3.3.1 Lyophilization of antimicrobial and/or antifungal agent
Material
 Culture filtrate of Bacillus subtilis ssp. shriramensis (MTCC-5674)
 Ammonium sulphate
 Freeze drier
Method
The antimicrobial and/or antifungal agent was produced and purified by the
methods explained in 3.1.1. A 800 ml aliquot culture filtrate was mixed with
382.18 g ammonium sulphate at 70% (w/v) saturation (modified protocol of
Jing et al., 2009) and solution was gently mixed by stirring for overnight at
4°C. The suspension was centrifuged at 10,000 rpm for 10 min at 4°C. The
pellet thus obtained was lyophilized for 24 hours in a freeze drier and the
dried pellet was stored at room temperature.
3.3.2 MIC of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial and/or
antifungal agent
Methods
3.3.2.1 Tube dilution method
3.3.2.2 Agar diffusion method
 Preparation of stock solution of lyophilized antimicrobial and/or
antifungal agent
The stock solution was prepared by dissolving 1 g of lyophilized powder
of antimicrobial and/or antifungal agent in 50 ml phosphate buffer (pH
7.0). The final concentration of the stock solution was adjusted to 20
μg/μl. This stock solution was used for making dilutions with PDB
media in different ratios as shown in Table-1.
3.3.2.1 Tube dilution method
Material
 1.5 ml tubes
 PDB medium
 Antimicrobial and/or antifungal agent stock
 Fusarium oxysporum spore suspension
‐ 26 ‐
Method
MIC assay of antimicrobial and/or antifungal agent was carried out in
1.5 ml tubes. Different dilutions of antimicrobial and/or antifungal
agent were prepared in PDB medium (Table -2) ranging from 10
μg/μl (1:1) to 198 ng/μl (1:100). The MIC assay was carried out
against Fusarium oxysporum by adding 30 μl (5x106 cfu/ml) of spore
suspension in all the tubes and were incubated at 28°C, for 2 days,
shaking at 180 rpm.
Three controls were used, one with undiluted antimicrobial and/or
antifungal agent stock, second with 70% ammonium sulfate in PDB
and third with only PDB. Medium in all the three tubes was
inoculated with 30 μl (5x106 cfu/ml) of Fusarium oxysporum spore
suspension and were incubated at 28°C, for 2 days, shaking at 180
rpm.
3.3.2.2 Agar diffusion method
Material
 PDA (Potato Dextrose Agar) plates
 PDB (Potato Dextrose Broth) medium
 Antimicrobial and/or antifungal agent stock
 Fusarium oxysporum
Method
MIC assay of lyophilized Bacillus subtilis ssp. shriramensis (MTCC-
5674) antimicrobial and/or antifungal agent was also carried out by
agar diffusion method. Four wells of 9 mm diameter each were made
at equal distances in PDA plates. Different dilutions of antimicrobial
and/or antifungal agent were prepared in PDB (Table-3) ranging
from 10 μg (1:1) to 198 ng (1:100). 200 μl of each dilution was
added in to the well labeled with the respective dilution. The test
fungus Fusarium oxysporum was inoculated in the center of the PDA
medium and the plates were incubated at 28oC for 4 days.
A plate with three controls, one containing undiluted antimicrobial
and/or antifungal agent stock, the second containing only PDB broth
and the third containing PDB with 70 % ammonium sulphate was
‐ 27 ‐
used as control. The activity was measured as inhibitory zone in
millimeters surrounding the well.
Result
Tube dilution method (Fig.12)
The samples were observed after 48 hours of incubation under light
microscope for spore germination. Spores did not germinate in the
tubes containing antimicrobial and/or antifungal agent in the ratios
1:1, 1:2, 1:3 and 1:4. Moderate spore germination was observed in
the tubes containing antimicrobial and/or antifungal agent in the
ratios 1:5, 1:6, 1:7, 1:8 and 1:9 dilutions, and normal spore
germination and mycelia formation was observed in the remaining
tubes containing the antimicrobial and/or antifungal agent in the
ratios 1:10 to 1:100 (Table 2).
Agar diffusion method (Fig.13)
Inhibition of fungal mycelium growth was observed around the wells
containing antimicrobial and/or antifungal agent in the ratios 1:1, 1:2,
1:3 and 1:4 (Table 3). Moderate inhibition was observed surrounding
the wells containing antimicrobial and/or antifungal agent in the
ratios 1:5, 1:6 and 1:7 dilutions and no inhibition were observed in
the remaining dilutions (from 1:8 to 1:100) (Table 3).
Conclusion
From the above experiment it is concluded that the antimicrobial
and/or antifungal agent in powder of crude extract is inhibiting spore
germination as well as mycelium growth upto a dilution of 1:4 (v/v),
in a concentration dependent manner.
Table-2 MIC of antimicrobial and/or antifungal agent by tube dilution method
‐ 28 ‐
S.No.
