KR910002204B1 - Cellular encapsulation of biological pesticides - Google Patents

Abstract

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Description

Manufacturing method of microorganisms containing toxic

The present invention relates to a method for preparing the pesticide composition and a method for preparing the cells so that the pesticide composition can be encapsulated in unproliferated cells.

A better understanding of farming methods, improved equipment, the use of fertilizers and improved pesticides have resulted in a surprising increase in agricultural productivity. However, the latter, pesticides, have had a detrimental effect on the environment. Therefore, it is desirable to develop pesticides that can be applied more effectively to the environment.

Ecologically useful insecticides include protein toxins produced by various microorganisms such as Bacillus thuringiensis, but there are significant disadvantages in using lysates or spores of B. thuringiensis as insecticides. Insecticides have a relatively short lifespan in the environment and require multiple applications to obtain adequate protection. Thus, these pesticides are uneconomical compared to conventional chemicals with long lasting residues. If the activity of biological insecticides or protein toxins as a main ingredient is extended, the field life will be extended.

As mentioned above, pesticides with unusual application methods have different requirements. For example, in many cases, pesticides that remain long in the field without accumulating in the environment are preferred. In addition, in consideration of economics, pesticides having a wide range of bactericidal action are preferable. Insecticides must also degrade into environmentally acceptable degradation products. Many other conditions, such as stability, insecticidal action, and easy formulation against environmental effects such as light, moisture, and organisms, are considered, and these conditions affect the useful organisms or non-toxic organisms microorganisms in the environment.

West, Soil Biol. Biochem, (1984) 16: 357-360 describe B. t. The results of studies on the persistence of toxins are recorded. This is also shown in West et al., J. of Invertebrate Pathology (1984) 43: 150-155. US Pat. No. 4,265,880 describes a live insecticidal pathogen present in coacervate microbeads. Japanese Patent No. 51-5047 describes a physico-chemical method of eradicating B. thuringiensis spores while retaining toxicity.

Methods and compositions for protecting crops and agricultural products from pests are disclosed herein. In one aspect of the invention, a microorganism that produces unlyzed toxin (pesticide) cells is treated with a reagent, which extends the action of the toxin produced in the cell when the cell is subjected to the target pest's environment. It is a substance to give. In another aspect of the present invention, the pesticide is prepared by inserting a heterologous gene into a host cell. Expression of heterologous genes enables direct and indirect intracellular production and maintenance of pesticides. As mentioned above, the cells are treated under conditions that prolong the action of toxins produced in the cells when the cells are applied to the environment of the target pest. The resulting product is toxic. Such naturally encapsulated pesticides may be formulated according to conventional techniques for application in environments such as rehabilitation, moisture, and leaves of plants that parasitic target pests.

According to the present invention, improved insecticides have an extended residual life by modifying naturally occurring insecticide producing microorganisms or insecticide producing microorganisms parasitic heterologous genes that can be expressed in a host. Expression of the gene in the host occurs either directly or indirectly. The present invention involves treating microorganisms with a reagent that prolongs the action of toxins produced in cells when the cells are applied to the environment of the target pest. The resulting product retains the toxicity of the toxin.

A wide range of pesticides can be prepared and are characterized in that they are produced intracellularly, in particular in single cell microbial hosts. Single cell microbial hosts are prokaryotes (eg bacteria); Or eukaryotes (eg, yeast and filamentous fungi as fungi, namely Neurospora and Aspergillus); Or protists, for example, protozoa, algae and the like.

The pesticide may be a toxin produced by the microorganism. Pesticides include insects such as coleoptera, lepidoptera, fly, stink wood, herbaceous locust and locust which are eukaryotic multicellular pests; Or template animals; Mollusks; Or a polypeptide having a toxic effect on worms, for example nematodes and worms. Examples of such insects include bees, moths, flies, grasshoppers, lice, and tongs.

The pesticide produced in the host cell may be a polypeptide of the active form or a polypeptide produced in the form of a precursor or a precursor or an active form which requires the progression of toxic action by the pest to the crystalline toxin of B. thuringiensis strain Kurstaki. . Thus, genes can code for enzymes that modify metabolites to produce pesticides. Spontaneous toxins include B. thuringiensis mutant Kurstaki active on Lepidoptera; B. t. Active in mosquitoes. Mutant israelensis; B. t. Active against coleoptera. M-7; Thuringiensis mutant aizawai that is active in spodoptera: polypeptides such as B. sphaericus are active in mosquito larvae. Other toxins include fungi, insect pathogens, for example beauverin from Beauveria basiana and destruxin from Metarhizium spp; Or a wide range of pesticidal compounds, for example Streptomyces avemitilus avermectins. The cultures exemplified above are shown below: Bacillus thuringiensis mutant Kurstaki HD-1-NRRL B-3972; Described in US Pat. No. 4,448,885. Bacillus thuringiensis strain israelensis-ATCC 35646 Bacillus thuringiensis M-7-NRRL B-15939 Bacillus thuringiensis strain tenebrionis-DSM 2803 The following B. thuringiensis cultures obtained from the US Department of Agriculture, Brownsville, Texas. Purchases are available from United States Texas 78520, Brownsville, P. Box 1033, Joe Garcia, USDA, ARS, and Cotton Insects Research Unit.

