JP2000316568A - New strain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a biologically pure cultured product of a new Bacillus thuringiensis strain excellent in insecticidal activity by producing CryIA(C) in a larger amount than that of CryIA(a) or in a large amount than that of CryIA(b) produced by the strain. SOLUTION: This biologically pure cultured product of Bacillus thuringiensis strain which is S197, S3158, S3159, S3212 or F810 capable of producing CryIA(c) in a larger amount than that of CryIA(a) or in a large amount than that of CryIA(b) produced by the strain. The CryIA(c) can be produced in an amount of at least 50% based on the total amount of the CryIA type proteins produced by the strain. An insecticidal factor selected from the group consisting of the Bacillus thuringiensis strain, a spore derived from the strain and a crystal toxin is preferably included to produce an insecticidal composition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a Bacillus thuringiensis having normal insecticidal activity.
ingiensis).

[0002]

BACKGROUND OF THE INVENTION Bacillus thuringiensis belongs to a large group of gram-positive, aerobic, endospore-forming bacteria. Unlike other very closely related Bacillus species such as B. cereus or B. anthracis, Bacillus thuringiensis (B. thuringiensis or B. anthracis)
t.) The majority of what is known so far is
During their sporulation process, they produce paraspore inclusions, which are commonly referred to as crystals due to their crystal structure. This crystal consists of a crystalline protoxin protein with insecticidal activity, the so-called δ-endotoxin.

[0003] These protein crystals contribute to the toxicity of B. thuringiensis to insects. δ-Endotoxin does not exert insecticidal activity after the crystals have been taken orally until they are dissolved in the intestinal fluid of the target insect. In most cases, the actual toxic components are released from protoxins as a result of proteolytic cleavage caused by the action of proteases from the gastrointestinal tract of insects. The crop can be treated by applying a pesticidally effective amount of B. thuringiensis, spore or protein toxin crystals, or a combination thereof. Unfortunately, such pesticidal compositions of B. thuringiensis are sensitive to such treatments, and will cause them to die easily.
It cannot be used on important crops in some markets such as spinach and mustard. In the United States, low volume sprays (1 lb / 10 gal / acre, or 1%) have been reported to kill Chinese cabbage and spinach, and are more damaging in Japan. Multiple sprays are even more damaging. Thus, the phytotoxicity of B. thuringiensis to certain plants is a clear and economically important consideration.

[0004] δ-Endotoxin of various B. thuringiensis strains has high specificity for particular target insects, particularly for various Lepidoptera, Coleoptera and Diptera larvae And by a high degree of activity against these larvae. A further advantage of the use of the B. thuringiensis strain δ-endotoxin lies in the fact that it is harmless to humans, other mammals, birds and fish.

[0005] Various insecticidal crystal proteins of B. thuringiensis have been classified on the basis of their similarity in activity spectrum and sequence. According to the classification published by Hofte and Whiteley (Microbiol. Rev., 53: 242-255 (1989)), known insecticidal crystal proteins were classified into four major classes. . In general, these major classes include activity spectra, namely CryI protein activity against Lepidoptera, CryII protein activity against both Lepidoptera and Diptera,
Defined by CryIII protein activity against Coleoptera and CryIV protein activity against Diptera.

[0006] In each major class, δ-endotoxins are grouped according to sequence similarity. Cr
The yI protein is typically produced as a 130-140 kDa protoxin protein, which is cleaved by proteolysis to produce an active toxin protein of about 60-70 kDa. The active portion of this δ-endotoxin is present in the NH ₂ -terminal portion of the full length molecule. Hofte and Whiteley (supra)
Converts known CryI proteins into six groups, namely IA
(a), IA (b), IA (c), IB, IC and ID (the proteins were therefore CryIE, CryIF, CryIG, CryIH and Cr
yIX).

[0007] Among the three types of CryIA proteins, CryIA (c)
Hofte and Whitele
y) (supra), identified as having the highest specific activity on nettle (Trichoplasia ni). Unfortunately, as exemplified by the B. thuringiensis strain HD-1, isolated and widely marketed in 1971, the known CryIA producing B. thuringiensis strain is preferentially To produce CryIA (b).

In conclusion, a new strain of B. thuringiensis has been identified which produces relatively high levels of CryIA (c), thereby exerting a high specific activity against lepidopteran pests, such as caterpillars. And the isolation and production of pesticidal compositions therefrom remains a long felt need in agricultural and crop production areas, but has not been met. Desirably, such improved strains exhibit reduced phytotoxicity.

[0009]

SUMMARY OF THE INVENTION The present invention relates to a Bacillus thuringiensis having insecticidal activity.
ringiensis).

[0010]

SUMMARY OF THE INVENTION The present invention has long been felt and fulfilled in the agricultural industry for a long time by providing new strains of B. thuringiensis that are useful for pesticidal purposes. Addressing the need.
One feature of the present invention is that the novel strains are derived from Trichoplastia ni (T.ni) and Spodoptera exigua (S.
exigua) exerts a significantly higher potency than the HD-1 strain. Another unique feature of the present invention is that these novel strains have a beneficial ratio of insecticidal proteins, especially CryIA
Significantly increased amount of CryIA (c) relative to the amount of type protein
Is to show the protein. Yet another unique feature of the present invention is that the new strain F810 is significantly better than strains present in plants that are not susceptible to mortality. An advantage of the present invention is that strains, their derivatives, variants and progeny, as well as pesticidal compositions containing such strains, and methods of using these strains and compositions in controlling pest erosion, In particular, it is a particularly effective insecticide against lepidopteran pests.

The present invention describes a novel CryIA-producing B. thuringiensis strain that expresses CryIA (c) in greater amounts than CryIA (a) or CryIA (b) expressed by this strain. A preferred embodiment of the present invention is a cottonseed flour substrate (cotto
(n seed flour substrate) when grown on a substrate such as CryIA (c).
(a), including a biologically pure culture of B. thuringiensis that produces an amount that is at least about 50% of the total amount of CryIA (b) and CryIA (c) proteins. . Desirably, this strain was grown under the same conditions
B. thuringiensis had at least 1.8 times greater insecticidal efficacy against Trichoplacina ni as measured by BIU / Qt than the potency of B. thuringiensis strain HD1, and was grown under the same conditions. BSU / Q rather than the efficacy of Ensis HD1
It has at least 1.6 times greater insecticidal efficacy against Spodoptera exigua as measured at t. Specific B. thuringiensis strains of the invention include S197, S3
158, S3159, S3212 and F810, and their derivatives, variants and progeny, as well as pesticidal compositions containing the strain and the use of the strain in controlling pest infestation.