Antimicrobial
and/or antifungal
agent : PDB
Concentration of
antimicrobial
and/or antifungal
agent after
dilution (μg/μl)
Total
concentration of
antimicrobial
and/or antifungal
agent used per
tube (μg/200μl)
Spore germination
1 1:1 10.000 2000.00 No germination
2 1:2 6.666 1333.20 No germination
3 1:3 5.000 1000.00 No germination
4 1:4 4.000 800.00 No germination
5 1:5 3.333 666.60
Germ tube
emergence
6 1:6 2.857 571.40 Germ tube growth
7 1:7 2.500 500.00
Germ tube
elongation
8 1:8 2.222 444.40 Mycelia growth
9 1:9 2.000 400.00 Mycelia extension
10 1:10 1.818 363.60 Compact mycelia
11 1:15 1.250 250.00 Compact mycelia
12 1:20 0.952 190.40 Compact mycelia
13 1:25 0.769 153.80 Compact mycelia
14 1:30 0.645 129.00 Compact mycelia
15 1:35 0.555 111.00 Compact mycelia
16 1:40 0.487 97.40 Compact mycelia
‐ 29 ‐
17 1:45 0.434 86.80 Compact mycelia
18 1:50 0.392 78.40 Compact mycelia
19 1:55 0.357 71.40 Compact mycelia
20 1:60 0.327 65.40 Compact mycelia
21 1:65 0.303 60.60 Compact mycelia
22 1:70 0.281 56.20 Compact mycelia
23 1:75 0.263 52.60 Compact mycelia
24 1:80 0.246 49.20
Compact mycelia
25 1:85 0.232 46.40 Compact mycelia
26 1:90 0.219 43.80 Compact mycelia
27 1:95 0.208 41.60 Compact mycelia
28 1:100 0.198 39.60 Compact mycelia
29
Only
antimicrobial
and/or antifungal
agent
Crude (20 μg/μl)
4000.00
No germination
30
Only PDB
medium
0 0 Compact mycelia
31
Only ammonium
sulphate 70%
0 0 Compact mycelia
‐ 30 ‐
Table-3 MIC of antimicrobial and/or antifungal agent by agar diffusion method
S.No. Antimicrobial
and/or antifungal
agent : PDB
Concentration of
antimicrobial
and/or antifungal
agent after dilution
(μg/μl)
Total concentration
of antimicrobial
and/or antifungal
agent used per well
in agar plate
(μg/200μl)
Percent of
fungus inhibited
(inhibition zone
in mm)
1 1:1 10.000 2000.00 92.30 (12)
2 1:2 6.666 1333.20 84.61 (11)
3 1:3 5.000 1000.00 76.92 (10)
4 1:4 4.000 800.00 61.53 (8)
5 1:5 3.333 666.60 46.15 (6)
6 1:6 2.857 571.40 38.46 (5)
7 1:7 2.500 500.00 23.07 (3)
8 1:8 2.222 444.40 15.38(2)
9 1:9 2.000 400.00 0 (0)
10 1:10 1.818 363.60 0 (0)
11 1:15 1.250 250.00 0 (0)
12 1:20 0.952
190.40 0 (0)
13 1:25
0.769 153.80 0 (0)
14 1:30
0.645 129.00 0 (0)
15 1:35
0.555 111.00 0 (0)
16 1:40
0.487 97.40 0 (0)
‐ 31 ‐
17 1:45
0.434 86.80 0 (0)
18 1:50
0.392 78.40 0 (0)
19 1:55
0.357 71.40 0 (0)
20 1:60
0.327 65.40 0 (0)
21 1:65
0.303 60.60 0 (0)
22 1:70
0.281 56.20 0 (0)
23 1:75
0.263 52.60 0 (0)
24 1:80
0.246 49.20 0 (0)
25 1:85
0.232 46.40 0 (0)
26 1:90
0.219 43.80 0 (0)
27 1:95
0.208 41.60 0 (0)
28 1:100
0.198 39.60 0 (0)
29 Only
antimicrobial
and/or antifungal
agent
Crude (20
μg/μl) 4000.00
100
(13)
30 Only PDB
medium 0
0
0 (0)
31 Only ammonium
sulphate 70% 0 0 0(0)
‐ 32 ‐
3.4 To test the antimicrobial and/or antifungal activity of cell lysate of the novel isolate
Bacillus subtilis ssp. shriramensis (MTCC-5674)
Material
 PDA agar plates
 LB Broth
 Bacillus subtilis ssp. shriramensis (MTCC-5674) cells
 Lysozyme
 Incubator
Methods
3.4.1 Bacillus subtilis ssp. shriramensis (MTCC-5674) cell lysis
The LB broth was prepared according to the method described in Annexure – I
(3). A single colony of Bacillus subtilis ssp. shriramensis (MTCC-5674) was
inoculated into the sterile LB broth and incubated at 30oC for 24 hours. After
24 hours of incubation the vegetative cells were collected by centrifugation at
6500 rpm, 4oC, the cells were washed thrice with sterile distilled water and
subjected to cell lysis by incubation in lysozyme at 37oC for 2 h. After lysis the
suspension was centrifuged at 10,000 rpm for 10 min at 4oC to remove the cell
debris. The supernatant was collected and passed through 0.22 μm filter and
stored at 40 C.
3.4.2 Antimicrobial and/or antifungal assay of the cell lysate
Two wells were bored in two diagonal ends of the PDA agar in the petri plate
and labelled one as “test” and the other as “control”. An aliquot of 500 μl of
the lysate was added to the test well and 500 μl of only lysozyme was added
to the control well. The test fungus Fusarium oxysporum was inoculated in
the middle of the PDA agar and incubated for 5 days at room temperature.
Result
The cell lysate did not exhibit antimicrobial and/or antifungal activity (Fig.
15) against the fungus Fusarium oxysporum, suggesting that the
antimicrobial and/or antifungal agent is primarily secreted out into the media.
Example 4
4.1 To test the antimicrobial and/or antifungal activity against other pathogenic fungi
Material
‐ 33 ‐
 Bacillus subtilis ssp. shriramensis (MTCC-5674)
 Plant Pathogenic fungi
1. Rhizoctonia solani (Causes sheath blight in members of family solanacea).
2. Sarocladium oryzae (Causes sheath rot in rice)
3. Colletotrichum capsicii (Causes anthracnose in chilli).
4. Exerohilum turcicum (Causes turcicum blight).
5. Macrophomina phaseolina (Causes charcoal rot).
 T3 – broth
 T3 – Agar plates
 PDA – Agar plates
 Incubator
Method
4.1.1 To test the antimicrobial and/or antifungal activity with Bacillus subtilis ssp.
shriramensis (MTCC- 5674) cells against various plant pathogenic fungi
A loop full of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells and a
loop full of test fungi were inoculated at the diagonally opposite ends of the
T3 plates labeled with the respective fungus and incubated at 28oC till the
growth of fungal mycelium was observed in the vicinity of the bacterial
colony.
4.1.2 To test the antimicrobial and/or antifungal activity with the filtrate against
various plant pathogenic fungi
An aliquot of 500 μl of the culture filtrate containing antimicrobial and/or
antifungal agent was added into the wells made in the PDA agar and a loop
full of test fungi were inoculated at the other corner of the respective plates
labeled with the respective fungus and incubated at 28oC till the growth of
fungal mycelium was observed in the vicinity of the well containing culture
filtrate.
