Title of Invention

A PROCESS FOR NEW ENTOMOPATHOGENIC PREPARATION FROM STREPTOMYCES AVERMITILIS FOR USE AS ROOT FEED TO CONTROL THE COCONUT ERIOPHYID MITE"

Abstract This invention in general relates to a process for new entomopathogenic preparation for use as root feed to control the coconut eriophyid mite from a single bacteria and the novelty l^s in the process of culture filtrate production and also in the process of root feeding to control the mite. Hie process of preparing the culture filtrate of single bacteria Streptomyces Avermitilis thi*ou^ fermentation pi*oces» which involves three steps namely in first Internal multlplicfftion process in which the bacteria are cultivated in a liquid medium composition to reach 10^^ bacterial counts. Hien secondly, the fiiU grown bacterial organism is heated at desired temperature and pressure^ which is normally 120®C with 201bs pressure to kill the microorganism, which leading to lysis of microorganisms. Then thirdly^ the mass multiplication process in which a formulation is prepared by impr^piating the culture filtrate in a suitable carrier composition. Then the culture filtrate is homogenized with the carrier composition through an agitator. The carrier com.position used in the hom.ogenization is non-toxic and neutral. Then the formulation is packed by known art. The method of root feeding the prepared formulation involves adding of water to the said formulation in the ratio of 5:1 and then it is submerged onto slantly cut root to allow tlie root to absorb the fai*mtilation.
Full Text

MnmmnfiiD OF THI xiwrnmon
The invention eeiates to a pmceas fyr entomopathoffenk prepafatton fur use as met fiBed to contm/tfm coconut aflofihykf mitB containfnp axu/atm fivm a sintfta bacteria through a prooeas offermenbition,
Aoerta guarrarwtis (Coconut Perfantfi Mite) is a known suddng patX. in cooonttt not only in India but also spraad all ovar tha world. Pnnanlly in India it has baoonrta
r
menace in all the coconut growing tract and creating havoc In coconut cuKtvattoa Consequently it has aoqulnad political Impoitinoe since coconut gnowerB ane having a powerful bbby with diffiarant state govemmenti.
Many attempts have been made to control the pest through a systemic poison i.a. poisoNng the plant arKl its sap so that the mita is killed in initlat stage. This poisoning is given as rxmt ffsading with difRsrsnt Organo-phosphorous and CNorlnated hydrocarbons. In additton spray of the Just opened spathe with fertiiiaad female flowers tending to daveiop further and also the young tender coconufci already infoctad with mites are sprayed with botanical rapeilenbi like neem oil preperattons couptad with well known eniomopethogenic spores like oonldial spores of httrautmUa thonTpaonli era recommended end prsctJoed but with certain raservattons these ramedtas have their own short comings. The pestfddes used presently for root feeding are not miticides and hence the control is not

sattefactory. Spray of antifaadant and antomopathogans on coconut tnHopaBconce ara cumbarsome and pofaw pracUcai appHcadon problams.
Tha present tnventioiv Involvas root fiMdlng mathodology wvtth a noval mMarial which is biological in origia Tha mathodology is a root (aad of axudatia of oaitain flingi and bacteria. Thase fungi or bacteria ara cultivated in Uquid madlum with fermentor (bHowIng the process of fiMmnartatton and once tha ftjngal/becterlal organism ara fully grown in tha cultura medium the antira medium It haated myoes avermltiiis and diffiwant fungal organisnrw belonging to the orxjars Dauteromycatas and Phycomyoates including the orgenism Ilka BemMerta bass^na, PaecUwnyces lUmenaoteus, MetartUzftan anlac^itUaei, M^/tAcMfcfn lecartK arxf the fungi belongirx) to the order Aspwgillalas which irtdude difRuwit spedas of Aspergillus and Penidlliura The cultura medium thet Is used in the praperallon ara common nutrients carrying salts dissolved In stiertte wMiar. Howevaff special carrier salts for the nnediuivv characteristic for individual cultura nMdium of tha respective orgenism ara also added. Normal formenUng conditions for the bactarium and the fungi ara nriaintoinad and onoe the muMpHcatkm of organisms raaches 10^^ bacterial counts or lOP CRTs for fUngI, the anbra cultura medium it

