CA2157904A1 - Biocontrol agents for use in agriculture - Google Patents
Biocontrol agents for use in agricultureInfo
- Publication number
- CA2157904A1 CA2157904A1 CA002157904A CA2157904A CA2157904A1 CA 2157904 A1 CA2157904 A1 CA 2157904A1 CA 002157904 A CA002157904 A CA 002157904A CA 2157904 A CA2157904 A CA 2157904A CA 2157904 A1 CA2157904 A1 CA 2157904A1
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- Canada
- Prior art keywords
- fruit
- biocontrol agent
- biocontrol
- starter
- yeast
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/32—Yeast
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- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Pest Control & Pesticides (AREA)
- Biotechnology (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A biocontrol agent which includes or is derived from a sourdough starter formulation a mixed culture of the yeast component a bacterial component and a substrate for the mixed culture. The agent can be used for reducing or alleviating fungal infection of plants or fruit.
Description
~WO 94/19950 ~ 1 5 7 9 0 I PCT/AUg4/oollo - Tl rLE
"BIOCONTROL AGENT~ FOR USE IN AGRICULTURE"
~1 LD OF T~IE INVENTION
THIS INTV~NTION relates to biocontrol agents for use in 5 ~ tllre.
PRIOR ART
- Convçnti~ n~l hi~ntrol agents for use in horticulture are sl-.. ~.ised in an article by J. M. Whipps entitled "Status of Biological Disease Control in Horticulture" Biocontrol Sci & Technol (1992) _ 3-24. In 10 this article Ç...l.h~ is placed on I~Se~ relating to the use of biological disease control agents in horticulture which may include vegetables, fruit, orn~ment~ , flowers and protected crops.
In this article it is est~hli~h~A that the three main direct biocontrol me~.h~ni.~m~ are:-(i) ~alaSiliSIll or predation of one or~;~ is.. by another;
(ii) ~ntihio~ie, where antagonists secrete met~holites harmful to plant p~thog~n~; and (iii) co.. ~ ;on where flf~.m~n~1 ~sY~eA!~; immeAi~te supply of m~tri~.nt~ or space.
These actions can take place outside or within the plant. Other ."ee~include pror3l~ctinn of cross protection or in~l~ceri r~si~t~n~e involving inoc~ tion of a plant with a micro organism which is non pathogenic or only mildiy p~thog~.nic which results in the plant becoming ~ to subsequent ~h~llen~. Also inoC1ll~tion of plants with p~thog~n~ of reduced 25 patho~,enicily carrying double stranded RNA or DNA pl~mitls may result indecreased virulence in the overall population of pathogens.
However, it is also pointed out in the Whipps article that until the modes of action can be shown to occur in soil or on or within plants growing in natural enviro~ , the signific~nce of any of the abovr~.~e..l;rn~
30 m~ must be viewed with ~t;se~valion.
It is also noted that a major problem facing all biological control agents is that they are generally expecte~ to match the efficacy of existing wo 94~19950 2 ~ ~ 7 9 ~ 4 . PCTIAU94/OOllO ~
.
~h~mir~l meth~ under all con~iition~ as 1PSC riheA in Powell & Faull Bioterhnology of Fungi for Impr~ving Plant Growth, Cambridge UniveL~ily Press (1989) p 259-275. This is pointed out in the Whipps artic~e as being an objeclive which may not always be possible. An .oY~mple of this is that the 5 eYi~t~n~e of spe~ific su~lessivè soils ill~L~a~es the i~ u~l~ce that soil conAition~ can have in biocontrol. .~imil~rly in~lltlm potential of the p~thogen is known to be ill.~l~ll to the degree of biocontrol achieved but is often ignored as ~ipscriheA in McQuilken et al., Plant Pathology ~2 452~62 (1990) and Budge and Whipps Plant Pathology Q 59-66 (1991).
Direct appli~ticn of biolog~ l control agents to aerial, root and soil microbiomes before, during and after plant growth has also been ~lle~ A as described in the Whipps article but relatively few of these techniques have been shown to be co.. ~-,ial in use.
In s~ .y ~lelerure~ bioc-ntrol techniques in horticulture have not 15 been colllll-er~ially ~ucce~rul and one reæon for this is that there is often a lack of ,e~rûdl~ hility b~lwèen trials in vitr~ and in the field. Also when colll~a~d to ~hPmi~ b ologjc~l techniques suffer because of in~ll1~m pro~tlction, application and cost. t'.h~mi~ are also often ~J~eÇélled on the grounds of efficacy bec~ e rhPmit~ generally work irrespective of
"BIOCONTROL AGENT~ FOR USE IN AGRICULTURE"
~1 LD OF T~IE INVENTION
THIS INTV~NTION relates to biocontrol agents for use in 5 ~ tllre.
PRIOR ART
- Convçnti~ n~l hi~ntrol agents for use in horticulture are sl-.. ~.ised in an article by J. M. Whipps entitled "Status of Biological Disease Control in Horticulture" Biocontrol Sci & Technol (1992) _ 3-24. In 10 this article Ç...l.h~ is placed on I~Se~ relating to the use of biological disease control agents in horticulture which may include vegetables, fruit, orn~ment~ , flowers and protected crops.
In this article it is est~hli~h~A that the three main direct biocontrol me~.h~ni.~m~ are:-(i) ~alaSiliSIll or predation of one or~;~ is.. by another;
(ii) ~ntihio~ie, where antagonists secrete met~holites harmful to plant p~thog~n~; and (iii) co.. ~ ;on where flf~.m~n~1 ~sY~eA!~; immeAi~te supply of m~tri~.nt~ or space.
These actions can take place outside or within the plant. Other ."ee~include pror3l~ctinn of cross protection or in~l~ceri r~si~t~n~e involving inoc~ tion of a plant with a micro organism which is non pathogenic or only mildiy p~thog~.nic which results in the plant becoming ~ to subsequent ~h~llen~. Also inoC1ll~tion of plants with p~thog~n~ of reduced 25 patho~,enicily carrying double stranded RNA or DNA pl~mitls may result indecreased virulence in the overall population of pathogens.
However, it is also pointed out in the Whipps article that until the modes of action can be shown to occur in soil or on or within plants growing in natural enviro~ , the signific~nce of any of the abovr~.~e..l;rn~
30 m~ must be viewed with ~t;se~valion.
It is also noted that a major problem facing all biological control agents is that they are generally expecte~ to match the efficacy of existing wo 94~19950 2 ~ ~ 7 9 ~ 4 . PCTIAU94/OOllO ~
.
~h~mir~l meth~ under all con~iition~ as 1PSC riheA in Powell & Faull Bioterhnology of Fungi for Impr~ving Plant Growth, Cambridge UniveL~ily Press (1989) p 259-275. This is pointed out in the Whipps artic~e as being an objeclive which may not always be possible. An .oY~mple of this is that the 5 eYi~t~n~e of spe~ific su~lessivè soils ill~L~a~es the i~ u~l~ce that soil conAition~ can have in biocontrol. .~imil~rly in~lltlm potential of the p~thogen is known to be ill.~l~ll to the degree of biocontrol achieved but is often ignored as ~ipscriheA in McQuilken et al., Plant Pathology ~2 452~62 (1990) and Budge and Whipps Plant Pathology Q 59-66 (1991).
Direct appli~ticn of biolog~ l control agents to aerial, root and soil microbiomes before, during and after plant growth has also been ~lle~ A as described in the Whipps article but relatively few of these techniques have been shown to be co.. ~-,ial in use.
In s~ .y ~lelerure~ bioc-ntrol techniques in horticulture have not 15 been colllll-er~ially ~ucce~rul and one reæon for this is that there is often a lack of ,e~rûdl~ hility b~lwèen trials in vitr~ and in the field. Also when colll~a~d to ~hPmi~ b ologjc~l techniques suffer because of in~ll1~m pro~tlction, application and cost. t'.h~mi~ are also often ~J~eÇélled on the grounds of efficacy bec~ e rhPmit~ generally work irrespective of
2 0 ellvi~lu~lelll or in~llllm potential. ('h~mit~lc also are eæier to apply to target plants and also a greater range of p~th~g~n~ may be controlled.
However, the use of l~h~mi~ such æ f~mgicitles e.g. in control of storage rot of fruit and veget~hlP,s which may be caused by sperific plant p~thogen~ is now l~eco.~ g lmpop~ r becau~e they are hazardous to the health 25 of hllm~n~ and are also crn~i~lered to be tl~l.;...~..l~l to the envi~ l Thisis speçifiç~lly ~ c~ eA in ~gri~llhlral Research, April 1990. I'his lerelènce describes a strain of yeast that may be used to control fruit rot. However, a~ain co~ e~ç;~ hon of such biocontrol agents is liffi~llt becau~e of the time, effort and e~l~n~e involved not only in finding biocontrol agents but also30 ve~ifying their errè~livel~ess.
In Neth. J. P1. Path 91 (1985) p 265-267 by Willi~m~on et al., there is descriheA the use of as biccontrol agents the yeasts Sporobolomyces roseus WO 94/l~s50 2 1 5 ~1 9 0 9~ PCT/AU94/00ll0 and Cryptococcus laul~nlii varfla~esce"s in relation to control of the pathogen Colletotrichum graminicola in maize plants. It was found that the yeasts reduced lesion density and necrosis from C graminicola by a~v~ ely 50%. This ~eÇe~ ce also made the ol)selvalion that naturally occ1~rring yeast 5 poplllAticn~ may have a moderating effect of maize anthracnose, Pspe~iAlly in conjunction with sPlectP~l fungi. However, the same problems described above in relation to bioccntrol agents also apply to this lerelt;l-ce.
R~re~ ce also may be made to U.S. Patent 5,041,384 which described various strains of Pichia guilliermondi which were j~ol~t~P~ from the 1 0 surface of citrus fruits and which are useful in controlling a variety of fruit rot p~thog~Pn~ in a variety of fruits.
Other references which describe the use of yeasts or fungi as well as ba~tPriA o~ ed from a natural source include (i) EP 485440 which ~P~çrihPs a new yeast strain obtained from the surface of citrus fruits and which may be 1 5 used to control fruit rot pAthogPn.~; (ii) U.S. Patents 5,047,239 and 4,764,371 which describes the use of a strain of Rncil~ subtilus for biological control offruit rot; (iii) U.S. .Cpe~ific~tir)n 4,377,571 which ~lescnhPs the use of Pseudomonas syringae for 1~ ll of ~utch elm ~ eA~e; (iv) U.S.
~pe~ifi~tion 4,950,472 which ~PSÇrihps the use of a new strain of Acremonium 2 0 breve in controlling grey mould infection of pome fruit; and (v) U.S.
Spe~ifi~tion 4,975,277 which describes an isolate of Pseudomonas cepnci~n for ~io]ogic~l control of post harvest disease in fruit.
In JAP~ e Patent JP 3077803 ~ ce is made to Pseudomonas b~ sP1ecte~1 from P c~pncin~ P gladioli, P picketti, P vorans, P dimunata and Rncillt~: bactPri~ selectçcl from B cereus, B mycoides, B anthracis and B
thuringiensis as biocontrol agents in relation to soil borne ~;~ÇA~ÇS.
U.S. Patent 4,878,936 describes the use of B cereus ATCC 53522 as having biocQntrol aclivily and which produces a filn~le active against Phytophera mega~", a.
In Russian Patent SU 237480 reference is made to a strain of B
cereus useful as a biocontrol agent for pr~te~ g plants against insects.
In U.S. Patent 4,661,351 ,efel~,lce is made to coml)os;l;ons WO 94/lg9so 2 15 7 ~ O ~ PCT/~U94/01)110 c~ n~ in~ biosynthetic pP~tiri~1 products obtained from Raci~ c b~c~et-i~
s~lecteA for B thuringiensis, B sphaericus, B popilliae, B cereus, B lentimorbusor B friboungensis. These bacteria may also be provided with a low m~1tin~
point polyester as ~P~rriheA in Patent EP 145087.
In Patent Spe~ific~tiQn JP 59082085 there is described a method of controlling harmful insects by use of a biocontrol agent cc~ ini-~g R(7c~
subtilus, R~ci~ coagulans, Micrococcus luteus, Racil~ stearothermophilus, Clostridium pasteurianum, Clostridium aminovalericum, Clostridum thermo-saccharolyticum and Thermoactinomyces vulgaris.
Rerel~nce also may be made to a pt1h1iraffon by Broadh~nt et al entitled "R~ct~ri~ and ~ctin~llllyceles Antagonistic to Fungal Root Pathogens inAustralian Soils" which inr1~ es B subtilus, B megaterium, Streptomyces spp, B cereus, B pumilus, B polynyxa, B badius, Pseudomonas putida, P
fluorescens and Pseudomonas spp as having biocontrol activity.