The inhibitory activity of the filtrate against the target fungus was recorded in
millimetres as the inhibitory zone formed surrounding the well.
Result
‐ 34 ‐
A range of fungal species causing diseases in plants were tested in the
antimicrobial and/or antifungal assay and all of them demonstrated complete
inhibition of growth in the presence of Bacillus subtilis ssp. shriramensis
(MTCC-5674) cells and also its culture filtrate.
4.2 Efficacy of antimicrobial and/or antifungal agent in protecting rice seed from fungal
attack
Rice seeds were treated with Fusarium oxysporum spores and Bacillus subtilis
ssp. shriramensis (MTCC-5674) culture filtrate and placed in the petri plates
containing plain agar to check the efficacy of Bacillus subtilis ssp. shriramensis
(MTCC-5674) antimicrobial and/or antifungal agent in inhibiting the fungal
attack on germinating seed.
Control seeds were treated only with Fusarium oxysporum fungal spores.
Result
In presence of Bacillus subtilis ssp. shriramensis (MTCC-5674) antimicrobial
and/or antifungal agent, fungus failed to infect the seeds and the rice seeds
germinated normally. However, the seeds treated only with fungus showed
severe infection and failed to germinate (Fig. 17).
4.3 To test the pathogenic nature of Bacillus subtilis ssp. shriramensis (MTCC-
5674) on plants
Material
 Bacillus subtilis ssp. shriramensis (MTCC-5674) in 1 % CMC in sprayable
form
 Rice, cotton, tobacco, corn and tomato plants
 Sprayer
Method
The Bacillus subtilis ssp. shriramensis (MTCC-5674) culture was extensively
tested for pathogenic behaviour, if any, on a range of plant species.
Bacillus subtilis ssp. shriramensis (MTCC-5674) was inoculated into 1 L
sterile LB broth in a 2 L conical flask and incubated at 30°C, for 24 hours,
shaking at 200 rpm. Following the growth of the bacteria, the cells were
harvested by centrifuging at 6,500 rpm, at 4°C for 10 min. The pellet was
washed twice in phosphate buffer (pH 7.0) and made into slurry in 1% CMC
(Carboxy Methyl Cellulose) in phosphate buffer (pH 7.0). This suspension
‐ 35 ‐
was used for spraying on crop plants like rice, tobacco, corn, tomato and
cotton.
Result
From the observations it has been concluded that, all the plant species (rice,
tobacco, corn, tomato and cotton) sprayed with Bacillus subtilis ssp.
shriramensis (MTCC-5674) did not exhibit any kind of disease symptoms
and their growth and development was equivalent to control plants indicating
that Bacillus subtilis ssp. shriramensis (MTCC-5674) is non-pathogenic for
plant species (Fig. 18).
Example 5
5.1 Formulation of antimicrobial and/or antifungal compositions containing Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells as a biological control agent
Material
 Bacillus subtilis ssp. shriramensis (MTCC-5674)
 LB - broth
 PDB (Potato Dextrose Broth)
 Phosphate buffer (pH 7.0)
 CMC (Carboxy Methyl Cellulose)
Method
5.1.1 Preparation of Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension
Bacillus subtilis ssp. shriramensis (MTCC-5674) was inoculated in 1 L sterile
LB broth in a 2 L conical flask and incubated at 30°C, for 24 hours, shaking at
200 rpm. Following the growth Bacillus subtilis ssp. shriramensis (MTCC-
5674), the culture was centrifuged at 6,500 rpm, at 4 °C for 10 min. The pellet
was washed twice in phosphate buffer (pH 7.0) and mixed with 1% CMC
(Carboxy Methyl Cellulose) in phosphate buffer (pH 7.0) to prepare a slurry
containing 6x107 cfu/ml. The slurry containing Bacillus subtilis ssp.
shriramensis (MTCC-5674) was used to spray on plants and treat plant
seedling roots by dipping.
‐ 36 ‐
5.1.2 To test the efficacy of formulate containing antimicrobial and/or antifungal agent
to inhibit the infestation of Rhizoctonia solani (NFCCI-3194) in the roots of tomato
plants
Material
 Slurry containing Bacillus subtilis ssp. shriramensis (MTCC-5674)
 Rhizoctonia solani (NFCCI-3194) fungus (causes sheath blight in members of
family solanacea).
 Soil rite
 Tomato seedlings
5.1.3 Preparation of Rhizoctonia solani (NFCCI-3194)
Rhizoctonia solani (NFCCI-3194) was grown in Potato Dextrose Broth
(prepared as per the method provided in the Annexure – I (V) medium for 6
days. Following the growth of the Rhizoctonia solani (NFCCI-3194), it was
thoroughly mixed with soil rite and incubated for 15 days at room temperature.
The soil rite containing the fungus was mixed with soil in 1:1 ratio.
 Tomato seedlings
Tomato seedlings of 10 cm height were used in this study
Method
The experiment was carried out as described below
A. Tomato seedlings were planted in the soil containing Rhizoctonia solani
(NFCCI-3194), but were not treated with Bacillus subtilis ssp. shriramensis
(MTCC-5674).
B. The roots of Tomato seedlings were treated with slurry containing Bacillus
subtilis ssp. shriramensis (MTCC-5674) and were planted in the soil
containing Rhizoctonia solani (NFCCI-3194).
C. Tomato seedlings without any treatment.
A. Seedling treatment with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells
and fungus Rhizoctonia solani (NFCCI-3194)
Tomato seedling roots were dipped in the Bacillus subtilis ssp. shriramensis
(MTCC-5674) cell formulate for 30 min. The treated seedlings were planted in
the pot containing soil mixed with the fungus Rhizoctonia solani (NFCCI-
3194).
‐ 37 ‐
Control seedlings
For inducing the disease in the seedlings, tomato seedlings (untreated) were
planted in the pot containing soil mixed with the fungus Rhizoctonia solani
(NFCCI-3194).
For negative control tomato seedlings (untreated) were planted in pot
containing soil which is not mixed with the fungus Rhizoctonia solani (NFCCI-
3194).