The present invention also encompasses insecticidal compositions containing the B. thuringiensis strains disclosed herein or containing spores or crystalline toxins derived therefrom. Further, the present invention is a recombinant of B. thuringiensis transformed with heterologous DNA, wherein the recipient Bacillus thuringiensis strain comprises CryIA (c),
The amount of CryIA (a) produced by this strain or CryIA
Recombinants that can be produced in an amount larger than the amount of (b) are also included.

In the culture according to the present invention, (1) Cr
It is possible to produce yIA (c) in a larger amount than the amount of CryIA (a) or CryIA (b) produced by this strain,
It is a biologically pure culture of a B. thuringiensis strain.

In the strain according to the present invention,
(2) The B. thuringiensis strain described in (1) above, which is capable of producing CryIA (c) in an amount of at least 50% of the total amount of CryIA type protein produced by this strain. It is characterized by.

Further, in the strain according to the present invention,
(3) S197, S3158, S3159, S3212 or F810,
The B. thuringiensis strain described in (1) above.

In the culture according to the present invention,
(4) B. thuringiensis strain grown under the same conditions against Trichoplusia ni as determined by BIU / Qt when grown in cottonseed flour substrate
B. chew, which has a pest control efficacy at least 1.8 times greater than that of HD-1 and is grown against Spodoptera exigua under the same conditions as measured by BSU / Qt It is a biologically pure culture of a B. thuringiensis strain having a pest control efficacy that is at least 1.6 times greater than that of Lingiensis strain HD-1.

In the strain according to the present invention,
(5) S197, S3158, S3159, S3212 or F810,
The B. thuringiensis strain described in (4) above.

Further, in the insecticidal composition according to the present invention, (6) CryIA (c) can be used to produce Cry produced by this strain.
B. thuringiensis strain capable of producing a larger amount than the amount of yIA (a) or CryIA (b), the spores derived from the B. thuringiensis strain, and the B. thuringiensis strain A pesticidal composition comprising a pesticidal factor selected from the group consisting of:

Further, in the composition according to the present invention,
(7) The composition according to the above (6), wherein the strain is capable of producing CryIA (c) in an amount of at least 50% of the total amount of the CryIA type protein produced by the strain. Features.

In the composition according to the present invention,
(8) When the strain is grown in cottonseed flour substrate, BI
Has an efficacy against pest control as measured by U / Qt which is at least 1.8 times greater than the efficacy of B. thuringiensis strain HD-1 grown under the same conditions, and (6) having a pest control efficacy against Spodoptera exigua that is at least 1.6 times greater than that of B. thuringiensis strain HD-1 grown under the same conditions, as measured by BSU / Qt. ).

In the composition according to the present invention,
(9) further comprising at least one drug selected from the group consisting of a carrier, a surfactant, an agricultural auxiliary, a diluent, and a spraying aid suitable for use in combination with B. thuringiensis; It is a composition according to the above (6).

Further, in the composition according to the present invention,
(10) The composition according to (6), wherein the insecticidal factor is the B. thuringiensis strain.

In the composition according to the present invention,
(11) The strain is S197, S3158, S3159, S3212 or F81
The composition according to the above (11), which is 0.

In the composition according to the present invention,
(12) The composition according to (6), wherein the insecticidal factor is a spore derived from B. thuringiensis.

Further, in the composition according to the present invention,
(13) The composition according to (6), wherein the insecticidal factor is a crystal toxin derived from B. thuringiensis.

In the strain according to the present invention, (1)
4) B. thuringiensis strain contains foreign DNA that is capable of producing CryIA (c) in greater amounts than the amount of CryIA (a) or CryIA (b) produced by the strain. It is a recombinant B. thuringiensis strain.

In the strain according to the present invention, (1)
5) When the strain is grown in cottonseed flour substrate, BIU /
Have a pest control potency as measured by Qt that is at least 1.8 times greater than the potency of B. thuringiensis strain HD-1 grown under the same conditions, and (14) having a pesticidal efficacy against Spodoptera exigua that is at least 1.6 times greater than that of B. thuringiensis strain HD-1 grown under the same conditions, as measured by / Qt. The recombinant B. thuringiensis strain described above.

In the Bt according to the present invention, (1)
6) The strain is the recombinant B. thuringiensis according to the above (14), wherein the strain is S197, S3158, S3159, S3212 or F810.

In the Bt according to the present invention, (1)
7) the foreign DNA encodes an insecticidal protein,
It is the recombinant B. thuringiensis according to the above (14).

In the method according to the present invention, (1)
8) CryIA (c) can be produced in a larger amount than the amount of CryIA (a) or CryIA (b) produced by the strain
An insecticidal composition comprising an insecticidal factor selected from the group consisting of a B. thuringiensis strain, a spore derived from the B. thuringiensis strain, and a crystal toxin derived from the B. thuringiensis strain, It is a method of treating a plant, comprising applying to the plant or the soil in which it grows in an effective amount.

In the method according to the present invention, (1)
9) The method according to the above (18), wherein the insecticidal factor is the B. thuringiensis strain.

In the method according to the present invention, (2)
0) The method according to (18), wherein the strain is S197, S3158, S3159, S3212 or F810.

In the method according to the present invention, (2)
1) The method according to the above (18), wherein the insecticidal factor is a spore derived from B. thuringiensis.

In the method according to the present invention, (2)
2) The method according to (18), wherein the insecticidal factor is a crystal toxin derived from B. thuringiensis.

In the method according to the present invention, (2)
3) The method according to the above (18), wherein the plant is susceptible to withering and the B. thuringiensis strain is F810.