Re~,~nce may also be made to Visser et al Applied and EllviL`~
Microbiology 52 552-555 (1986) which ~lesr. rih~s a variety of lactic acid bact~-ri~ i~o1~t~d from plant surfaces and plant ~ori~teA. products which were found to be antagonistic to the ph~l~aL~Iogens ~nthomonas campestris, Erwinia cafotovora and Pseudomonas syringae. In pot trials, ~ l of bean plants with a Lactobacillus planetarium strain before inc~ll~tion with Pseudomonas syringae caused a signific~nt reAllcti~n of the in~1~nre of tli~e~e From a review of the abov~ -.l;oneA prior art, it is clear that it is well known to take natural isolates which may be found on leaf surfaces of fruit and cultivate such natural isolates for their use as biocontrol agents when applied to crops or fruit and other plants. The natural isolates may be removed from the leaf surfaces and cultivated in suitable media. In some cases culture media may be dried and ground into a powder before being i~col~laLed in a carrier. In some cases the culture media may be mixed with wax such as a water or p~arr~/mineral oil base. Som~tim~ the infected fruit may be dipped into a sol1-tion co..l;.;~ g the natural isolate.
However, it would seem that one i~ l disadvantage of the use ~WO 94/19950 215 7 9 0 4 PCT/AU94/001l0 of natural isolates is that this is often a~l~ached in a random ~ ner and that the relevant antagonist must be irl.ontifi~ and i~lit~i before being used in field trials. There is also no evidence that the use of natural isolates has heen adopted on a co.,...~..;ial scale.
It would also seem clear that the main disadvantage of use of biocontrol agents such as P~acil~ thuringiensis is the cost because of their need to he applied more often than rh~mi~ and also because of loss of ~lcn~;y within a relatively short time.
It is also clear when use is made of genetic ~n~ g techniques to produce hiocontrol agents such as r~mhinint strains or p~leills obtained from recl~mhin~nt strains that such techniques are eA~ensive and also cannot be sold on the market until obl~ g appr~vdl from the re~ tory- authorities such as the U.S. Food and Drug ~ alion. Also ex~cl~sive field trials are required before co~ ;ial ev~ t~on can be con~ ered.
SUMMARY OF THE INVENTION
It is tl-c.cfc)lc an object of the present invention to provide a biocontrol agent which may alleviate the abov~m~ntion~ problems of the prior art.
The biocontrol agent of the invention may inciude or be derived 2 0 from:
(i) a mixed culture of a yeast co,..~onc~.l and a bacterial cc,..-ponent; and (ii) a substrate for the mixed culture a).
The yeast c~ pQ~ is suitably useful for pro~ in~ a leavcl~ g or 25 rising action in a sourdough which are doughs in which flour (for example, rye flour) is ~e.-l-c -lcd by lactic acid b~c1P~ ;~ rather than by bakers yeast.
Sourdoughs are ~les~ for ~Y~mple, by Oura et al. in F~onomi~
Microbiology, Volume 7 entitled "Fe....~..l~ Foods" edited by A. H. Rose pages 123-134. Such yeasts may be select~d from Cn~ n krusei, 30 Saccharomyces c~"G~iae, ~oc~hnromyces exiguss and Pichia saitoi.
However, any yeast could be utilised which is erîc,~ivc in combination with an a~r~,iale bacterial co-n~l~enl and a snit~hle substrate to produce æ main 2 ~ a l WO 94/I9950 PCT/AU94/00ll0 products lactic acid, acetic acid, ethanol and carbon dioxide which is the h~llm~rk of sourdough fe~ n- In sourdough fe....~ n a glycolytic paLllway is chara~-lP~;~eA by homore...~ ;ve lactic acid ba~ ;a via ~ ol~se and a h te~re~ l;ve lactic acid bact~ri~ pal~,way for gl~ ses and ~.loses 5 via phosrhokPtol~e Saccharomyces exiguss is ~lt~.rn~tively known as Torulopsis holmii.
Other yeasts that may be used include yeasts of the genus ,Snccharomyces ~.ner~lly which include as mentionPA above S. cerevisiae, S. exiguss, S.
in~it~ and S. uvarum.
The bacterial component of the symkioti~ mixed culture may be sP.l~ted from Lactobacilli æ well æ m~mhers of the genera Leuconostoc, Pediococcus and Streptococcl~s of the family ~treptococcoce~e. Dirre~
species of Lactobacilli may be used ~:iep~n~ling on th eir ability to use dirre ~nl substrates. Typical lactic acid bacteri~ may be sPlected from L. plantarum, L.
casei, L. delbreuckii, L. IeichP~ mii, L. brevis (especially var lin~nP.ri), L.
fermentum, L. pastorianus, L. bucheri, L. acidophilus, L. farciminis, L.
alim~,.lu,.~, L. fructivorans, L. viridescens, L. cellobiosus and L. solivarius.Other bacteria that may be used include Pe~iococc~ cerevisiae, P. aci~ ctici æ well æ Citroba.,ter spp or Micrococcus spp.
An espPc-i~lly pl~,L,ed bactP.ri~ for use in the biocontrol agent of the invention are Lactobacillus sanfrancisco which are used in the San Fl~cisco so~lough "French bread" process. These bacteria are indirrt;~ t to oxygen and do not use carbollyL~Les other than m~lt~se æ a carbon source. L.
sanfrancisco form a symbiotic r~ tion~hip with Sacchar,.yces exiguss which mainly ~r~ ls the leavenillg fim~i-~n Further infol,l,alion on sourdough cultures may be obtained from:
(i) Nout Int. J of Food Microbiology 12 217-224 (1991);
(ii) Nout et al., J of Applied RaCtPri~10gY S~"~osium Supplem~.nt 1992, 73 1365-1475;
(iii) S~lln~lP~ et al., Cereal (~hPmi~ 49 86-91 (1972); n (iv) Cooke et al., FEMS Microbiology Reviews 359-379 (1987);
(v) Srir~n~ nl~ . et ûl., Applied Microbiology 25 461-470 (1973);
~ WO 94119950 21 S 7 9 Q ~ ~IAU94/OOllO
(vi) Kline and Sugihara Applied Microbiology 21 459-465 (1971);
and (vii) Ng Applied and Ellvh~Jn~ l Microbiolo_y ~ 395-398 (1976).
In a spe~fic form of the biocontrol agent of the invention, which has been ~pO~il~ at the Australian Co v.~ n~ Analytical Labola~olies as ~i~c~ e~ in more detail he~ arl~l (herein referred to as SDBS), the T ~tobaci1li which vere located in the deposited mixed culture in~h~dPrl (i) L.
parabuchneri which vas cl dl ~1r.;7~ by Phillips and Collins in 1988, (ii) L.
1 0 parabuchneri/brevis st~ain, and (iii) L. casei ssp casei which was Cllal<~ ;7~d by Rogose et al. in 1953. However, difr~ tinn b~weel- L. buchneri and L. parabuchneri can be very l1iffi~)h and it is within the scope of the invention to provide a mixed starter culture which inf~ ps L. buchneri in s1lhætit ltion of L. parabuchneri and other strains of L. casei for eY~ le in substitution of L.
casei ssp casei. Difr~ l;on may also be extremely ~limc llt l~lwæn L.
parabuchneri, L. buchneri andL. brevis andL. brevis may also be s1~hsl;lul~A
for L. parabuchneri or L. buchneri.
A sample of the mixed culture wæ rolwa~ed to Kluyver Laboldlo,y of Te~hnolopy Delft Te hni~l Univt;~sily in The Ne~ and Lactobacilli strains (i), (ii) and (iii) were i(lPntifi~P~ The yeæt co.~ of the mixed culture wæ also dele.ll-illed as Sncchoromyces cerevisiae Meyen ex Hansen by CBS Yeæt k~Pnti~c~tion Service Delft, The NethPrl~n~
The su~sll~d~e utilised in the mixed culture may colllplise any ~l)~dLion derived from cereals such as flour and in particular rye flour or white flour which may include unblP~h~P~ white wheat flour. MO1~S~PS may also be utilised as a SU~ ld~
The substrate may also be mixed wi~ w~ter which is suitably "pure water" CG.,.l,t;~ bottled l~lwdler or sterilised Milli-Q filtered water.
The pH of the mixed starter is suitably in the range 3.0-6.0 and more suitably 3.04.5.
Fe.~ n may be initi~te~ by mixing equal parts flour and water to form a paste, and this is then added at a suitable ratio (e.g. 1:1) to starter WO 94/19950 21~ 7 9 ~ 4 PCT/AU94/OOllo culture. Tnrt1b~tion may occur over 548 hours but more suitably 24 hours.
Advantages of the bico~trol agent of the invention over cOIl~ r.l;nn~l pesti~ s and biocontrol agents include the following:
(i) ease of registration as a filngi~e because a)m~n~nt~ of the mixed culture have been utilised in food pro~lu~on for a relatively long period;
(ii) the b~ l co~ l such as the lactobacilli are cl~imeA to have probiotic effects on the o~ which ccn~...e them.
This means that health is ~nh~n~e~i by i-l-~r~vi.lg the balance of b~ct~ri~ and yeasts or microflora of the gut;
(iii) the biocontrol product of the invention is co...p~l;hle with s~ in~hle agriculture and free from health risks which is not the case wi1h converlional h~.mi~l or ~ntihioti~ fi)ngi~le,s as c~ eA above which are disadv~nt~Eeo~ , of:
(a) high oncc,geliic risks;
(b) loss of utility beça~ce of the devel~ pm~nt of fi1ngi~e resistant strains of many p~tho~PnS; and (c) p~èssllle from envilo~ l groups for los~eninp dep~n~1~n-~e on toxic r.h.omi~ 3~.e of dangers to the elvi,~,nll-ent and the development of ~ hle cropping ~y~l~.ns.
The bioc~nt~ol agent of the invention may also include other co..~poIl~nt~ so as to farilit~te its mode of action in a desired applic~tit~n Thus one c~-nlk~ I may include a carrier which may help to 25 ~stahli~h and Il~h;~ nt~gnni~tic pop~ tion~ of the binc~ntrol micr~
o~ on the target surface. This carrier may be a lipid such as wax or oils l~ive of coc~nut oil, veget~hle oil, olive oil, canola oil or emIll~ion~
lusive of fish oil ~mlIl~ion .S~lspPn~ion~ or slurries may also be used ~ with the ahove ~iteri~ One s~it~hle carrier is an çmIll~ifi~
30 veget~hle oil i~lentifieA by the trade mark CODACIDE. Another lipid carrier is itlentifi~A by the trade mark NU-FILM 17. The use of the lipid may also enh~n~e ~ hility and may also be used as an adhesive to the target ~ WO 94/19950 2 1 ~ 7 ~ 0 4 PCT/AU94/QQll0 Another co-l-pol~el-~ that may be utilised is a thirL-PnPr which is suitably a gum such as natural or moAifiP~ gums i--elusive of algin, PPn~n, Y~nth~n and trAg~rAntil gums. A gum may also be used as an 5~m~ ifiPr or stAhili~Pr.
F.Y~mplPs of p~ths)gPn~ of aerial su,races of crop plants or crop products which may be ~ul);~d to the biocontrol agent of the invention include mainly fungal pathogens i.e. Colletotrichum (&l,LL.~c,~se), Cercospora (spots and blights), Botrytis (grey mould and rots), Alternaria (leaf spot), 10Monilini~ (brown rot), Penicillium (blue mould) and Rl~pus (soft rot).
Other co~ )ol~el,L~ that may be include~ in the bioc~-ntrol agent of the invention include micro-or~is--l ~nt~g~ni~m Pnh~nr~r(s) which may include MgO, MgCO3 and/or CaCl2, p~thogpn inhihitoes which may include tea polyphPnol~ (esperi~lly epi~tPrhin gallate and epig~lloc~tPrhin gallate) as well15as ~ntioxiA~nt~ and s~hsl~i-~s to f~rilit~te proAl~cticnl storage and/or applicAtion of the agent.
It will also be app.N ialed that the sol,l~ugh hioc~ntrol products of the invention may be foemlll~tPA for col.. e~ial p~u~oses in any s~it~hle .......................... al~ner and thus may be freeze dried to .mpl~ve storage life.
20The bioc~-ntrol agent of the invention may be applied to the target before, during and after haevest.
FERMENTATION. The SDBS starter culture was stored at 4C
before use. To l~ a~ ale, equal parts of "Kialla" or~nic~lly grown 25lmhlP~achP~A white flour and Milli-Q filtered water were mixed to form a smooth paste. The paste was then comhinPA with the starter culture (1:1) so~.~el;...~,s it may be nP~P~ to fully decant off the "clear layer" of acc~lml-l~tP~A acetic acid, lactic acid and ethanol from the top of the starter culture when this is more than 1/3 of the total volume. The culture was then 30 i~ eA in a loosely covered glass jar at room tC~ al~ (23-30C) for mostly 24 hours but up to five days.
MANGOES. In E~. il~lr.~ naturally infected green fruit were wo 94,l9g50 2 ~ 5 ~ 9 ~ ~ PCT/AU94/OOllO
de~a~)l,ed and then graded into two groups acccl-ling to the density of lesions,i.e. mild natural infection and severe natural infection. In E~ ,el~l 2, the naturally infected fruit were de~apyed and then graded accc,nlillg to the level of natural infection. Fruit were then Ai~rih1)ted to 6 groups, each group 5 co~-s~ g of 3 fruit displaying a similar range of infection levels.