All the pots containing tomato seedlings were transferred to the green house
and maintained till fruiting stage.
Result
From the observations it was concluded that the seedlings treated with
combination of Bacillus subtilis ssp. shriramensis (MTCC-5674) and the
fungus Rhizoctonia solani (NFCCI-3194) grew very well, equivalent to control
plants, whereas the seedlings (untreated) planted in the pot containing fungus
Rhizoctonia solani (NFCCI-3194) exhibited retarded growth, poor flowering
and fruit formation, as compared with control. Hence, it can been concluded
that Bacillus subtilis ssp. shriramensis (MTCC-5674) inhibited the growth of
the fungus Rhizoctonia solani (NFCCI-3194) in the rhizosphere area of tomato
seedlings and protected the seedlings from disease causing fungus (Fig. 19).
Example 6
6.1 In vitro evaluation of minimum number of Bacillus subtilis ssp. shriramensis
(MTCC-5674) cells which can control the infection of germinating corn seeds by soil and
seed borne fungal pathogen Penicillium oxalicum (NFCCI-1997).
6.1.1 Preparation of bacterial and fungal suspension cultures
Materials
a. Bacillus subtilis ssp. shriramensis (MTCC-5674)
b. Penicillium oxalicum (NFCCI-1997)- Plant pathogenic fungus
c. Carbendazim WP50 (commercial fungicide)
d. Luria Bertani Broth (LB)
‐ 38 ‐
Method
6.1.1.1 Preparation of suspension culture of Bacillus subtilis ssp.
shriramensis (MTCC-5674)
A pure colony of Bacillus subtilis ssp. shriramensis (MTCC-5674) (Fig. 20-
1) was inoculated in 10 ml LB broth and incubated at 30°C for 24 h at 180
rpm. For preparation of bio-control formulation 1 ml of fresh culture was
inoculated in 100 ml of LB broth and incubated at 30°C for 24 h at 180 rpm.
Growth of culture was monitored by periodic measurement of absorbance of
culture at 625 nm. The bacterial cells were harvested by centrifugation and
washed with sterile phosphate buffer, by centrifugation at 5500 rpm for 10
min at 4°C. The cells were finally suspended in 5 ml of sterile phosphate
buffer. This concentrated suspension was used for preparation of bio-control
formulations.
6.1.1.2 Preparation of suspension culture of Penicillium oxalicum (NFCCI-
1997) (fungal pathogen)
Pure colony of Penicillium oxalicum (NFCCI-1997) (Fig. 20-2) was
inoculated on PDA plate and incubated at 28°C till spore formation. A loop
full of the fungal spores were inoculated in 100 ml of PDB and incubated at
28°C for 7 days at 180 rpm. The aqueous part of the culture containing fungal
spores was collected in 50 ml polypropylene tubes. The spores were washed
with sterile phosphate buffer by centrifugation at 8000 rpm for 10 min at 4°C.
The spores were suspended in required volume of sterile phosphate buffer to
obtain a cfu of 6x104 ml-1.
6.1.2 Soil infestation with P. oxalicum (NFCCI-1997) (fungal pathogen)
To maintain adequate fungal spore load in the soil medium, 50 ml fungal spore
suspension (6x104 cfu/ml) was mixed with 1 kg of autoclaved soilrite and incubated
for 10 days at 28°C. The soilrite colonized with fungus was uniformly mixed with
soil in 1:1 ratio and filled in 96 cup trays.
6.1.3 Preparation of formulation of Bacillus subtilis ssp. shriramensis (MTCC.
5674) for biological control of soil borne plant disease
‐ 39 ‐
Material
a. CMC (Carboxy Methyl Cellulose)
b. Sucrose
c. Red polymer (without fungicide)
d. Bacillus subtilis ssp. shriramensis (MTCC-5674) cell suspension (bio-control
agent)
e. Carbendazim (commercial fungicide)
To assess effective concentration of Bacillus subtilis ssp. shriramensis (MTCC.
5674) cells which can suppress growth and pathogenicity of P. oxalicum (NFCCI-
1997) on germinating corn seed, four different formulations were designed (details
are provided in table below). Formulations containing only bio-control agent, only
commercial fungicide, and one without bio-control agent or fungicide were used as
controls. All the formulations contain a binding material - CMC (Carboxy Methyl
Cellulose), carbon source (sucrose) and a red polymer (without fungicide).
1. Control-1 (Formulation without fungal pathogen and bio-control agent)
This formulation is composed of 1% CMC, 2% sucrose, and red polymer. This
formulation has no bio-control agent, disease causing agents and fungicide. Seeds
treated with this formulation were used as normal control seeds.
Composition
S. No. Components Weight/Volume/Number Final Concentration
1 CMC 1.71 μg 1.00 % w/v
2 Sucrose 3.42 μg 2.00 % w/v
3 Red Polymer 34 μl 19.88 %v/v
4 Water 31.87 μl -
Total 171 μl -
‐ 40 ‐
2. Control-2 (Formulation with fungal pathogen but no bio-control agent)
This formulation is composed of 1% CMC, 2% sucrose, and red polymer. It has no
bio-control agent/commercial fungicide, but the seeds treated with this formulation
were sown in the soil inoculated with P. oxalicum (NFCCI-1997) fungus. As there is
no biological or chemical protection around the seeds, the fungus grows profusely,
infects the seeds and develops disease in the seedlings. The seeds treated with this
formulation are used as diseased controls.
Composition
S.
No.
Components Weight/Volume/Number Final
concentration
1 P. oxalicum (NFCCI-1997)
fungal spores
Present in the soil -
2 CMC 1.71 μg 1.00 % w/v
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34 μl 19.88 % v/v
5 Water 31.87 μl -
Total 171 μl -
3. Control-3 (Formulation with commercial fungicide “Carbendazim WP50”)
This formulation is composed of 1% CMC, 2% sucrose, red polymer and a
commercial fungicide Carbendazim WP50 (trade name Bavistin) was used at a
concentration of 500 μg/ml (Mohiddin et al., 2013). This formulation is used to
compare the efficacies of both bio-control agent and the commercial fungicide in
suppressing the fungal growth in the vicinity of the germinating seed.
