Bacterial insecticide and production thereof

A bacterial insecticide containing an insecticidal substance produced by a practically reversionless asporogenous mutant of Bacillus thuringiensis, especially Bacillus thuringiensis serovar. kurstaki 290-1 and the process for the production in addition to a practically reversionless asporogenous mutant of Bacillus thuringiensis, especially Bacillus thuringiensis serovar. kurstaki 290-1 and the process for the production.

This invention relates to an insecticide containing an insecticidal 
substance produced by a practically reversionless asporogenous mutant of 
Bacillus thuringiensis and production thereof. Furthermore, it relates to 
a practically reversionless asporogenous mutant or Bacillus thuringiensis 
and the process for the production. 
Chemical insecticides generally used have many disadvantages in, for 
example, having adverse influence on men, beasts and fishes, toxic 
residue, induction of drug-resistant insects and the like. Many research 
and development programs have been carried out to find an alternative 
insecticide. Research for bacterial agricultural chemicals utilizing 
Bacillus thuringiensis has been conducted as alink in the chain of the 
programs and the research has been partly put to practical use. It is 
based on utilization of insecticidal activity of .delta.-endotoxin 
(abbreviated to as .delta.-toxin hereinafter). In general, Bacillus 
thuringiensis produces crystals of .delta.-toxin and spores in the cells. 
The crystals and spores are usually released out of the cells when cell 
walls naturally rip at the final stage of cultivation and strengthen 
activity available as insecticide. The whole culture broth is usually 
dried and applied directly without separating the crystals from the spores 
when Bacillus thuringiensis is used as an insecticide because the 
separation procedure is very complicated. Unfortunately, however, the 
application is extremely restricted since there is the possibility that 
the spores in the preparation will be multiplied in the natural world 
after application and cause hazzard on the sericulture. The creation of an 
asporogenous strain of Bacillus thuringiensis was attempted to remove the 
defect and an insecticide containing Bacillus thuringiensis but not 
causing secondary contamination to silkworms was developed (Japan. Pat. 
Pub. (unexamined) No. 50/125022). 
The inventors of this invention, however observed in their investigation 
that 1-10 or more spores/ml were present in a fermented broth of Bacillus 
thringiensis asporogenous mutant discovered by the present inventors. The 
spores are likely to be derived from reverse mutation of the mutant. The 
same reverse mutation is confirmed in all reported asporogenous mutants. 
The inventors, thereupon, aimed to create a completely reversionless 
asporogenous mutant which had completely lost sporulation and renaturation 
abilities and develop an insecticide giving no adverse secondary 
contamination to silkworms, i.e. no secondary multiplication. As the 
result, a practically reversionless asporogenous mutant was obtained by 
mutation with ethyl methanesulfonate. The inventors have completed this 
invention by confirming that the fermented broth of the mutant does not 
contain any detectable spore and is obviously utilizable as safe 
insecticidal substance. Thus, a reversionless asporogenous mutant was 
created by the present inventors for the first time. This invention 
provides a novel and useful bacterial insecticide using the said mutant 
and the process for the production in addition to a novel practically 
reversionless asporogenous mutant and the process for the production. 
The above practically reversionless asporogenous mutant can be obtained by 
treating the spore of an asporogenous mutant with a mutagen inducing 
deletion mutation, e.g. alkyl alkanesulfonate, especially ethyl 
methanesulfonate. The parent strains to be used are asporogenous mutants 
of Bacillus thuringiensis, e.g. Bacillus thuringiensis serovar. kurstaki, 
aizawai, thuringiensis, alesti, galleriae, tolworthi, subtoxicus and the 
like; kurstaki being preferred. The asporogenous mutants are disclosed in 
Japan. Pat. Pub. (unexamined) No. 50/125022, J. Invertebr. Pathol. 25, 
355-361 (1975), Eur. J. Biochem. 18, 226-239 (1971) and Can. J. Microbiol. 
24, 492-494 (1978). An example for preparing a practically reversionless 
asporogenous mutant is shown below. 
EXPERIMENT 1 
A suspension of spores of Bacillus thuringiensis serovar, kurstaki IK (10 
ml, ca. 10.sup.8 spores/ml, 0.1M phosphate buffer solution, pH 7.0) was 
heated at 70.degree. C. for 15 minutes. After cooling to room temperature, 
ethyl methanesulfonate.sup.1) (0.25 ml) was added thereto. The mixture was 
shaken at 28.degree. C. for 15-18 hours and centrifuged under aseptic 
conditions (.times.4000, 20 minutes, 5.degree. C.). The precipitate was 
washed with a sterile saline solution by centrifugation two times and then 
suspended in 0.1M sterile phosphate buffer solution (pH 7.0, 5.0 ml). The 
suspension was used to rub the surface of 30 Petri dishes into which 
CL-agar medium.sup.2) (15 ml) had been poured in the proportion of 100 
.mu.l of the suspention to a Petri dish. After incubation at 28.degree. C. 
for 2-5 days, the resultant colonies were isolated about 200-300 per lot 
by random isolation method. 