~ REATME~NTS. The 1~ applied in F.x~ ; 1 and 2 are sl,,..",~.ised in Table 1. In both e-Yl~l;...Pnt~, fruit were dipped in Milli-Q
filtered water (control) or SDBS (Tlr~l...e~-~ /i additives) for 1 minute and in~1h~teA on a self draining rack for 6 (FY1~ l 2) or 7 (E~p~lilllt;lll 1) 10 days in the lal~ral(,l~ at room lt;~ alurt; (23C). In E~lhllelll 2a, in which fruit were dipped 3 times, dipping was carried out on days 1, 2 and 3 of the e~ The addilives tested in FY~ Ç~I 2b, Nufilm 17 (Miller ChPmir~l and Fertiliser CGI~.aLion USA) and Cod~de (Spraytech Australasia Pty. Ltd.) were added to the dipping Illi~C at the rate and in the 15 manner l~o. ... ..~n-leA in the in~stru~it~n~
DISEASE ~ ;~ENT. An~.l acllose disease development on the fruit was ~ s~eA after 6 days (FY1~ 1 2) or 7 days ~FY~k;l;...~l.~ 1) according to the following diseæe rating scale:
Diseæe Rating Yo Fruit Surface Infected
However, the use of l~h~mi~ such æ f~mgicitles e.g. in control of storage rot of fruit and veget~hlP,s which may be caused by sperific plant p~thogen~ is now l~eco.~ g lmpop~ r becau~e they are hazardous to the health 25 of hllm~n~ and are also crn~i~lered to be tl~l.;...~..l~l to the envi~ l Thisis speçifiç~lly ~ c~ eA in ~gri~llhlral Research, April 1990. I'his lerelènce describes a strain of yeast that may be used to control fruit rot. However, a~ain co~ e~ç;~ hon of such biocontrol agents is liffi~llt becau~e of the time, effort and e~l~n~e involved not only in finding biocontrol agents but also30 ve~ifying their errè~livel~ess.
In Neth. J. P1. Path 91 (1985) p 265-267 by Willi~m~on et al., there is descriheA the use of as biccontrol agents the yeasts Sporobolomyces roseus WO 94/l~s50 2 1 5 ~1 9 0 9~ PCT/AU94/00ll0 and Cryptococcus laul~nlii varfla~esce"s in relation to control of the pathogen Colletotrichum graminicola in maize plants. It was found that the yeasts reduced lesion density and necrosis from C graminicola by a~v~ ely 50%. This ~eÇe~ ce also made the ol)selvalion that naturally occ1~rring yeast 5 poplllAticn~ may have a moderating effect of maize anthracnose, Pspe~iAlly in conjunction with sPlectP~l fungi. However, the same problems described above in relation to bioccntrol agents also apply to this lerelt;l-ce.
R~re~ ce also may be made to U.S. Patent 5,041,384 which described various strains of Pichia guilliermondi which were j~ol~t~P~ from the 1 0 surface of citrus fruits and which are useful in controlling a variety of fruit rot p~thog~Pn~ in a variety of fruits.
Other references which describe the use of yeasts or fungi as well as ba~tPriA o~ ed from a natural source include (i) EP 485440 which ~P~çrihPs a new yeast strain obtained from the surface of citrus fruits and which may be 1 5 used to control fruit rot pAthogPn.~; (ii) U.S. Patents 5,047,239 and 4,764,371 which describes the use of a strain of Rncil~ subtilus for biological control offruit rot; (iii) U.S. .Cpe~ific~tir)n 4,377,571 which ~lescnhPs the use of Pseudomonas syringae for 1~ ll of ~utch elm ~ eA~e; (iv) U.S.
~pe~ifi~tion 4,950,472 which ~PSÇrihps the use of a new strain of Acremonium 2 0 breve in controlling grey mould infection of pome fruit; and (v) U.S.
Spe~ifi~tion 4,975,277 which describes an isolate of Pseudomonas cepnci~n for ~io]ogic~l control of post harvest disease in fruit.
In JAP~ e Patent JP 3077803 ~ ce is made to Pseudomonas b~ sP1ecte~1 from P c~pncin~ P gladioli, P picketti, P vorans, P dimunata and Rncillt~: bactPri~ selectçcl from B cereus, B mycoides, B anthracis and B
thuringiensis as biocontrol agents in relation to soil borne ~;~ÇA~ÇS.
U.S. Patent 4,878,936 describes the use of B cereus ATCC 53522 as having biocQntrol aclivily and which produces a filn~le active against Phytophera mega~", a.
In Russian Patent SU 237480 reference is made to a strain of B
cereus useful as a biocontrol agent for pr~te~ g plants against insects.
In U.S. Patent 4,661,351 ,efel~,lce is made to coml)os;l;ons WO 94/lg9so 2 15 7 ~ O ~ PCT/~U94/01)110 c~ n~ in~ biosynthetic pP~tiri~1 products obtained from Raci~ c b~c~et-i~
s~lecteA for B thuringiensis, B sphaericus, B popilliae, B cereus, B lentimorbusor B friboungensis. These bacteria may also be provided with a low m~1tin~
point polyester as ~P~rriheA in Patent EP 145087.
In Patent Spe~ific~tiQn JP 59082085 there is described a method of controlling harmful insects by use of a biocontrol agent cc~ ini-~g R(7c~
subtilus, R~ci~ coagulans, Micrococcus luteus, Racil~ stearothermophilus, Clostridium pasteurianum, Clostridium aminovalericum, Clostridum thermo-saccharolyticum and Thermoactinomyces vulgaris.
Rerel~nce also may be made to a pt1h1iraffon by Broadh~nt et al entitled "R~ct~ri~ and ~ctin~llllyceles Antagonistic to Fungal Root Pathogens inAustralian Soils" which inr1~ es B subtilus, B megaterium, Streptomyces spp, B cereus, B pumilus, B polynyxa, B badius, Pseudomonas putida, P
fluorescens and Pseudomonas spp as having biocontrol activity.
Re~,~nce may also be made to Visser et al Applied and EllviL`~
Microbiology 52 552-555 (1986) which ~lesr. rih~s a variety of lactic acid bact~-ri~ i~o1~t~d from plant surfaces and plant ~ori~teA. products which were found to be antagonistic to the ph~l~aL~Iogens ~nthomonas campestris, Erwinia cafotovora and Pseudomonas syringae. In pot trials, ~ l of bean plants with a Lactobacillus planetarium strain before inc~ll~tion with Pseudomonas syringae caused a signific~nt reAllcti~n of the in~1~nre of tli~e~e From a review of the abov~ -.l;oneA prior art, it is clear that it is well known to take natural isolates which may be found on leaf surfaces of fruit and cultivate such natural isolates for their use as biocontrol agents when applied to crops or fruit and other plants. The natural isolates may be removed from the leaf surfaces and cultivated in suitable media. In some cases culture media may be dried and ground into a powder before being i~col~laLed in a carrier. In some cases the culture media may be mixed with wax such as a water or p~arr~/mineral oil base. Som~tim~ the infected fruit may be dipped into a sol1-tion co..l;.;~ g the natural isolate.
However, it would seem that one i~ l disadvantage of the use ~WO 94/19950 215 7 9 0 4 PCT/AU94/001l0 of natural isolates is that this is often a~l~ached in a random ~ ner and that the relevant antagonist must be irl.ontifi~ and i~lit~i before being used in field trials. There is also no evidence that the use of natural isolates has heen adopted on a co.,...~..;ial scale.
It would also seem clear that the main disadvantage of use of biocontrol agents such as P~acil~ thuringiensis is the cost because of their need to he applied more often than rh~mi~ and also because of loss of ~lcn~;y within a relatively short time.
It is also clear when use is made of genetic ~n~ g techniques to produce hiocontrol agents such as r~mhinint strains or p~leills obtained from recl~mhin~nt strains that such techniques are eA~ensive and also cannot be sold on the market until obl~ g appr~vdl from the re~ tory- authorities such as the U.S. Food and Drug ~ alion. Also ex~cl~sive field trials are required before co~ ;ial ev~ t~on can be con~ ered.
SUMMARY OF THE INVENTION
It is tl-c.cfc)lc an object of the present invention to provide a biocontrol agent which may alleviate the abov~m~ntion~ problems of the prior art.
The biocontrol agent of the invention may inciude or be derived 2 0 from:
(i) a mixed culture of a yeast co,..~onc~.l and a bacterial cc,..-ponent; and (ii) a substrate for the mixed culture a).
The yeast c~ pQ~ is suitably useful for pro~ in~ a leavcl~ g or 25 rising action in a sourdough which are doughs in which flour (for example, rye flour) is ~e.-l-c -lcd by lactic acid b~c1P~ ;~ rather than by bakers yeast.
Sourdoughs are ~les~ for ~Y~mple, by Oura et al. in F~onomi~
Microbiology, Volume 7 entitled "Fe....~..l~ Foods" edited by A. H. Rose pages 123-134. Such yeasts may be select~d from Cn~ n krusei, 30 Saccharomyces c~"G~iae, ~oc~hnromyces exiguss and Pichia saitoi.
However, any yeast could be utilised which is erîc,~ivc in combination with an a~r~,iale bacterial co-n~l~enl and a snit~hle substrate to produce æ main 2 ~ a l WO 94/I9950 PCT/AU94/00ll0 products lactic acid, acetic acid, ethanol and carbon dioxide which is the h~llm~rk of sourdough fe~ n- In sourdough fe....~ n a glycolytic paLllway is chara~-lP~;~eA by homore...~ ;ve lactic acid ba~ ;a via ~ ol~se and a h te~re~ l;ve lactic acid bact~ri~ pal~,way for gl~ ses and ~.loses 5 via phosrhokPtol~e Saccharomyces exiguss is ~lt~.rn~tively known as Torulopsis holmii.
Other yeasts that may be used include yeasts of the genus ,Snccharomyces ~.ner~lly which include as mentionPA above S. cerevisiae, S. exiguss, S.
in~it~ and S. uvarum.
The bacterial component of the symkioti~ mixed culture may be sP.l~ted from Lactobacilli æ well æ m~mhers of the genera Leuconostoc, Pediococcus and Streptococcl~s of the family ~treptococcoce~e. Dirre~
species of Lactobacilli may be used ~:iep~n~ling on th eir ability to use dirre ~nl substrates. Typical lactic acid bacteri~ may be sPlected from L. plantarum, L.
casei, L. delbreuckii, L. IeichP~ mii, L. brevis (especially var lin~nP.ri), L.
fermentum, L. pastorianus, L. bucheri, L. acidophilus, L. farciminis, L.
alim~,.lu,.~, L. fructivorans, L. viridescens, L. cellobiosus and L. solivarius.Other bacteria that may be used include Pe~iococc~ cerevisiae, P. aci~ ctici æ well æ Citroba.,ter spp or Micrococcus spp.
An espPc-i~lly pl~,L,ed bactP.ri~ for use in the biocontrol agent of the invention are Lactobacillus sanfrancisco which are used in the San Fl~cisco so~lough "French bread" process. These bacteria are indirrt;~ t to oxygen and do not use carbollyL~Les other than m~lt~se æ a carbon source. L.
sanfrancisco form a symbiotic r~ tion~hip with Sacchar,.yces exiguss which mainly ~r~ ls the leavenillg fim~i-~n Further infol,l,alion on sourdough cultures may be obtained from:
(i) Nout Int. J of Food Microbiology 12 217-224 (1991);
(ii) Nout et al., J of Applied RaCtPri~10gY S~"~osium Supplem~.nt 1992, 73 1365-1475;
(iii) S~lln~lP~ et al., Cereal (~hPmi~ 49 86-91 (1972); n (iv) Cooke et al., FEMS Microbiology Reviews 359-379 (1987);
(v) Srir~n~ nl~ . et ûl., Applied Microbiology 25 461-470 (1973);
~ WO 94119950 21 S 7 9 Q ~ ~IAU94/OOllO
(vi) Kline and Sugihara Applied Microbiology 21 459-465 (1971);
and (vii) Ng Applied and Ellvh~Jn~ l Microbiolo_y ~ 395-398 (1976).
In a spe~fic form of the biocontrol agent of the invention, which has been ~pO~il~ at the Australian Co v.~ n~ Analytical Labola~olies as ~i~c~ e~ in more detail he~ arl~l (herein referred to as SDBS), the T ~tobaci1li which vere located in the deposited mixed culture in~h~dPrl (i) L.
parabuchneri which vas cl dl ~1r.;7~ by Phillips and Collins in 1988, (ii) L.
1 0 parabuchneri/brevis st~ain, and (iii) L. casei ssp casei which was Cllal<~ ;7~d by Rogose et al. in 1953. However, difr~ tinn b~weel- L. buchneri and L. parabuchneri can be very l1iffi~)h and it is within the scope of the invention to provide a mixed starter culture which inf~ ps L. buchneri in s1lhætit ltion of L. parabuchneri and other strains of L. casei for eY~ le in substitution of L.
casei ssp casei. Difr~ l;on may also be extremely ~limc llt l~lwæn L.
parabuchneri, L. buchneri andL. brevis andL. brevis may also be s1~hsl;lul~A
for L. parabuchneri or L. buchneri.