‐ 41 ‐
Composition
S. No. Components Weight/Volume/Number Final concentration
1 Carbendazim 85.50 μg
2 CMC 1.71 μg 1.00 % w/v
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34 μl 19.88 % v/v
5 Water 31.87 μl -
Total 171 μl -
4a. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674) (5x104 cfu)
This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5x104 cfu/ml
(50,000 cells/ml of carrier). This formulation has minimum number of Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells.
Composition
S.
No.
Components Weight/Volume/Number Final
concentration
1 Bacillus subtilis ssp.
shriramensis cell
suspension
100.00 μl 5x104 cfu/ml
2 CMC 1.71 μg 1.00 % w/v
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34.0 μl 19.88 % v/v
‐ 42 ‐
5 Water 31.87 μl -
Total 171.0 μl -
4b. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5x105
cfu)
This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus
subtilis ssp. shriramensis (MTCC-5674)cells at a concentration of 5x105 cfu/ml (0.5
million cells/ml of carrier).
Composition
S.
No.
Components Weight/Volume/Number Final concentration
1 Bacillus subtilis ssp.
shriramensis (MTCC-5674)
cell suspension
100.00 μl 5x105 cfu/ml
2 CMC 1.71 μg 1.00 % w/v
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34 μl 19.88 % v/v
5 Water 31.87 μl -
Total 171 μl -
4c. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5x106
cfu)
This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5x106 cfu/ml (5
million cells/ml of carrier).
‐ 43 ‐
Composition
S.
No.
Components Weight/Volume/Number Final
concentration
1 Bacillus subtilis ssp.
shriramensis (MTCC-
5674) cell suspension
100.00 μl 5x106 cfu/ml
2 CMC 1.71 μg 1.00 % w/v
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34 μl 19.88 % v/v
5 Water 31.87 μl -
Total 171 μl -
4d. Formulation containing Bacillus subtilis ssp. shriramensis cells (5x107 cfu)
This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5x107 cfu/ml (50
million cells/ml of carrier). This formulation has maximum number of Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells.
Composition
S.No. Components Weight/Volume/Number Final
concentration
1 Bacillus subtilis
ssp. shriramensis
(MTCC-5674) cell
suspension
100.00 μl 5x107 cfu/ml
2 CMC 1.71 μg 1.00 % w/v
‐ 44 ‐
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34 μl 19.88 % v/v
5 Water 31.87 μl -
Total 171 μl -
5. Formulation with only Bacillus subtilis ssp. shriramensis (MTCC-5674) cells
(5x107 cfu)
This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus
subtilis ssp. shriramensis (MTCC-5674)cells at a concentration of 5x107 cfu/ml (50
million cells per ml of carrier). This formulation has maximum number of Bacillus
subtilis ssp. shriramensis(MTCC-5674) cells and is used to study the effect of
biocontrol agent on seed germination and plant growth.
Composition
S.
No.
Components Weight/Volume/Number Final
Concentration
1 Bacillus subtilis ssp.
shriramensis (MTCC-5674)
cell suspension
100.00 μl 5X107 cfu/ml
2 CMC 1.71 μg 1.00 % w/v
3 Sucrose 3.42 μg 2.00 % w/v
4 Red Polymer 34 μl 19.88% v/v
5 Water 31.87 μl -
Total 171 μl -
‐ 45 ‐
Table-4 Experiment plan in tabular form
Treatment Material
used
Bacillus
subtilis
ssp.
shriramens
is (MTCC-
5674) cells
(cfu/ml)
P.
oxalicu
m
(NFCCI
-1997)
spores
in soil
(cfu/g)
Carbendazi
m
(in μg)
No. of corn seeds per
treatment
Origin
al
Duplica
te
triplicat
e
*1 Corn seed
+ Red
polymer +
CMC +
Sucrose
(Control
seed)
Nil Nil Nil 20 20 20
*2 Corn seed
+ Red
polymer +
CMC +
Sucrose
(seed were
sown in
soil
containing
P.
oxalicum
(NFCCI-
1997))
(Control
seed)
Nil 6x104 Nil 20 20 20
*3 Corn seed
+ Red
polymer +
CMC +
Sucrose +
Nil 6x104 85.50 20 20 20
‐ 46 ‐
Carbendazi
m (Control
seed)
*4a
Corn seed
+ Red
polymer +
CMC +
Sucrose +
Bacillus
subtilis
ssp.
shriramens
is (MTCC-
5674) cells
(Concentra
tion-1)
5x104 6x104 Nil 20 20 20
*4b
Corn seed
+ Red
polymer +
CMC +
Sucrose +
Bacillus
subtilis
ssp.
shriramens
is (MTCC-
5674)
cells
(Concentra
tion-2)
5x105 6x104 Nil 20 20 20
*4c
Corn seed
+ Red
polymer +
CMC +
Sucrose +
Bacillus
subtilis
ssp.
shriramens
5x106 6x104 Nil 20 20 20
‐ 47 ‐
is (MTCC-
5674)
cells
(Concentra
tion-3)
*4d
Corn seed
+ Red
polymer +
CMC +
Sucrose +
Bacillus
subtilis
ssp.
shriramens
is (MTCC-
5674)
cells
(Concentra
tion-4)
5x107 6x104 Nil 20 20 20
*5 Corn seed
+ Red
polymer +
CMC +
Sucrose +
Bacillus
subtilis
ssp.
shriramens
is (MTCC-
5674)
cells
5x107 Nil Nil 20 20 20
*1(Control-1)- Formulation without fungal pathogen and antifungal agent; *2(Control-2)-
Formulation with fungal pathogen but no bio-control agent; *3(Control-3)- Formulation with
commercial fungicide “Carbendazim WP50”; *4a- Formulation with Bacillus subtilis ssp.
shriramensis (MTCC-5674) (5x104 cfu); *4b- Formulation with Bacillus subtilis ssp. shriramensis
(MTCC-5674) cells (5x105 cfu); *4c- Formulation with Bacillus subtilis ssp. shriramensis(MTCC-
5674) cells (5x106 cfu) ; *4d- Formulation containing Bacillus subtilis ssp. shriramensis (MTCC-
5674) cells (5x107 cfu) and *5- Formulation with only Bacillus subtilis ssp. shriramensis (MTCC-
5674) cells (5x107 cfu).
