The present invention relates to a novel bacterium belonging to B& il s family, designated as
Bacillus s s p . s !s exhibiting nti micr bia and/or anti-&ngal and plant growth
promoting t v , isolation and identification of extract of the novel microbe exhibiting
antimicrobial and/or ant f gal proteolytic, a lo ytie activities, composition comprising the
novel bacie iu and/or extract, method of inhibiting th growth of pathogenic microbes and/or
ongi by contacting the pathogenic microbes a d/or fungi with an effective amount of the novel
bacterium and/or a antimicrobial and/or antifungal mid plant growth promotion composition
and/or agent and 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 scarcity of
water, the environment unsuitable for microbial growth. Biological material may
contribute about 20%, 22% and % to the total airborne particulate matter by volume in remote
continental, populated continental and remote maritime environments, respectively. Most of
them originate from natural source such a 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, prokaryoiic microorganisms. Typically a few
micrometres in length, bacteria hav 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, a well a in organic matter and the live bodies of plants and
animals. Th bacilli are rod-shaped, gram-positive, ti aerobic or facultative -anaerobic
bacteria. Most bacilli r saprophytes. Each bacterium creates only one spore, which i 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 the desert sands, hot springs, and Arctic s B v i s s p s can b
thermophilic, psyehrophiiie, acidophilus, a!kaiiphi!ic, halotolerant, or halophilie and are capable
of growing at various p values, temperatures, and salt concentrations.
Production o antimicrob l agents seems to be a genera! phenomenon for most bacter These
bacteria produce an admirable arra of microbial defence systems
:
including broad-spectrum
classical antibiotics., metabolic by-products such as organic acids, and lytic agents swell as
lysozyme. n addition, several types of protein exotoxins, and bact o ins which are
biologically active peptide moieties with bactericidal mode of action, ar also produced. Th
biological arsenal microbes s 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 ala ing rate. Current solutions involve
development of .mo e rational approach to antibiotic-use an discovery of new aiitiniicrobiais-
Highly re vant patents
. Novel bacterial strains and methods of controlling fungal pathogens ( O 2 7 ) ,
OBJECTIVES OF THE INVENTION
The objective of the. present Invention is to provide a novel bacter exhiMtlng anti rObial
and/or antifungal and plant growth promotion 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 plant growth promotion activity.
Th objective of the present invention is also to provide an antimicrobial nd or antifungal and
plant growth promotion composition or agent wherein th composition or the agent co prise
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 o
pathogemc microbes and/or fungi by contacting th pathogenic microbes and/or fe gi with an
effective a ount of the novel bacteri urn and/or an antimicrobial and/or antifungal and plant
growth promotion composition and/or agen wherein the composition or the agent comprises the
novel bacterium and/or the extract of the novel bacterium and/or a mixture of th novel
bacteri urn and Its extract.
Th other objective of the present invention is to rovide use of the novel bacterium, ar
antimicrobial and/or antifungal composition or agent wherein the composition or the agent
comprises the novel bacterium and/or the extract of the novel bacteri ur n and/or a mixture: of the
novel bacterium and it extract, for inhibiting the growth of pathogenic microbes and/or fungi.
SUMMARY OF THE
An aspect of the present invention is to provide an isolated, .novel bacterium wM s useful in
producing antimicrobial and/or anti&ngal metabolites or agents.
One aspect of the present invention is to provide e form of bacterium belonging to
Bacillus species whi h i designated as Bacillus subtilis ssp shrirainemis .having secession
number (MTCG-S674), .In particular, the novel bacterium disclosed in the present work is
capable of exhibiting distinct antit i robia and/or antifungal and plant growth promotion
property.
Another aspect of the present invention is to provide a process for the production of a
antimicrobial and or anti&ngai and plant growth promotion composition or agent wherein the
composition or the -agent comprises Bacillus subtilis ssp, :$ rir sls and/Or the
extract of the Bacillus subtilis ssp. s riy is (MTCC-5674).
There is provided co positio comprising Bacillus subtUis ssp, shriramensis (MTCC-5674).
The composition may further comprise pharmaceutically acceptable excipients, diluents and/or
carriers.
There s provided composition containing an extract of Bacillus subtilis ssp, shrira e i
(MTCC-5674), There is also provided a composition comprising an aqueous extract of Bacillus
subtUis ssp. s rin ensi (MTCC-5674). The composition ma er comprise
phar ac ai y acceptable xcipie ts, 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 wit a effective amount ύΐ Bacillus subtilis
ssp. shriramemis (MTCC-5674) or th extract of Bacillus subtUis ssp. sl r is (MTCC-
3674). The Bacillus subtil® ssp. skriramensis (MTCC-5674) and/or the extract of Bacillus
subtilis ssp. shriramemis (MTCC-5674) may optionally contain one or more additional
antimicrobial and/or antifungal an plant growth promoting agents.
There is provided in the present invention the use of Bacillus subtilis ssp. shrirainemis (MTCCS674)
and/or the extract of the Bacillus subtilis ssp. skriramensis (MTCC-5674} i the
formulation of an antimicrobial and/or antifungal and plant growth promotion composition r
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 subtil s s p ri me i .and having an accession number ( T G-567 4 and a method
of producing an antimicrobial and/or antifungal and plant growth promotion composition or
agent wherein the composition or the agent comprises Bacillus subiiUs ssp, s rir m& i
(MT 74 and/or the extract of the Bacillus subtilis ssp. sh m m i TCC 5 4).