The isolated colonies were inoculated on a nutrient agar disks and after 
incubation at 28.degree. C. for 3-5 days the presence of spore and 
.delta.-toxin was checked. On the other hand, the colonies were incubated 
on CL-agar disks, incubated at 28.degree. C. for 1 day and then kept at 
5.degree. C. 
In the above test, 42 colonies did not form any detectable spore and had 
good productivity to .delta.-toxin. They were incubated on both 10 ml of M 
medium.sup.3) and S medium.sup.4) and incubated at 28.degree. C. for 3-4 
days under shaking. About 5 ml each of incubated media were heated at 
70.degree. C. for 20 minutes and number of spores per 1 ml were counted. 
The rest of the heated media was centrifuged, washed with water and 
lyophilized. Insecticidal activity of the thus-obtained cell to silkworms 
was measured. Besides, the steadiness of .delta.-toxin production by the 
test strains was also observed. 
Four strains were selected owing to their nature, i.e. no formation of 
spore, abundant production of .delta.-toxin and stability. The strains 
were inoculated on S medium (100 ml) in Sakaguchi flasks and incubated at 
28.degree. C. for 3-4 days. Incubation was repeated from 7 to 17 times and 
number of spores and producibility of .delta.-toxin per 5 ml of medium 
were measured. As the result, strain 290-1 was obtained, of which the 
spore formation was not observed during the incubation over 17 
generations. 
Notes: 
(1). Ito, J. and J. Spizizen: Radiation Research 13, 93-96 (1971) 
(2). CL-agar medium: 0.25% glycerol, 0.5% polypeptone, 0.5% casein, 0.3% 
sodium chloride, 1.25% agar. 
(3). M medium: 0.5% glucose, 1.0% polypeptone, 0.5% lactocasein, 1.0% cane 
molasses, 0.3% sodium chloride. 
(4). S medium: 3.0% starch, 3.0% soybean meal, 1.5% corn steep liquor, 0.1% 
sodium carbonate, 0.3% ammonium sulfate. 
The above strain 290-1 was named Bacillus thuringiensis var. kurstaki 290-1 
and has been deposited in Agency of Industrial Science & Technology, 
Fermentation Research Institute and 1--1, Higashi 1-cho, Yatabemachi, 
Tsukuba-gun, Ibaraki Pref. Japan under FERM-P No. 5794 since Dec. 3, 1980. 
The strain also has been deposited in American Type Culture Collection in 
12301, Parklawn Drive, Rockville, Md., U.S.A. under accession number 31813 
since Feb. 25, 1981. Besides, the name is correctly revised to Bacillus 
thuringiensis serovar. kurstaki 290-1 in this specification according to 
rule of nomenclature. 
The parent strain of strain 290-1 was isolated in a test field of Shionogi 
& Co., Ltd. in Japan and identified to be the same as a strain HD-1 being 
recognized as Bacillus thuringiensis serovar. kurstaki from the 
description in J. Invertebr. Pathol. 11, 335-347 (1968), ibid. 15, 139-140 
(1970), ibid. 15, 232-239 (1970) and ibid 22, 273-277 (1973). 
Strain 290-1 has the same properties as the parent strain except for the 
irreversibility of spore formation. Strain 290-1 produces almost the same 
amount of .delta.-toxin as the parent strain and does not form any spore 
even in 17 times of subinoculations. Additionally, no spore was detected 
neither in 30.5 ml of S medium after 3 days incubation nor 45 ml of M 
medium after the same incubation period. The results of irreversibility 
test of strain 290-1 are shown below. 
EXPERIMENT 2 
Strain 290-1 was inoculated on S medium (100 ml) in a Sakaguchi flask and 
incubation at 28.degree. C. for 4 days was repeated 17 times. The 
fermented media of each time were heated at 70.degree. C. for 15 minutes 
and transferred to a nutrient agar medium. After incubation at 28.degree. 
C. for 3-4 days, formation of spore was checked. Simultaneously, 
insecticidal activity was measured with lyophilizate of precipitate of 
centrifugation. The assay for measuring the activity has been disclosed by 
Nishiitsutsuji-Uwo et al. in J. Invertebr. Pathol. 29, 162-169 (1977). The 
result is shown in Table 1. 