loatBd laading to lysis of micaD-orsanism. Thoy ara homooantaMi and stendsftHaBad on tha basis of danslty and caiorfmatrfc vaiua.
1. The process of the culture filtrate production Is novel.
2. Tha usage of culture filtrate to control Eriophyid mites Is novel.
3. Tha praoass of root fsading such cufajra ftttrafea to oonbol mite aa a systemic action is novel.
4. Tha organisms selected to produce the culture filtrate Is unique.
5. Mixing of difl^pant culture filtrate is novel.
6. The stendanli2Btion of culture flltrvte ttvough density and calorlmablc method is novel.
D^CRIPTTOM OF THE IMVBKTIOK
The procass as per the inventkm is a root ilsad of exudetes of e sat of ft«igi
and bacteria comixlsing of steps as fi9ilows:
I. Tha ^MH|i or bacteria ana cultivated in a specMc liquid culture medium with tiie process of fm 11 lentetion.
II. The full grown fungal bactariel oigenism in culture medium elonywiili medium is iieated to a tamparature of aCP-9GP C wNch process kills the orgmiisnrt

m. TTi« cvtturmUkn/tm indudinp the dUsirtlmgmimi oiyiitoiii i» homogiiiaBid IKI standandized and is ready to be used as roct feed.
A prooBss for mskHiii a ouHoure fiHrata of diflarant fnicraoipwriifnB tfvoiiBha process cf fermentatioiv which contains the exudates cf the mkrooroanlsms and ateo living iiacDaila, wherein in
a. Onjanisms means different strains of BadNus spp« different species of
Stnaptomyoa% dtfferant fungirf organisms belonoing to ontars DeubBromycatas and
Phyoomycetes and fungi iMslonging to tiw order Aspergilialas.
b. And whentin cidtura medium means addition of special carrier setts to
medium in acoordanoe with tlie respective organism to adiieve a desired
characteristics for hidividuBl culture medium;
c. And wtiereinr *^ initial cultivation means multlpiication of organisms to
reach 1&° badertai counts or KF CPU'S for (lingi and wherein fermentation means
normal femrMnting conditions for the bacterium and fungi.
d. And wherein heating of entire culture medium means heattng such as lead
to lysis of micno-organism.
Rjtther as per the invention the cuhrure flitralM are of organisnra like different strains of Bsciltus spp including Bedlius megattiertum and different