The present invention also provides a method of inhibiting the pathogenic mi robe and/or fu ng
by contacting the microbes and/or fungi with a v of the novel bacterium.
Bacillus subtilis ssp, shrir m is (MTCC-5674) and/or the composition comprising the novel
bacterium or its extract.
The present Invention also provides use of Bacillus subtilis ssp. s i si (MT€C-5674),
arsd/or a antimicrobiai and/or antifungal and plant growth promotion composition- or agent
comprising the novel bacterium Bacillus subtilis ssp, shriramemis {MTCC-5674} and/or the
extract of the Bacillus subtilis ssp. shiramensis ( T€ C 674) for inhibi i g-th pathogenic
microbes -and/or fungi
The novel Bacillus subtilis ssp. shri m ns (MTCC-5674) may be Utilised for the mass
production of antimicrobial and/or antifungal and plant growth promotion
ro pos on/prep r iion/agen b c lt ring Bacillus subtilis ssp. shriramemis i the. s l
.growth medium under favourable conditions.
Through deep and caref l researches, the inventors have surprisingly found, isolated and
cultured ;a novel bacterium, which can produce a novel agent. Through detailed experimental
researches, th inventors have also invented a method of producing the said novel agent fro
the said novel microorganism.
Brief r i i of Accompanying n
Fig. I - Plate showing isolation and purification of BaciUus- bti ssp shriramemis (MTCC-
5674) (A) Mother culture plate showing bacterial growth along with fungal mycelium; (B
Purification ϋ Bacillus subtilis ssp. shrimmensis (MTCC-5674) from bacterial eo o y( ) in (A).
Arrow e putative bacterial colony.
Fig, 2 - Clous of o e of the purified colonies of BaciUus subtilis ssp s riram r s (MTCC-
5674} showing inhibition of growth of Fusarium oxyspo i mycelium.
Fig. 3 - Microscopic picture of vegetative bacterial cells of Bacillus subtilis ssp. s rir ens is
TCC ?4) along with spores.
Fig, 4 - Plate showi g actively growin colonies of Bacillus subtilis g s ri a sis TCC
56 4
Fig, 5 - Rod shaped acili subtilis ssp. s rl m ns ts (MTCC-5674) under light microscope.
Fig, 6 - Picture showing results of ati las test; (A) Negative control; ( Positive control and
(C) Bacillus s btl ii ssp. shriramemis (MTCC-5674) showing positive result-? for eatalase
activity,
Fig 7 - Plate showing anxiolytic activity of BaciUus subtilis ssp, shriramemis (MTCC-5674)
culture .titrate.
Fig, 8 - Picture test (A) Negative control; .(B)
Bacillus subtilis ssp. skriramensis (MTCC-5674) showing color change only at the top portion
of the medium; (C) Positive control.
Fig. 9 - Picture showing results of Hydrogen sulphide production test (A Negative control; (B)
Bacillus subtilis ssp. shriramemis (MTCC-5674) and (C) Positive control
Fig. J O- Pictur showing results of 8DS-PAGB of the concentrated culture filtrate Bacillus
subtilis ssp. shri s is (MTCC-5674),
Fig - Culture plates showing antimicrobial and/or antifungal activity displayed by (A)
Bacillus subtilis ssp. shriramemis (MTCC-5674) colony and (B) Bacillus subtilis ssp.
shriramemis (MTCC-5674) culture filtrate. Fusarium oxysporum culture was use as test
fungus.
Fig 12 - Picture showing results of MIC of antimicrobial and/or; antifungal compound by
the tube dilution method, 1 to C-2; Pictures of Fusarium oxysporum spores (observed under
light microscope), after incubating i P B containing different concentrations of antimicrobial
and/or antifungal agent 1-28 (Dilutions 1 1 to 1:100), C- - Spores u antimicrobial and/or
antifungal agent (Crude); C-2 - Control (Spores in PDB broth without antimicrobial and/or
antifungal agent).
Fig, 13 - Picture showing results of MIC assay of antimicrobial and/or antifungal agent by agar
diffusion method. 1 to C-2: Pictures of Fusarium oxysporum mycelium growing on test plates.
1-28 Dilutions of antimicrobial and/or antifungal agent at 1:1 to 1:100 (v/v), CI - Well
containing antimicrobial and/or antifungal agent (Crude); C2 - Control well containing PDB; C3
- Control well containing 70% saturated ammonium sulfate.
Fig. 14 - Picture showing effect of antimicrobial and or antifungal agent on spores of
Aspergillus niger. (A, B & C) Spores of Aspergillus niger showing normal germination in PDB
media; (D) Spores of Aspergillus niger failed to germinate in PDB media containing Bacillus
subtilis ssp. shriramensis (MTCC-5674) extract.
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).
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. A. Fusarium oxysporum, B. Sarocladium oryzae C. Trichoderma viridae D.
Colletotrichum capsicii E. Exerohilum turcicum F. Rhizoctonia solanii G. Macrophomina
phaseolina H.Xanthomonas oryzae
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. (A) Rice seed treated with fungus Fusarium oxysporum spores; (B & C) Rice seeds
treated with fungus Fusarium Oxysporum and Bacillus subtilis ssp. shriramensis (MTCC-5674)
extract.