A sample of the mixed culture wæ rolwa~ed to Kluyver Laboldlo,y of Te~hnolopy Delft Te hni~l Univt;~sily in The Ne~ and Lactobacilli strains (i), (ii) and (iii) were i(lPntifi~P~ The yeæt co.~ of the mixed culture wæ also dele.ll-illed as Sncchoromyces cerevisiae Meyen ex Hansen by CBS Yeæt k~Pnti~c~tion Service Delft, The NethPrl~n~
The su~sll~d~e utilised in the mixed culture may colllplise any ~l)~dLion derived from cereals such as flour and in particular rye flour or white flour which may include unblP~h~P~ white wheat flour. MO1~S~PS may also be utilised as a SU~ ld~
The substrate may also be mixed wi~ w~ter which is suitably "pure water" CG.,.l,t;~ bottled l~lwdler or sterilised Milli-Q filtered water.
The pH of the mixed starter is suitably in the range 3.0-6.0 and more suitably 3.04.5.
Fe.~ n may be initi~te~ by mixing equal parts flour and water to form a paste, and this is then added at a suitable ratio (e.g. 1:1) to starter WO 94/19950 21~ 7 9 ~ 4 PCT/AU94/OOllo culture. Tnrt1b~tion may occur over 548 hours but more suitably 24 hours.
Advantages of the bico~trol agent of the invention over cOIl~ r.l;nn~l pesti~ s and biocontrol agents include the following:
(i) ease of registration as a filngi~e because a)m~n~nt~ of the mixed culture have been utilised in food pro~lu~on for a relatively long period;
(ii) the b~ l co~ l such as the lactobacilli are cl~imeA to have probiotic effects on the o~ which ccn~...e them.
This means that health is ~nh~n~e~i by i-l-~r~vi.lg the balance of b~ct~ri~ and yeasts or microflora of the gut;
(iii) the biocontrol product of the invention is co...p~l;hle with s~ in~hle agriculture and free from health risks which is not the case wi1h converlional h~.mi~l or ~ntihioti~ fi)ngi~le,s as c~ eA above which are disadv~nt~Eeo~ , of:
(a) high oncc,geliic risks;
(b) loss of utility beça~ce of the devel~ pm~nt of fi1ngi~e resistant strains of many p~tho~PnS; and (c) p~èssllle from envilo~ l groups for los~eninp dep~n~1~n-~e on toxic r.h.omi~ 3~.e of dangers to the elvi,~,nll-ent and the development of ~ hle cropping ~y~l~.ns.
The bioc~nt~ol agent of the invention may also include other co..~poIl~nt~ so as to farilit~te its mode of action in a desired applic~tit~n Thus one c~-nlk~ I may include a carrier which may help to 25 ~stahli~h and Il~h;~ nt~gnni~tic pop~ tion~ of the binc~ntrol micr~
o~ on the target surface. This carrier may be a lipid such as wax or oils l~ive of coc~nut oil, veget~hle oil, olive oil, canola oil or emIll~ion~
lusive of fish oil ~mlIl~ion .S~lspPn~ion~ or slurries may also be used ~ with the ahove ~iteri~ One s~it~hle carrier is an çmIll~ifi~
30 veget~hle oil i~lentifieA by the trade mark CODACIDE. Another lipid carrier is itlentifi~A by the trade mark NU-FILM 17. The use of the lipid may also enh~n~e ~ hility and may also be used as an adhesive to the target ~ WO 94/19950 2 1 ~ 7 ~ 0 4 PCT/AU94/QQll0 Another co-l-pol~el-~ that may be utilised is a thirL-PnPr which is suitably a gum such as natural or moAifiP~ gums i--elusive of algin, PPn~n, Y~nth~n and trAg~rAntil gums. A gum may also be used as an 5~m~ ifiPr or stAhili~Pr.
F.Y~mplPs of p~ths)gPn~ of aerial su,races of crop plants or crop products which may be ~ul);~d to the biocontrol agent of the invention include mainly fungal pathogens i.e. Colletotrichum (&l,LL.~c,~se), Cercospora (spots and blights), Botrytis (grey mould and rots), Alternaria (leaf spot), 10Monilini~ (brown rot), Penicillium (blue mould) and Rl~pus (soft rot).
Other co~ )ol~el,L~ that may be include~ in the bioc~-ntrol agent of the invention include micro-or~is--l ~nt~g~ni~m Pnh~nr~r(s) which may include MgO, MgCO3 and/or CaCl2, p~thogpn inhihitoes which may include tea polyphPnol~ (esperi~lly epi~tPrhin gallate and epig~lloc~tPrhin gallate) as well15as ~ntioxiA~nt~ and s~hsl~i-~s to f~rilit~te proAl~cticnl storage and/or applicAtion of the agent.
It will also be app.N ialed that the sol,l~ugh hioc~ntrol products of the invention may be foemlll~tPA for col.. e~ial p~u~oses in any s~it~hle .......................... al~ner and thus may be freeze dried to .mpl~ve storage life.
20The bioc~-ntrol agent of the invention may be applied to the target before, during and after haevest.
FERMENTATION. The SDBS starter culture was stored at 4C
before use. To l~ a~ ale, equal parts of "Kialla" or~nic~lly grown 25lmhlP~achP~A white flour and Milli-Q filtered water were mixed to form a smooth paste. The paste was then comhinPA with the starter culture (1:1) so~.~el;...~,s it may be nP~P~ to fully decant off the "clear layer" of acc~lml-l~tP~A acetic acid, lactic acid and ethanol from the top of the starter culture when this is more than 1/3 of the total volume. The culture was then 30 i~ eA in a loosely covered glass jar at room tC~ al~ (23-30C) for mostly 24 hours but up to five days.
MANGOES. In E~. il~lr.~ naturally infected green fruit were wo 94,l9g50 2 ~ 5 ~ 9 ~ ~ PCT/AU94/OOllO
de~a~)l,ed and then graded into two groups acccl-ling to the density of lesions,i.e. mild natural infection and severe natural infection. In E~ ,el~l 2, the naturally infected fruit were de~apyed and then graded accc,nlillg to the level of natural infection. Fruit were then Ai~rih1)ted to 6 groups, each group 5 co~-s~ g of 3 fruit displaying a similar range of infection levels.
~ REATME~NTS. The 1~ applied in F.x~ ; 1 and 2 are sl,,..",~.ised in Table 1. In both e-Yl~l;...Pnt~, fruit were dipped in Milli-Q
filtered water (control) or SDBS (Tlr~l...e~-~ /i additives) for 1 minute and in~1h~teA on a self draining rack for 6 (FY1~ l 2) or 7 (E~p~lilllt;lll 1) 10 days in the lal~ral(,l~ at room lt;~ alurt; (23C). In E~lhllelll 2a, in which fruit were dipped 3 times, dipping was carried out on days 1, 2 and 3 of the e~ The addilives tested in FY~ Ç~I 2b, Nufilm 17 (Miller ChPmir~l and Fertiliser CGI~.aLion USA) and Cod~de (Spraytech Australasia Pty. Ltd.) were added to the dipping Illi~C at the rate and in the 15 manner l~o. ... ..~n-leA in the in~stru~it~n~
DISEASE ~ ;~ENT. An~.l acllose disease development on the fruit was ~ s~eA after 6 days (FY1~ 1 2) or 7 days ~FY~k;l;...~l.~ 1) according to the following diseæe rating scale:
Diseæe Rating Yo Fruit Surface Infected
3 51-75
4 76- 100 RESULIS. In the first e~ lelll (FIG 1), the amount of 25 ~l~ cllo~e diseæe developmPnt in bo~ mildly and severely infected groups of m~n~oes wæ ~duced by one disease rating level by SDBS llrdl~..f-.l re, on the SDBS treated m~ng~S, typically lesions were loc~ e~
and necrotic ra~er than the sunken and s~ adh~g type which pre~o~ e~ on the control fruit.
I2epeateA dipping on each of the first three days of the e~erill~e (F..pf;.;...~nt 2a), reduced the diseæe rating for the fruit fr~!m level 3 to level 1, so that the fruit ripPned with only ~e lor~ necrotic lesions from ~e ~ Wo s4/lss50 215 7 9~0 ~ ~/AU94/OOllO
natural infection.
In F~l~r;.~ 2b, llr~ with one dip of SDBS achieved a single re~luc ti~n in the a,~-ow~l of ~.LLlacllose developing on the m~nEo-p~s~ The ~lAition of the spreader - sticker form~ ti~n.~ Nuf~ 17 and Co~l~ci~le rPduced
and necrotic ra~er than the sunken and s~ adh~g type which pre~o~ e~ on the control fruit.
I2epeateA dipping on each of the first three days of the e~erill~e (F..pf;.;...~nt 2a), reduced the diseæe rating for the fruit fr~!m level 3 to level 1, so that the fruit ripPned with only ~e lor~ necrotic lesions from ~e ~ Wo s4/lss50 215 7 9~0 ~ ~/AU94/OOllO
natural infection.
In F~l~r;.~ 2b, llr~ with one dip of SDBS achieved a single re~luc ti~n in the a,~-ow~l of ~.LLlacllose developing on the m~nEo-p~s~ The ~lAition of the spreader - sticker form~ ti~n.~ Nuf~ 17 and Co~l~ci~le rPduced
5 disease ratings by 2 levels (FIG 2), to give much the same level of disease contr~l achieved with 3 dips. SDBS with Nufilm 17 achieved the best and most c~ disease control.
When the SDBS lllixlweS were washed off the treated fruit and the clean fruit in~lh~te 1 for a further three days in the labo~dlul~y at r~om 10 lelllpeldlwe~ the anthracnose developed in the same manner obse- ved on contr~l fruits.
The SDBS mixture appea,~ to act fimgi~htic~lly to inhibit or retard the devPlopmPnt of anthracnose on naturally infected comm~-n mango fruits.
The fimEi~t~tir effect of the L~ n--~-l can be Pli---il~led when the Illix~we is15 removed from the fruit surface by wæhing. The effect can be ~nh~n.~eA. by prolcnEeA ~Ypos~J~e to the actively growing (wet) l~ixLwe by ~pe~,leA rlipI)ing or by the ~ itic-n of the spreader - sticker formlll~ti~n~ Nufilm ~7 and Cod~de.
The SDBS product has now been ~le~sil~d at the Australian 20 Gov~ ..1 Analytical Labo,dLolies, Suakin Street, Pymble, New South Wales, Australia on March 5, 1993 and has been allocated ~ccPs~ion mlmher N93/9578. In 1 g of SDBS starter culture there is 0.37 g flour, 0.63 g water up to 1012 cfu/g of lactobacilli and 2 x 103 cfu/g .S~7c~h~romyces cerevisiae.
The SDBS starter culture origin~teA from a sourdough starter which 25 was developed by Mrs Edith Aylward of Roys Road, Palmwoods, QllPPn~1~n-l Australia. In bread m~kin~ operaffons carried out by Mrs Aylward, the starter iniffally c~ eA a u~mhin~tion of intact grapes of the C~hernet variety and a cc,.. -e~cial starter obt~in-PA from Germany called "BACK FERMENT" which was utilised in rye bread m~mlfactl1re and such co..~...P~ial starter was mixed 30 with water and lmhle~rheA organic white flour ("KIALLA") in equal volumes.
A r~ ;on occurred and the ~e~ n vessel was lightly covered and left st~n-ling at RT for up to seven days. After bl~bbling or frothing took place Wo 941l9950 21~ ~ ~ 0 4 PCT/AU94100110 the fw~l~enl was ~lec~ntP~l to remove glApe~ x and staLIcs and was placed in a refri~rAtor at 4C in a sealed jar for a resting period of 1-2 days.
Sul)sequel-lly 50~o of the pl~alion was used for bread making where yeast, salt and flour was added and ~e r~mAin(lPr ~llrpl~m~nteA with flour and water 5 and ,el~ ed in the refrigerator at 4C for the next [e,...~la~;on. Fxp,lnQion Of the starter by Çe~ ;on of organic lmhlPAch~A white flour with culture from the previous week occul,ed weekly over a period of five years and also in a totally non-sterile fashion so ~at the starter may have also in-h1~A micro o~ ix.~.x introduced f~m the flour. Mrs Aylward, from the e operations, 10 has produced a sourdough bread which has been unaltered in flavour or texture and smell.
CHARACTERISTICS OF SDBS - RELATIVE DEN~ ~ OF MICRO-ORGANISMS IN MIXTURE.
It is not po~xihle to Aistin~lish between the dirr~L~nl species of 15 lactobacilli of the SDBS starter culture on plates. We always obtain best growth of all the species i~ 7t~A on MRS so we have used this m-qAi~m for total l~ob~illi counts. Lactobacillus casei ssp caesi grows well on T~^tQSe LB m~Ail1m, while the other isolates either do not grow or only grow very slowly and poorly on this m~Ail1m We have used this ...~A;~.... to ~ e the ~lo~Llion of L. casei ssp casei in the total count.