[0036]

DETAILED DESCRIPTION OF THE INVENTION B. thuringiensis strains are isolated from soil and screened to determine their CryIA-type protein expression profiles. The soil isolate is cultured using media components and fermentation techniques common in the art of B. thuringiensis. For example, B. thuringiensis describes the method described in Lee et al. (Microbial Biomass (1979, AH
Rose (eds.), Academic Press, pp. 91-114)
It is produced in large quantities by submerged culture fermentation using such processes. A typical growth medium contains one or more of the following combinations suitable for growing B. thuringiensis in culture: fishmeal, cottonseed flour, soy flour, molasses, diammonium phosphate and starch. .

After fermentation, the broth can be sprayed directly onto the plant or soil. Alternatively, bacteria, spores and crystalline toxins are separated and collected by standard methods, such as, for example, centrifugation, filtration, spray drying, or vacuum drying. as a result,
The resulting isolated spore-crystal fraction is very stable and can be stored, analyzed and used directly as pesticides or incorporated into other pesticide products. For example, CryIA (a), CryIA (b) by methods routinely used by those skilled in the art, such as biochemical, (enzymatic) assays, or biological assays to determine insecticidal efficacy.
And the relative amounts of CryIA (c) were measured. Figures 1 and 2
The chromatographic profiles for the CryIA proteins of strain HD-1 and S3159, respectively, are shown.

[0038] A level greater than the amount of CryIA (a) or CryIA (b), preferably at least 50% greater than the amount of either CryIA (a) or CryIA (b), more preferably CryIA (a).
Or at least 100% of any amount of CryIA (b)
Many, more preferably expressed CryIA (a) and CryIA
C expressing CryIA (c) at a level greater than the sum of the amounts of (b)
A ryIA-producing B. thuringiensis strain is selected. FIG.
Represents the control strain HD-1, and S3158, S3159, S3212, respectively.
1 shows the chromatographic profile of the CryIA protein of strains F810 and F810 (also called SAN 1401).

The spores and crystalline toxins produced by the B. thuringiensis strains of the present invention are useful as insecticides. These can be used, for example, for wet granulation, or for liquid concentrates, or surfactants, to facilitate application to plants or soil, or to improve the efficiency of the formulation.
Formulated into other formulations by the addition of dispersants, inert carriers or other ingredients or combinations thereof.

Thus, in a first embodiment, the invention relates to the ARS Patent Culture Collection on March 19, 1997 in accordance with the Budapest Treaty.
Provided is the novel B. thuringiensis strain S197, deposited under number B-21666, and their taxonomic equivalents.

In a second embodiment, the invention is based on the Budapest Treaty, as defined in the ARS Patented Culture Collection.
New B. deposited on March 19, 7 as NRRL number B-21667.
Provides Thuringiensis strain S3212, and their taxonomic equivalents.

In a third embodiment, the present invention is based on the Budapest Treaty, and has been incorporated into the ARS Patented Culture Collection by 199
New B. deposited on March 19, 7 as NRRL number B-21668.
Provides Thuringiensis strain S3159, and their taxonomic equivalents.

In a fourth embodiment, the invention relates to the ARS Patented Culture Collection, which complies with the preceding Budapest Treaty.
New B. deposited with NRRL number B-21669 on March 19, 7
Provides Thuringiensis strain S3158, and their taxonomic equivalents.

In a fifth embodiment, the present invention relates to the ARS patent culture collection according to the preceding Budapest Treaty.
New B. deposited on March 19, 7 as NRRL number B-21670.
Provides Thuringiensis strain F810, and their taxonomic equivalents.

These new strains exhibit the following characteristics:
Colony morphology-typical for B. thuringiensis,
Large colony, dull surface proliferative cell morphology-typical for B. thuringiensis, rod culture method-typical for B. thuringiensis flagella serotype 3a3b, Kurustaki encapsulation Body-two bipyramidals and cubes.
Alkali-soluble protein-SDS polyacrylamide gels show two major protein bands, approximately 130
And 70KD. Insecticidal activity-a novel B. trichoplastia ni using the third instar of Trichoplastia ni larvae as target insects.
The average potency of the Thuringiensis strain is two times that of HD-1.
Greater than twice. Protein expression: Chromatographic analysis of CryIA protein expression indicates that these strains express significantly higher levels of CryIA (c) compared to CryIA (a) or CryIA (b). (See FIGS. 1-3).

[0046] Strain F810 has another beneficial property that mortality-sensitive plants such as Chinese cabbage, spinach and mustard do not die. As used herein, the term "susceptible to mortality" refers to B. thuringiensis fermentation broth, which, when applied to a plant, causes browning, damage or necrosis of plant tissue, especially leaf tissue, and Plants that are susceptible to insecticide products derived from them. For example, such plants are damaged by the application of fermentation broth or insecticidal products of strain HD-1.

Derivatives, variants or progeny of such strains, including the deposited isolates or taxonomic equivalents, are genetically modified, for example, through the introduction of foreign DNA or by mutagenesis. Genetic engineering of B. thuringiensis is well known in the art and
No. 5,530,195 to Er et al., which is incorporated herein by reference in its entirety. By way of example, the novel deposited strain of the present invention is used as a host for receiving the CryIE (c) gene disclosed in the aforementioned US Patent No. 5,530,195 as foreign DNA.
The resulting recombinant B. thuringiensis expresses a unique pesticidal protein profile in which CryIA (c) is predominant compared to either CryIA (a) or CryIA (b), as well as recombinant Expresses the CryIE (c) gene product introduced by E. Li et al. (Nature, 353: 815-821 (1991))
It further details that genetic engineering of B. thuringiensis is well known in the art. Each of these references is incorporated herein by reference in its entirety. Methods for introducing nucleic acid sequences into host cells, such as one of the new deposited strains, are described in Do
U.S. Patent No. 5,186,800 to Schwer and Schurter et al. (Mol. Gen. Genet., 218: 177-181 (1989)), both of which are hereby incorporated by reference in their entirety. ing.