Fig. 18 - Plate showing results of experiments to show absence of pathogenicity of Bacillus
subtilis ssp. shriramensis (MTCC-5674) on various plant species. (A) Rice, (B) Cotton, (C)
Tobacco, (D) Corn, and (E) Tomato.
Fig. 19 - Picture showing results of experiments to show action of Bacillus subtilis ssp.
shriramensis (MTCC-5674) as bio-control agent. (A) Tomato plant infected with Rhizoctonia
solani (NFCCI-3194) fungus. (B) Tomato plant ith Rhizoctonia solani (NFCCI-3194) and
Bacillus subtilis ssp. shriramensis (MTCC-5674) and (C) Control tomato plant (without

subiilis p shriramensis cells (5 0 cfu) a d 5, Seeds treated with the fo u!ation which
has only Bacillus s b i s ssp, shriramensis cells- (Sx cfu).
F g. 24 - Assay of antifungal and/or antimicrobial activity against a ar iet of pathogenic
fungal species. A, B and C - Assay of antifungal and/or antimicrobial activity against
P i i m sp , (A) P n ci i m spp. f¾ngal colonies B) Mycelium an (C) Effect of B&ci!lw
subiilis ssp. rir & is ( ~S674 culture filtrate, D E and Assay of antifungal, and/or
antimicrobial activity against Aspergillus v Fungal colonies E) Mycelium an (F)
Effect of Bacillus subiilis p s i nsls (M C€ S 74 culture filtrate. G and I Assay of
antifungal a d/or antimicrobial activity against Aspergillus niger, ( ) Fungal colonies (11)
Mycelium and ( Effect of cil subiiiis ssp, shriramensis MTCC- 74) culture .filtrate,.
and L - Assay of antifungal and/or antimicrobial activit against unknown fungus causing
ski infection. (J) Fungal colonies ( Conidia and (L Effect of Bacillus subiilis ..ssp.
shriramensis (MTCG-5674) culture filtrate.
Fig.25 - Effect of Bacillus bt is ssp, shriramensis (MTCC-5674) formulation on growt and
development of Corn. The com seed with formulation containing Bacillus ilis .ssp.
shriramensis (MTCC-5674) showed higher growth rate, biomass and grain yield.
ISOLATION AND fi T CAT OF T E NOVEL BACTERIUM
The inventors collected a r samples fro 18 different locations i Hyderabad and atan her .
(Telangana, India.) while conducting a study o air flora. Disposable petr plates containing
edia (T3 Medium, Travers, t 98?) were prepared in the laboratory . exposed to air at
dif ferent locations. he exposed plates were sealed and incubated at 30° C in ab incubator, n
o e of the plates exposed to air in Pa n he area* a bacteria! colony surrounded by fungal
mycelium was observed (Fig. 1A), Despite continued incubation, the clearance zone was
maintained and. growth o 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. B) The individual colonies were tested against a common
fungus n nm x sp ar m (Fig, 2),
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 th culture medium the bacteria produced
spores. The colonies of the bacteria were mucoid, raised, circular, smooth, and creamy to offwhite
n ;color (Fig. 4), and cells showed variable gram staining (Fig, 5).
A range of biochemical tests including carbohydrate fermentation, eatalase activity, oxidationfermentation
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, possess amylase activity, strongly aerobic, does not produce hydrogen sulfide,
oxidase positive and gram variable.
For identification of bacteria 6 DNA sequencing and FAME analysis was earned out The
results of both studies showed that the bacteria is showing 0.37% difference i 6S DNA
sequence and. FAME similarity index of 0,827; with Bacillus s b i -ssp, s bi ls and 0.84%
diflference in 16$ DNA sequence and FAME similarity index of 0.749 with Bacillus
a p e . ms, the results suggest that- this bacterium is related t Bacillus subiilis and
B i r ph s but not identical to any of the catalogued bacterial species in ATCC
collection.
The isolated bacterium is a new member of sub-species of the .genus ci According to
bacterial nomenclature convention, the novel bacterial species was named as Bacillus subtiiis
ssp. s riran is The bacterium is deposited i the Microbial Type Culture Collection (MTCC)
at IMTECH, Chandigarh, India, The deposition number of this novel species s (MTCC~56?4)
colonies are smooth, mucoid, off-white to ereamish 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 spoliation takes place in. 4 days of incubation in 10 n l mediu
containing 100 mί of 5x eells inoculum in a 25x 0 m culture tube at 30 °C and shaking a
200 rp.rn,
The novel bacterium, Bacillus subtiiis ssp. shri i s having number (MTCC-
5674} exhibits antimicrobial and/or antifungal activity, The extract of the novel baeterium,
Bacitiusi subtiiis ssp. shrimmemis having accession number (MTCC-5674) exhibits
antimicrobial and/or antifungal activity, Th range of potential applications- and uses o the
bacterium- are extensive.
The present invention provides method of producing the antimicrobial -and/or antifungal
extract from the novel baeterium, Bacillus subtiiis ssp. i r m s having accession number
(MTCC0674).

bacterium, Bacillus b i s ssp, s riran s.having accession number ( TC 56 4 .
In one embodiment of the present invention there is provided an extract of the novel bacterium,
Bacillus xubtilis ssp. shriramemis having accession number ( . CC 56 4) wherein the extract
exhibits antimicrobial and/or antifungal activity.