spades of SCraptDmyoos induding Staraptomyoos avomnitJIis and difllMiant funo^ onianisms tMrfonging to the onJors OmMmmmfoatms and Phycomyoaias Induding the organisms lite Beauveria bassiana, Paadlonntyoes Fumenosoreus^ MetarWzlum anisopiiaa, \Awtidliium locanil and fungi bolonging to the oider AspenBiliales which indude difRw«nk spedes of A^MNigiiius and PenidNiunn.
A process Ibr making ciftura fHtrate containing exudates ftDfn a single bacteria or mixtora of bacteria and fungi through e process of fiBrmentatlon for use as root feed to control coconut erloph^ mite which comprises the following steps:
SUBl' Multiplication
aL selecting a single cultura bacteria Streplomyoes mmnnMiOs (Bl)
a2. selecting e mixture thereof from a group comprising difforent strains
of badlius species and difforent orders of fungal organisms, the said mixture
coraisting of Streptomyces avermlOtls (Bl), BacH/us megatxsrkjm (B2),
\fiseudofnofms fhreaoenoe (B3), BacMjs pofyrrUxa (B4), B/KMUB UAO^ (BS),
' VertkHttum hcan^ (£1), Metartizkjm anbopOae (Bi), fiaedtomyces fUmoaoreaus
(£3), Baawaria baaafana (E4) and f^nutaOa thompaontt (ES))
bL for step a 1 preparing a single culture imidiuni
setodjon to achtev« tha pturality of spodfk cultura medium compmiition fbr each bacteria/fiingai organism;
c. Cultiine of each selected iaacteria and/or fungal organism ie
Streptomycxs av&mtit^ (Bl), BSKMJB megatmkm (B2), Psmjchmonas
fhrescenoe (B3), BadUus pofymbea (B4), Badfkjs suttiOs (BS), Vertidltium hcmhii
(£1), MetarNzitMn anisop/fae (EZX Paedfomyoss fiMnosoresus (£3), Beawerta
bassfana (E4) and Htrsutella tftompsontt (£S)) separstjily and respecttveiy in
prepared cidture mediurTVis (Cl-ClO) so as ID adiieve 10^° bactaanal count or l(f
CPU's fungal count in each cirtture;
fituUI: Fiitration
d. heating the cultured orgar^sm/s at 120^ with 20lbs pressure so as
to kiii organism and to obtain cuituned filtrate (CFO wNch indudes exudates and
1^*^' living bactarla;
Step III : Permuletion
e. Pneparfng carrier medium (Cal) or pnsperfng carrier medium (Ca2)
respedix^y fix step al or stop a2;
f. Adding and homogenizfrig the cultured flitrate (CfO to carrier medium
(Cal or Ca2} so prepared to achieve a formulation (FO;
g. Packing the (iMmulation (F) in sealed bag In d«^red quantity.
The process (br making a culture flitrate conteining exudates from a i^ngle culture bacteria Stnplomyoes avenr^OSs (Bl) through a prooess of fermertotion wherein cultire medium (CI) means chemical oompositton as given below:

Cultura medium CI for Streptotnyces ayemOM (Bl) i. Primaiy cultur*:
Pdwdered OBtmeai 40 gm
fiQur 200m
DMIM WMar - 1000 mi
ii. Secondary multiplication:
Growth condition - aerobic
Temperatura 25-27 OC
IncutMtion - 3 days
pH - 6-7

The process for meldng a culture flttratB contBining exudates from mbctura of bacteria and fungue through a procass of formantation wherein culture medium (C^ means diamicai composition as given beiow:

CuHura MwJium (C2) fbr BmMus mmgmtBrtum(JBa)

TNi prooMS fbr maidno a oiltura filtnite containino axudates from mixtura of iNictNMla and ftingus ttwouoh a prooeas of fBrmantatton wharain cultura madium (C3) means chemical composition as given below:
Culture Medium (C3) (br Psaudomonas floraaeenop(B3)

1
TTiQ proofiss Ibr makfno d cutture flttretB contBfnino sxudBtBS fhsni mixtunB of badwia and fungus through a praoass of fumantBtJon wharain ojitura nrwoKum (C4) means chemicai composition as given below:
Culture Medium (C4) for Bmdllus po/ymbm (B4)



ThQ prooass for maidno a cultura flltratB containino sxudatas from mbctura of bactaria and fungus through a procmaa of fermentation wharain cultura medium (C5) means chemicai compaction as given below:
Culture Medium (C5) for Bacmjs subtOs {B^



Ths praooss Ibr msklng B culture flitFste oontetninB wcuctotes frafn mixture C3f bacteria and flinguB through a process of farmontatton wherein culture medium (C6) meare chemicai composition as givan below:
Culture Medium (C6) for Verticlllium lecanlKEl}


Ihm prooMS fbr matdno a cutoini flltrata contafning axudatas from mbctura of bacteria and fungus ttvough a process of fermentation wherein culture medium (C7) means chemicai composition as given below:


The pnxass for maldng a culture filtrate contairting exudates from mfxture of badmla and fungut tfirough a procase of fiMmantition wherein culture medium (CGD means chemical composition as given bekm:
Cutture Medium (CS) for PaecSbmyces AjmosoresuBiESy

The procett for making a culture filtrate oonteinlng exudates from nr^xtiR^e of bacteria and fungus through a process of fenmentBtkan wherein culture medium (C9) nneans chemicai composition as given iMiow:
Culture Medium (C^ for BeaiAWia baati»fm (E^