In another embodiment, the invention relates to an insecticidal composition comprising a B. thuringiensis strain according to the preceding description, in particular a spore of such a strain, for example an agriculturally acceptable carrier, An insecticidal composition is provided that is combined or combined with adjuvants such as diluents, surfactants, or adjuvants that facilitate application. The composition optionally comprises a fertilizer, a micronutrient supply, a plant growth agent,
Including other biologically active compounds selected from herbicides, pesticides, fungicides, bactericides, nematicides and molluscicides, and mixtures thereof. The composition comprises from 0.1 to 99% by weight of the B. thuringiensis strain of the invention, or a derivative or variant thereof, from 1 to 99.9% by weight of a solid or liquid adjuvant, and from 0 to 25% of a surfactant. % By weight. The composition exhibits pesticidal activity when administered to a plant or soil in a pesticidally effective amount.

In horticulture as well as in farming, crops are vulnerable to attack or damage caused by pests and / or infectious diseases, and also to competition with weeds.
In order to improve crop yields, measures have been taken to protect growing plants from harmful conditions such as reducing weeds, plant diseases, pests, nematodes and other crops. Mechanical measures such as plowing, removal of infected plants or weeds, pesticides such as herbicides, fungicides, reproductive agents, nematicides, growth regulators, ripening agents and pesticides can reduce crops. Widely used to protect. New isolated B. thuringiensis strains and insecticides derived therefrom have been used beneficially as pesticides.

A particularly useful use of the strains of the present invention is CryIA
(c) the amount of CryIA (a) produced by this strain or C
B. thuringiensis strain capable of being produced in an amount larger than the amount of ryIA (b), a spore derived from the B. thuringiensis strain, and a crystal toxin derived from the B. thuringiensis strain A method of treating a plant or soil comprising applying a pesticidal composition containing a selected pesticidal agent to a plant or the soil in which it grows, in an effective amount.

In a preferred embodiment of the present method, the plant is a plant important in the market, as exemplified below. In a preferred embodiment, the insecticidal agent is B. thuringiensis S197, S3158, S3159, S3212 or F81.
It is 0. In another preferred embodiment, the insecticidal agent is
Spores derived from one or more of these B. thuringiensis strains. In yet another embodiment, the insecticidal agent is a crystalline toxin from one or more of these B. thuringiensis strains. The preferred method is
Strains that are particularly useful in plants that are susceptible to withering
Use F810. This method is useful for plants that are susceptible to mortality such that their leaves would otherwise show substantial mortality (eg, using fermentation broth of strain HD-1 would kill approximately 20% of the leaves). Essentially does not cause death.
According to this method, very robust and marketable plants are obtained, and crop yields are substantially improved.

In another embodiment, the novel B. thuringiensis strains, or compositions containing them, are used to control the pesticides or pesticides of a particular species on the plants or crops to be protected or on the soil in which they grow. Along with chemicals (1993
Crop Protection C
chemicals Reference), Chemical and PharmaceuticalPr
ess, Canada), administered without loss of efficacy. It is compatible with most other commonly used agricultural spray materials, but should not be used in extreme alkaline spray liquors. It is administered in a pesticidally effective amount as a powder, suspension, wettable powder, or in any other material suitable for agricultural use.

Target crops to be protected within the scope of the present invention include, for example, the following plant species: cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beets ( Sugar beet and forage beet), forage (ducklings, fescue, etc.), stone fruits, fruits and small fruit trees (apple, pear, plum, peach, almond, cherry, strawberry, raspberry and blackberry),
Leguminous plants (green beans, lentils, peas, soybeans), oily plants (rapeseed, mustard, poppy, olives,
Sunflower, coconut, castor bean, cacao bean, Jerusalem artichoke, plants of the cucumber family (cucumber, pepo squash, melon), fibrous plants (cotton, flux, indoorsa, jute), citrus (orange, lemon,
Grapefruit, mandarin mandarin, vegetables (spinach, lettuce, asparagus, cabbage and other cruciferous plants, onions, tomatoes, potatoes, paprika), camphor trees (avocado, carrots, cinnamon, camphor), deciduous and coniferous trees (Eg, linden, yew, oak, alder, poplar, birch, fir tree, larch, pine), or corn, tobacco, nuts, coffee, sugar cane, tea tree, grape (vine), hops, banana and natural rubber trees, and Plants such as house plants (including Asteraceae plants).

Insecticidal Composition Containing Recombinant Bacillus thuringiensis Strain The novel B. thuringiensis strain of the present invention, or a recombinant B. thuringiensis strain derived therefrom, is usually used in the insecticidal composition. It can be applied in form and applied to the cultivated area or the plants to be treated, simultaneously or successively, with other biologically active compounds. These compounds may be fertilizers or micronutrient feeds or other preparations that affect the growth of other plants. These are more selective herbicides, pesticides, fungicides,
It may be a bactericide, a nematicide, a molluscicide or a mixture of some of these preparations and, if necessary, furthermore a carrier, surfactant or surfactant commonly used in the art of pharmacy. It can be used in combination with an auxiliary agent that promotes spraying. Suitable carriers and adjuvants can be solid or liquid, and include materials commonly used in the art of pharmacy, such as natural or regenerated minerals, solvents, dispersants,
Corresponds to wetting agents, tackifiers, binders or fertilizers. An active strain, spore, crystal or spore-crystal complex,
Or the formulations of combinations thereof can also be combined with other active ingredients and, where appropriate, solid or liquid auxiliaries can be added in a known manner, e.g.
It can be prepared by intimately mixing and / or pulverizing a solid carrier, optionally with an extender such as a surface-activating compound (surfactant). Formulations and methods of formulation are disclosed in U.S. Patent No. 5,501,852, which is incorporated herein by reference in its entirety.

Suitable solvents are: aromatic hydrocarbons, preferably fractions containing from 8 to 12 carbon atoms, for example xylene mixtures or substituted naphthalenes, phthalic esters such as dibutyl or dioctyl phthalate, for example cyclohexane or Aliphatic hydrocarbons such as paraffins, alcohols and glycols and their ethers and esters, for example ethanol, ethylene glycol monomethyl ether or ethylene glycol monoethyl ether, ketones such as cyclohexanone, N-methyl-2-pyrrolidone, dimethylsulfoxide or dimethyl Strong polar solvents such as formamide and the like, as well as vegetable oils or epoxidized vegetable oils, for example epoxidized coconut oil or soybean oil; or water.