The data in Table 2 show that the total number of lactobacilli illc,ease from about 1 million per ml of fe,n-elll to a peak of about 7 million per ml after 24 hours in~u1lation- The total b~cten~l population seems to remain at around this leve} for the following 24 hours.
The size of the popnl~tion of L. casei Illeæ~d in these exl,ellll.~"lL~, in~1ic~te that it is an i~ L~ cc,~ ~n~ of this mixture.
Ul~,Lullately, we were unable to find a ..~eA;~I~" sl~it~hl^ for di~,~ g beL~n L. parabuchneri and the L. parabuchneri/brevis strain.
As ~ sed below yeast population growth seems to be very 30 se~siLive to both oxygen availability and organic acid and ethanol cC~n~ntr~ti~n If yeæt numhers are low in the starter at the be~inning of the Ç~ l--e~ n (in our ~Y~rience due to prolonged growth and/or storage under ~WO 94/1gg50 215 7 9 0 I PCT/AU94/001l0 anaerobic cnn~iticn~), the pop~ tion will not grow and very little ethanol prod~1Gtinn will occur. When re~on~hle m1mhers are present > 103 per ml, the population seems to be active in the first 24 hours of fe~l..P~ ;nn proclt~ing eth~noi This can be seen in the f~ mix as it becomP-~ very 5 frothy. As the fe~ l;nn proceeds, acid ~ccllmlll~tinn and possibly oxygen l~lepletinn will limit yeast growth and activity. When this occurs the I~A~
stops frothing and dies down again.
Yeast mlmhP~ show enormous variation be~ eA~.i-l-en~. In Table 2, when we realised that their m1mbPr~ were very low after Run 2, we 10 altered fermPnt~tion cnn~ition~ to make cnntlition~ more aerobic to encou~ageyeast growth. The steady in~,~ase in their m1mhPrs over the three runs would suggest r~voulillg aerobic con~itinn~ early in Çel..~P..I~l;on allows them to grow, before their activity is su~l~sed by i~cl~asing acidity and ethanol con~-entration of the rr~
Table 3 illL~IIal~s media on which the isolates of SDBS will grow as pure cultures. In Table 3, LLA meAium col,l~,ised Lactic Acid R~ct~-ri~
Media using lactate. This l..~Aill." co~ eA 0.03% Tween 80, 1.5% fresh yeast extract, 0.6% I,.y~tone and 2.0% lactose which was adjusted to pH 5.45 with agar. SLA meAillm is the same as LLA ...e~li ... except sucrose is utlised 20 instead of lactose at the same col~cP..I~alion. MLS ,..Pfl;,..,. is the same as LLA mPAil1m except m~ltose is used instead of lactose at the same cc n~pntration.
RSYM mPAillm oc~ eA. 10% raw sugar, 0.05% (NH4) 2HPO4 1.5~o fresh yeast extract and 2~o agar. Water is added to 100~o and the pH
25 adj~ed with lN HCl to pH 6. The ...~I;,.." should be autoclaved before use.
SYM .,.PAi,.... is the same as RSYM with sucrose being used instead of raw sugar. MEA is a col-v~l;on~1 malt extract ...PA;.~.. obt~inp~A from Oxoid Australia. PDA is a co~ ;cn~l potato dextrose m~Ail1m also obt~in~hle from Oxoid Australia. RAA is a collv~ ;nn~l rogosa agar ~-.P~ .. obt~in~hle from Oxoid Australia. Mol~es media comrriP-c~ 1%, 2%, 5%, 10~o or 20%
mol~Ps in water which is autoclaved before use. MRS l..eAil~" is also obtainable from Oxoid Australia where 52 g of MRS broth is dissolved in 1000 Wo 94tlsg50 21~ 7 ~ PCT/AU94/OOllO
ml of MilliQ H2 0 and 20 g of agar is added. The mL IUI~ is autoclaved at 121C for 15 ...;n~
During the rf~ ;ons shown in Table 4, ~lthr,ugh total L. casei and yeast numbers end up nc~ g to a~ 1e1y the same levels in each of the four ~ lulcs after 48 hours inrl~h~tion~ total lactobacilli mlmhe~ do notseem to recover as well.
In the ~L~Idal~ fel-~ l;cn, 50% of the Illixlule is the starter culture. When we carried out fçrment~ti~n~ in which the starter culture colll~ elll was reduced to 20%, 10% and 5% as shown in Table S, we found that after 24 hours fermentation normal levels of efh;~nrJ1, lactic and acetic acid were pro~ ce~ ~1th~t1~h pH tended to be slightly higher after 24 hours, pH
was normal in all the f~rm~nt~tic-n~ after 24 hours.
Table 6 shows pH profiles of the ~ ddll re~ l;on at 30C.
All data ~c~prcsenl means of Illea~u,cmcn~ made in trip1irate from 1 5 t vo or three rep1ir,~te f~rment~tic~n~. Ethanol l.leasulclllell~ made using Boehringer ~nnh~.im test kit, Acetic and Lactic acids Boehringer ~i nnheim test kits and HPLC (Biorad Aminex HPX-87H co1~mn).
~ Table 6, the freshly pncl~d cd mixed culture has a pH around 3.8 beco~ slightly more acid over time with a 48 hour pH around 3.4 Lowest pH recorded with wheat flour s~ dle wæ pH 3.2.
In Table 7, which shows typical profiles of ethanol pro~luc~ion, the f,~ n starts with a residual level of ethanol in the mix from the starter at around 2 g/L. Pro1uction peaks after 24 hours at around 7 g/L, then tapers off as acid levels illc,case.
Fth~nol l)r~ n relates d~cclly to yeast density in the miX. In Runs I and 2 yeast n~lmh~rs were very low at the beginnin~ of fe....~ n Fern^ent~ti~n in Table 8 starts with about 1 g/l acetic acid residual frcm the starter, which ap~e~ to be metabolized as levels drop con~^id~tably after 17 hours then slowly LllCl~caScS to reach oriEin~l levels after 48 hours.
l~ 1 levels lactic acid in ~e starter in Table 9 are relatively hi~h (around 3.5 g/L) and rise steadily during ~r..,..~...l~l;on to reach levels double the 0 time con~entrations after 48 hours.
~ WO 94/lgg50 215 7 9 Q 4 PCTIAU94100110 After f~ n, it is noted that m~ e is produced as an end product.
In a standard protocol for carrying out the r...,~ 1;on.~ shown in Tables 7-10, 200 ml of the standard mixture (1 part starter culture: 1 part 5 flour/w~ paste) was p~;p~d in triplicate in 250 ml pyrex bottles and in~-h~t~A for 48 hours in the dark at 30C. The 'oottles were loosely covered with ~ ;,,." foil to allow aerobic fel..-~ n After 0, 17, 24 and 48 hours, the pH of each relll-en~ was measured with a digital stick pH meter (Hanna Ins~ .en~). F.th~n~l and organic acid 1 0 profiles were descrihed using I-eas~ len~ obtained from the following W
metho~l A~lr. ..~ ;on kits, accol.l,ng to the manufacturers instructions:
1. F.th~nol - Boehrin~r ~nnheim F.th~nol test kit (No. 176290);
2. Acetic Acid - RoPhringer ~nnh~.im Acetic Acid test kit (No.
148261);
1 5 3. Lactic Acid - Boehringer ~nnheim test kit (No. 1112821).
Acetic and lactic acid c- n-~ntrations were c~ r;~---eA with a Waters/Applied Bio~y~n.s HPLC using a Biorad Aminex HPX-87H column acco,~ g to the m~m-fa~t...~ ;on.c.
In relation to the Çr~ n~ shown in Tables 4-5, these were 20 carried out exactly æ described above except that the ~mo1~nt of starter in the ult; wæ varied i.e. starter contributed to 5, 10, 20 or 50% (50% =
standard nli~ ) of the total volume of the mlxture.
In relation to the fe~ nc shown in Tables 2 and 4, Aencities were initially carrying out serial Aill~ti~n~ of each rw...~ n sample were ~;d in sterile water. Viable counts of microflora were made at 160,000 Ailuti~n Yeæt m1mhçrs were ~ ecl by plating out 100 microlitres of dilut~d sample onto DRBC plates (DRBC ln~ is obt~in~hle from Oxoid and hæ 0.1 g/l chlol.J-..~ ;cQl added thereto). Total l~-~tob?~eilli were colulled by plating out 50 microlitres of diluted sample onto MRS
30 r..~ ...., Lactobacillus casei mlmher~ were esl;,-~leA by plating out 50 microlitres of diluted sample onto T a~tose LB 1~
Plates were in~-b~teA in the dark at 30C for appr~xim~tely five Wo g4/lg95o 21~ 16 ~cr/AU94/OOllO
days.
Each plating was ~,l~l",ed in triplic~te and the data repl~el~L the mean numbers of each bug c~ teA from the numbers growing on the three plates. Data are e*)ressed as CFlJ/ml of fe~
In regard to Tables 10 and 11, such Tables illustrate e~ "el,~
which were c~n~ te~ using a pl~ ~ocol similar to that described in regard to Table 1 which measures in Table 10 the mean ~ ...elPr of l~.~sion~ in cm on mango fruit naturally infected with ~ acl~se over 17 days post l~
with the biocontrol agent of the invention which was p,e~a~ed as set out in the 10 ~l~locol co"esponding to Table 1 above. Table 11 ill~ll~les the il~,t;ase in density of lesions on naturally infected mango fruit over a 17 day period after ~a~
Table 12 refers to ret~ on~ r~lh~r~ose dev~lopm~nt in mangoes and avocado after post harvest ll~dl-neol with SDBS.
~3RIEF DESCRIPIION OF THE DRAWINGS
FIG 1 refers to the development of ~,~"a~l,ose on naturally infected mangoes after 7 days in~lh~ti~ n at RT as ~ c~l~se~l above in relation to Table l;
FIG 2 refers gr~phi~1ly to the effect of sticker-spreaders on the 20 development of ~,lll.acl~ose on naturally infected co..~ ol~ mangoes;
FIG 3 is a flow diagram ill..~ l;ng a p~osed method of .-r~ lu~t; of the biocontrol agents of the invenffon on a cG..~ ial scale.
FIG 4 is a graph illli~a~ing in graphical form the data set for~ in Tables 11-12.
FIG. 5 is a graph illu~llal~lg in graphical form the data set forth in Tables 6-9.
It will be appreciated from the foregoing that the Lvel-live co~
refers to the rP~ ti~n th2t sourdough sta~ter formt-l~tion~ ~uplisingly have a biocc)nttol capability ~per~lly in relation to fungal ~ e~ses of plants. This iseYemplifie~ by applir~tion of the biocontrol agent of the invention to mangoes and avocado which were infected with Colletotrichum gloeosporioides and Collectotrichum ~cuf~7fr~.
~WO 94/199~0 21 S 7 9 0 ~1 PCT/AU94/OOllO
. i In another aspect of the invention there is provided a process for pro~1u~tit~n of a biocontrol agent for use in bioc~ntrol of fungal ~ e~es in fruit or plants inc~ ing the steps of:
(i) obtaining a sourdough starter culture in~hl~ing a yeast ccnll)onelll and a b~ct~ri~
(ii) adding said starter culture to a Çe~ l;on substrate to form a f~""~ ;.l;on broth;
(iii) in~lh~tin~ said fe~ l;nn broth for a period of time which is desirably 12~8 hours; and (iv) forming a liquid from said rç~ e~ l;nn broth into a biocontrol form~ tic~n and optionally adding a sl~it~hle carrier which may include or is selected from a spreader or sticker, dis~L~ L, thi~ n~ m~ ifi~r or stabiliser.
WO 94/l9950 ~ 1 5 7 9 ~ ~ PCr/AU94/00ll0 TABLE l. ~ of the ~ design of E~ nenls 1 and 2 (a & b) TREATMENTNUMBER OF Fl~Urr ~;Xl ~lMENT 1 Mild Infection Severe Infection Contr~l (water dip) 5 5 SDBS l~ . (dip) 5 5 ~;~ ~KIMENT 2 (a) Z
Control (3 dips in water) 3 SDBS L~ra~ .l (3 dips) 3 (b) Z
Control (l water dip) 3 SDBS tre~tnlf~nt (1 dip) 3 SDBS + Nufilm 17 (1 dip) 3 SDBS + Corl~r~ (l dip) 3 ~ wo 94,lgg50 2 1 5 7 9 ~ 4 PCT/AU94100110 TABLE 2. Densities of T~ctob~illi and yeasts in ~e mix.
Group Run No CFU/ml over ~me (hours) Total 1 1.23 4.58 4.70 5.44 Lactobacillii 2 1.74 5.34 9.12 5.01 3 1.~4 - 6.26 8.60 L. casei 1 2.78 3.03 2.32 12.30 `(x 104) 2 2.75 2.92 1.92 5.20 3 2.64 - 2.83 7.36 Total Yeast 1 < 102 < lo2 < lo2 < lo2 (x 103~ 2 1.13 0.58 1.05 0.47 3 312.00 - 35.20 67.20 Wo 94/19950 2 1 ~ 4 TABLE 3. Media on which ~e isolates of SDBS will grow as pure cultures.