In another embodiment of the present invention there is provided a extract of the nove
bacterium. Bacillus subiilis ssp. shriramensis having accession number ( T C 5674 wherein
the extrac exhibiting antimicrobial and/or antifungal activity is an aqueous extract. In et
another embodiment of the present invention there is provided process for the production of
the extract of the novel bacterium, Bacillus subiilis ssp. shrira s is having accession
MTCG 56 4) where in the process comprises growing the novel bacterium, Bacillus subiilis
ssp shriramensis having accession number (MTCC-5674) in a nutrient medium and recovering
the extract having antifungal, ct vity by using conventional methods..
In another embodiment of the .present invention there is provided process for the production of
the extract of the novel bacterium, Bacillus s t ssp, shriramemis having accession number
5674) wherein the process comprises growing the novel bacterium, Bac s subiilis
ssp, shriramemis havin accession umber (MTCC-5674) under aerobic conditions.
In ye another embodiment of the present invention there is provided a . process for the
production of the extract of the novel bacterium, i s subiilis ssp, skri ra s having
accession number ( X C 5674) wherein the process comprises growing the novel bacterium,
Bacillus: subiilis ssp, shriramensis in a nutrient medium, recovering the extrac having
nti e bial.and/or 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 subiilis ssp. shriramensis having accession number (MTCC-5674)
wherein the composition has antimicrobial and/or antifungal activity.
n another embodiment of the present invention, there is provided a composition comprising the
extract of th novel bacterium. Bacillus subiilis ssp. shriramensis having accession number
( T GV56 4) wherein the composition has antimicrobial and/or antifungal activity,
in another embodiment of the present invention, there is provided a composition comprising the
nove bacterium, BaciUiis subtilis ssp, shriramensis having accession number (MTCC-56 4 a
the extract of t iTOve! i i subtil is ssp shriramensis having accession number
(MTCC-5674) wherein the composition: has .antimicrobial and/or antifungal activity.
n on embodiment of the present invention, there is provided a composition comprising the
novel bact erium i u ti is ssp, shri r m is having accession n ber (MTCC-5674}
and/or an extract of the said novel bacterium., BaciUiis. subtilis ssp shriramensis, or a
combination thereof tha optionally comprises one or more antimicrobial and/or antifungal
agents.
n another embodiment of th present invention, there is provided a composition comprising
extract of the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number
(MTCC-5 4 that optionally comprises one or more antimicrobial an d/o antifungal agents.
n yet another embodiment of the present invention there is. provided a c m os on comprising
the combination of the novel bacterium, .Bacillus subtilis ssp, shriramensis having accession
number .(MTCC-5674) and it extract that optionally comprises one or mor antmiicrobial
and/or antifungal agents.
In one embodiment of the present invention there s provided composition comprising the
novel bacterium, Bacillus subtilis ssp. shr im sis having accession number (MTCC-5674) or
an extract of the novel bacterium. Bacillus subtilis ssp. shriramensis having accession number
(MTCC'5674) or combination thereof that optionally comprises agri t r l or
p arina en i a iy acceptable earner.
n another embodiment of the present invention there is provided a composition comprising the
nove bacterium, Bacillus subtilis ssp, shrirmmnsis havin accession number (MTCC-5674)
that optionally comprises agriculturally or pharmaceutically acceptable earner.
yet another embodiment of the present invention there is provided a composition containing
an extract of the novel bacterium. Bacillus subtilis ssp. shriramensis having accession number
(MTCC-5674) thai optionally comprises agriculturally or pharmaceutically acceptable carrier.
In still another embodiment of the present there is provided a composition comprising
the combination of the novel bacterium, S i u subtilis ssp. shriramensis -hating · s o
number -(MTCC 7 ) and an extract of the said novel bacterium, Bacillus subtilis ssp,
nr m r which optionally comprises agriculturally acceptable carrier (See Annexure HI),
on embodiment of the present invention there Is provided a method for inhibiting growth o
pathogenic fungi and/or bacteria, wherein said ethod comprises ta ift g the. pathogenic
fungi and/or bacteria w h an effective amount of the novel bacterium, Bacillus s l ssp.
s fi r r ns s havin accession number (MTCG-5674) or a composition comprising th said
novel bacterium, or its extract o a combination thereof.
In o embodiment :of the present invention there is provided a method for inhibiting growth of
pathogenic fungi and/or bacteria, wherei said method comprises contacting the pathogenic
fangs a d/or bacteria with an effective amount of the novel bacterium, Bacillus- subtilk ssp
r r i having accession number (MTCC-5674),
In another embodiment o the present invention there is pro vided a method for iiu¾biting gn th
of pathogenic fungi and/or bacteria, wherein said method comprises contacting th pathogenic
fungi and/or. bacteria with an effective amount of a composition comprising the novel bacterium,
B cii subtilis ssp. s n e is having accession number (MTCC-5674),
In yet another embodiment of the present invention there is provided a method for Inhibiting
growth of pathogeme fungi and/or bacteria, wherein said method comprises contacting the
pathogenic fungi and/or bacteria with an ti amount of a composition comprising an
extract of the novel bacterium, Bacillus subtilis ssp. shriratmmis (MTCC.-5674} wherein the
extract has antimicrobial and/or antifungal activity.
In yet another embodiment of the present invention there is provided a . n tho i for inhibiting
growth of pathogenic fung and/or bacteria, wherein said method comprises contacting thepathogenic
fungi and/or bacteria with an effective amoun of a composition co pri sin the
novel bacterium, B ci s s f is ssp. shdramemis (MTCC-5674) and an extract f the- said
novel bacterium. Bacillus b s ssp. s n m s, wherein the extract has antimicrobial a d or
antifungal activity.