The solid carriers used, for example, in powders and wettable powders, are usually natural mineral fillers such as calcite, talc, kaolin, montmorillonite or attapulgite. It is also possible to add highly dispersed silicic acids or highly dispersed absorbent polymers to improve the physical properties. Suitable granular adsorbent carriers are of the porous type, for example pumice, brick debris, sepiolite or bentonite; and suitable non-adsorbent carriers are materials such as calcite or sand . In addition, a large number of pre-granulated inorganic or organic substances can also be used, examples being especially dolomite or finely ground plant residues.

Depending on the nature of the active ingredient to be incorporated, suitable surface-activating compounds are nonionic, cationic and / or anionic surfactants having good emulsifying, dispersing and wetting properties. is there. Also, it will be understood that the term "surfactant" includes mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-activating compounds. Suitable soaps are alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C ₁₀ -C ₂₂ ), such as oleic acid or stearic acid, or for example coconut oil or tallow Can be the sodium or potassium salt of a natural fatty acid mixture obtainable from Further suitable surfactants are also methyltaurine salts of fatty acids, as well as modified or unmodified phospholipids.

However, more often, so-called synthetic surfactants are used, in particular fatty acid sulphonates, fatty acid sulphates, sulphonated benzimidazole derivatives or alkylaryl sulphonates. Fatty acid sulphonates or sulphates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and generally comprise a C ₈ -C ₂₂ alkyl radical which also comprises the alkyl part of the acyl radical. For example, sodium or calcium salts of lignosulfonic acid, dodecyl sulfate, or a mixture of fatty alcohol sulfates derived from natural fatty acids. These compounds further include fatty alcohol / ethylene oxide adduct sulfates and salts of sulfonic acids. The sulfonated benzimidazole derivatives preferably contain two sulfonic acid groups and one fatty acid radical having about 8 to 22 carbon atoms.
Examples of alkylaryl sulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzene sulfonic acid, dibutyl naphthalene sulfonic acid, or the naphthalene sulfonic acid / formaldehyde condensation product. Also suitable are the corresponding phosphates, for example the phosphate ester salts of p-nonylphenol with an adduct of 4 to 14 mol of ethylene oxide. Preferred as non-ionic surfactants are aliphatic or cycloaliphatic alcohols or polyglycol ether derivatives of saturated or unsaturated fatty acids and alkylphenols,
These derivatives have 3 to 30 glycol ether groups,
And 8-20 carbon atoms in the (aliphatic) hydrocarbon portion and 6-18 in the alkyl portion of the alkylphenol.
Contains carbon atoms.

Further suitable nonionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycols having 1 to 10 carbon atoms in the alkyl chain, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol, Additives are 20
~ 250 ethylene glycol ether groups and 10-100
Propylene glycol ether groups. These compounds are generally used in an amount of 1 to 1 per unit of propylene glycol.
Contains 5 ethylene glycol units. Representative examples of nonionic surfactants are nonylphenol polyethoxyethanol, polyglycol ether of castor oil, polypropylene / polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan, such as polyoxyethylene sorbitan trioleate, are suitable non-ionic surfactants.

The cationic surfactants comprise, as N-substituents, at least one C8-substituted C22-substituted alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, Quaternary ammonium salts containing a benzyl or hydroxy lower-alkyl radical are preferred. These salts are halides, methylsulphonates or ethylsulphonates, for example stearyltrimethylammonium chloride or benzyldi- (2-chloroethyl) ethylammonium bromide.

These surfactants conventionally used in the art of pharmacy are described, for example, in McCutcheon's Detergents and Emulsifiers Yearbook.
ulsifiers Annual) (MC Publishing Corp, Ridgewood, NJ, 1979); Dr. Helmut Stache's "Surfactants Handbook (Tensid Taschenbuch)" (Carl Hanse
r Verlag, Munich / Vienna).

Another particularly preferred property of the insecticidal compositions of the present invention is the persistence of the active ingredient when applied to plants or soil. Possible causes of loss of activity include:
Inactivation by UV light, heat, leaf exudates and pH is included. For example, at high pH, especially in the presence of a reducing agent, the δ-endotoxin crystals are solubilized and consequently more susceptible to proteolytic inactivation. High leaf pH is equally important, especially when the leaf surface is pH 8-10
Is important. Formulations of the pesticidal compositions of the present invention may contain additives to help prevent loss of the active ingredient, or may encapsulate the substance in such a way that the active ingredient is protected from inactivation. Either of these can address these issues. Encapsulation is achieved chemically (McGuire and Shasha, 1992) or biologically (Barnes and Cummings, 1986).
Chemical encapsulation relates to the method of coating the active ingredient with a polymer, while biological encapsulation relates to the expression of the delta-endotoxin gene in microorganisms. Biological encapsulation uses intact microorganisms containing the δ-endotoxin protein as the active ingredient in the formulation. The addition of UV inhibitors effectively reduces damage from UV irradiation. Thermal inactivation can also be controlled by including appropriate additives.

The pesticidal composition usually contains at least one
0.1 to 99%, preferably 0.1 to 95%, of a recombinant B. thuringiensis strain, comprising a novel gene of a recombinant of the species or in combination with their other active ingredients, in solid or liquid form 1-99.9%, and surfactant 0-25%,
Preferably, the content is 0.1 to 20%. Commercially available products are preferably formulated as concentrates, but the end user will typically use substantially lower concentrations of the diluted formulation. The insecticidal composition further comprises a stabilizer, an antifoaming agent,
In addition to components such as viscosity modifiers, binders, tackifiers and the like, it can also include fertilizers or other active ingredients for achieving a particular effect.

[0064]

The following examples further illustrate the materials and methods used in the practice of the present invention, and the results. These are provided by way of illustration, and the details should not be deemed to limit the scope of the invention.

Example 1-Culture of a novel B. thuringiensis isolate A subculture of the novel B. thuringiensis isolate, or a mutant thereof, was used as a substrate for the cottonseed flour described below. Inoculate (Table 1).

