The present invention relates to a novel bacterium belonging to B&cilhts family, designated as Bacillus mht ts sp. shmmm !s exhibiting anti-microbiai and/or■■anti-&ngal and plant growth, promoting activity;, isolation and identification of extract of the novel microbe exhibiting antimicrobial and/or antlfiragal, proteolytic, amyloiytie activities,, compositio comprising the novel bacierium and/or extract, method of inhibiting the growth of pathogenic microbes and/or fongi by contacting the pathogenic microbes and/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 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 arid 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 source 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, prokaryoiic microorganisms. Typically a few micrometres, in. length, bacteria have a wide range of shapes, ranging from cocci to rods and spirals. Bacteri 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, specul tin , aerobic or facultative -anaerobic bacteria. Most bacilli ar saprophytes. Each bacterium creates only one spore, which is resistant to heat, cold, radiation, desiccation, and disinfectants. The bacilli exhibit an array of physiological abilitie that allow them to live in a wide range of habitats, including many extreme habitats such s the desert sands, hot springs, and Arctic soih. Buv ius s pcies can be thermophilic, psyehrophiiie, acidophilus, a!kaiiphi!ic, halotolerant, or halophilie and are capable of growing at various pH values, temperatures, and salt concentrations. Production of antimicrobial agents seems to be a genera! phenomenon for most bacteria. 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. In addition, several types of protein exotoxins, and bactenocins, 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 alamiing rate. Current solutions involve development of a ..more rational approach to antibiotic-use and discover of new aiitiniicrobiais-

Highly relevant patents

1. Novel bacterial ^' strains and methods of controlling fungal pathogens (WO 2000 Qf 5761),

OBJECTIVES OF THE INVENTION

The objective of the _. present Invention is to provide a novel bacterium exhiMtlng anti erObial 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.

The ^■ objective of the present invention is also to provide an antimicrobial and or ^■ antifungal and plant growth promotion composition or agent wherein the composition or the agent comprises the no vel 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 pathogemc microbes and/or fungi by contactin the pathogenic microbes and/or fengi with an effective amount 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 the novel bacteri urn and Its extract.

The other objective of the present invention is to rovide use of the novel ^' bacterium, art 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 urn. and/or a mixture: of the novel bacterium and i ts extract, for inhibiting the growth of pathogenic microbes and/or fungi. SUMMARY OF THE I ENTION

An aspect of the present invention is to provide an isolated, .novel bacterium wMe Js useful in producing antimicrobial and/or anti&ngal metabolites or agents.

One aspect of the present invention is to provide no e form of bacterium belonging to Bacillus species which, is 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 antitnicrobial and/or antifungal and plant growth promotion property.

Another aspect of the present invention is to provide a process for the production of an antimicrobial and or anti&ngai and plant growth, promotion composition or agent wherein the composition or the -agent ^' comprises Bacillus subtilis ssp, _: $kriramzrisls and/Or the extract of the Bacillus subtilis ssp. shriyamemis (MTCC-5674).

There is provided compositio comprisin Bacillus subtUis 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, shrirame is (MTCC-5674), There is also provided a composition comprising an aqueous extract of Bacillus subtUis ssp. shrinmensis .(MTCC-5674). The composition may ^: tober comprise pharmaceuiieaily 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 aa effective amount ύΐ Bacillus subtilis ssp. shriramemis (MTCC-5674) or the extract of Bacillus subtUis ssp. slmrotmmis (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 and plant gro wth promoting agents.

^' There is provided in the present, invention the use of Bacillus subtilis ssp. shrirainemis (MTCC- S674) and/or the extract of the Bacillus subtilis ssp. skriramensis (MTCC-5674} in 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 subtilis-s$p< skrimmemis. and having an accession number (MTCG-5674) 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 rirqm&nsis (MTCC-S674) and/or the ^' extract of the Bacillus subtilis ssp. shrimmemis ( TCC-5674).

The present invention also provides a method of inhibiting the pathogenic mierobes and/or .fung by contacting the microbes and/or fungi with an ei&edve amoun of the novel bacterium. Bacillus subtilis ssp, shriramemis (MTCC-5674) and/or the composition comprisin the novel bacterium or its extract.