Tha process for making a cuitune fittrats contafning exudates fiftMn rrrixCuna of bacteria and fungus tfvough a process of fiBrmentation wherein cirfture medium (CIO) means chemicai composition as given betow:
CuKxire Medium (CIO) for HimubsHa thomp»nll (E5)



Tho prooass (br makino a culture fittratia containing axudatas from i^ single cultura bacteria Str^3tx>myces aveimkUis (Bl) through a pfooeas of fiMTnantatk>n wherein ibrmulation(fO meens impregwHliig the culture flltrBte(CI^ of Stre/Xomyces 9¥enniti0S singly used as a broth in a carrier medium (Cal) wHch is made up of :-

The process for maldng a cuitisti fHtrata containing exudirtas from a mixture of bacteria & fungus through a process of femwmtation wherein fixTnuiatlon(FO means impregneting the cultuna fHtrato (Cf) of StrsptMnycBS a\mrmiOtts (Bl), Badllus megaterium (B2), Pseudomonas florescmnoB (B3), Baciffus pofymixa (B4), Baditus subtunB (BS), VertxUSum lecanH (El), MetarNiHjm

atmsopSae (G), PBecMosnyces fkimosoresuB (£3), Beaw&ia tassiana (E4) and Hifsutefia Ihomftaontt (ES^ combiraKJiy UMMJ as a bvdfh in a carrfar macfium (Ca2) wNch is mode up of :-

A process fbr making a cuitune flitnitie containing exudaties from a single bacteria or nrtixtuna of bactaria and fungus, wtianain tiw praparation of culture fJltTBtia is by using a medium in wNch additton of certain additionai oon^munds in

the mediiffn, is done when^^ the n^croorgartisms growth is er^noed and that which is chennicai and aasiiy available.
A process for nratdng a cidtura filtrate amtaining exudates from a single brctaria or nnixture of bacAaria and fungus, whmwn the initial multiplication of diffisrent micnoorganlsms is in a medium and; tiiereaftier the formutatton is achieved from culture flitrate of different organisms is in a farmendng medium either separately or in combination as described in titm preceding paras.
A process fbr maldng a culture ffltratis containing exudat»s from a single bacteria or mixture of bacteria and fiingus, wherein the Impregnation of the Cloture flltnite in desined ratio in the said carrier, which is neutral and not toxic
A process fbr maldng a culture filtrate containing «Kudates from a ^ngie bacteria or mixture of bacteria and fungus, wherein the culture flHrates Is carried in a unique medium and a iqaedfic metixxlology of root feeding is done.
Accondingiy the Invention Is a process Ibr entomopathogenic preparatJon fbr use as root feed to contrx^ tiie coconut erlopl^id mite containing exudates from a single bacteria through a process of fermentation, wNch comprises the fbliowing Steps:
a. sheeting a single cidture bacteria Stneptx>myces mmrmkMa {31}}
b. pr^saring a culture medium (CI);

c. culture of each setectsed bactena organism ie Streptamyces
avermitilis (Bl), in prepared culture medium ^Cl) so as to achieve 10^^ bacterial
count;
d. heating the cultured organism at 120**C with ZOJbs pr^ure so as to
kill organism and to obtain cultured filtrate (CF) which includes exudates and non
living bacteria;
e. pnef^ring carrier medium (Cal) ;
f. adding and homogenizing the cultured fiib^te (CF) to carri^^ medium
(Cal) so prepared to achieve a formulation (F);
g. packing the formulation (F) in sealed bag in desired quantity,
Accordingly the \n\ention is a process ior nnaking a culture filtrate
containing exudates from a single culture bacteria Streptomyces avermititis (Bl)
through a process of fermentation wherein culture medium (CI) means generally
a chemical composition as given below;
i. Primary culture :
Powdered Oatmeal - 40 gm
Agar - 20 gm
Distilled Water - 1000 mi
ii. Secondary multiplication :
Growth condition - aerobic
Temperature - 25-27 ^C
Incubation - 3 days
pH - 6-7