B. thuringiensis HD2

B. thuringiensis mutant finitimus HD3

B. thuringiensis mutant alesti HD4

B. thuringiensis 〃 kurstaki HD73

B. thuringiensis 〃 sotto HD770

B. thuringiensis 〃 dendrolimus HD7

B. thuringiensis 〃 kenyae HD5

B. thuringiensis 〃 galleriae HD29

B. thuringiensis mutant canadensis HD224

B. thuringiensis mutant entomocidus HD9

B. thuringiensis 〃 subtoxicus HD109

B. thuringiensis 〃 aizawai HD11

B. thuringiensis 〃 morrisoni HD12

B. thuringiensis 〃 ostriniae HD501

B. thuringiensis 〃 tolworthi HD537

B. thuringiensis 〃 darmstadiensis HD146

B. thuringiensis 〃 toumanoffi HD201

B. thuringiensis 〃 kyushuensis HD541

B. thuringiensis 〃 thompsoni HD542

B. thuringiensis 〃 pakistani HD395

B. thuringiensis 〃 israelensis HD567

B. thuringiensis 〃 indiana HD521

B. thuringiensis 〃 dakota

B. thuringiensis 〃 tohokuensis HD866

B. thuringiensis 〃 kumanotoensis HD867

B. thuringiensis 〃 tochigiensis HD868

B. thuringiensis variant colmeri HD847

B. thuringiensis mutant wuhanesis HD525

Bacillus cereus-ATCC 21281

Bacillus moritai-ATCC 21282

Bacillus popilliae-ATCC 14706

Bacillus lentimorbus-ATCC 14707

Bacillus sphaericus-ATCC 33203

Beauveria bassiana-ATCC 9835

Metarhizium anisopliae-ATCC 24398

Metarhizium flavoviride-ATCC 32969

Streptomyces avermitilus-ATCC 31267

Toxins need not be identical to naturally occurring toxins. Polypeptide toxins are fragments of naturally occurring toxins; Is a transgenic product of missing, displaced or metastasized, with up to two amino acids changed; It may be a repeat sequence that can be delivered by the pest host of interest. In addition, the fusion product can provide a protein degradation reduced toxin at the N-terminus. In some cases, most of the same or different toxins from each other can be encoded and expressed. The cleavage site is inserted between each toxin site in the polytoxin.

Examples of host cells are prokaryotic or eukaryotic cells and are generally limited to cells that do not cause toxic to body components in higher animals such as mammals. However, in higher animals, microorganisms that make body components toxic can be used, provided that the toxin is applied at a level low enough to preclude unstable or toxic potential in mammalian hosts. Hosts include lower eukaryotes such as prokaryotes and fungi. As gram negative and gram positive eukaryotes, Enter-obacteriaceae such as Escherichia, Erwinia, Shigella, Salmonella Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae such as phosphorus fungi, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Peseudomonadaceae such as Pseudomonas and Acetobacter; Includes Azotobacteraceae and Nitrobacteraceae. Eukaryotic hosts include euthanasias such as Saccharomyces and Schizasaccharomyces, rather than eukaryotes such as Phycomycetes and Ascomycetes; Basidiomycetes yeast such as Phodotorula, Aureobasidium, Sporobolomyces, and the like.

Features of selecting host cells for production purposes include the ease of incorporating heterologous genes into the host, the usefulness of the expression system, the efficiency of expression, the stability of the pesticides in the host, and the presence of adjuvant gene activity. .

Advantages of being used as insecticide microcapsules include the protection of insecticides such as thick cell walls, pigmentation, intracellular arrays or body formation; Affinity for leaves; Mammalian toxin deficiency; Pest digestibility; To facilitate eradication and fixation without damaging toxins; And the like. Other features include ease of formulation and handling, economics, storage stability, and the like.