I one embodiment of the present Invention there is provided a use of the novel bacterium,
B ci subtilis ssp, skrirmmnsfs having accession .number ( CC 674) or a composition
comprising the sa d novel bacterium or its extract or a combination thereof, for tire preparation
of an an icrobia and/or antifungal composition for inhibiting the growth of pathogenic fungi
and/or bacteria.
In another e bodi ent of the presen invention there is provided a s of the nove bacterium,
Bacillus siditilis ssp, shriramemis having accession number (MTCC-5674) for the preparation
of an antimicrobial and/or antifungal composition for inliibiting the growth of pathogenic fungi
and/or bacteria.
in another embodiment of the present invention there provided a us of the composition
comprising the novel bacterium, Bacillus siibtilis ssp, shriramemis (MTCC-5674) for the
preparation of an antimicrobial and/or antifungal composition fo inhibiting the growth of
pathogenic fungi and/or bacteria.
In another embodiment of th present invention there is provided a use of the composition
comprising; an extract of the novel bacterium, Bacilhis siibtilis ssp, shrimmensis MTGC- 74)
for the preparation of an antimicrobial a d/or antifungal composition fo inhibiting the growth
of pathogenic fungi and/or bacteria.
In another embodiment of the present invention there is provided use of the composition
comprising the extract of the novel bacterium, Bacillus sub is ssp, shriramemis -(MTCC-5674)
and an extract o the said novel bacterium, Bacillus sti is ssp. shrirammsis, for the
preparation of an antimicrobial and/or antifungal composition fo inhibiting th growth of
pathogenic fungi and/or bacteria.
I another embodiment, there is provided a pharmaceutical and agriculturally "effective
composition comprising the novel bacterium. Bacillus btil ssp. shriramemis having
accession number (MTCC-5674).
n another embodiment, there is provided pharmaceutical and agriculturally effective
composition comprising the extract of the novel bacterium, Bacilhis subtilis ssp. shriramemis
(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 suhiilis ssp, shriramemis havin
access!on. number (MTCC-5674):,

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 offwhite
n color (Fig, 4) an the cells showed variable gra staining.
2
2, Characterization an Identi f a io of novel . roo ani
2.1-1 ,2 C itur Character i i s
Bacillus tib ili ssp. shri & : ( TCC 5674) shows optimum growth at
30 C (can gr f m C to 55 . As it is an aerobic b eteri , it requires
adequate oxygen for its growth, needs continuous shaking for cuituriag in
broth.
Colony Cream to off-white Off-white to Dark brown to black
Color brown
.1.1.4 Catalase test
Material
• Culture tubes of Bacillus sabtilis ssp. shriramensis (MTGC-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 tubes respectively. Following incubation at 30°C 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" 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
• Incubator
Method
The starch agar medium was prepared as per the method provided in the
Annexure - I (VI). Two wells were made at equal distances in the plate
containing starch agar medium and labelled as "test" and "negative control".
An aliquot of 500 mΐ 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 50°C for 4
hours.
Result
After hours of incubation, the blue color su ound ng the test we l
disappeared indicating that the Bacillus subtilis ssp. s n nsis (MTCC-
5674) culture filtrate has a ol t c activity. No change in the blue color was
observed In the area surrounding control well (Fig, 7),
1.6 O F r ntat¾c ) Test
Material
» gs ifs n s OF Basal Medium
Test tubes
* . i cultur
* Bacillus subtilis ssp. s i s (MTCC-5674)
* Incubator
Method
Three tubes containing Bugs Leifson's OF basal mediu (OFBM
(A exur I (V )) were labelled as "negative control "positive control"
and t s and a loo full of A ig ms f e ti$ Escherichia ' and
Bacillus subtilis ssp, s mis (MTCC-5674) was Inoculated in the tubes
respectively. The tubes were incubated at for 48 hours and observed for
change of color.
Result
I has been concluded fr o th observations that the test organism (Bacillus
subtilis ssp. shr r sis (MTCC-5674) is strictly aerobic as it did not
ferment carbohydrate (neither gas formation nor the color change) deep
inside the mediu Du to availability of oxygen the surface of n d r
some color change was observed, Whereas E. coli grew very e deep inside
the medium and fe nent 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,
1 7 y ¾ri S lf d¾ od tion Test
SIM (Sulfide Indole Motility) medium
* Culture tubes
* Exoli culture
* Bacillus subtilis ssp, n r nsi (MTCC-5674)
* Incubator
Method
The tubes containing SIM [Sulfide Indole Motility. {A ne r - I (V )}]
med ns were labelled as "negative control" and "test" and a loop foil of £
¾ and Bacillus subtilis ssp. s ri r me is (MTCC-5674), were inoculated
in the tubes respectively, n incubated at 30°C for 24 hours and observed
for color change.
Result
From the observations, it has bee concluded that th test organism s
negative or ¾ S production as the medium did not turn black. The same
result was observed in the negative control (Fig. 9).
. Effect of p on th growth of Bacillus subtiUs ssp, sh a is iMTCG
Culture tubes containing standard culture medium (LB) adjusted to different
pH values ranging from 3,4 to 11,0 (acidic to basic) were used t grow
Bacillus subtilis ssp, hri me is (MTCC-5674) under standard conditions.