Accordingly as per ttie invention is for a process fisr maidnB a cuituni fUtratNi contBinino exudates fnom a single culture bacteria Stnptomyass mfermltffla (Bl) through a pnxess of farmontation wherein formulation(F) nwans impragnaUng the culture filtratie(O0 of Streptomyces avermiUrm singly used as a broth in a canier medium (Cal) wNdi Is macte up (rf: -




1 A pnKMm for mitomopKthogmic prmimnitkm for m^
the oooonut eriopliyid mtte oontairtno exudates from a single bacteria tNfDush a procQss of fermentation wNch comprises the foitowing steps:
a. selecting a single culture ixctaria Sireptomyces avermitiSB (Bl)
(MTXX:345A
b. preparing a single culture mediiMn (CI),
c cultupe of single selecfiad bacteria StreptDmyoes wymrnlOOs (Bl) (MTOC 345^ in prepered culture medium(Cl} so as tt> achieve 10^° becteriel count;
d. heating the ciritured onienism et 120^ with 20n» pressure so es to
kill onjar^sm and to obtain cultunad ffltnitB (CF) wNch includes exudatw and non
living bacteria,
e. preparing carrier medium (Cal),
f. adding and iKMDogenizing the cultured flhrete (CF^ ta cerrier medium
(Cal) so pr^iared to achieve a formuietion (fO» and
g. packing the fbrmuletkNi (F) in seeled beg in desirad quenUty.
2 The process for entomopethogenk prepenitkNi for use es root foed to
control the coconut ertophyid mitB containing exudMas from a sinole bectarte
Streptomyces avemMSs (Bl) through a pnocess of fiarmentatton es cMmed in
cielni 1, wherein culture medium (CI) meens e compositton es given beksw :-
i. Primary culture:
Powdered Oatmeal - 40gm
Agar 20 gm
l^istilled Water - 1000 ml

ii. Secondary multiplicatiofi:
GkxMwth condition - aerobic
Temperature - 25-27 «C
Incutxatjon - 3 days
pH - 6-7

3. The process for entonfupathogenic praparaiion for use aa root foed to axioroU the coconut ehophytd nrtlte containing exudates from a single bacteria through) a process of formentatfon as dafmed in claim i, wherein lbfmulatlon(F) coTT^rlsing of irr^sregnating the cidtura filtratB(CF^ of Stneptxunyass mmrmiuns as a broth in a carrier medium (Cai) contains :-



4. A profw for «rftDmopathog«itk prtparatton for UM as root faod t» control tKt oooonut «rk>phyi S A prooaw for entDmopathogenic preparation for um as root faad to control the coconut eriophyid mite containino exudates from a single bacteria throuoh a process of fennentation and wherein the said carrier medium (Cal) as claimed In the Claini 1 & 3 » neiAral and non-toxic.
6. A process for enb3mopathogenic preparation for use as root feed to control the coconut eriophyid mite corttaimng exudates firom a single bacteria through a process of fermenbition, wherein the said culture fiitrates

Documents:

797-che-2003-abstract.pdf

797-che-2003-claims.pdf

797-che-2003-correspondnece-others.pdf

797-che-2003-correspondnece-po.pdf

797-che-2003-description(complete).pdf

797-che-2003-form 1.pdf

797-che-2003-form 19.pdf

797-che-2003-form 26.pdf

797-che-2003-form 3.pdf

797-che-2003-form 5.pdf


Patent Number 241671
Indian Patent Application Number 797/CHE/2003
PG Journal Number 30/2010
Publication Date 23-Jul-2010
Grant Date 19-Jul-2010
Date of Filing 01-Oct-2003
Name of Patentee T. STANES AND COMPANY LIMITED
Applicant Address 8/23-24, RACE COURSE ROAD, COIMBATORE-641 018, TAMILNADU, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 SANTHANAM RAMARETHINAM EXECTIVE DIRECTOR (PROJECTS), 8/23-24, RACE COURSE ROAD, COIMBATORE-641 018, TAMILNADU, INDIA.
PCT International Classification Number A01N63/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA