Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
10853Z6 ~
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METHOD ~OR ~HE ~IOSYNTH~SIS OF A
MICROBIA~ I~SE~TICIDE
______________________ ___________________________________________
. . : '
The present invention relates to a novel method for the bio-
synthesis of a microbial insecticide containing spores and
crystalline endotoxin, said method being characterized in that
the bacillus thuringiensis microorganism or a related bacillus is
cultivated in a nutrient medium under formation of spore~ in such I -
a manner that the early ly~is of cells caused in the course of
the submerged culti~ation by the ventilation prior to the termi-
nation of the formation of spores is prevented.
. .
It is known that the early lysis of cells in the course of the
cultivation leads to low yields and a reduced effectiveness of the
bioinsecticide as well as to an increase of its toxicity.
,. '
The present i~vention pro~ides for an improved method of
producing spores and crystalline endotoxin in the process in which
the early lysis of bacteria cell~ normally occurs as a result o~
enzymatic actions which are induced by the ventilation.
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~08S3Z6
-- 2
The delay in the formation of spores during the vegetative
growth of bacteria is attributed to the effect of one or
several inhibitors of an albuminous nature. The proteolytic
decomposition of suggested inhibitors initiates the formation
of the spores. Furthermore, various theories relating to the -
function of the protease have been submitted which is one of --
the early manifestations connected with the begin of spore
formation (J. MANDELSTAM and W.M. WAITES, Biochem. J., volume
109, pp. 793 to 801, 1968). According to SCHAEFFER, it has
been suggested that in the course of growth of bacteria in a
quickly metabolized nutrient substrate and in the presence of
a usuable nitrogen source, metabolic products occur which
would suppress the synthesis of extracellular proteases and an
enzym that is specific to the formation of spores, possibly in
a manner similar to what is happening in the cancer cycle
(P. SCHAEFFER, Bacteriol. Rev., volume 33, pp. 48 to 71,
1969). The result of such a suppression is the accumulation
of acetic acid and pyroracemic acid which causes a decline of
the pH and the early lysis of bacteria cells in general.
Now, according to a preferred execution of the present
invention it is possible in connection with the sporulation to
prevent the early lysis of bacteria cells by carrying out the
biosynthesis for 4 to 6 hours under anaerobic conditions, and
maintaining the p~l in the course of the biosynthesis within
the range of 6.3 to 7.
With the help of the present invention it was possible to
recover, among other things, the parasporal protein crystal
bodies (~-endotoxin), and to purify the same by removing
108S3Z6
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therefrom the water-soluble exotoxin ( ~ -exotoxin) which must be
removed because of its toxicity to mammals. ~he best method for
removing the exotoxin, according to the present invention, com-
prises the use of semi-permeable diaphragmq.
It is known that the bacillus thuringiensis-group of micro-
organisms is characterized by the ~ormation of protein-containing
parasporal endotoxin crystals. ~he insect-pathogenic nature of
bacillus thuringiensis on a large row of lepidopterous larvae
by ingestion is primarily to be attributed to the e~fect of endo-
toxin. Certain strains o~ bacillus thuringiensis, in addition to
intracellular endotoxin, form an extracellular, water-soluble
exotoxin which is stable unaer heat. The term exotoxin relates in
this case to an active substance of living cells which is dis-
charged or secreted into the medium while the endotoxin contrary
thereto is set free only after the cells have been completely
dissolved.
So as to facilitate a better understanding of the present in-
vention the characteristics of the above-mentioned toxins are
deæcribed in greater detail as follows:
,
~he Cr~stalline Endotoxin
A protein-containing crystalline inclusion has been isolated
f~om the sporulated culture of bacillus thuringiensis by HANNAY
and ~I~Z-JAMES (C.~. EANNAY and P. ~ITZ-JAMES, Can J. Microbiol.,
volume 1, pp. 694 to 710, 1955). ~he electron-microscopic studies
have sho~m that in the course of the formation of spores of
bacillus thuringiensis bacteria, an asporal crystalline body was
developed together with the spores~ It was concluded that a new
cont~d.
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~8S326
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relation exists between the formation of the~ spores and the
endotoxin crystals. Recent research indicated that the crystalline
endotoxin is composed of a high molecular protein component and
a Rilicon sl~eleton. ~he spores and the endotoxin crystals may be
recovered from the culture medium in the sediment of a high-speed
centrifuge. ~he separation of the crystalline endotoxin from the
spores was achieved in different ways, e.g. by fractionated sedi-
mentation, step-by-step sedimentation, and separation in a two-
phase system.
The Exotoxin
, . .
In addition to the crystalline endotoxin, certain bacillus
thuringiensis strains secrete in the course of the vegetative
gro~ing phase a toxic fraction into the medium which is chemically
related to the adenine nucleotides. No relation exists between
the generation of crystalline endotoxin and e~otoxin. According
to tests, exotoxin can be formed only by defined generlc types
of special bacillus thuringienæis varieties. ~he method of pro-
ducing exotoxin has been developed by De ~ARJAC. According to
said method, bacillus thuringiensis is cultivated in a medium
containing mineral salts in addition to 0.75~ peptone and 1%
glucose, namely for 70 hours at 30~.
~ he preparation of microbial insecticides according to the
present invention is preferably carried out in the following
stages:
A) Cultivation
~ he cultures are obtained based on the spores of a strain
of bacillus thuringiensis bacteria. (ATCC 10792 E.A. STEINHAUS-
O. MATTES) (N.R. SMITH USDA 996~,FROM MEDITERRANEAN FLOUR MOTH~
EPHESTIA KUEHNIELA, USDA MISC., PUBL. 559:51(1946) USDA AGR.
MONOGR. 16:67 (1952).
cont~d.
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. The nutrient mediu~ for preparing the vegetative inoculum in
shaking bottles contains the following components:
bacto-trypton (T7Difco") 0.8 % by weight
glucose 2.5 % " ~'
NgS04. 7H20 0.2 % " "
4 7 2 0.2 % ~
. ~e2(S04)3 0.2 ~ " "
~nS04. 5H20 0.1 ~0 " '~ . - . .
The medium for agar slant cultures was the same as above but
including also an addition of 2% agar.
.
Good results have been obtained with the cultivation in the
following media:
. Medium I:
molasses (50~ solid material) 1~4 ~0 by weight
yeast 0,3 ~O~t "
(~H4)2S04 0.1 ~0'' "
C.S.~. (maize-macerating liquid) 0.1 % " "
CaC03 0.1 ~o 11 n
The vH after the sterilization was 6.8.
Or with the medium containing starch instead o~ molasses:
edium II:
starch 1.3 ~0 by weig~t
yeast 1.0 ~o " "
2 4 0.4 % ll ~
C.S.~. (maize-maceratin~ liquid) 0.2 % " "
CaC03 0.8 ~0 "
The pH after sterilization was 6.8.
: cont'd.
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The nutrient medium for the vegetative inoculum (300 ml)
was contained in sterilizable bottles with flat bottoms and
1 liter filling capacity. The nutrient medium contained in
said bottles was sterilized for 45 minutes at 121C. The
glucose was sterilized separately according to the Tindali-
zation method and aseptically added to the sterilized medium.
The nutrient medium for the cultivation in the fermentators
was prepared and sterilized in portions in the fermentator for
45 minutes at 121C. Cultivation was carried out in fermen-
tators made of stainless steel. The culture was rotated at150 r.p.m.; ventilated with 0.5 liter per liter of culture per
minute; the incubation temperature was from 28 to 30C. The
application of an "anaerobic shock" was carried out by dis-
continuing the ventilation for 3 to 6 hours in the 12th hour
of submerged cultivation. The timing for interrupting the
ventilation was achieved with the help of an oxygen analyzer.
The cultivation may be carried out without any early lysis of
cells and without applying the afore-stated "anaerobic shock"
if in the course of the cultivation the pH is regulated in
such a way that it will not fall below 6.3. The formation of
spores and the simultaneous setting free of endotoxin crystals
ends after 35 to 40 hours of cultivation.
B. Separation and Purification
After the bacteria have completely dissolved while spores
and endotoxin crystals have been set free at the same time,
the separation of the spores and the crystalline endotoxin
from the exotoxin is carried out. Water-soluble exotoxin is
preferably removed by means of dialysis. This method results
in a mixture of spores and crystalline endotoxin which is not
toxic to mammals. The presence or absence of water-soluble
exotoxin was determined by the biological test described by
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BOND et al. A nutrient medium for the biological test was
prepared by heating the mixture comprising 10 g pulverized
agar, 500 ml fresh milk, and 7 g yeast. A 10 ml-sample was
subsequently placed into a Petri dish having a diameter of
9 cm and filled already with 250 u liter of the test solution.
As soon as the gels had solidified, 20 eggs of musca domestica
were added. The plates were kept for 48 hours in an incubator
at 30C, and the development of larvae and the disturbance of
the agar gel were observed (R.P.M. BOND, C.B.C. BOYCE and
10 S.J. FRENCH, Biochem. J., volume 114, pp. 477 to 488, 1969).
The resulting product, which is freed from water-soluble -
exotoxin, may be processed and used in the customary manner
for the preparation of insecticides.
C. Drying
The drying step may be carried out according to any one of
the following methods:
By admixing the resulting mixture comprising spores and
endotoxin crystals with Bentonitic clays and placing the blend
in the form of thin films in a ventilated dryer equipped with
plates, at a temperature of about 40 to 45C;
by suspending the resulting mixture in water and drying in
a rotating or pneumatic dryer;
by spraying the resulting mixture in a spray dryer.
D. Crushing and Screening
If the mixture is dried in a spray dryer the resulting
product requires no further crushing. When drying the mixture
in ventilated dryers on plates, the product is crushed in a
device of the Alpina type and screened using a sieve with a
fine mesh conforming at least to mesh No. 180.
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E. Determination of the Effectiveness
The effectiveness was determined according to the following
methods: by counting the living spores present according to
the plate method and by carrying out bio-tests on test
insects.
The resulting products are normed according to the
standards established by the French Laboratoires de Lutte
Biologique et de Biocinetique de la Miniere (I.N~R.A.).
The present invention will be more clearly understood with
the help of the description of the following examples which,
however, do not in any way constitute any limitation of the
present invention:
EXAMPLE 1
Based on the Berliner variety of the bacillus thuringiensis
culture, the cultivation is carried out according to the method
herein described using a liquid, ventilated medium, namely
medium I (see above). The incubation temperature applied in
the course of the submerged cultivation is about 28C. The
culture is put into motion at 150 r.p.m.; the rate of venti-
lation is 0.5 liter per liter per minute. The ventilation isinterrupted for 3 to 6 hours in the 12th to 14th hour of
cultivation. The timing for interrupting the ventilation was
determined with the help of an oxygen analyzer; ventilation
was interrupted as soon as the percentage of solubility and
the oxygen consumption decreased, and the pH fell to about
5.8. After from 3 to 6 hours, the ventilation was continued
at the same rate of 0.5 liter per liter per minute. The culti-
vation without early lysis may be carried out without anaero-
biosis if the pH of the medium is maintained within the range
of 6.3 to 6.5~ The submerged cultivation is continued until
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~0853269
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the spores and the crystalline endotoxin are yielded or trans-
ferred into the medium (35 to 40 hours on the average). ~he
culture medium is subsequently placed into the re^eptacle in whic~
the purification is carried out by removing the water-soluble exo
toxin. Said purification takes place by means of dialysis. Eor
this purpose it is possible to use a device as shown in the en-
closed figure. ~he culture medium is transferred from the fermen-
tator to receptacle A which is equipped ~lith an agitator e and a
dialysis diaphragm a. This diaphra~m may be colloidal or of any
type as customarily used in practical life. ~he dialysis ta~es
place more rapidly if deionized water b is added to receptacle ~.
lhe presence of absence of water-soluble eYotoxin is determined
by the afore-described bio-test. A powdery fillin~ material is
added to the resulting mixture of spores and cr~stalline endo-
toxins, and the mixture is then dried in a drying kiln on plates.
~he resulting product is gro~md in a crusher of the Alpina type,
and then screened in order to obtain a powder.
. '
Example 2
~ he method is carried out as defined in Example 1, however,
the nutrient medium II (see above) is used for the submerged cul-
tivation in the fermentator.
..: .
` ~ Example 3
.~
~ he method is carried out as defined in Example 1, however~
after the submerged cultivation of the bacteria and execution of
¦ the dial~sis, i.e. after the exotoxin has been removed, the resul
ting product in dried in a centrifugal dryer and subsequently
ground in an Alpina mill.
. .
cont'd.
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XAMPLE 4
The method is carried out as defined in Example 1, however,
the product obtained after the submerged cultivation of the
bacteria and execution of the dialysis is sus~ended again in a
defined amount of water, and this suspension is spray-dried in
a spray dryer. The temperature within the dryer was from 90
to 100C. The best suitable devices for this purpose are
spray dryers equipped with disks which rotate at from 10,000
to 15,000 r.p.m. The product is a very fine powder which
needs neither grinding nor screening. The exotoxin is not
present in this end product.
The present invention may be applied or utilized also for
other varieties of the same type of bacteria, for example the
sotto variety of bacillus thuringiensis, the subtoxicus
variety of bacillus thuringiensis. All these bacteria are
described in detail in the relevant literature.
One advantage of the present invention lies in the higher
effectiveness of the resulting product and the excellent
purity of this product with respect to exotoxin combined with
high yields of spores and crystalline endotoxins.
Ii 108S326
Insecticidal Activity of the Active Com~onent, BACTUCIDE
The insecticidal activity of the active component embracing
the spores of the Bacillus thuringiensis bacteria bacteria and the
crystal endotoxin can be regarded in two ways: ¦
- as the number of spores of Bacillus thuringiensis bacteria i
a gram of the active component, and
- as inte~national units (IU).
The active component as a part of our formulations, shows the
following activity:
101 spores/g of active component;
6000 and 15,000 IU/mg of the active component.
1000 IU/mg is the value of the product showing the same
insecticidal activity as a standard sample E-61 (a mixture of
spores and crystals of endotoxin of Bacillus thuringiensis
bacteria tested on the insect Ephestia Kuhniella). With the
active component the following formulations are prepared:-
Powdered formulation "BACTUCIDE-P"
4.0% of the active component 15,000 IU/mg
25.0% aerosil (vesalon)
5.0% jugopon
66.0% china clay
Liquid formulati_n ''BACTUCIDE-S"
8.0% of the acti~e component 6000 IU/mg
1.0% carboxymethyl cellulose
0.3 emulgator
3.0~ petrol-hydrocarbon
87.7% water
pH 6, 8-7, 2
cont'd.
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Formulation of BACTUCIDE-G granules
98.0% of granule (calcium carbonate)
2.0~ of the active component (2.4 109 spores/g)
The biologic insecticide "~ACTUCIDE" is used especially against
insects of the Lepidoptera group.
The quantity of liquid formùlation of BACTUCIDE is 0.8-1.5 litres
per ha, in that the dilution of this has to occur ~before the use)
in water quantity of 500-1000 litre. The dilution depends on the
mode of application, the power of insects and the thickness of
plants. The product can also be applied from air.
The powder formulation of BACTUCIDE-P has the same application as
a liquid formulation in quantity of 0.6-l.O kg~ha.
The formulation in form of granule (BACTUCIDE-G) is exclusively
applied against insects Pyrausta nubilalis (corn). The quantity
for application is 30 kg/ha.
BACTUCIDE is easily miscible and is compatible with chemical
insecticides.
The examples of mixing the chemical insecticides with biologic
insecticide BACTUCIDE are as follows:
Malathio~ 0.2% + Bactucide 0.12%
Dimethoate O.1-0.2% + Bactucide 0.15%
Sevin O.1-0.2% + Bactucide 0.15%
The same effects can be obtained with lower concentrations of
chemical insecticide in that the toxicity is lowered. Also
other chemical insecticides are compatible with Bactucide, as
for example methylparathion, diazinon, DDT, Pyrethrum, Phosdrin
etc.
We tested approximately 50 insects. The following tables list the
insects sensitive to BACTUCIDE and the results obtained on these
insects.
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The activity of the liquid formulation of BACTUCIDE-S on
some important insects.
Table_l.
The activity of BACTUCIDE on the on the insect Trycho~lusia ni
(cabbage).
. _
Quantity% of killed larva Total of larva
lit/haTESTING in days
tested
1-5 5-7 10
0.2 20 27 90 180
0.5 63 80 112 220
1.0 75 92 200 222
Control -- -- --- 650
Table 2.
The activity of BACTUCIDE on the insect Hyponomeuta mallinellus
(apple).
- . .
Quantity Insectarium Field Tests
- -- . - .
llt/ha ~ mortality corr. mort. ~ mort. corr. mort.
.. . ... ... _ ,
0.4 15 13.8 12 11.05
0.6 32 31.2 31 30.2
1.0 85 84.2 86.0 85.2
1.5 100 100 100 100
' ' ' ' " '
cont'd.
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Table 3.
The activity of BACTUCIDE on the insect Heliothis virescens
and Heliothis zea (tobacco and cotton).
.. . . . _ _ _ , .. .. .... . _
Concentration Days Cotton Tobacco
lit/ha
_ __ %_mortal_ y _ % mortality
0.2 4 30 75
0.5 6 50 100
1.0 8 95 100
1.5 10 10~
The activity of formulation of granule BACTUCIDE-G
Table 4.
:
- The activity of BACTUCIDE-G on the insect Pyrausta nubilalis
(corn).
Material Dose N %of plant Average Effective-
with living N of ness of
(formulation) kg/ha plant larva larva/ BACTUCIDE- .
plant G in %
.
Bactucide-G 30 80. 41.0 0.9 82 ~ ~
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: ' ' , ' ~ ' . : -