The present Invention also provides use of Bacillus subtilis ssp. shrimm sis ( T€C-5674), arsd/or an 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 (MT€C~5674) for inhibiii g- the pathogeni 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 eoroposi lon/preparaiion/ageni by culturing Bacillus subtilis ssp. shriramemis in the. suitable .growth medium under favourable conditions.

Through deep and caref l researches, the inventors have surprisingl found, isolated and cultured ^; a novel bacterium, which can produce a novel agent. Through detailed experimental researches, the inventors have also invented a method of producing the said novel agent from the said novel microorganism. Brief scripiioii of Accompanying Drawings

Fig. I - Plate showing isolation and purification of BaciUus- subtitte ssp, shriramemis (MTCC- 5674) (A) Mother culture plate showing bacterial growth along with fungal mycelium; (B) Purification ϋϊ Bacillus subtilis ssp. shri mensis (MTCC-5674) from bacterial eolony(s) in (A). Arrow indica es putative bacterial colony.

Fig, 2 - Clous of one of the purified colonies of BaciUus subtilis ssp, skriramerts (MTCC- 5674} showing inhibition of growth of Fusarium oxysponi mycelium.

Fig. 3 - Microscopic picture of vegetative bacterial cells of Bacillus subtili ssp. skrira ensis ( TCC-56?4) along with spores.

Fig, 4 - Plate showing, actively growin colonies of Bacillus subtilis gsp.- s riram ttsis ( TCC- 5674).

Fig, 5 - Rod shaped acilim subtilis ssp. shrlmmensts (MTCC-5674) under light microscope. Fig, 6 - Picture showing results of catialase test; (A) Negative control; (B) Positive control and (C) Bacillus subtllii 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. JO - Pictur showing results of 8DS-PAGB of the concentrated culture filtrate of Bacillus subtilis ssp. shri m sis (MTCC-5674),

Fig. I I - Culture plates showing antimicrobial and/or antifungal activity displayed by (A) Bacillus -I subtilis ssp. shriramemis (MTCC-5674) colony and (B) Bacillus subtilis ssp. shriramemis (MTCC-5674) culture filtrate. Fusarium oxysporum culture was used as test fungus.

Fig, 12 - Picture showing results of MIC ssa 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 PDB containing different concentrations of antimicrobial and/or antifungal agent 1-28 (Dilutions 1 :1 to 1 :100), C-l - 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 with Rhizoctonia solani (NFCCI-3194) and Bacillus subtilis ssp. shriramensis (MTCC-5674) and (C) Control tomato plant (without subiilis $sp> shriramensis cells (5x 0 ^? cfu) and 5, Seeds treated with the fomiu!ation-S which has only Bacillus subtiMs ssp, shriramensis cells- (SxlO ⁷ cfu).

Fig. 24 - Assay of antifungal and/or antimicrobial activity against a variety of uman pathogenic fungal species. A, B and C - Assay of antifungal ^' and/or antimicrobial activity against Pmimiimm spp, (A) PeniciUium spp. f¾ngal colonies (B) Mycelium and (C) Effect of B&ci!lw subiilis ssp. shrir &mis ( iTCO~S674} culture filtrate, D _> E and F ~ Assay of antifungal, and/or antimicrobial activity against Aspergillus lav (P) Fungal colonies (E) Mycelium and (F) Effect of Bacillus subiilis $$p, s timmensls (MTC€-S674) culture filtrate. G, H and I - Assay of antifungal and/or antimicrobial activity against Aspergillus niger, (Q) Fungal colonies (11) Mycelium and (i) Effect of B cilh s subiiiis ssp, shriramensis (MTCC-5674) culture .filtrate,. ! _* and L - Assay of antifungal and/or antimicrobial activit against unknown fungus causing skin infection. (J) Fungal colonies ( Conidia and (L) Effect of Bacillus subiilis ..ssp. shriramensis (MTCG-5674) culture filtrate.

Fig.25 - Effect of Bacillus btUis ssp, shriramensis (MTCC-5674) formulation on growt and development of Corn. The com seed reated- with formulation containing Bacillus mhtilis .ssp. shriramensis (MTCC-5674) showed higher growth rate, biomass and grain yield.