TABLE 1 
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Incubation Frequency 
1 2 3 4 5 6 
______________________________________ 
Number of Spore/ 
0/1 0/5 0/5 0/5 0/5 0/2 
Number of Petri dish 
Relative Activity/ml 
874 1036 645 1065 870 2152 
______________________________________ 
Incubation Frequency 
7 8 9 10 11 12 
______________________________________ 
Number of Spore/ 
0/2 0/2 0/2 0/2 0/2 0/3 
Number of Petri dish 
Relative Activity/ml 
1122 999 2088 2034 1533 733 
______________________________________ 
Incubation Frequency 
13 14 15 16 17 
______________________________________ 
Number of Spore/ 
0/3 0/3 0/3 0/3 0/3 
Number of Petri dish 
Relative Activity/ml 
647 508 1357 1164 1067 
______________________________________ 
EXPERIMENT 3 
The broth of strain 290-1 preliminarily incubated on a CL-agar medium at 
28.degree. C. for 1 day was inoculated on a nutrient broth and incubated 
at 28.degree. C. for 1 day under shaking. The fermented broth was 
inoculated on an M medium (60 ml) and an S medium each in a 300 ml 
Erlenmeyer flask at the final concentration 2% and incubated at 28.degree. 
C. for 3 days. Both 15 ml of the fermented media were transfered into 
non-necked test tubes with caution not to touch the wall and heated at 
70.degree. C. for 30 minutes. The fermented M medium was portioned into 1 
ml each on 5 Petri dishes, 1.5 ml each on 20 Petri dishes and 2.0 ml each 
on 5 Petri dishes respectively. The fermented S medium was portioned into 
0.5 ml each on 11 Petri dishes and 1.0 ml each on 25 Petri dishes 
respectively. All dishes were incubated at 28.degree. C. for 2.4 days and 
no formation of colony was observed. 
Accordingly, it was confirmed that spore was not formed in 45 ml of 
fermented M medium nor in 30.5 ml of fermented S medium. The result of the 
test was converted into as the ratio to number of living cells and cell 
weight, which are shown in Table 2: In the same manner, a practically 
reversionless asporogenous mutant can be obtained from other varieties by 
the usual method. 
TABLE 2 
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M medium S medium 
______________________________________ 
Number of Spore/ 0/2.1 .times. 10.sup.10 
0/5.1 .times. 10.sup.9 
Number of Living Cell 
Number of Spore/ 0/90 mg 0/549 mg 
Cell Weight 
______________________________________ 
As shown above, the reversionless asporogenous mutant prepared by this 
inventors does not form any spore practically. Thus, the fermented broth 
is utilized as insecticide in itself. Besides, "insecticidal substance" of 
this invention includes not only the free crystalline .delta.-toxin above 
mentioned but also the living cells of practically reversionless 
asporogenous mutant of Bacillus thuringiensis, especially Bacillus 
thuringiensis serovar. kurstaki 290-1, containing the above crystals, the 
dead cells and the mixture, fermented broth containing bacteria cells 
and/or the free crystals, condensate of the fermented broth, their dried 
correspondents and the like. 
The production of the insecticidal substance of the above practically 
reversionless asporogenous strain follows the methods of producing the 
insecticidal substance of the parent strain. The insecticidal substance 
can also be produced by the method disclosed in Japan. Pat. Pub. 
(unexamined) No. 50/125022 and the method by Dulmage, H. T. in J. 
Invertebr. Pathol. 22, 237-277 (1971). More practically, a practically 
reversionless asporogenous mutant of Bacillus thuringiensis, especially 
Bacillus thuringiensis serovar. kurstaki 290-1 is inoculated on an organic 
medium being abundant in nitrogen sources and carbon sources. The yield of 
.delta.-toxin is remarkably increased when the medium contains potassium 
ion at the final concentration being about 2-50 mM, more favorably 3-30 
mM. Additionally, production of .delta.-toxin is largely affected by 
aeration volume, especially oxygen supply. The fermentation under 
sufficient oxygen supply brings about rich production of .delta.-toxin. 
Fermentation may be continued at about 25.degree.-30.degree. C. for about 
2-5 days under heavy aerobic condition. There are exemplified sucrose, 
maltose, glucose, fructose, can molasses, beet sugar, corn steep liquor 
and the like as carbon sources. Nitrogen sources are, for example, 
ammonium sulfate, ammonium chloride, cotton seed powder, soybean meal, 
casein hydrolysate and the like. Minerals and vitamins may be added, if 
necessary. Fermentation is effected under aerobic condition as noted 
above. Submerged aeration fermentation is preferred in a large scale 
production. 