IDENTl~ BEST GROWTH SUBOPTIMAL
(slow & poor) ,Sacct~nromyces MEA
cerevisiae PDA
RSYM
SYM
%-~o Molasses Lactobacillus MRS LLA
parabuchneri MLA
SLA
RAA
Lactobacillus MRS LLA
parabuchneri MLA
Lactobacillus MRS MLA
parabuchneri SLA LLA
Lactobacillus MRS SLA
parabuchneri/brevis MLA
Lactobacillus LLA MLA
caseissp casei MRS SLA
Lactobacillus LLA MLA
casei ssp casei MRS SLA
~WO 94/lg950 2 15 7 9 0 4 PCT/AU94/00110 TABLE 4. Densities of the micro-o~ in ~e minim~l starter culture ~ ",~"~ n Group % StarterCFIJ (x 105) per ml starter over in mix Time (hours) Total 50 15.35 62.60 86.00 Lactobacilli 20 2.84 7.36 11.20 2.62 6.10 9.02 1.65 4.10 8.98 L. casei 50 0.26 2.83 0.74 2.70 1.97 0.98 2.16 1.70 0.91 1.65 1.89 1.44 Total Yeasts 50 3.12 0.35 0.67 1.52 0.13 0.45 0.98 0.16 0.37 0.26 0.24 0.43 WO 94/19950 2 ~ 5 7 9 0 ~ PCT/AU94/00110 ABLE ~;. Fe....~ l;nn chara~ten~ti~ using 50%, 205~o, 10% and 5%
starter culture.
Parameter % Starter Time (hours) in n~ix 0 24 48 pH 50 3.22 3.71 3.53 3.88 3.65 3.30 4.45 3.51 3.27 5.04 3.60 3.27 F.th~nol 50 2.29 3.03 1.56 (g ) 20 1.33 3.05 2.96 0.64 3.02 2.84 O.lS 3.02 1.94 Acetic Acid 50 - 0.96 3.21 ) 20 - 1.13 2.87 - 1.25 2.54 - 0.89 2.81 Lactic Acid 50 2.74 7.46 4.95 (g ) 20 1.52 8.07 9.21 0.70 7.41 9.01 0.49 7.95 6.37 ~ wo 94/lgg50 . 2 1 ~ 7 9 0 4 Pcr/Aug4loollo TAiBLE 6. Typic~ pH pno~e Run No l~ne (hours) 1 3.87 - 3.43 3.40 2 3.73 3.45 3.37 3.29 3 3.73 3.45 3.44 3.43 Mean 3.78 3.45 3.41 3.37 T~IBLE 7. Typic~ pnof~es of Fth~nol pro~uction ~/L) Run No l~me (hours) 1 - 1.34 2.40 1.50 2 0.87 1.36 2.60 1.00 3 2.31 2.90 7.06 5.74 4 2.29 - 3.03 1.56 WO 94/19g50 21 ~i 7 ~ O 4 PCT/AU94100110 TABLE 8. Typical profiles of Acetic Acid pro~u~ion (g/L) Run No Time (hours) - 1.92 2.6g 2.80 2 0.76 0.39 0.65 1.29 3 0.99 0.28 0.44 1.08 4 - - 0.96 3.21 T~BLE 9. Typical profile of Lactic Acid pr~t~ n (g/~,) Run No Time (hours) --2 4.69 7.36 8.39 9.45 3 3.30 6.15 5.90 7 45 4 2.74 - 7.46 4.95 ~WO 94/lss50 215 7 9 ~ 4 PCT/AU94/OOllO
TABLE 10. Mean ~ mPt~r of lesions (cm~ on Mango fruit naturally infected with anthracnose over 17 days post ~alllelll (Mean 20 lesions per 16 fruit per l~
Days after Treatments treatment Control SDBS SDBS SDBS +
(water dip) (1 dip) (3 dips) Yeast (l dip) 2 1.48 1.39 0.70 0.32 4 3.15 1.68 1.15 0.76 7 8.66 2.40 2.56 2.73 9 11.35 4.12 4.65 4.50 11 25.39 6.94 7.46 6.25 14 36.40 18.32 23.3 15.4 17 44.15 27.02 24.4 24.2 TABLE 11. Increæe in density of lesions on naturally inÇe~d Mango fruit over 17 day period after h~ (Mean no. per 6, 4 cm2 quad~al~s per fruit, 16 fruit per ~
Days after Treatrnents treatment Control SDBS SDBS SDBS +
(water dip) (1 dip) (3 dips) Yeast (1 dip) 2 0.19 0.20 0.16 0.01 4 0.85 0.20 0.19 0.19 7 2.33 0.52 0.23 0.77 9 2.70 1.4 1.03 1.65 11 3.74 2.86 1.85 2.37 14 5.29 3.52 3.46 3.38 17 6.32 6.01 4.71 6.11 WO 94/l99~iO ~ 1 ~ 7 9 (~ 4 PCT/AU94/00110 ABLE 12. pcp~ n~ in anthracnose development in mangoes and avoc~doP,s after post harvest treAtmpnt wit~ SDBS. Fruit green (few, small or no lesions) or in semi-ripe (many lesions) con-1ition Varie~ No. of No. of Disease Msease fruit in days in Rating' of Ratingl of treatment ~I~.~,e Controls SDBS fruit Common ~; 7 3 i 1 2 i 1 mangoes (few lesions) Common 5 7 4 i 0 3 i 1 mangoes (many lesions) Common 3 6 3 + 1 1 i 0 mAngoPS (3 on c n~e~tive days) Common 3 6 3 i 1 1 i 1 mAngoes ~n~ing~orl10 6 2 i 1 1 i 1 mangoes3 n~ti-~n 10 8 3 i 1 2 i 1 mangoes (small lesions) Sensation 10 8 4 i 0 2 i 1 mAngl~P~3 (no lesions) Fuerte 10 10 60% 10%
avoc~ es2 1 Disease rating scale for rnAn~o~: 1 = no infection to Y4 of surface with lesions, 2 = ~ to l/2 of surface with lesions, 3 = ~c2 to 3h of surface wimle~ion~ 4 = 3h to whole of surface wi~ lesions 2 Disease rating for avocadoes e~ ed as a ~,c~n~ge of fruit with lesions 3 fruit fly inr~ ion LEGENDS
Disease rating scale for mangoes: 1 = no infection to l~ of surface with lesions, 2 = lh to l~2 of surface with lesions, 3 = ~2 to 3~ of surface withlesic~n.~, 4 = 3~i to whole of surface with lesions 2 Disease rating for avocadoes expressed as a pelc~llage of fruit with lesions 3 Fruit fly il~fe~L~lion 10 The effects of SDBS post harvest dip on the development of ~~ acnose on naturally infected mangoes after 7 days incuhation at room tem~e-a~e.
(a) Natural infection mild (b) Natural infection severe 15 The effect of 3 dips and sticker-spreader on the development of anthracnose on naturally infected common mangoes.
Diagr~mm~tic repr~s~nt~tion of fermentation process.
20 pH and organic acid profiles of the standard ferm~n~tion at 30C (Mean of 3 replic~te ferm~nt~tions~ Run 2N).
Increase in mean diameter of anthracnose lesions on naturally infected mango fruit over a 17 day period after tre~tm~nt (mean 20 lesions on each of 15 fruit 25 per tre~tment).
:iVk~1lUl~ SHE~T (Rule 26)
When the SDBS lllixlweS were washed off the treated fruit and the clean fruit in~lh~te 1 for a further three days in the labo~dlul~y at r~om 10 lelllpeldlwe~ the anthracnose developed in the same manner obse- ved on contr~l fruits.
The SDBS mixture appea,~ to act fimgi~htic~lly to inhibit or retard the devPlopmPnt of anthracnose on naturally infected comm~-n mango fruits.
The fimEi~t~tir effect of the L~ n--~-l can be Pli---il~led when the Illix~we is15 removed from the fruit surface by wæhing. The effect can be ~nh~n.~eA. by prolcnEeA ~Ypos~J~e to the actively growing (wet) l~ixLwe by ~pe~,leA rlipI)ing or by the ~ itic-n of the spreader - sticker formlll~ti~n~ Nufilm ~7 and Cod~de.
The SDBS product has now been ~le~sil~d at the Australian 20 Gov~ ..1 Analytical Labo,dLolies, Suakin Street, Pymble, New South Wales, Australia on March 5, 1993 and has been allocated ~ccPs~ion mlmher N93/9578. In 1 g of SDBS starter culture there is 0.37 g flour, 0.63 g water up to 1012 cfu/g of lactobacilli and 2 x 103 cfu/g .S~7c~h~romyces cerevisiae.
The SDBS starter culture origin~teA from a sourdough starter which 25 was developed by Mrs Edith Aylward of Roys Road, Palmwoods, QllPPn~1~n-l Australia. In bread m~kin~ operaffons carried out by Mrs Aylward, the starter iniffally c~ eA a u~mhin~tion of intact grapes of the C~hernet variety and a cc,.. -e~cial starter obt~in-PA from Germany called "BACK FERMENT" which was utilised in rye bread m~mlfactl1re and such co..~...P~ial starter was mixed 30 with water and lmhle~rheA organic white flour ("KIALLA") in equal volumes.
A r~ ;on occurred and the ~e~ n vessel was lightly covered and left st~n-ling at RT for up to seven days. After bl~bbling or frothing took place Wo 941l9950 21~ ~ ~ 0 4 PCT/AU94100110 the fw~l~enl was ~lec~ntP~l to remove glApe~ x and staLIcs and was placed in a refri~rAtor at 4C in a sealed jar for a resting period of 1-2 days.
Sul)sequel-lly 50~o of the pl~alion was used for bread making where yeast, salt and flour was added and ~e r~mAin(lPr ~llrpl~m~nteA with flour and water 5 and ,el~ ed in the refrigerator at 4C for the next [e,...~la~;on. Fxp,lnQion Of the starter by Çe~ ;on of organic lmhlPAch~A white flour with culture from the previous week occul,ed weekly over a period of five years and also in a totally non-sterile fashion so ~at the starter may have also in-h1~A micro o~ ix.~.x introduced f~m the flour. Mrs Aylward, from the e operations, 10 has produced a sourdough bread which has been unaltered in flavour or texture and smell.
CHARACTERISTICS OF SDBS - RELATIVE DEN~ ~ OF MICRO-ORGANISMS IN MIXTURE.
It is not po~xihle to Aistin~lish between the dirr~L~nl species of 15 lactobacilli of the SDBS starter culture on plates. We always obtain best growth of all the species i~ 7t~A on MRS so we have used this m-qAi~m for total l~ob~illi counts. Lactobacillus casei ssp caesi grows well on T~^tQSe LB m~Ail1m, while the other isolates either do not grow or only grow very slowly and poorly on this m~Ail1m We have used this ...~A;~.... to ~ e the ~lo~Llion of L. casei ssp casei in the total count.
The data in Table 2 show that the total number of lactobacilli illc,ease from about 1 million per ml of fe,n-elll to a peak of about 7 million per ml after 24 hours in~u1lation- The total b~cten~l population seems to remain at around this leve} for the following 24 hours.
The size of the popnl~tion of L. casei Illeæ~d in these exl,ellll.~"lL~, in~1ic~te that it is an i~ L~ cc,~ ~n~ of this mixture.
Ul~,Lullately, we were unable to find a ..~eA;~I~" sl~it~hl^ for di~,~ g beL~n L. parabuchneri and the L. parabuchneri/brevis strain.
As ~ sed below yeast population growth seems to be very 30 se~siLive to both oxygen availability and organic acid and ethanol cC~n~ntr~ti~n If yeæt numhers are low in the starter at the be~inning of the Ç~ l--e~ n (in our ~Y~rience due to prolonged growth and/or storage under ~WO 94/1gg50 215 7 9 0 I PCT/AU94/001l0 anaerobic cnn~iticn~), the pop~ tion will not grow and very little ethanol prod~1Gtinn will occur. When re~on~hle m1mhers are present > 103 per ml, the population seems to be active in the first 24 hours of fe~l..P~ ;nn proclt~ing eth~noi This can be seen in the f~ mix as it becomP-~ very 5 frothy. As the fe~ l;nn proceeds, acid ~ccllmlll~tinn and possibly oxygen l~lepletinn will limit yeast growth and activity. When this occurs the I~A~
stops frothing and dies down again.
Yeast mlmhP~ show enormous variation be~ eA~.i-l-en~. In Table 2, when we realised that their m1mbPr~ were very low after Run 2, we 10 altered fermPnt~tion cnn~ition~ to make cnntlition~ more aerobic to encou~ageyeast growth. The steady in~,~ase in their m1mhPrs over the three runs would suggest r~voulillg aerobic con~itinn~ early in Çel..~P..I~l;on allows them to grow, before their activity is su~l~sed by i~cl~asing acidity and ethanol con~-entration of the rr~
Table 3 illL~IIal~s media on which the isolates of SDBS will grow as pure cultures. In Table 3, LLA meAium col,l~,ised Lactic Acid R~ct~-ri~
Media using lactate. This l..~Aill." co~ eA 0.03% Tween 80, 1.5% fresh yeast extract, 0.6% I,.y~tone and 2.0% lactose which was adjusted to pH 5.45 with agar. SLA meAillm is the same as LLA ...e~li ... except sucrose is utlised 20 instead of lactose at the same col~cP..I~alion. MLS ,..Pfl;,..,. is the same as LLA mPAil1m except m~ltose is used instead of lactose at the same cc n~pntration.