ISOLATION AND ffiEMTiF!CATiO 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 t, 198?) were prepared in the laboratory .and exposed to air at different. locations. The exposed plates were sealed and incubated at 30° C in lab incubator, in one of the plates exposed to air in Paianehem area _* a bacteria!, 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. IB). The individual colonies were tested against a common fungus Fumnnm oxysparum (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 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, _, eatalase 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, possess 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 earned 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 subiiUs -ssp, subiUls and 0.84% diflference in 16$ DNA sequence and FAME similarity index of 0.749 with Bacillus air ophaeMs. Jims, the results suggest that- this bacterium is related t Bacillus subiilis and B cill airophams, 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 Bacitl According to bacterial nomenclature convention, the novel bacterial species was named as Bacillus subtiiis ssp. shriranwmis. The bacterium is deposited in the Microbial Type Culture Collection (MTCC) at IMTECH, Chandigarh, India, The deposition number of this novel species is (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 nil medium containing 100 μί of 5x1 ⁸ eells inoculum in a 25x130 mm culture tube at 30 °C and shaking at 200 rp.rn,

The novel bacterium, Bacillus subtiiis ssp. shrimme is having ccession; 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 of the bacterium- are extensive.

The present invention provides a method of producing the antimicrobial -and/or antifungal, extract from the novel baeterium, Bacillus subtiiis ssp. sbirame is having accession number ( TCC0674).

bacterium, Bacillus- subtiHs ssp, shriranmmis. having accession number ( TCG-5674).

In one embodiment of the present invention there is provided an extract of the novel bacterium, Bacillus xubtilis ssp. shriramemis having accession number ( .TCC-5674) wherein the extract exhibits antimicrobial, and/or antifungal activity.

In another embodiment of the present invention there is provided an extract of the novel bacterium. Bacillus subiilis ssp. shriramensis having accession number (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 productio of the extract of the novel bacterium, Bacillus subiilis ssp. shrirar isis having accession nu ber (MTCG 5674)- 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, act vity 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 subtUte ssp, shriramemis having accession number (MTC 5674) wherein the process comprises growing the novel bacterium, BaciUus subiilis ssp, shriramemis having accession number (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, MaciUus subiilis ssp, skrira ms!s having accession number (M3X2C-5674) wherein the process comprises growing the novel bacterium, Bacillus ^' : subiilis ssp, shriramensis in a nutrient medium, recovering the extrac having antimie bial. and/or ant fungal 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.

In another embodiment of the present invention, there is provided a composition comprising the extract of the novel bacterium. Bacillus subiilis ssp. shriramensis having accession number ( TCGV5674) 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, BaciUiis subtilis ssp, shriramensis having accession number :(MTCC-5674} and the extract of tlie iTOve! haGt um _t .BciciUus- subtil is ssp, shriramensis having accession number (MTCC-5674) wherein the composition: has _. antimicrobial and/or antifungal activity. in one embodiment of the present invention, there is provided a composition comprising the novel bacterium* B ciilm -subtilis ssp, shrir me is having accession nuniber (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. in another embodiment of the present invention, there is provided a composition comprising air extract of the novel bacterium, Bacillus subtilis ssp. shriramensis having accession number (MTCC-5674) that optionally comprises one or more antimicrobial and/o antifungal agents.

In yet another embodiment of the present invention there is. provided a campos on comprisin the combination, of the novel bacterium, .Bacillus subtilis ssp, shriramensis having accession number .(MTCC-5674) and its : extract that optionally comprises one or more antmiicrobial and/or antifungal, agents.

In one embodiment of the present invention there is provided a composition comprising the novel bacterium, Bacillus subtilis ssp. shrimm sis- having accession number (MTCC-5674) or an extract of the novel bacterium. Bacillus subtilis ssp. shriramensi having accession: number (MTCC'5674) or combination thereof that optionally comprises agrieiuturalfy or pharinaceniieaiiy. acceptable earner. in another embodiment of the present inventio there is provided a composition comprising the novel bacterium, Bacillus subtilis ssp, shrirmmnsis havin accession number (MTCC-5674) that optionally comprises agriculturally or pharmaceutically acceptable earner. in 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 invention; there is provided a composition comprising the combination of the novel bacterium, Saci us subtilis ssp. shriramensis -hating · accession number - _; .(MTCC-5674) and an extract of the said novel bacterium, Bacillus s btilis ssp, ^' shnr mert , which optionally comprises agriculturally acceptable carrier (See Annexure HI), la one embodiment of the present invention there Is provided a method for inhibiting growth of pathogenic fungi and/or bacteria, wherein said -method comprises contactiftg the. pathogenic fungi and/or bacteria wi h, an effective amount of the novel bacterium, Bacillus subtilk ssp. s firarmnsis havin accession number (MTCG-5674) or a composition comprising the said novel bacterium, or its extract o a combination thereof.