48
Seed coating
The bio-control formulations as per the compositions given above were coated on corn seeds
(Fig. 21). Twenty corn seeds in triplicates (total 60 seeds) for each treatment were surface
sterilized with 0.1% HgCl2 for 10 min and rinsed with 95% ethanol, and washed with sterilized
water for 10 min each. Dry seeds were coated with 171 μl/60 seeds of different formulations and
air dried for 2 h.
Seed sowing
All the treated seeds were sown in 96 cup trays with three replicates per treatment. All trays were
kept in glasshouse and maintained under controlled conditions. From seed germination onwards
the trays were monitored till 5 weeks.
Data Recording
Germination percentage
Germination percentage of all the seed treatments was recorded after 1 week of seed sowing.
Disease Incidence
Disease incidence was recorded as percentage after 4 weeks of seed sowing. The formula used for
recording disease incidence (Hoffman et al., 2002) is as follows:
No. of diseased seedlings
% disease incidence = X 100
Total No. of seedlings
49
Results
Seed germination and seedling survival
Optimum seed germination i.e., 93.33%, 96.66%,100.00%, 100.00%, 100.00% and 100.00% was
recorded in seeds treated with formulations - 3(control-3), 4c, 1(control-1), 4b, 4d and 5,
respectively, followed by 83.33% 33.33% in the seeds treated with formulations – 4a and
2(control-2). The seedling survival rate after 4 weeks of sowing was recorded as 100.00 % in the
seeds treated with all the formulations mentioned above except in the seeds treated with the
formulation-3 (which has commercial fungicide), this clearly indicates that the commercial
fungicide “Carbendazim WP50”, though it was efficient in suppressing fungal growth, but was
not 100.00 % efficient. The formulations containing different concentrations (except formulation
4a- which has least number of cells) of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells
proved to be 100.00 % effective in protecting seeds from P. oxalicum (NFCCI-1997) present in
the soil.
The drop in germination rate of seeds treated with formulation – 4a (formulation with least
concentration of bacterial cells, 50,000 cells/ml carrier) is clear indication that a basal dose of
bacterial cells is required to confer protection to the germinating seeds against P. oxalicum
(NFCCI-1997) present in the soil. Thus, formulation-4b, which has a bacterial concentration of
5x105 cfu/ml (0.5 million cells/ml) conferred good protection against P. oxalicum (NFCCI-1997)
and gave 100% seed germination and seedling survival rate, same as control seeds.
50
Table-5 Detection of effective concentration (cfu/g carrier) of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells for suppressing P.
oxalicum (NFCCI-1997) growth and pathogenicity.
Treatment Concentration
of Bacillus
subtilis ssp.
shriramensis
(MTCC-5674)
cells (cfu/g)
Concentration
of P. oxalicum
(NFCCI-1997)
(cfu/ml)
Concentration
of Carbendazim
( μg)
No. of
seed
sown in
tray
No. of seed
germinated
(Values in %)
Data recorded after 2 weeks of sowing Data recorded after 4 weeks of sowing Percentage
variation
(Values in %)
No. of seedlings
survived
(Values in %)
No. of seedlings
died because of
disease
(Values in %)
No. of
seedlings
died
without
disease
(Values in
%)
No. of seedlings
survived
(Values in %)
No. of
seedlings
died because
of disease
(Values in
%)
No. of
seedlings
died
without
disease
(Values in
%)
1* - - - 60 60 (100.00%) 60 (100.00%) 0 0 60 (100.00%) 0 0 0
2* - 6x104 - 60 20 (33.33%) 0 (00.00%) 20 (100.00%) 0 0 0 0 -100.00
3* - 6x104 85.50 60 56 (93.33%) 54 (96.42%) 2 (3.33%) 0 54 (90.00%) 0 0 -10.00
4a* 5x104 6x104 - 60 50 (83.33%) 14 (28.00%) 36 (72.00%) 0 9 (18.00%) 5 (10.00%) 0 -82.00
4b* 5x105 6x104 - 60 60 (100.00 %) 60 (100.00%) 0 0 60 (100.00%) 0 0 0
4c* 5x106 6x104 - 60 59(96.66%) 59 (100.00%) 0 0 59(100.00%) 0 0 0
4d*
5x107 6x104 - 60 60 (100.00%) 60 (100.00%) 0 0 60 (100.00%) 0 0 0
5* 5x107 - - 60 60 (100.00%) 60 (100.00%) 0 0 60 (100.00%) 0 0 0
51
*1- Corn seed + red polymer + CMC + Sucrose; *2- Corn seed + red polymer + CMC + Sucrose + P. oxalicum (NFCCI-1997); *3- Corn seed +
red polymer+ CMC + Sucrose + P. oxalicum (NFCCI-1997) + Carbendazim; *4a to 4d - Corn seed + red polymer+ CMC + Sucrose + P. oxalicum
(NFCCI-1997) + different concentrations of Bacillus subtilis ssp. shriramensis (MTCC. 5674) cells; *5- Corn seed + red polymer + CMC +
Sucrose + Bacillus subtilis ssp. shriramensis (MTCC-5674).
52
Disease incidence
The results of the study showed that there was significant difference between the
treatments. The seeds treated with the formulations 1, 4b, 4c, 4d and 5 did not exhibit
any disease symptoms and displayed healthy growth, similar to control seedlings,
indicating that the bio-control agent Bacillus subtilis ssp. shriramensis (MTCC-5674)
present in the formulations greatly suppressed growth and pathogenicity of the fungus
P. oxalicum (NFCCI-1997), and thus protected the seeds from getting infected with
the fungus. The seeds treated with the formulation-3 (which has a commercial
fungicide Carbendazim 50WP) showed a disease incidence of 3.57 % indicating that
though the commercial fungicide was effective in suppressing the fungal growth, but
not as good as bio-control agent used in this study.