TABLE 1 Ingredients% drug loading medium (Pharmamedia) 6 Starch 4 yeast extract 0.5 Bacto - Peptone _{0.5 MgSO 4 · 7H 2 O 0.05} FeSO 4 · 7H 2 O 0.002 ZnSO 4 · 7H 2 O 0.002 CaCO 3 0.1

The medium was autoclaved and the flask was incubated at 30 ° C. on a rotary shaker at 360 rpm for 48 hours. For commercial purposes, the above methods are scaled up by standard methods.

Example 2 Activity of a Novel B. thuringiensis Isolate Against Lepidoptera 20 ml of an appropriately diluted fermentation broth of the above B. thuringiensis was mixed with 180 g of diet. The diet was injected into the holes of the jelly tray (50 holes / tray). After consolidation and cooling of the diet, 25 larvae of 3 weeks old Trichoplacia ni or Spodoptera exigua were placed in the pits and the pits were sealed with Mylar membrane. The bioassay was performed in a 27 ° C. incubator. After 3 days, efficacy was assessed by comparing mortality and comparing to the activity of a widely used and well-characterized commercial B. thuringiensis isolate HD-1 as a control. It was determined. A comparison of potency is shown in Table 2.

TABLE 2 Comparison of potency of four B. thuringiensis cultures grown on cottonseed substrate with HD-1 Culture T. ni S. exigua BIU / QT SD BSU / QT SD HD1 2.6 1.2 3.3 0.7 S197 5.4 1.0 5.4 1.0 S3158 5.2 1.0 5.6 1.0 S3159 4.5 0.9 5.2 0.9 S3212 4.9 1.0 5.7 0.5 F810 4.8 1.4 2.3 0.9 * Average of 4 fermentations

Example 3 Analysis of CryIA Protein Expression The fermentation broth is used to analyze the CryI type protein component of the crystals produced by the B. thuringiensis strain included in the present invention. Exactly 1 ml of each fermentation broth is mixed with 10 ml of 500 mM NaCl (pH 8) buffered with 10 mM Tris-HCl, and 1 ml
Suspend in mM EDTA solution and centrifuge at 15,000 rpm for 15 minutes. The precipitate generated by centrifugation is resuspended in the same NaCl solution as described above. This step is repeated three times to remove proteinase from the B. thuringiensis sample. The last precipitate is suspended in water to a total volume of 1.20 ml. PMSF (phenylmethylsulfonyl fluoride, serine proteinase inhibitor, 5 mM final concentration), EDTA (ethylene dinitrotetraacetic acid, metalloproteinase inhibitor, 10 mM), and E6
4 (N- [N- (L-3-transcarboxylan-2-carbonyl)-
[L-leucyl-L-leucyl] -agmatine, a cysteine proteinase inhibitor, 20 μg / ml) to suppress residual proteinase and to transform B. thuringiensis crystals with 20 μl β-mercaptoethanol and 2N NaOH 150
Dissolve in μl. 15,000 rpm of dissolved crystal protein
Centrifuge at for 30 minutes and collect as supernatant. About 1 ml of supernatant
Collect.

Next, the dissolved crystal protein was separated by Sephad
Separation from small molecules such as proteinase inhibitors by ex G25 (Pharmacia) column chromatography. The fractions eluted from Sphadex G25 are combined and the crystalline protein in the fraction is recovered by acid precipitation. The crystalline proteins precipitate effectively at their isoelectric point. The precipitated protein is dissolved in 700 μl of 100 mM Tris-HCl (pH 8) containing 1 mM EDTA and digested with 50 μg of trypsin at 37 ° C. for 30 minutes. The digestion is terminated by adding another 50 μg of trypsin and incubating for another 30 hours. Trypsin converts 130 kdal crystal protein (also called protoxin) to 66 kdal
Digests to a core fragment (activated toxin). Next, trypsin was inactivated with 25 μl of 100 mM PMSF,
The trypsin-digested crystal protein was added to the solution at pH 1.
220 μl of 8M urea with the addition of several μl of 2N NaOH to make 0.5
Dissolve completely in. Transfer the volume of this solution to Amicon Mi
Using a crocon-30 rotary concentrator, reduce to 150 μl and inject 100 μl of the concentrated solution into a 10 × 300 mm Superdex-75 (Pharmacia) column to remove small peptide fragments. This chromatography was performed at 20 mM containing 2 M urea.
Perform with Tris-HCl (pH 8.8). Next, 1.5 ml of the column eluate containing the protein digested with 66 kdal trypsin
The fraction is injected onto a 5 × 100 mm Mono-Q (Pharmacia) column. For the Mono-Q column, the same buffer used for Superdex-75 is used. Elution was carried out with NaCl and the concentration was increased from 150 mM to 200 mM in 20 ml (10 to 50 minutes), followed by 15 ml of 400 mM (50 to 80 minutes).
Min). Under these conditions, the protein elutes in the order of CryIA (a), CryIA (b) and CryIA (c) within 50 minutes.

The expression of CryIA is further exemplified below in the B. thuringiensis strain of the present invention. Strain S1
97, S3158, S3159, S3212, F810 and HD1 (control strain) are cultured and treated essentially as described above. CryIA (a), Cr
The amounts of yIA (b) and CryIA (c) are quantified for each strain. Each of the new strains is significantly different from the control strain HD1. For all new strains, CryIA (a), Cr
The proportion of yIA (b) and CryIA (c) increases, and CryIA (c)
More than 50% of the total amount of yIA type expressed protein. In contrast, HD-1 has relatively more CryIA (b) than CryIA (a) or CryIA (c). The results are detailed in Table 3.

Table 3 CryIA protein expression profile samples CryIA (a) CryIA (b) CryIA (c) S197 13% 28% 60% S3158 14% 29% 58% S3159 17% 27% 56% S3212 13% 29% 58% F810 15% 27% 58% HD-1 27% 40% 33%

Example 4-Phytotoxicity of strain F810 To determine the phytotoxicity of strain F810, F810 and HD-1 (control) are grown under similar fermentation conditions. The spore count at the end of fermentation is typically about 5 × 10 ⁹ / ml. Fermentation broth from each strain culture is collected and agriculturally important,
Applies to plants, Chinese cabbage that are susceptible to withering.
The leaves are sprayed with undiluted fermentation broth having a solids content of approximately 7% until dripping. The leaves of Chinese cabbage treated with the F810 strain fermentation broth do not die,
More than 20% of the leaves of Chinese cabbage treated with HD-1 fermentation broth die. The phytotoxicity present in the fermentation broth of HD-1 is maintained when this fermentation broth is further processed to produce pesticidal products. Consequently, insecticide products from HD-1 are not suitable for use in methods of treating plants that are susceptible to withering, while products from F810 are.