After fermentation is completed, generally used methods such as 
centrifugation, filtration and the like may be applied in the case that it 
is preferred to isolate insecticidal substance such as free .delta.-toxin 
crystals, cells and the like. It is also appropriate to apply ordinary 
concentration and drying methods in order to pulverize the fermented broth 
containing cells and/or free crystals. Practically, spray-drying method 
popularly used in these days can be preferrably be applied because fine 
powder can be obtained without decrease of activity. In the method 
wettable powder can be obtained at one operation by adding a fixing agent, 
a spreader, a diluent and the like to a cell floating liquid at 
spray-drying. Ordinary sterilizing method can be used, if necessary, when 
the product obtained by the above methods contains living cells. There are 
exempified heating, supersonic treatment, irradiation with X ray and the 
like as physical sterilization, treatment with formalin, hydrogen 
peroxide, sulfites, nitrofurylacrylamide, furylfuramide, chlorine 
compounds, .beta.-propiolactone, nitrites, nitrates, surfactants, ethylene 
oxide, propylene oxide and the like as chemical sterilization and 
autolysis, treatment with phage, treatment with lysozyme as biological 
sterilization. Heating and chemical sterilization are convenient for 
industrial scale production among the above sterilization methods. 
Spray-drying method is particularly useful for pharmaceutical preparation 
in no necessity of sterilization process since the product is 
simultaneously heated. 
Thus obtained insecticidal substance is formulated into pharmaceutical 
preparations, for example, tablets, granules, powders, wettable powders, 
suspensions, emulsions, pastes and the like. There is added filler, for 
example, kaolin, bentonite, talc, diatomaceous earth, wheat meal and 
sugars, with spreaders, sufactants, stabilizers and the like during the 
preparation procedure, if necessary. Other insecticides, sterilizers, 
herbicides, plant growth regulatores, flavours, nitrients, and the like 
may be added, if desired, as long as it does not give adverse influence on 
the insecticidal activity. 
The insecticide obtained by this invention is effective against laravae of 
Lepidoptera such as Plutella maculipennis Curtis, Chilo suppressalis 
Walker, Cnidocampa flavescens Walker, Pieris rapae crucivora Boisduval, 
Momestra brassicae Linne, Papilio machaon hippocrates Felder et Felder, 
Chalcosia remota Walker, Stauropos fagi persimilis Butler, Arctia caja 
Linne, Pernara guttata Bremer et Grey and the like. The insecticide can be 
sprayed freely to rice fields, fields, forests and waste lands without 
regard for the hazzard to silkworms because it does not cause the 
secondary multiplication. 
The invention is exemplified more in detail in the following examples.

EXAMPLE 1 
KB-5 medium: 1.0% potato starch, 0.5% glucose, 1.5% soybean meal, 2.0% 
Pharmamedia.RTM., 1.0% powdery pork meat, 0.2%, polypeptone, 0.03% 
magnesium sulfate heptahydrate, 1.0% calcium carbonate, 0.002% zinc 
sulfate heptahydrate, and 0.002% ferrous chloride. 
Bacillus thuringiensis serovar. kurstaki 290-1 (FERM-P No. 5794) was 
cultured on a nitrient agar medium at 28.degree. C. for one day, and one 
platinum-loopful of the resultant broth was inoculated on a nutrient 
medium (700 ml) in a 2 L Meyer flask, and cultured with shaking (180 
r.p.m.) at 28.degree. C. for 12 hours. The resultant culture broth was 
inoculated on a KB-5 medium (15 L) of the above mentioned composition in a 
30 L jar, and after culturing under aeration and agitation (400 r.p.m.) at 
28.degree. C. for 52 hours, the fermented broth was treated by sharples 
centrifugation (.times.13,000 g) to give 15.2 mg/ml (dry weight) of 
insecticidal material (activity: 3070/ml). The activity is measured by the 
method as described in J. Invertebr. Pathol. 29, 162-169 (1977) 
(Nishiitsutsuji-Uwo, J., et al.). 
EXAMPLE 2 
The culture was carried out in the same manner as in Example 1 employing 
the S medium (its composition is mentioned above) instead of the KB-5 
medium for fermentation to give 14.7 mg/ml (dry weight) of insecticidal 
material (activity: 2279/ml). 