RSYM mPAillm oc~ eA. 10% raw sugar, 0.05% (NH4) 2HPO4 1.5~o fresh yeast extract and 2~o agar. Water is added to 100~o and the pH
25 adj~ed with lN HCl to pH 6. The ...~I;,.." should be autoclaved before use.
SYM .,.PAi,.... is the same as RSYM with sucrose being used instead of raw sugar. MEA is a col-v~l;on~1 malt extract ...PA;.~.. obt~inp~A from Oxoid Australia. PDA is a co~ ;cn~l potato dextrose m~Ail1m also obt~in~hle from Oxoid Australia. RAA is a collv~ ;nn~l rogosa agar ~-.P~ .. obt~in~hle from Oxoid Australia. Mol~es media comrriP-c~ 1%, 2%, 5%, 10~o or 20%
mol~Ps in water which is autoclaved before use. MRS l..eAil~" is also obtainable from Oxoid Australia where 52 g of MRS broth is dissolved in 1000 Wo 94tlsg50 21~ 7 ~ PCT/AU94/OOllO
ml of MilliQ H2 0 and 20 g of agar is added. The mL IUI~ is autoclaved at 121C for 15 ...;n~
During the rf~ ;ons shown in Table 4, ~lthr,ugh total L. casei and yeast numbers end up nc~ g to a~ 1e1y the same levels in each of the four ~ lulcs after 48 hours inrl~h~tion~ total lactobacilli mlmhe~ do notseem to recover as well.
In the ~L~Idal~ fel-~ l;cn, 50% of the Illixlule is the starter culture. When we carried out fçrment~ti~n~ in which the starter culture colll~ elll was reduced to 20%, 10% and 5% as shown in Table S, we found that after 24 hours fermentation normal levels of efh;~nrJ1, lactic and acetic acid were pro~ ce~ ~1th~t1~h pH tended to be slightly higher after 24 hours, pH
was normal in all the f~rm~nt~tic-n~ after 24 hours.
Table 6 shows pH profiles of the ~ ddll re~ l;on at 30C.
All data ~c~prcsenl means of Illea~u,cmcn~ made in trip1irate from 1 5 t vo or three rep1ir,~te f~rment~tic~n~. Ethanol l.leasulclllell~ made using Boehringer ~nnh~.im test kit, Acetic and Lactic acids Boehringer ~i nnheim test kits and HPLC (Biorad Aminex HPX-87H co1~mn).
~ Table 6, the freshly pncl~d cd mixed culture has a pH around 3.8 beco~ slightly more acid over time with a 48 hour pH around 3.4 Lowest pH recorded with wheat flour s~ dle wæ pH 3.2.
In Table 7, which shows typical profiles of ethanol pro~luc~ion, the f,~ n starts with a residual level of ethanol in the mix from the starter at around 2 g/L. Pro1uction peaks after 24 hours at around 7 g/L, then tapers off as acid levels illc,case.
Fth~nol l)r~ n relates d~cclly to yeast density in the miX. In Runs I and 2 yeast n~lmh~rs were very low at the beginnin~ of fe....~ n Fern^ent~ti~n in Table 8 starts with about 1 g/l acetic acid residual frcm the starter, which ap~e~ to be metabolized as levels drop con~^id~tably after 17 hours then slowly LllCl~caScS to reach oriEin~l levels after 48 hours.
l~ 1 levels lactic acid in ~e starter in Table 9 are relatively hi~h (around 3.5 g/L) and rise steadily during ~r..,..~...l~l;on to reach levels double the 0 time con~entrations after 48 hours.
~ WO 94/lgg50 215 7 9 Q 4 PCTIAU94100110 After f~ n, it is noted that m~ e is produced as an end product.
In a standard protocol for carrying out the r...,~ 1;on.~ shown in Tables 7-10, 200 ml of the standard mixture (1 part starter culture: 1 part 5 flour/w~ paste) was p~;p~d in triplicate in 250 ml pyrex bottles and in~-h~t~A for 48 hours in the dark at 30C. The 'oottles were loosely covered with ~ ;,,." foil to allow aerobic fel..-~ n After 0, 17, 24 and 48 hours, the pH of each relll-en~ was measured with a digital stick pH meter (Hanna Ins~ .en~). F.th~n~l and organic acid 1 0 profiles were descrihed using I-eas~ len~ obtained from the following W
metho~l A~lr. ..~ ;on kits, accol.l,ng to the manufacturers instructions:
1. F.th~nol - Boehrin~r ~nnheim F.th~nol test kit (No. 176290);
2. Acetic Acid - RoPhringer ~nnh~.im Acetic Acid test kit (No.
148261);
1 5 3. Lactic Acid - Boehringer ~nnheim test kit (No. 1112821).
Acetic and lactic acid c- n-~ntrations were c~ r;~---eA with a Waters/Applied Bio~y~n.s HPLC using a Biorad Aminex HPX-87H column acco,~ g to the m~m-fa~t...~ ;on.c.
In relation to the Çr~ n~ shown in Tables 4-5, these were 20 carried out exactly æ described above except that the ~mo1~nt of starter in the ult; wæ varied i.e. starter contributed to 5, 10, 20 or 50% (50% =
standard nli~ ) of the total volume of the mlxture.
In relation to the fe~ nc shown in Tables 2 and 4, Aencities were initially carrying out serial Aill~ti~n~ of each rw...~ n sample were ~;d in sterile water. Viable counts of microflora were made at 160,000 Ailuti~n Yeæt m1mhçrs were ~ ecl by plating out 100 microlitres of dilut~d sample onto DRBC plates (DRBC ln~ is obt~in~hle from Oxoid and hæ 0.1 g/l chlol.J-..~ ;cQl added thereto). Total l~-~tob?~eilli were colulled by plating out 50 microlitres of diluted sample onto MRS
30 r..~ ...., Lactobacillus casei mlmher~ were esl;,-~leA by plating out 50 microlitres of diluted sample onto T a~tose LB 1~
Plates were in~-b~teA in the dark at 30C for appr~xim~tely five Wo g4/lg95o 21~ 16 ~cr/AU94/OOllO
days.
Each plating was ~,l~l",ed in triplic~te and the data repl~el~L the mean numbers of each bug c~ teA from the numbers growing on the three plates. Data are e*)ressed as CFlJ/ml of fe~
In regard to Tables 10 and 11, such Tables illustrate e~ "el,~
which were c~n~ te~ using a pl~ ~ocol similar to that described in regard to Table 1 which measures in Table 10 the mean ~ ...elPr of l~.~sion~ in cm on mango fruit naturally infected with ~ acl~se over 17 days post l~
with the biocontrol agent of the invention which was p,e~a~ed as set out in the 10 ~l~locol co"esponding to Table 1 above. Table 11 ill~ll~les the il~,t;ase in density of lesions on naturally infected mango fruit over a 17 day period after ~a~
Table 12 refers to ret~ on~ r~lh~r~ose dev~lopm~nt in mangoes and avocado after post harvest ll~dl-neol with SDBS.
~3RIEF DESCRIPIION OF THE DRAWINGS
FIG 1 refers to the development of ~,~"a~l,ose on naturally infected mangoes after 7 days in~lh~ti~ n at RT as ~ c~l~se~l above in relation to Table l;
FIG 2 refers gr~phi~1ly to the effect of sticker-spreaders on the 20 development of ~,lll.acl~ose on naturally infected co..~ ol~ mangoes;
FIG 3 is a flow diagram ill..~ l;ng a p~osed method of .-r~ lu~t; of the biocontrol agents of the invenffon on a cG..~ ial scale.
FIG 4 is a graph illli~a~ing in graphical form the data set for~ in Tables 11-12.
FIG. 5 is a graph illu~llal~lg in graphical form the data set forth in Tables 6-9.
It will be appreciated from the foregoing that the Lvel-live co~
refers to the rP~ ti~n th2t sourdough sta~ter formt-l~tion~ ~uplisingly have a biocc)nttol capability ~per~lly in relation to fungal ~ e~ses of plants. This iseYemplifie~ by applir~tion of the biocontrol agent of the invention to mangoes and avocado which were infected with Colletotrichum gloeosporioides and Collectotrichum ~cuf~7fr~.
~WO 94/199~0 21 S 7 9 0 ~1 PCT/AU94/OOllO
. i In another aspect of the invention there is provided a process for pro~1u~tit~n of a biocontrol agent for use in bioc~ntrol of fungal ~ e~es in fruit or plants inc~ ing the steps of:
(i) obtaining a sourdough starter culture in~hl~ing a yeast ccnll)onelll and a b~ct~ri~
(ii) adding said starter culture to a Çe~ l;on substrate to form a f~""~ ;.l;on broth;
(iii) in~lh~tin~ said fe~ l;nn broth for a period of time which is desirably 12~8 hours; and (iv) forming a liquid from said rç~ e~ l;nn broth into a biocontrol form~ tic~n and optionally adding a sl~it~hle carrier which may include or is selected from a spreader or sticker, dis~L~ L, thi~ n~ m~ ifi~r or stabiliser.
WO 94/l9950 ~ 1 5 7 9 ~ ~ PCr/AU94/00ll0 TABLE l. ~ of the ~ design of E~ nenls 1 and 2 (a & b) TREATMENTNUMBER OF Fl~Urr ~;Xl ~lMENT 1 Mild Infection Severe Infection Contr~l (water dip) 5 5 SDBS l~ . (dip) 5 5 ~;~ ~KIMENT 2 (a) Z
Control (3 dips in water) 3 SDBS L~ra~ .l (3 dips) 3 (b) Z
Control (l water dip) 3 SDBS tre~tnlf~nt (1 dip) 3 SDBS + Nufilm 17 (1 dip) 3 SDBS + Corl~r~ (l dip) 3 ~ wo 94,lgg50 2 1 5 7 9 ~ 4 PCT/AU94100110 TABLE 2. Densities of T~ctob~illi and yeasts in ~e mix.
Group Run No CFU/ml over ~me (hours) Total 1 1.23 4.58 4.70 5.44 Lactobacillii 2 1.74 5.34 9.12 5.01 3 1.~4 - 6.26 8.60 L. casei 1 2.78 3.03 2.32 12.30 `(x 104) 2 2.75 2.92 1.92 5.20 3 2.64 - 2.83 7.36 Total Yeast 1 < 102 < lo2 < lo2 < lo2 (x 103~ 2 1.13 0.58 1.05 0.47 3 312.00 - 35.20 67.20 Wo 94/19950 2 1 ~ 4 TABLE 3. Media on which ~e isolates of SDBS will grow as pure cultures.
IDENTl~ BEST GROWTH SUBOPTIMAL
(slow & poor) ,Sacct~nromyces MEA
cerevisiae PDA
RSYM
SYM
%-~o Molasses Lactobacillus MRS LLA
parabuchneri MLA
SLA
RAA
Lactobacillus MRS LLA
parabuchneri MLA
Lactobacillus MRS MLA
parabuchneri SLA LLA
Lactobacillus MRS SLA
parabuchneri/brevis MLA
Lactobacillus LLA MLA
caseissp casei MRS SLA
Lactobacillus LLA MLA
casei ssp casei MRS SLA
~WO 94/lg950 2 15 7 9 0 4 PCT/AU94/00110 TABLE 4. Densities of the micro-o~ in ~e minim~l starter culture ~ ",~"~ n Group % StarterCFIJ (x 105) per ml starter over in mix Time (hours) Total 50 15.35 62.60 86.00 Lactobacilli 20 2.84 7.36 11.20 2.62 6.10 9.02 1.65 4.10 8.98 L. casei 50 0.26 2.83 0.74 2.70 1.97 0.98 2.16 1.70 0.91 1.65 1.89 1.44 Total Yeasts 50 3.12 0.35 0.67 1.52 0.13 0.45 0.98 0.16 0.37 0.26 0.24 0.43 WO 94/19950 2 ~ 5 7 9 0 ~ PCT/AU94/00110 ABLE ~;. Fe....~ l;nn chara~ten~ti~ using 50%, 205~o, 10% and 5%
starter culture.