Example 5-Recombinantly Improved Strains An example illustrating the advantages of the recombinant B. thuringiensis isolates covered by this specification is described herein. The newly discovered Bt kurstaki strains described above include Trichoplacia ni (Cabbage shrimp beetle), Tumexaga (He
It has been shown to have a higher insecticidal activity against certain insects, such as liothis virescens (tobacco green worm), than previously known Bt kurstaki strains, such as strain HD-1. . However, there was no significant difference in activity against Spodoptera species. The present inventors have found that the pest control activity of these strains is further improved by recombinant DNA technology. This recombinant strain is a hybrid B. thuringiensis crystal protein gene.
H04 was obtained by introducing it into the chromosome of Bt Klustachy strain S3158. The H04 protein has been reported to be highly active against Spodoptera exigua (de Maagd et al., Domain III in Bacillus thuringiensis δ-endotoxin CryIAb showing excellent toxicity to Spodoptera exigua. Substitution and alteration of membrane protein recognition, Applied Environmental Microb
iology, 62: 1537-1543 (1996)). The H04 gene was prepared in a three-step
Arrange on 158 chromosomes. First, construct the integration plasmid. Next, the integrated plasmid harboring H04 is introduced into the chromosome of Bt Klustachy strain W4D23 (without crystals of the derivative of Bt Klustachy strain HD73). In the last step, the inserted sequence was transformed from the chromosome of W4D23 with S3158 using the generalized transducing phage CP51.
Transfer to the chromosome.

1. Construction of Integration Vector pSB1217 As shown in FIG. 4, plasmid pSB1217 contains four DNAs.
Has fragments. This includes (i) the E. coli origin of replication and the ampicillin resistance gene from plasmid pBR322, (i
i) the erythromycin resistance gene ermC of B. subtilis, which acts as a selection marker in Bt,
(iii) Bt Kurusutaki strain Bt Aisawai (aizawai) 7.21
Amino acid residues 1-4 encoded by the cryIA (b) gene
60, and c of Bt entomocidus strain 60.5
a hybrid crystal protein gene H04 comprising residues 461 to 1189 encoded by the ryIC gene, and
(iv) It has a DNA fragment derived from Bt kurstaki HD73 chromosome that encodes phospholipase C (PLC) specific for phosphatidylinositol and provides a site for integrating H04 into the Bt chromosome.

The plasmid pSB1217 is composed of the following three steps. First, the tetracycline resistance gene of plasmid pBR322 is replaced with the B. subtilis erythromycin resistance gene ermC. In the second step, a 2.2 kb PLC fragment derived from HD73 is added. In a third step, the hybrid crystal H04 gene is added to the integration vector.

2. Transformation of Bt kurstaki W4D23 The integration plasmid pSB1217 was placed into the Bt kurstaki strain W4D23 by transforming competent cells using electroporation. The W4D23 strain is a partially cured derivative of HD73, which is a Kurstak
Was isolated as AP77BX17. W4D23 was selected because it is relatively easy to transform and does not have the gene for the crystal protein to be integrated into pSB1217. Integration of pSB1217 occurred when the PLC region of this plasmid was aligned with the PLC region of W4D23 chromosome. The entire plasmid was integrated by a single transfer. As a result of this integration, S3158 :: H04 has two PLC regions; both of which have the PLC region encoding pSB1217 and
It is a hybrid of W4D23 chromosome PLC. The obtained strain is
Called W4D23 :: H04. Plasmid pSB1217, PL of W4D23
Integration into the C region was confirmed by PCR analysis.

3. Transfer of H04 from W4D23 to S3158 Attempts to transform strain S3158 by incorporation of plasmid pSB1217 yielded no transformants due to the limited transformation efficiency of this strain. To circumvent this technical obstacle, we used the common transducing phage CP51 to transfer H04 from the W4D23 :: H04 chromosome to the S3158 chromosome (Thorne, CB, Bacillus cereus). Bacteriophage for transduction, J. Viol., 2: 657-
662 (1968)). Phage lysate was washed with phage CP51 W
It was produced by infection of strain 4D23 :: H04. Next, the lysate was used to infect S3158. Selection by growth on erythromycin resulted in strain S3158 :: H04. Plasmid
Integration of pSB1217 into the PLC region of S3158 was confirmed by PCR.

4. Pest control activity of recombinant S3158 strain Wild type and recombinant strains were grown until sporulation was completely terminated, and sufficient whole culture dilutions were mixed into the artificial insect diet. The dilution was infested by insects and mortality was recorded after 4 days. As shown here,
The S3158 strain showed significantly improved pest control activity against Spodoptera exigua as compared to the hitherto known Bt crustachy HD-1 strain. Insects HD1 S3158 :: H04 EC50 EC50 Trichopracia ni 164ppm 92ppm (Cabbage shrimp) Lepidoptera Spodoptera exigua 1475ppm 194ppm (Syringo spp.) Lepidoptera Plutera xylostella 9ppm 8ppm ( Lepidoptera ) Lepidoptera

The present invention has been disclosed and described herein above by way of non-limiting detailed description and accompanying drawings. Various modifications and further aspects will be apparent to those skilled in the art in light of the present disclosure. Such modifications and embodiments are intended to be included within the scope of the present invention.

[0079]

According to the present invention, the expression level of CryIA (c) is
(a) or a novel CryIA-producing B. thuringiensis strain greater than the expression level of CryIA (b), its products, recombinant strains derived therefrom, and methods of using them, and insecticidal compositions comprising the strain A recombinant Bacillus thuringiensis transformed with heterologous DNA, wherein the recipient host Bacillus thuringiensis strain is produced by the strain. A Bt strain capable of producing CryIA (c) in an amount larger than that of CryIA (a) or CryIA (b) was provided. In a preferred embodiment, the B. thuringiensis strain and the insecticidal product can be applied to plants that are susceptible to mortality in an insecticidally effective amount without substantially causing plant mortality.