In one 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 and/or bacteria with an effective amount of the novel bacterium, Bacillus- subtilk .ssp. shrirm mis having accession number (MTCC-5674),

In another embodiment o the present invention there is pro vided a method for iiu¾biting gnswth 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, B ciiim subtilis ssp. shnramemis 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 effective 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. ntethoii 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, Becitttts s bf is ssp. shdramemis (MTCC-5674) and an extract, of the- said no vel bacterium. Bacillus ubtilis ssp. shnmm ss, wherein the extract, has antimicrobial artd or antifungal activity.

In one embodiment of the present Invention there is provided a use of the novel bacterium, BaciHm subtilis ssp, skrirmmnsfs having accession .number ( XCC-S674) or a compositio comprising the said novel bacterium or its extract or a combination thereof, for tire preparation of an anumicrobiai 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 novel 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 is provided a use 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 the present invention there is provided a use of the composition ^• comprising; an extract of the novel bacterium, Bacilhis siibtilis ssp, shrimmensis (MTGC-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 use of the composition comprising the extract of the novel bacterium, Bacillus subiUis ssp, shriramemis -(MTCC-5674) and an extract of the said novel bacterium, Bacillus stihti!is ssp. shrirammsis, 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 subtilte ssp. shriramemis having accession number (MTCC-5674). in another embodiment, there is provided a 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 off- white in color (Fig, 4) and the cells showed variable gram staining.

Exam le 2

2, 1 Characterization and Identification of the, novel .ffl icroomanisTO

2.1 -1 ,2 C iture Character i s ies

Bacillus stibtilis ssp. shri m&m : (MTCC»5674) shows optimum growth at

30 ^* C (can grow from IS ^' C to 55 ^' C). As it is an aerobic baeterium, 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 μΐ 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 4 hours of incubation, the blue color sutrounding the test well disappeared indicating that the Bacillus subtilis ssp. skn m nsis (MTCC- 5674) culture filtrate has am lol tic activity. No change in the blue color was observed In the area surrounding control well (Fig, 7),

1.6 Q/ ^' F iO datkffl »Ferm¾ntat¾ cm) Test

Material

» Hugsh Leifson's OF Basal Medium

« Test tubes

* B.coli cultur

* Bacillus subtilis ssp. ski me is (MTCC-5674)

* Incubator

Method

Three tubes containing Bugsh Leifson's OF basal medium (OFBM) (Asmexure - I (VO)) were labelled as "negative control "positive control" and "tes ^* and a loo full of Akaiigems faeeati$ _t Escherichia CQ ^' H and Bacillus subtilis ssp, skH memis (MTCC-5674) was Inoculated in the tubes respectively. The tubes were incubated at W°C for 48 hours and observed for change of color.

Result

If has been concluded from th observations that the test organism (Bacillus subtilis ssp. shrlram sis (MTCC-5674) is strictly aerobic as it did not ferment carbohydrate (neither gas formation nor the color change) deep inside the medium Due to availability of oxygen an the surface of niediur some color change was observed, Whereas E. coli grew very weli deep inside the medium and fennented 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 BydF ^¾ri..S.ulfid¾.Ffoduotion Test

M terial

* SIM (Sulfide Indole Motility) medium

* Culture tubes * Exoli culture

* Bacillus subtilis ssp, sknrwmnsis (MTCC-5674)

* Incubator

Method

* The tubes containing SIM [Sulfide Indole Motility. {Annexure - I (VII)}] medians, were labelled as "negative control" and "test" and a loop foil of £ <¾> . and Bacillus subtilis ssp. shrir me is (MTCC-5674), were inoculated in the tubes respectively, and incubated at 30°C for 24 hours and observed for color change.

Result

From the observations, it has been concluded that the test organism is negative For ¾S production as the medium did not turn black. The same result was observed in the negative control (Fig. 9).

.8 Effect of pH on the growth of Bacillus subtiUs ssp, shmamemis iMTCG» ill

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, shrimmemis (MTCC-5674) under standard conditions. Growth of Bacillus subtiUs ssp, shriramemis (MTCC-5674) was observed in a pH range of 6.4 to 7.2 and the optimtsrn pH was found to be 7.0.