Host organisms include Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., Sporobo1omyces sp. Same yeast; Phylloplane organisms such as Pseudomonas sp., Erwinia sp., Flavobacterium sp; Or other organisms such as Escherichia Lactobacillus sp., Bacillus sp., And the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus Subtilis and the like.

Cells are generally intact, complete, and have a form of proliferation when they are substantially eradicated. In some cases spore forms may be used, but proliferative forms are preferred over spore forms.

Cells can be inhibited by a variety of methods if they do not adversely affect the properties of the pesticides and do not reduce cell capacity in preserving the pesticides. This technique includes physicochemical treatments that do not alter the physical properties of the cell or cause changes in the original physical properties of the cell.

Various techniques for inactivating host cells include heat at 50 ° C-70 ° C; Freezing; UV radiation; Freeze drying; Antibiotic-like toxins; Phenol; Anilides such as carbaanilides and salicylates; Hydroxyurea; Quaternaries; Alcohol ; Bactericidal dyes; EDTA and Amidine; Nonspecific organic and inorganic chemicals such as halogenating agents such as chlorinating agents, brominating agents or iodinating agents; Aldehydes such as glutaraldehyde or formaldehyde; Toxic gases such as ozone oxide and ethylene oxide; Peroxide; Psoralens;

Desiccant; And the like, which may be used alone or in combination. The choice of reagents includes specific pesticides, the characteristics of the host cell, and changes in the cell structure with high information on the cell walls with crosslinkers; And the like.

Cells generally enhance structural stability which can enhance resistance to environmental degradation products in the field. If the pesticide is a preform, the inert process must be chosen so that the preform does not inhibit the progression of the pesticide to the aging form of the pesticide by the target pest pathogen. For example, formaldehyde crosslinks proteins and interferes with the pro form of polypeptide pesticides. Inactivation or eradication methods retain at least a substantial site of bioavailability or bioavailability of toxins.

Heterologous genes can be inserted in a convenient manner that provides extrachromosomal persistence or binding to the host genome. Heterologous refers to genes that do not exist in the host to be inserted or are generally not found in such host. In other words, even if heterologous genes and host microorganisms exchange information, heterogeneous genes are not found in wild-type host cells by their nature. In general, the term heteromorphic means that the host and the gene source are different genera.

Various structures can be used, which include their own replication segments and replication systems of plasmids, viruses or centromeres for stable persistence. If binding is required, the construct is replicable and it is either a transposon or provides an analog to the host genome or has an insert action such as a transposon. DNA sequences with heterologous genes are used between sequences homologous to sequences in the host genome, such as chromosomes or plasmids. Preferably, the heterologous gene comprises a large number of copies. This is shown in US Pat. No. 4,399,216. Therefore, conjugation, transduction, transfection, transformation and the like can be used for heterologous gene insertion. Numerous vectors that rely on eukaryotic and prokaryotic systems are available. For example, Co 1E1, P-1 incompatible plasmids, ie pRK290 yeast 2 mM plasmid, lambda and the like.

When components other than chromosomes are used, the DNA constructs are required to include markers capable of selecting host cells containing the constructs. Markers provide fungicide resistance. That is, antibiotic resistance or heavy metal resistance, and complements that provide prototrophy to an axoxotroph host. Replication systems provide features such as breakaway replication and include cos cells or other features.

When the heterologous gene has a transcriptional and translational initiation and termination control signal recognized by the host cell, it is preferable to use a regulatory gene conjugated with the heterologous gene. However, if a heterologous gene is modified, e.g., removing the leader sequence or providing a sequence encoding the mature form of the pesticide, where the total gene encodes a precursor, it is necessary to manipulate the DNA sequence and thus initiate transcription. Regulatory sequences may be provided differently from natural sequences; Extensive transcription initiation sequences exist in a wide variety of hosts. The sequence may provide for constitutive or controlled expression of the pesticide. Modulation here can be induced by chemical methods such as metabolism or by heat or regulatory inhibitors. This is shown in US Pat. No. 4,374,927. The choice of promoter is determined by a number of factors. For example, the strength of the promoter, the influence of endogenous regulatory mechanisms on the cell in the interference promoter of the promoter with the viability of the cell, and the like. Many promoters are available from a variety of sources, including commercial sources.

Cell hosts containing heterologous insecticidal genes are cultivated in nutrient media with DNA constructs with selective advantages to provide selective media so that all cells are retained in the heterologous gene. Such cells can be harvested according to conventional methods and modified by the methods described above. The cells can also be harvested after fixation.

