Note: Descriptions are shown in the official language in which they were submitted.
~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
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
1 0-25
2 26 - 50
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
1 1
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
12
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
1 3
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
14
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
17
. 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
1 8
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
1 9
TABLE 2. Densities of T~ctob~illi and yeasts in ~e mix.
Group Run No CFU/ml over ~me (hours)
- 0 17 24 48
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
3 PCT/AU94/00110
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
21
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)
0 24 48
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
22
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
23
TAiBLE 6. Typic~ pH pno~e
Run No l~ne (hours)
- O 17 24 48
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)
0 17 24 48
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
24
TABLE 8. Typical profiles of Acetic Acid pro~u~ion (g/L)
Run No Time (hours)
0 17 24 48
- 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)
0 17 24 48
--
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
26
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
WO 94/19950 2 15 7 9 0 4 PCT/AU94/00110
27
LEGENDS
TABLE 12
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
FIGURE 1
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
FIGURE 2
15 The effect of 3 dips and sticker-spreader on the development of anthracnose on
naturally infected common mangoes.
FIGURE 3
Diagr~mm~tic repr~s~nt~tion of fermentation process.
FIGURE 4
20 pH and organic acid profiles of the standard ferm~n~tion at 30C (Mean of 3
replic~te ferm~nt~tions~ Run 2N).
FIGURE 5
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)