1.9 Antibiotic Sensitivity Test οΐ Bacillus subtilis ssp. shriramemis (MTCC- 5674) ■

A 24 hours old Bacillus subtffis ssp. sh ^' rir mensis (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 pistes were incubated at 30 ^'3 C For 24 hours. Table- 1 Observations on antibiotic sensitivity of Bacillus suhtt!is ssp _t shrirame is {MTCC.-5674) RES - Resistant, INT - Intem ediate, SE - Sensitive

Result

It has been concluded from the observations that BaciU ^' s suhtiiis ssp. shrir menxis (MTCC-5674) is resistant to the antibiotics - ampieillia, carbe eillin _s kaaaraycin, earofloxaein, lincpmyoin, amoxicillm, clindamycin, neomycin, ithiotmyci , The test bacterium is sensitive to gentamicin, tohrartiicin, vancomycin, novobiocin, suifisoxazole, C ^' e halothid, chloramphenicol, ceftriaxone and bacitracin and showed intermediate resistance to oxacillin, amikacin, streptomycin and erythromycin,

CflaracterizatioR of ; antimicrobial and/or antifungal agent

The iuitimicrobia! -and/or antifungal activity associated with Bacillus suhtilis ssp, shriramemis was Investigated to ascertain the nature of agent causing antimicrobial -and/or antifungal activity .

Material

T3 broth ~ 1 L

Conical Fl sk - 2 L capacity

Kanamycin (30 .pg ml)

BaeiU s suhtili ssp. shrimmemis- (MTCC-3674) inoeulum

Shaking incubator

Method

The ΠΓ3 broth as prepared according the method described i Armemsre - I (1), A 1 ml aliquot, of 24 horns old Bacillus suhtilis ssp, shri tm sis (MTCC»5 ?4) v s inoculated into the sterile T3 broth and Incubated in the shaking incubator at 30°C for 60 hours, while shakin at 200 xpm, Following growth of Bacillus suhiiiis ssp, shrirantensis (MTCC-S674) in the T3 broth for 60 hours* the culture- medium was eentrlfuged at 12000 rpm and 4°C for 10 rain. The supernatant was collected and passed through 0.22 μηι Filters to remove an remaining bacterial cells.

3.2.2 Antimicrobial and/or antifungal assay with the Bacillus subtilis ssp. shriramensis (ΜΤ^-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 μΐ 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. 1 IB) 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 382.18 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

- 77 - Agar diffusion .method _.. (Fig, 13)

Inhibition of iiingal mycelium growth was observed around the wells containing antimicrobial and/or antiiliiigal agent in the ratios 1 :1, 1 :2, 1 :3 and 1 :4 (Table 3). Moderate ^' inhibition was observed sunmmdirig th 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 dilution of 1 :4 (v/V), in a concentration dependent 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 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 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 sUbtili ssp. shriramensis (MTGC-5674) is non-pathogenic for plant species (Fig. 18)..

Example 5

5 - 1 FQrmiilation of antimicrobial and/or antifungal compositions convening Bacillus subiiiis ssp. shriramensis (MTC S674) cells as a biological control agent

Material

» Bacillus subiiiis ssp. shriramensi ( TCC-5674)

* LB - broth

* FOB (Potato Dextrose Broth)

* Phosphate buffer (pH 7.0)

» CMC (Carboxy Methyl Cellulose)

Method

5.1:1 Preparation o f Bacillus subtiHs ssp. shrimmensis. (K COC-5674) cell suspension

Bacillus subiiiis ssp. shrimmensis (MTCC-5S74) was inoculated i 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 s-ubtilis ssp, shrimmensis (MTCC-5674), the culture was eentrifuged at 6,500 rpm, at 4 "C- for 10 min. The pelle 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 6x10' ^' ciu/ml. The slurry containing Bacillus sybtilis ssp, shriramensis (MTC05674) was used to spra on plants and treat plant seedling roots by dipping.

5. .2 To test the efficacy of formulate containing antimicrobial and/or antifungal agent to inhibit the infestatio of Rhimctonta sol ni {NFCCI-3194) in the roots of tomato plants

Material

* Slurry containing Bacillus suhtUis ssp. shriramensis (MTCC-5674)

* R izQ ^' ctonia solani (NFCCI-3194) fungus (causes sheath blight in members of family soianacea),

* Soil ite

* Tomat 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 : 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-3194).