Growth of Bacillus subtiUs ssp, s r e is (MTCC-5674) was observed in
a pH range of 6.4 to 7. and the pt rn p was found to be 7.0.
1.9 Antibiotic Sensitivity ΐ Bacillus subtilis ssp. shriramemis (MTCC-
5674)
A 24 hours old Bacillus t s ssp. shrir nsis (MTCC-5674) 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
p ste were incubated at 30 C For 2 hours.
Table- Observations on antibiotic sensitivity of Bacillus s h !is ssp
shriramemis MTCC-5 74) RES Resistant, NT - nte iate SE
Sensitive
Result
It has been concluded from the observations that B ci s s tiii ssp. shrir enxi
(MTCC-5674) is resistant to the antibiotics - a pi illi carbe illin ka a y in,
earofloxaein, linc in, a xicil m clindamycin, neomycin, Th test
bacterium is sensitive to gentamicin, t ra i in vancomycin, novobiocin, suifisoxazole,
Ce hal thi chloramphenicol, ceftriaxone and bacitracin d showed intermediate
resistance to oxacillin, amikacin, streptomycin erythromycin,

Cflaracteriz ati of ;antimicrobial and/or antifungal agent
The iu ti icro i -and/or antifungal activity associated with Bacillus suhtilis ssp,
hrir me i was Investigated to ascertain the nature of agent causing antimicrobial -and/or
antifungal activity .
Material
T3 broth 1 L
Conical F sk - 2L capacity
an my in ( g l)
i s suhtilis ssp. s ri m mis (MTCC 36 4 inoe um
Shaking incubator
Method
The G3 broth s prepared according the method desc bed r I ( ), A ml
aliquot of 24 horns old Bacillus suhtilis ssp, s ri tm si s (MTCC 5 ?4) inoculated into
the sterile T3 broth an Incubated n th shaking incubator at 30°C for 6 hours, while
h at 200 p , Following growth of Bacillus su iiii ssp, s rir nsi (MTCC-S674)
in the T3 broth for 0 hours* th culture- medium was eentrlfuged at 12000 p and 4 C for
10 rain. The supernatant was collected and passed through 0.22 mh Filters to remove an
remaining bacterial cells.
3.2.2 Antimicrobial and/or antifungal assay with the Bacillus subtilis ssp. shriramensis
(MT^-5674 culture filtrate
To test the nature of antimicrobial and/or antifungal agent in the filtrate, a well was
made in the PDA agar plate and 500 mΐ of the filtrate was placed 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. 1IB) 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
3.3.1 Lvophilization 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 382.1 8 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

- 7 7 -

Agar diffusion metho .. Fig 1 )
Inhibition of ngal mycelium growth was observed around the wells
containing antimicrobial and/or antiiliiigal agent in the ratios 1 :1 :2 :3 an
1:4 (Table 3). Moderate inhibition was observed sun dirig th wells
containing antimicrobial and/or antifungal agent in the ratios :5, 1:6 an 1:7
.dilutions and no inhibition were observed in th 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 dilution of :4 (v/V in a concentration
d d manner,

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) of
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 0 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 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 (MTCC5674)
did not exhibit any kind of disease symptoms and thei growth and
development was equivalent t o control plants indicating that: Bacillus s b ili ssp.
shriramensis (MTGC-5674) is non-pathogenic for plant species (Fig. 8)
Example
- FQrmiilation of antimicrobial and/o antifungal compositions Bacillus subiiiis
ssp. shriramensis MTC 74) cells as a biological control agent
Material
Bacillus subiiiis ssp. shriramensis ( T C 5674)
* LB - broth
* FOB (Potato Dextrose Broth)
* Phosphate buffer (pH 7.0)
CMC (Carboxy Methyl Cellulose)
Method
5.1:1 Preparation of Bacillus ubt s ssp. shrimmensis. ( 56 4) cel suspension
Bacillus subiiiis ssp. shrimmensis ( T C 5 74 was inoculated i 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 s b ilis ssp, shrimmensis (MTCC-5674), the culture
was eentrifuged at 6,500 rpm, a 4 for 10 min. The pelle was washed twice in
phosphate buffer (pH 7.0) and with 1 CMC (Carboxy Methyl Cellulose) in
phosphate buffer (pH 7.0) t prepare a slurry containing 6 ' u/ . The slurry
containing Bacillus s bt lis ssp, shriramensis (MTC05674) was used to spra on
plants and trea plant seedling ro s by dipping.
5. .2 T test the efficacy of formulate containing antimicrobial and/or antifungal agent to
inhibit the infestation of hi c on a s l ni {NFCCI-3194) in the roots of tomato
plants
Material
* Slurry containing Bacillus suh is ssp. shriramensis (MTCC-5674)
iz ctonia solani (NFCCI-3 94) fungus (causes sheath blight in members of family
soianacea),
* Soil ite
To at seedlings
.3 Preparation of Rhizoctonia solani fNFCCl-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 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 (NFCCl-3 94).
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) .
Tor 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 R izoct ni so ni ( FCC 4
exhibited retarded growth, poor flowering and fruit foi ation a compared withcontrol
Hence, it can bee -concluded that i s s bti li s sh ir en (M CC-
5674) inhibited; the growth of the funps R i ei i ni PCC - 4) in the
r pn r area of tomato seedlings and protected the seedlings from disease causing
fungus (Fig* ) .
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 6x104ml 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 (6xl0 4 cfu ) 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.