Table-6 Percentage disease Incidence of corn seedlings treated with different
formulations.
Treatment Concentration
of bacterial
cells
Concentration of
fungal spores in soil
Total
No. of
seedlings
No. of
diseased
seedlings
Per cent
Disease
incidence
1(control-
1)
Nil Nil 60 0 0.00
2(control-
2)
Nil 6x104 20 20 100.00
3(control-
3)
Nil 6x104 56 2 3.57
4a 5x104 6x104 50 41 82.00
4b 5x105 6x104 60 0 0.00
4c 5x106 6x104 59 0 0.00
4d 5x107 6x104 60 0 0.00
5 5x107 Nil 60 0 0.00
53
Disease incidence of 82 % and 100 % was recorded in the seeds treated with the
formulations 4a and 2, respectively. The results indicate that the Bacillus subtilis ssp.
shriramensis (MTCC-5674) cell density present in the formulation 4a was not
effective in suppressing growth of fungus and hence the germinating seeds were
infected with the fungus and died after 2 weeks of germination. As expected, the
seeds treated with formulation -2 which has neither bio-control agent nor commercial
fungicide showed 100% disease incidence indicating that the fungus infected the
germinating seeds and killed the seedlings within 2 weeks of germination.
Conclusion
The above results clearly indicate that Bacillus subtilis ssp. shriramensis (MTCC-
5674) at a concentration of 5x105 cfu/ml (formulation-4b) is effective in suppressing
growth of fungal pathogen and gives 100% protection to germinating seedlings of
corn. Hence, the bio-control agent can be successfully used in coating seeds for
effective control of soil borne pathogenic fungus P. oxalicum (NFCCI-1997).
Example 7
7.1 Screening the efficacy of antifungal/antimicrobial agent to inhibit the growth of
human pathogenic fungi
A range of fungal species causing diseases in human beings were isolated from
the people suffering from various skin and lung infections. The antifungal and/or
antimicrobial activity was tested against all the isolated human pathogenic fungi.
Material and Methods
Material
1. Penicillium ssp.
2. Aspergillus flavus
3. Aspergillus niger
4. Aspergillus nidulans
5. PDA plates
54
6. Antifungal/antimicrobial agent isolated from Bacillus subtilis ssp. shriramensis
(MTCC-5674)
Method
An aliquot of 500 μl of the culture filtrate containing antimicrobial and/or
antifungal agent was added into the wells made in the PDA agar and a loop full
of test fungi were inoculated at the other corner of the respective plates labeled
with the respective fungus and incubated at 28oC till the growth of fungal
mycelium was observed in the vicinity of the well containing culture filtrate.
The inhibitory activity of the filtrate against the target fungus was recorded in
millimetres as the inhibitory zone formed surrounding the well.
Result
A range of fungal species causing diseases in human beings were isolated from
the people suffering from various skin and lung infections tested in the
antimicrobial and/or antifungal assay and all of them demonstrated complete
inhibition of growth in the presence of Bacillus subtilis ssp. shriramensis
(MTCC-5674) culture filtrate (Fig. 24).
Conclusion
From the observations it has been concluded that the Antifungal/antimicrobial
agent isolated from Bacillus subtilis ssp. shriramensis (MTCC-5674) can be used
in pharmaceutical applications also.
55
References
1. Mena-Violante, H.G. and Olalde-Portugal, V. 2007. Alteration of tomato fruit quality by
root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus
subtilis BEB-13bs. Sci. Hort., Vol.1 (113), pp. 103–106.
2. Mohiddin, F. A. and Khan, M. R. 2013. Tolerance of fungal and bacterial bio-control
agents to six pesticides commonly used in the control of soil borne plant pathogens.
Global J. Pests, Dis. Crop Prot., Vol. 1 (1), pp. 001-004.
3. Hoffmann, W.A. and Poorter, H. 2002. Avoiding Bias in Calculations of Relative
Growth Rate. Ann. Bot., Vol.90 (1), pp. 37-42.
4. Li, J. Yang, Q. Zhao, L-H, Zhang, S.M., Wang, Y.X. Xiao-yu and Zhao, X.Y. 2009.
Purification and characterization of a novel antifungal protein from Bacillus
subtilis strain B29. J. Zhejiang Univ. Sci. B., Vol.10 (4) pp. 264–272..
5. Malusá, E. Sas-Paszt, L. and Ciesielska, J. 2012. Technologies for beneficial
microorganisms inocula used as biofertilizers. Sci. World J., Vol 2012, pp. 001-012.
56
Annexure I
Composition of Culture Media used
Note: A general method of media preparation is provided below. All the media
compositions given below are for 100 ml volume. Compositions changes depending
upon the quantity required.
(I) Preparation of T3 broth (pH – 6.8)
Composition of T3 medium
S. No. Component Quantity
1 Tryptone 0.32 % (w/v)
2 Tryptose 0.24 % (w/v)
3 Yeast Extract 0.18 % (w/v)
4 NaH2PO4. H2O 0.044 M
5 Na2HPO4 0.062 M
6 MnCl2.4H2O 0.0005 % (w/v)
Method
All the media components were weighed and taken in a glass bottle and dissolved in
distilled water. The container with the medium was autoclaved at 1210C for 15 min.
(II) Preparation of T3 agar plates (pH – 6.8)
Composition of T3 medium
S. No. Component Quantity
1 Tryptone 0.32 % (w/v)
2 Tryptose 0.24 % (w/v)
3 Yeast Extract 0.18 % (w/v)
4 NaH2PO4 0.044 M
5 Na2HPO4 0.062 M
6 MnCl2 0.0005 % (w/v)
7 Agar agar 2.0 % (w/v)
Method
All the media components were weighed and taken in a glass bottle and dissolved in
distilled water. The glass bottle with the medium was autoclaved at 1210C for 15 min.
After autoclaving, the medium was poured in to sterile petri plates.