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 Rhizoctoni sol ni (NFCCi*31 4) exhibited retarded growth, poor flowering and fruit foiTtiation as compared with- control Hence, it _. can been -concluded that iltus stibtilis ssf shrirawensk (M CC- 5674) inhibited; the growth of the funps Rhi eionia l ni OIPCCI-31 ^' 94) in the rhifcospnere area of tomato seedlings and protected the seedlings from disease causing fungus (Fig _* 1 ).

Pure colony of Penicillium oxalic m (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 6x10 ⁴ ml ^*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/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.

.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 μg/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 μg

2 CMC 1.71 μ ₈ 1.00 % w/v

3 Sucrose 3.42 μg 2.00 % w/v 4 Red Polymer 34 μΐ 19.88 % v/v

5 Water 31.87 μΐ -

Total 171 μΐ - a. Formulation with Bacillus subtilis ssp. shriramensis (MTCC-5674^ (5x10 ⁴ 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 (5x10 ^s 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, red 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 arbendazim 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. shriramensis(MTC -5674) cells (5x10* cfa) ; *4d- Formulation containing BacUlus subtilis ssp. shriramensis (MTCC-567 cells (5 l0 ⁷ cfu) and *5- Formulation with only Bacillus subtilis ssp. shriramensis (MTCC-5674) cells (5xl0 ⁷ 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 μ1/60 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 ^s 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 ( FCCf-1 97) *3- Com seed + r polymer* CM + Sucrose 4 /' oxalicum ( FCCl-1997) - Carbendazlm; *4a to 4d - Com seed - red polymer CMC. + Sucrose P. oxalic (NFCCi~1997) - different amceniratkms o Bactilm sublilis ss . shrlratnemis (MTCC.5674} cells; *5~ Cora seed + red polymer -f CMC + Sucros

+ Bacim mbti ^' lis ssp shri m mts (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 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.

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 ^s 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). Testing the efficacy of the Bacillus subtilis ssp. shriramensis (MTCC-567<fl 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.

. Aspergillus flavus .

. Aspergillus niger

. Aspergillus nidulans

. PDA plates Antifungal/antimicrobial agent isolated from Bacillus subtilis ssp. shriramensis (MTCC-5674)

Method

An aliquot of 500 μΐ 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 subtili 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 Prot, Vol. 1 (1), pp. 001-004.

Relevant Patents

1. A novel strain of Bacillus for controlling plant diseases and corn rootworm. (EP981540A1).

2. Strain of Bacillus subtilis for agricultural use. (WO2009031874A1). ;

3. Antifungal Bacillus subtilis and a microorganism wettable powder containing the same (KR2011075132A).

water. The container with the medium as autoekved at I21°C for 15 mm.

•au.tooiav.mg» the medium was poured In to sterile culture tubes.

(YD Preparation of SIM f Sulphide Indole Motility) medium (pH - 7,3) Composition of SIM medium

S. No,. Component Quantity

1 Peptone 3.0 % (w v)

2 Beef Extract 0,3 % (w/v)

3 Ferrous Ammonium Sulphate 0.02 % (w/v)

4 Sodium thiosulhphate 0.0025 % (wV)

7 Aga agar 2.0 % (w v) All the media . components were weighed and taken in a glass bottle and dlssoived in distilled, water. The glass bottle with the medium was autociaved at 121 ¾ 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

bbr yjaii M

S,No, ■ Short ^' orm Full Fo m

1 American T Culture Collection

CMC Csrboxy Methyl Cellulose

3 CFU Colony I¾ hng Units

4 L Liter

S LB turia-Berimi

5 ≠ Micro Lifer

7 Mice Microbial Type Calt«re Collection

8 Ml Mill] Liter

9 MIC Minimal Inhibitory .Concentration

10 in Minutes

11 M Molar

12 NFCCI National Fungal Culture Collection of India

13 OFBM ^Ί Oxidation I½rrn niatkB Basal .Medium

14 PDA Potato Dextros® Agar

15 PDB Potato P¾x os8 Broth

16 KFM Revolutions Per Minue

Π Ssp Sub-species

18 SIM Sulphide Indole Motility

19 v/v · Volume b volume

20 v Weight by volume

21 ww Weight by weight

22 WP Wettab!e Powder