EXAMPLE 3 
The cells of bacterium obtained in Example 2 (the insoluble precipitate 
after centrifugation) were suspended in water so that the concentration of 
the insoluble component was about 5% w/v. The resultant suspension was 
spray-dried with Minispray (made by Yamato Scientific Co. Ltd., model 
DL-21). Minispray is maintained under the following conditions: inlet 
temperature: 190.degree. C.; outlet temperature: 90.degree. C. or lower; 
flow rate of solution: 3-4 ml/min.; flow rate of hot air: 0.4-0.5 m.sup.3 
/min.; spraying pressure at the binary nozzle: 3.0-1.0 Kg/cm.sup.2 ; air 
rate at the nozzle: 13-9 L/min. 
The activity and particle size of the resultant powder specimen are shown 
in Table 3 in comparison with the result of a powder specimen prepared by 
lyophilization of a part of the above suspension followed by pulverization 
with a fluid energy mill (alias jet mill). Powder specimens prepared by 
the above two methods respectively are almost equal in activity and 
particle size. 
TABLE 3 
______________________________________ 
The 
Jet Mill 
The Spray-Drying Method Method.sup.(1) 
______________________________________ 
A. Binary Nozzle 
Spraying Pressure 
3.0 2.5 2.0 1.5 1.0 
kg/cm.sup.2 
Air Rate L/min. 
13 13 11 11 9 
B. Dried Powder 
Moisture % 8.7 7.3 6.9 4.9 6.2 5.5 
LC.sub.50.sup.(2) .mu.g/ml 
87 59 66 71 71 58 
Particle Size (R) %.sup.(3) 
&gt;10.mu. 8.6 8.8 6.9 8.4 8.9 6.0 
&gt;4.mu. 13.5 14.0 12.5 14.4 13.9 12.0 
&gt;2.mu. 25.6 26.3 23.8 28.0 26.8 24.7 
&gt;1.5.mu. 39.7 40.0 35.0 43.3 39.7 33.7 
&gt;1.2.mu. 53.7 52.5 49.0 56.3 54.3 49.5 
&gt;1.0.mu. 64.2 64.0 60.7 66.2 65.8 54.7 
&gt;0.8.mu. 75.2 75.3 73.2 76.8 76.4 66.4 
&gt;0.6.mu. 85.5 85.4 84.0 86.5 85.3 79.0 
______________________________________ 
Note: 
.sup.(1) The lyophilizate contained 2.7% moisture and the activity was 
LC.sub.50 67 .mu.g/ml. 
.sup.(2) LC.sub.50 shows 50% lethal concentration (.mu.g/ml) for 
silkworms. 
.sup.(3) Particle size was measured with an apparatus (CP50; Shimazu Co., 
Ltd.) of phototransmission type for measuring particle size distribution 
on the basis of centrifugal sedimentation. 
EXAMPLE 4 
In the same manner as in Example 2, the culture was made under aeration and 
agitation on the S medium for 54 hours to give insecticidal material 
specimen. And the resultant insecticidal specimen was suspended in water 
so that the concentration of the insoluble solid component was about 3%. 
The additives as shown in Table corresponding to one third amount of the 
insoluble solid component were added to the suspension and spray-dried. 
Calculated value of LC.sub.50 in this case is 86. 
The activity of the obtained specimens and the effect of sterilization are 
shown in Table 4. 
TABLE 4 
______________________________________ 
Sam- 
ple LC.sub.50 
Number of live 
No. Additive .mu.g/ml 
bacteria/mg 
______________________________________ 
1 Non 53 0 
2 Non 45 0 
3 Gum arabic powder 62 0 
4 Methyl cellulose (500 cps) 
57 0 
5 Polyvinylalcohol 75 0 
6 Carboxymethyl cellulose 
78 0 
7 Hydroxypropylmethyl cellulose 
84 0 
phthalate (pH 5.0) 
8 New Cargen NX 405H* 72 0 
9 Talc 70 0 
Lyophilized specimen 
65 -- 
______________________________________ 
*Made by Takemoto oil & fat Co. Ltd. 
EXAMPLE 5 
Powdery preparation prepared by combination of spray-dry powder (25 parts) 
from Bacillus thuringiensis var. kurstaki 290-1 obtained in Example 3 with 
talc (75 parts). The preparation may be used in an amount of 100 g or more 
per 10 are. 
EXAMPLE 6 
Spray-dry powder (25 parts) obtained in Example 3, sodium 
dodecylbenzenesulfonate (2 parts), sodium dinaphthylmethanedisulfonate (2 
parts) and a mixture (71 parts) of kieselguhr and clay are mixed and 
pulverized to give wettable powder. 
EXAMPLE 7 
Powdery preparation prepared by combination of the powder (30 parts) 
obtained by spray-drying together with carboxymethyl cellulose in Example 
4 with talc (70 parts).