Parameter % Starter Time (hours) in n~ix 0 24 48 pH 50 3.22 3.71 3.53 3.88 3.65 3.30 4.45 3.51 3.27 5.04 3.60 3.27 F.th~nol 50 2.29 3.03 1.56 (g ) 20 1.33 3.05 2.96 0.64 3.02 2.84 O.lS 3.02 1.94 Acetic Acid 50 - 0.96 3.21 ) 20 - 1.13 2.87 - 1.25 2.54 - 0.89 2.81 Lactic Acid 50 2.74 7.46 4.95 (g ) 20 1.52 8.07 9.21 0.70 7.41 9.01 0.49 7.95 6.37 ~ wo 94/lgg50 . 2 1 ~ 7 9 0 4 Pcr/Aug4loollo TAiBLE 6. Typic~ pH pno~e Run No l~ne (hours) 1 3.87 - 3.43 3.40 2 3.73 3.45 3.37 3.29 3 3.73 3.45 3.44 3.43 Mean 3.78 3.45 3.41 3.37 T~IBLE 7. Typic~ pnof~es of Fth~nol pro~uction ~/L) Run No l~me (hours) 1 - 1.34 2.40 1.50 2 0.87 1.36 2.60 1.00 3 2.31 2.90 7.06 5.74 4 2.29 - 3.03 1.56 WO 94/19g50 21 ~i 7 ~ O 4 PCT/AU94100110 TABLE 8. Typical profiles of Acetic Acid pro~u~ion (g/L) Run No Time (hours) - 1.92 2.6g 2.80 2 0.76 0.39 0.65 1.29 3 0.99 0.28 0.44 1.08 4 - - 0.96 3.21 T~BLE 9. Typical profile of Lactic Acid pr~t~ n (g/~,) Run No Time (hours) --2 4.69 7.36 8.39 9.45 3 3.30 6.15 5.90 7 45 4 2.74 - 7.46 4.95 ~WO 94/lss50 215 7 9 ~ 4 PCT/AU94/OOllO
TABLE 10. Mean ~ mPt~r of lesions (cm~ on Mango fruit naturally infected with anthracnose over 17 days post ~alllelll (Mean 20 lesions per 16 fruit per l~
Days after Treatments treatment Control SDBS SDBS SDBS +
(water dip) (1 dip) (3 dips) Yeast (l dip) 2 1.48 1.39 0.70 0.32 4 3.15 1.68 1.15 0.76 7 8.66 2.40 2.56 2.73 9 11.35 4.12 4.65 4.50 11 25.39 6.94 7.46 6.25 14 36.40 18.32 23.3 15.4 17 44.15 27.02 24.4 24.2 TABLE 11. Increæe in density of lesions on naturally inÇe~d Mango fruit over 17 day period after h~ (Mean no. per 6, 4 cm2 quad~al~s per fruit, 16 fruit per ~
Days after Treatrnents treatment Control SDBS SDBS SDBS +
(water dip) (1 dip) (3 dips) Yeast (1 dip) 2 0.19 0.20 0.16 0.01 4 0.85 0.20 0.19 0.19 7 2.33 0.52 0.23 0.77 9 2.70 1.4 1.03 1.65 11 3.74 2.86 1.85 2.37 14 5.29 3.52 3.46 3.38 17 6.32 6.01 4.71 6.11 WO 94/l99~iO ~ 1 ~ 7 9 (~ 4 PCT/AU94/00110 ABLE 12. pcp~ n~ in anthracnose development in mangoes and avoc~doP,s after post harvest treAtmpnt wit~ SDBS. Fruit green (few, small or no lesions) or in semi-ripe (many lesions) con-1ition Varie~ No. of No. of Disease Msease fruit in days in Rating' of Ratingl of treatment ~I~.~,e Controls SDBS fruit Common ~; 7 3 i 1 2 i 1 mangoes (few lesions) Common 5 7 4 i 0 3 i 1 mangoes (many lesions) Common 3 6 3 + 1 1 i 0 mAngoPS (3 on c n~e~tive days) Common 3 6 3 i 1 1 i 1 mAngoes ~n~ing~orl10 6 2 i 1 1 i 1 mangoes3 n~ti-~n 10 8 3 i 1 2 i 1 mangoes (small lesions) Sensation 10 8 4 i 0 2 i 1 mAngl~P~3 (no lesions) Fuerte 10 10 60% 10%
avoc~ es2 1 Disease rating scale for rnAn~o~: 1 = no infection to Y4 of surface with lesions, 2 = ~ to l/2 of surface with lesions, 3 = ~c2 to 3h of surface wimle~ion~ 4 = 3h to whole of surface wi~ lesions 2 Disease rating for avocadoes e~ ed as a ~,c~n~ge of fruit with lesions 3 fruit fly inr~ ion LEGENDS
Disease rating scale for mangoes: 1 = no infection to l~ of surface with lesions, 2 = lh to l~2 of surface with lesions, 3 = ~2 to 3~ of surface withlesic~n.~, 4 = 3~i to whole of surface with lesions 2 Disease rating for avocadoes expressed as a pelc~llage of fruit with lesions 3 Fruit fly il~fe~L~lion 10 The effects of SDBS post harvest dip on the development of ~~ acnose on naturally infected mangoes after 7 days incuhation at room tem~e-a~e.
(a) Natural infection mild (b) Natural infection severe 15 The effect of 3 dips and sticker-spreader on the development of anthracnose on naturally infected common mangoes.
Diagr~mm~tic repr~s~nt~tion of fermentation process.
20 pH and organic acid profiles of the standard ferm~n~tion at 30C (Mean of 3 replic~te ferm~nt~tions~ Run 2N).
Increase in mean diameter of anthracnose lesions on naturally infected mango fruit over a 17 day period after tre~tm~nt (mean 20 lesions on each of 15 fruit 25 per tre~tment).
:iVk~1lUl~ SHE~T (Rule 26)
Claims (12)
1. A biocontrol agent which includes or is derived from a sourdough starter formulation comprising:
(i) a mixed culture of the yeast component and a bacterial component; and (ii) a substrate for the mixed culture (i).
(i) a mixed culture of the yeast component and a bacterial component; and (ii) a substrate for the mixed culture (i).
2. A biocontrol agent as claimed in Claim 1 wherein the yeast component comprises yeasts of the genus Saccharomyces.
3. A biocontrol agent as claimed in Claim 1 wherein the bacterial component comprises Lactobacilli.
4. A biocontrol agent as claimed in Claim 2 wherein the yeast component comprises Saccharomyces cerevisiae.
5. A biocontrol agent as claimed in Claim 3 wherein the Lactobacilli component comprises L. parabuchneri, L. parabuchneri/brevis strain and L.
casei ssp casei.
casei ssp casei.
6. A biocontrol agent as claimed in Claim 3 wherein Lactobacilli component comprises L. buchneri, L. casei and L. brevis.
7. A biocontrol agent as claimed in Claim 1 having been deposited at the Australian Government Analytical Laboratories on March 5, 1993 and having been allocated Accession No. N93/9578.
8. A process for reducing or alleviating fungal infection of plants or fruit including the step of application of a biocontrol agent as claimed in Claim 1 to such plants or fruit.
9. A process as claimed in Claim 8 wherein the starter sourdough formulation is mixed with cereals or molasses and also mixed with water at a pH in the range of 3.0-6.0 and thereafter allowed to ferment to form a fermentation broth which was then applied to plants or fruit infected with a fungus.
10. A process as claimed in Claim 9 wherein the fungus comprises Colletrotrichum gloeosporioides or Colletotrichum acutatum.
11. A process as claimed in Claim 9 wherein the fermentation broth is applied to mangoes or avocado.
12. A process for production of a biocontrol agent for use in biocontrol of fungal diseases in fruit or plants including the steps of:
(i) obtaining a sourdough starter culture including a yeast component and a bacterial component;
(ii) adding said starter culture to a fermentation substrate to form a fermentation broth;
(iii) incubating said fermentation broth for a period of time which is desirably 12-48 hours; and (iv) forming a liquid from said fermentation broth into a biocontrol formulation and optionally adding a suitable carrier which may include or is selected from a spreader or sticker, dispersant, thickener, emulsifier or stabiliser.
(i) obtaining a sourdough starter culture including a yeast component and a bacterial component;
(ii) adding said starter culture to a fermentation substrate to form a fermentation broth;
(iii) incubating said fermentation broth for a period of time which is desirably 12-48 hours; and (iv) forming a liquid from said fermentation broth into a biocontrol formulation and optionally adding a suitable carrier which may include or is selected from a spreader or sticker, dispersant, thickener, emulsifier or stabiliser.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPL7721 | 1993-03-10 | ||
| AUPL772193 | 1993-03-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2157904A1 true CA2157904A1 (en) | 1994-09-15 |
Family
ID=3776760
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002157904A Abandoned CA2157904A1 (en) | 1993-03-10 | 1994-03-10 | Biocontrol agents for use in agriculture |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0688169A4 (en) |
| JP (1) | JPH08507497A (en) |
| CA (1) | CA2157904A1 (en) |
| NZ (1) | NZ262853A (en) |
| WO (1) | WO1994019950A1 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI97148C (en) * | 1994-07-14 | 1996-10-25 | Lahden Polttimo Ab Oy | Process for the treatment of plants to improve the quality properties of seeds |
| AUPM840794A0 (en) * | 1994-09-26 | 1994-10-20 | State Of New South Wales, The | A method of controlling moth and other insect pests |
| AU6169396A (en) * | 1995-06-07 | 1996-12-30 | Mycogen Corporation | Materials and methods for inoculating plants with microbes |
| AUPN907296A0 (en) * | 1996-04-02 | 1996-04-26 | Commonwealth Scientific And Industrial Research Organisation | Biocontrol agents for use in treatment of opportunistic infections |
| AU756354B2 (en) * | 1998-06-05 | 2003-01-09 | Biotechnology Research And Development Corporation | Biological coating with a protective and curative effect for the control of postharvest decay |
| IT1306716B1 (en) * | 1999-06-21 | 2001-10-02 | Mendes S U R L | ASSOCIATION OF LACTIC BACTERIA AND ITS USE FOR THE PREVENTION AND / OR THERAPEUTIC TREATMENT OF INFECTIONS AND INFLAMMATORY STATES. |
| FR2841436B1 (en) | 2002-07-01 | 2006-01-27 | Univ Bourgogne | MEANS FOR BIOLOGICAL CONTROL OF CRYPTOGAMIC DISEASES IN PLANT VEGETABLES |
| US8591926B2 (en) | 2004-07-13 | 2013-11-26 | William Brower | Formulation and method for treating plants to control or suppress a plant pathogen |
| NZ552836A (en) * | 2004-07-13 | 2010-05-28 | William Brower | Microbial mixture for treating plants to control or suppress a plant pathogen, particularly blight |
| CA2770957C (en) * | 2011-03-15 | 2021-07-06 | William Brower | Formulation and method for treating plants to control or suppress a plant pathogen |
| WO2013063658A1 (en) * | 2011-11-04 | 2013-05-10 | International Marketing Partnerships Pty Ltd | Microbial inoculants and fertilizer compositions comprising the same |
| ES2710435T3 (en) | 2013-03-13 | 2019-04-25 | Novozymes As | New strains of Lactobacillus and their uses |
| CN105517557B (en) * | 2013-04-23 | 2021-11-30 | 特雷根控股有限公司 | Bacterial strains having antimicrobial activity and biocontrol compositions comprising said strains |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR8900938A (en) * | 1989-03-01 | 1990-10-09 | Fundacao Oswaldo Cruz | PROCESS OF OBTAINING LINES OF AMYLOLYTIC AND PROTEOLYTIC BACTERIA OF THE GENERATION BACILLUS THAT WAVE SPECIFIC LETHAL ACTIVITY FOR SOME ORDERS OF INSECTS; COMPOSITION OF CULTIVATION MEDIA FOR SEEDING OF THE DRAWN BACTERIA LINES; PROCESS OF OBTAINING THE ACTIVE BIOMASS OF BACILLUS ENTOMOPATOGENICOS; COMPOSITION FOR BACTERIAL CULTIVATION INTENDED FOR THE PRODUCTION OF THE ACTIVE BIOMASS AND BIOINSECTICIDE COMPOSITION BASED ON AMYLOLYTIC AND PROTEOLYTIC BACTERIA OF THE GENERIC BACILLUS |
| NZ240576A (en) * | 1990-11-16 | 1992-11-25 | Crop Genetics Int | Controlling weeds by treating with a herbicide in combination with a culture medium in which microorganisms have grown |
| US5525132A (en) * | 1991-04-11 | 1996-06-11 | Daratech Proprietary Limited | Yeasts as a biocontrol for microbial diseases of fruit |
| EP0517378A1 (en) * | 1991-06-03 | 1992-12-09 | Mycogen Corporation | Novel process and compositions for delivery of foliar herbicides |
| US5270059A (en) * | 1992-08-17 | 1993-12-14 | The United States Of America As Represented By The United States Department Of Agriculture | Method and preparation for inhibiting mold growth on pome fruit |
-
1994
- 1994-03-10 JP JP6519390A patent/JPH08507497A/en active Pending
- 1994-03-10 EP EP94910276A patent/EP0688169A4/en not_active Withdrawn
- 1994-03-10 CA CA002157904A patent/CA2157904A1/en not_active Abandoned
- 1994-03-10 NZ NZ262853A patent/NZ262853A/en unknown
- 1994-03-10 WO PCT/AU1994/000110 patent/WO1994019950A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994019950A1 (en) | 1994-09-15 |
| EP0688169A1 (en) | 1995-12-27 |
| EP0688169A4 (en) | 1998-01-28 |
| NZ262853A (en) | 1996-11-26 |
| JPH08507497A (en) | 1996-08-13 |
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