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(III) Preparation of LB broth (pH – 7.0)
Composition of LB medium
S. No. Component Quantity
1 Tryptone 1.0 % (w/v)
2 Yeast Extract 0.5 % (w/v)
3 NaCl2 1.0 % (w/v)
Method
All the media components were weighed and taken in a glass bottle and dissolved in
distilled water. The container with the medium was autoclaved at 1210C for 15 min.
(IV) Preparation of LB agar plates (pH – 7.0)
Composition of LB medium
S. No. Component Quantity
1 Tryptone 1.0 % (w/v)
2 Yeast Extract 0.5 % (w/v)
3 NaCl2 1.0 % (w/v)
4 Agar agar 2.0 % (w/v)
Method
All the media components were weighed and taken in a glass bottle and dissolved in
distilled water. The glass bottle with the medium was autoclaved at 1210C for 15 min.
After autoclaving, the medium was poured in to sterile petri plates.
(V) Preparation of PDB broth (pH – 7.0)
Composition of PDB medium
S. No. Component Quantity
1 Potato Powder 2.0 % (w/v)
2 Dextrose 2.0 % (w/v)
3 Spectinomycin
(Optional)
100 μg/ml
Method
Potato powder (2.0 g) was weighed and taken in a 250 ml glass bottle containing 50 ml
of distilled water and boiled for 5 min. The boiled potato water was filtered using
muslin cloth. The filtrate was collected in a fresh 250 ml glass bottle and 2.0 g of
dextrose was added to it. After making the total volume to 100 ml, the bottle with the
58
medium was autoclaved at 1210C for 15 min.
(VI) Preparation of PDA agar plates (pH – 7.0)
Composition of PDA medium
S. No. Component Quantity
1 Potato Powder 2.0 % (w/v)
2 Dextrose 2.0 % (w/v)
3 Agar agar 2.0 % (w/v)
4 Spectinomycin
(Optional)
100 μg/ml
Method
Potato powder (2.0 g) was weighed and taken in a 250 ml glass bottle containing 50 ml
of distilled water and boiled for 5 min. The boiled potato water was filtered using
muslin cloth. The filtrate was collected in a fresh 250 ml glass bottle and 2.0 g of
dextrose and 2.0 g of agar were added to it. After making the total volume to 100 ml,
the bottle with the medium was autoclaved at 121 0C for 15 min. The molten PDA was
poured into the sterile petri plates.
(VII) Preparation of Hugh and Leifson OF Basal Medium (OFBM) (pH – 7.1)
Composition of OFBM medium
S. No. Component Quantity
1 Casein Peptone (Tryptone) 0.2% 0.2 % (w/v)
2 NaCl2 0.5 % (w/v)
3 K2HP04 0.03 % (w/v)
4 Agar agar 2.0 % (w/v)
5 Bromothymol Blue 0.004% (w/v)
Method
All the media components were weighed and taken in a glass bottle and dissolved in
distilled water. The glass bottle with the medium was autoclaved at 1210C for 15 min.
After autoclaving, the medium was poured in to sterile culture tubes.
(VI) Preparation of SIM (Sulphide Indole Motility) medium (pH – 7.3)
Composition of SIM medium
S. No. Component Quantity
1 Peptone 3.0 % (w/v)
59
2 Beef Extract 0.3 % (w/v)
3 Ferrous Ammonium Sulphate 0.02 % (w/v)
4 Sodium thiosulhphate 0.0025 % (w/v)
7 Agar agar 2.0 % (w/v)
Method
All the media components were weighed and taken in a glass bottle and dissolved in
distilled water. The glass bottle with the medium was autoclaved at 1210C for 15 min.
After autoclaving, the medium of molten stage was poured in to sterile culture tubes.
60
Annexure II
Preparation phosphate buffer
S. No. Component Quantity Weight for 100 ml
4 NaH2PO4.H2O 1 M 5.38 g
5 Na2HPO4 1 M 8.66 g
pH 7.0
Method
Both the phosphate salts were taken in a glass beaker and added 50 ml distilled water.
After ensuring that the phosphate salts are completely dissolved, the solution was made
up to 100 ml with distilled water and displaying pH 7.0.

CLAIMS:We Claim:
1. An extract of the novel bacterium belonging to Bacillus species exhibiting antimicrobial and/or antifungal activity.
2. The extract of the novel bacterium as claimed in claim 1, wherein the novel bacterium is isolated Bacillus subtilis ssp. shriramensis having the accession number (MTCC-5674).
3. A pure culture of the novel bacterium as claimed in claim 2.
4. The extract as claimed in claim 1, wherein the process of its production comprises
a. Growing the Bacillus subtilis ssp. shriramensis having the accession number (MTCC-5674) in a T3 medium having pH 6.8 in a shaking incubator at 30°C for 60 h.
b. Recovering the extract having antimicrobial and/or antifungal activity
5. The process as claimed in claim 4, wherein the Bacillus subtilis ssp. shriramensis is grown under aerobic conditions.
6. The process as claimed in claim 4 optionally comprises concentrating the extract using conventional methods.
7. A composition comprising the extract as claimed in claim 1, wherein the composition has antimicrobial and/or antifungal activity at a concentration of 4 µg/µl to 20 µg/µl of the extract.
8. A composition comprising the novel bacterium as claimed in claim 2, wherein the composition has antimicrobial and/or antifungal activity at a concentration 5x105 cfu/ml to 5x107 cfu/ml of the bacterium.
9. A composition comprising the extract as claimed in claim 1 and the novel bacterium as claimed in claim 2, wherein the composition has antimicrobial and/or antifungal activity.
10. The composition as claimed in any of the preceding claims optionally comprises one or more antimicrobial and/or antifungal agents.
11. The composition as claimed in any of the preceding claims optionally comprises agriculturally acceptable carrier.
12. A method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said method comprises contacting the pathogenic fungi and/or bacteria with an effective amount of 5x105 cfu/ml to 5x107 cfu/ml of the novel bacterium as claimed in claim 2 or the composition as claimed in any of the preceding claims.
13. The extract or the novel bacterium as claimed in claims 1 and 2 or the composition as claimed in any of the preceding claims whenever used for the preparation of an antimicrobial and/or antifungal composition for inhibiting the growth of pathogenic fungi and/or bacteria.