.3 Preparation of formulation of Bacillus subtilis. ssp. shriramensis (MTCC-5674')
for biological control of soil borne plant disease
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-
1 97) 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 ed polymer. This
formulation has no bio-control agent, disease causing agents and fungicide. Seeds
treated with this formulation were used as control seeds.
Composition
Control-2 (Formulation with fungal pathogen but no bio-control agenf)
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
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 mg/ml (Mohiddin et ai, 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.
Composition
S. No. Components Weight/Volume/ Final concentration
Number
1 Carbendazim 85.50 m
2 CMC 1.71 1.00 % w/v
3 Sucrose 3.42 mg 2.00 % w/v
4 Red Polymer 34 mΐ 19.88 v/v
5 Water 31.87 mΐ -
Total 171 mΐ -
a. 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 5xl0 4 cfu/ml.
This formulation has minimum number of Bacillus subtilis ssp. shriramensis
(MTCC-5674) cells.
Composition
b. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-56741 cells 5x 0s cfu
This formulation is composed of 1% CMC, 2% sucrose, red polymer and Bacillus
subtilis ssp. shriramensis (MTCC-5674) cells at a concentration of 5xl0 5 cfu/ml.
Composition
This formulation is composed of 1% CMC, 2 sucrose, polymer and Bacillus
This formulatioti has maximum- number of Bacillus subtilis ssp.
shriramensis(MTCC-5614) cells and is used to study the effect of biocontrol agent
on seed germination and plant growth.
Composition
Table-4 Experiment plan in tabular form
* 1(Control- 1)- Formulation without fungal pathogen and antifungal agent; *2(Control-2)- Formulation with fungal pathogen but no bio-cont
agent; *3(Control-3)- Formulation with commercial fungicide Xarbendazim WP50"; *4a- Formulation with Bacillus subtilis ssp. shriramen
(MTCC-5674) (5xl0 4 cfu); *4b- Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5xl0 5 cfu); *4c- Formulation w
Bacillus subtilis ssp. shr iramens is -567 ) cells (5x10* cfa) ; *4d- Formulation containing BacUlus subtilis ssp. shriramensis (MTCC-567
cells (5 l 07 cfu) and *5- Formulation with only Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5 l07 cfu).
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% HgCh for 10 min and rinsed with 95% ethanol, and washed
with sterilized water for 10 min each. Dry seeds were coated with 171 m1/60 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
Results
Seed germination and seedling survival
Optimum seed germination i.e., 93.33%, 96.66%, 100.00%, 10.0.00%, 100.00% and
100.00% was recorded in seeds treated with formulations - 3(control-3), 4c, l(control-l),
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 5x10 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.
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.
*].- Corn Seed · ted polymer ·(·· CMC · Sucrose; *2- Com seed red polymer CMC + Sucrose + . oxalicum ( FCC - 97) *3- Co seed + r
polymer* CM + Sucrose / ' oxalicum FCC - 997 - Car enda ; *4a to 4d - Co seed red p l er CMC + Sucrose P. oxalic
(NFCCi~1997) - different n rat s ac l sublilis ss . s r r ne is (M CC 5674} cells; *5~ Cor seed red polymer - CMC + Sucros
+Baci m t 'li ssp shri s (MTCC-5674).
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 biocontrol
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.
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 5x10 cfu/ml (formulation-4b) is effective in suppressing growth of fungal
pathogen and gives 100% protection to germinating seedlings of corn. Hence, the biocontrol
agent can be successfully used in coating seeds for effective control of soil borne
pathogenic fungus P. oxalicum (NFCCI-1997).
Testing the efficacy of the Bacillus subtilis ssp. shriramensis MTCC-567 cells
formulation in promoting the growth and yield in plants
Materials and methods
Materials
. Seeds of Corn, Tomato and Brinjal treated with the formulation as mentioned in 6.1.3
(4b).
. Control seeds of Corn, Tomato and Brinjal.
Methods
Seed coating
Seeds of Corn, Tomato and Brinjal were treated with the formulation mentioned in 6.1.3
(4b) and air dried.
Seed sowing
Treated and untreated (control) seeds of Com, Tomato and Brinjal, each in three
replicates and each replicate containing 23 seeds were sown in the field of 4 meters area.
The standard spacing measurements like 20 cm plant to plant and 60 cm row to row
distance were maintained. Appropriate agronomy practices were followed to grow these
crops to maturity.
Results
There were significant increase in plant growth parameters and yield under field
conditions. The seeds coated with formulate containing Bacillus subtilis ssp.
shriramensis (MTCC-5674) cells increased the yield in Corn, Brinjal and Tomato by
17.60, 37.15 and 1.58%, respectively (Table -7). The Corn, Brinjal and Tomato plants
showed higher rate of growth, development and biomass accumulation (a representative
picture of difference in treated and untreated Corn is given in Fig 25). Earlier reports on
plant growth promoters have also proved that the formulation containing Bacillus
subtilis enhanced the growth of plants and induced systemic resistance to disease
protection by producing 60 different types of secondary metabolites (Compant et ai,
2005 and Mohan Kumar et al., 2015).
Table- 7 Effect of Bacillus subtilis ssp. shriramensis (MTCC-5674) cells formulation on
the yield of Corn, Tomato and Brinjal.
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.
Materials and Methods
Materials
. Penicillium ssp.
. Aspergillusflavus .