[Brief description of the drawings]

FIG. 1 shows the chromatographic profile of HD1 common to common B. thuringiensis var. Kurstaki strains. This chromatogram is
Three CryIA proteins are shown. The peaks from left to right correspond to CryIA (a), CryIA (b) and CryIA (c). As can easily be seen, the preferentially expressed protein was CryIA (b).

FIG. 2 shows a chromatogram of the protein produced by the deposited strain S3159. The peaks from left to right correspond to CryIA (a), CryIA (b) and CryIA (c). As can easily be seen, the preferentially expressed protein was CryIA (c).

FIG. 3. Each of the newly deposited isolates and strain HD
FIG. 3 shows the chromatographic profile of CryIA protein expressed by -1. The peaks from left to right correspond to CryIA (a), CryIA (b) and CryIA (c). As can be readily seen, the preferentially expressed proteins were each of the new isolates (A) = S197; (B) = S315
8; (C) = S3159; (D) = S3212; and (E) = F810, whereas control (F) = HD-1 preferentially expressed the protein CryIA (c). b).

FIG. 4. pSB121, including H04, ErmC, PLC and pBR322.
FIG. 7 is a diagram showing a map of 7 integration vectors.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1:07) (71) Applicant 597066223 81 Wyman Street, Walt ham, Massachusetts 02254-9046, United States of AmericaF Term (reference) 4B024 AA07 BA38 CA04 DA05 EA04 GA14 HA01 4B065 AA20X AA20Y AB01 AC12 AC13 AC14 BA02 BA03 BA22 BB26 BC03 BC26 CA24 CA48 4H011 AC01 BA01 BB21 BC23 DA01 DG05 DG13

Claims (23)

[Claims] 1. Biology of a B. thuringiensis strain capable of producing CryIA (c) in greater amounts than the amount of CryIA (a) or CryIA (b) produced by this strain Pure culture. 2. The B. thuringiensis strain according to claim 1, wherein it is capable of producing CryIA (c) in an amount of at least 50% of the total amount of CryIA type protein produced by this strain. 3. S197, S3158, S3159, S3212 or F810
The B. thuringiensis strain according to claim 1, which is 4. When grown in a cottonseed substrate, the BI
Measured by U / Qt, Trichoplusia n
i) has a pest control efficacy that is at least 1.8 times greater than the efficacy of B. thuringiensis strain HD-1 grown under the same conditions, and is determined by BSU / Qt to be Spodoptera. B. thuringiensis strain HD grown under the same conditions against excigua (Spodoptera exigua)
A biologically pure culture of a B. thuringiensis strain having a pest control efficacy that is at least 1.6 times greater than that of -1. 5. S197, S3158, S3159, S3212 or F810
The B. thuringiensis strain according to claim 4, which is: 6. A B. thuringiensis strain capable of producing CryIA (c) in an amount larger than the amount of CryIA (a) or CryIA (b) produced by this strain. An insecticidal composition comprising an insecticidal factor selected from the group consisting of a spore derived from Thuringiensis strain and a crystal toxin derived from the B. thuringiensis strain. 7. The strain comprises CryIA (c) which is at least 50% of the total CryIA type protein produced by this strain.
The composition of claim 6, wherein the composition is capable of being produced in an amount of 8. The B. thuringiensis strain HD grown under the same conditions against Trichoplacina as measured by BIU / Qt when the strain is grown in cottonseed flour substrate.
-1 has at least 1.8 times greater pest control efficacy than that of Spodoptera, measured by BSU / Qt.
7. The composition of claim 6, wherein the composition has a pest control efficacy against exigua that is at least 1.6 times greater than that of the B. thuringiensis strain HD-1 grown under the same conditions. 9. At least one selected from the group consisting of a carrier, a surfactant, an agricultural adjuvant, a diluent, and a spraying aid suitable for use in combination with B. thuringiensis.
7. The composition of claim 6, further comprising a species agent. 10. The composition according to claim 6, wherein the insecticidal factor is the B. thuringiensis strain. 11. The strain is S197, S3158, S3159, S3212.
Or the composition of claim 11, which is F810. 12. The composition according to claim 6, wherein the insecticidal factor is a spore derived from the B. thuringiensis. 13. The composition according to claim 6, wherein the insecticidal factor is a crystal toxin derived from B. thuringiensis. 14. The B. thuringiensis strain is CryI.
A (c) is the amount of CryIA (a) produced by the strain or Cr
A recombinant B. thuringiensis strain containing foreign DNA, which is capable of producing a larger amount than yIA (b). 15. The B. thuringiensis strain HD grown under the same conditions against Trichoplacia ni as determined by BIU / Qt when the strain is grown in cottonseed flour substrate.
B. thuringiensis strain HD, which has pest control efficacy at least 1.8 times greater than that of -1 and has been grown against Spodoptera exigua under the same conditions as measured by BSU / Qt 15. The recombinant B. thuringiensis strain of claim 14, which has a pest control efficacy that is at least 1.6 times greater than the efficacy of -1. 16. The strain may be S197, S3158, S3159, S3212.
15. The recombinant B. thuringiensis according to claim 14, which is F810. 17. The recombinant B. thuringiensis of claim 14, wherein the foreign DNA encodes an insecticidal protein. 18. CryIA (c) is produced by a strain
B. thuringiensis strain capable of producing a larger amount than the amount of CryIA (a) or CryIA (b), spores derived from the B. thuringiensis strain, and from the B. thuringiensis strain Applying an insecticidal composition comprising an insecticidal agent selected from the group consisting of crystalline toxins to a plant or soil in which it grows in an insecticidally effective amount.
How to treat plants. 19. The method according to claim 18, wherein the insecticidal factor is the B. thuringiensis strain. 20. The strain is S197, S3158, S3159, S3212.
20. The method of claim 18, wherein the method is F810. 21. The method of claim 18, wherein the insecticidal agent is a spore from B. thuringiensis. 22. The method according to claim 18, wherein the insecticidal factor is a crystal toxin from B. thuringiensis. 23. The plant is susceptible to withering,
And the B. thuringiensis strain is F810.
The described method.