. Aspergillus niger
. Aspergillus nidulans
. PDA plates
Antifungal/antimicrobial agent isolated from Bacillus subtilis ssp. shriramensis
(MTCC-5674)
Method
An aliquot of 500 m 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 28°C 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.
References
1. Compant , S., Duffy, B., Nowak, J., Clement, C. and Barka, E.A. (2005). Use of plants
growth promoting bacteria for biocontrol of plant diseases: Principles, Mechanisms of
action, and future prospects. Appl. Environ. Microbiol. 71:4951-4959. Sci. World J., Vol
2012, pp. 001-012.
2. Hoffmann, W.A. and Poorter, H. 2002. Avoiding Bias in Calculations of Relative Growth
Rate. Ann. Bo , Vol.90 (1), pp. 37-42.
3. 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.
4. Malusa, E. Sas-Paszt, L. and Ciesielska, J. 2012. 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.l (113), pp. 103-106.
6. Mohan Kumar, S.P., Chowdappa, P. and Krishna, V. (2015). Development of seed coating
formulation using consortium of Bacillus subtilis OTPB1 and Trichoderma harzianum
OTPB3 for plant growth promotion and induction of systemic resistance in field and
horticultural crops. Indian Phytopath. 68 (l):25-31.
7. 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 Pro , Vol. 1 (1), pp. 001-004.
Relevant Patents
1. Anovel strain of Bacillus for controlling plant diseases and corn rootworm. (EP981540A1).
2. Strain of Bacillus subtilis for agricultural use. (WO200903 874A1). ;
3. Antifungal Bacillus subtilis and a microorganism wettable powder containing the same
(KR2011075132A).
water. The container with the medium autoe v d at I21°C for 15 mm.

au.tooiav.mg» the medium wa poured In to sterile culture tubes.
(YD Preparation of SIM Sulphide Indole Motility) medium p - 7,3)
Composition of SIM medium
S. No Component Quantity
Peptone 3.0 % (w v)
2 Beef Extract 0,3 % (w/v)
3 Ferrous Ammonium Sulphate 0.02 % (w/v)
4 Sodium th osulhpha e 0.0025 % w V)
Aga agar 2.0 % (w v
All the media components wer weighed and taken a glass bottle and d s iv d n distilled
water. The glass bottle with the medium was autociaved at 121¾ for 15 min. After
autoclavmg, the medium of molten stage was poured in to sterile culture tubes.
P T/IN2015/000294
Annexure II
Preparation phosphate buffer
Method
Both the phosphate salts were taken in a glass beaker, SO ml distilled water was added to salts
and stirred on a magnetic stirrer using a magnetic bar. After ensuring that the phosphate salts
are completely dissolved, the solution was made up to 100 ml with distilled water.
Annexure HI
Carriers and other agents used in the experiment
yj i
S,No, Short orm Fu l
1 American Culture Collection
CMC C r ox Methyl Cellulose
3 CFU Colony I hng Units
4 Liter
S LB r a Beri i
5 Micro Lifer
7 Mce Microbial Type C lt«r Collection
8 Ml Mi l Liter
9 MIC Minimal hibitory Concentration
0 i Minutes
1 M Molar
2 NFCC National Fungal Culture Collection of India
13 FB Oxidation rrn niat B Basal .Medium
4 PDA Potato ®Agar
P B Potato ¾ Broth
16 M Revolutions Per
Ssp Sub-species
SIM Sulphide Indole Motility
19 v/v · Volume b volume
2 Weight by volume
2 1 w Weight by weight
22 P tt b!e Po der

We Claim:
1. A novel bacterium belonging to Bacillus species exhibiting antimicrobial and/or
antifungal and plant growth promoting activity.
2. 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. An extract of the novel bacterium belonging to Bacillus species as claimed in claim 1,
exhibiting antimicrobial and/or antifungal activity.
4. A pure culture of the novel bacterium as claimed in claims 1 and 2.
5. The extract as claimed in claim 3, 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
6. The process as claimed in Claim 5, wherein the Bacillus subtilis ssp. shriramensis is
grown under aerobic conditions.
7. The process as claimed in claim 5 optionally comprises concentrating the extract using
conventional methods.
8. A composition comprising the novel bacterium as claimed in claim 1, wherein the
composition has antimicrobial and/or antifungal and plant growth promoting activity at a
concentration 5x10 cfu/ml to 5xl0 7 cfu/ml of the bacterium.
9. A composition comprising the extract as claimed in claim 3, wherein the composition
has antimicrobial and/or antifungal activity at a concentration of 4 mg/ to 20 mg ml of the
extract.
10. A composition comprising the novel bacterium as claimed in claim 1 and the extract as
claimed in claim 3, wherein the composition has antimicrobial and/or antifungal and plant
growth promoting activity.
11. The composition as claimed in any of the preceding claims optionally comprises one or
more antimicrobial and/or antifungal and plant growth promoting agents.
12. The composition as claimed in any of the preceding claims optionally comprises
agriculturally acceptable carrier.
13. 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 5x10s cfu/ml to 5xl0 7 cfu/ml of the novel bacterium as claimed in claim 1 or the
composition as claimed in any of the preceding claims.
14. The novel bacterium or the extract as claimed in claims 1 and 3 or the composition as
claimed in any of the preceding claims whenever used for the preparation of an antimicrobial
and/or antifungal and plant growth promotion composition for inhibiting the growth of
pathogenic fungi and/or bacteria.