Methods of treating host microbes that contain toxins fulfill several functions. First, structural integration can be promoted. Second, it is possible to enhance the proteolytic stability of the toxin by modifying the toxin to reduce its sensitivity to proteolysis or by reducing the protein action of proteases that are naturally present in the cell. The cells are modified in the undigested stage so that the toxin protein is present. Such modifications are made in a number of ways, for example by extensive chemical reactions. Undigested cells are mixed with a liquid reagent medium containing a chemical reagent and stirred at a temperature in the range of -10 ° C to 60 ° C with or without stirring. The reaction time is determined consistently and varies widely depending on the reactant and reaction time. The concentration of cells varies from 10E ² -10E ^l0 per ml.

The chemical reactant is preferably a halogen reactant of elements 17-80. In particular, iodine can be used under normal conditions for a sufficient time by obtaining the desired results. Still other suitable techniques include aldehydes such as formaldehyde and glutaraldehyde; Anti-infective agents such as zepiran chloride and cetylpyridium chloride; Alcohols such as isopropyl and ethanol; Various fixatives such as Bouin fixative and Helly fixative (Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); Or treatment with a combination of physical reagents (heat) and chemical reagents that maintain the action of toxins produced in the cells when the cells are applied to the target pest environment.

To halogenate using iodine, the temperature can range from 0 ° C. to 50 ° C., but the reaction can be conveniently carried out at room temperature. Conveniently, the iodization can be carried out using 0.5-5% triiodine or iodine in acidic aqueous medium. As acidic aqueous medium, an aqueous carboxylic acid solution which can vary from 0.5-5 _M is suitable. Other carboxylic acids of C _1-4 may also be used but acetic acid may also be used. The reaction time is changed to 1 minute-24 hours or less, preferably 1-6 hours. Residual iodine can be removed by reaction with a reducing agent, which includes dithionite, sodium thiosulfate, or any other reducing agent that is finally used. In addition, the modified cells may be formulated with washing, separation in dry form, general adhesives, dispersants, auxiliaries and the like used in agriculture to remove the reaction medium as is known in the art.

Reagents capable of crosslinking cell walls are preferred and numerous reagents are known. The treatment must increase the stability of the pesticide. In other words, it is necessary to enhance the persistence and residual activity of the insecticide in the field conditions, and therefore, under the condition that the insecticidal action of the untreated cells is reduced, the activity of the treated cells remains 1 to 3 times.

Cells can be formulated in a variety of ways. Cells can be used as wettable powders, particulates or dusts by mixing various inerts, for example inorganics (phyllosilicates, carbonates, sulphates, phosphates, etc.) or vegetable materials (corn powder, rice husks, walnut husks, etc.). This formulation includes a dispersant-adhesive aid, stabilizer, other pesticide, or surfactant. Liquids are aqueous or oily and can be used as foams, gels, suspensions, emulsifiable concentrates and the like. The component includes a flow agent surfactant, emulsifier, dispersant or polymer.

Pesticide concentrations can vary widely depending on the condition of the formulation, particularly whether it is a concentrate or directly available. Pesticides may be present at least 1% -100% by weight. Dry formulations have 1-95% by weight of pesticides while liquid formulations have 1-60% solids in the liquid phase. The formulations have 1E ² -lE ⁴ cells per mg. Such formulations are administered at 2 oz (liquid or solid) to at least 1 b / ha.

The formulation is applied by spraying, spraying, sprinkling or the like to a pest environment such as plants, soil or moisture.

The following examples are for illustrative purposes only and are not limited to these.

Example 1

After treatment with B. thuringiensis containing undissolved spores with 1ugol's iodine (prior to autolysis), cells are killed; However, the cells are toxic to Trichoplusia ni larvae.

Immediately before the spore-forming cells autolyse by centrifugation, undissolved cells of Bacillus thuringiensis (HD-1) are collected and the cell pellet is suspended in deionized water to a concentration of 6.0 × 10E ⁹ cells per ml. An aliquot of the cell suspension was diluted to 1.5 x 10E ⁸ cells / ml and contacted with 1% 1ugol's iodine for 4 hours at room temperature. (1% of (lugol) solution was 1.0 g of calcium iodide per liter, 0.5 g of Iodine, 1.0 ml of glacial acetic acid). The treated cells are washed and resuspended in sterile deionized water to bring the cell concentration to 6.0 × 10 E ⁹ . After 4 hours of treatment with iodine, the plate was counted on agar and there were no living cells. Cells treated with Lugol solution and untreated control cells were biologically present to determine virulence against T. ni larvae.

Since the cells of the present invention are natural cells, it is not necessary to treat the cells under discrimination conditions in order to realize the advantages of the present invention. Therefore, as described above, cell treatment is suitable for the person skilled in the art to realize the longest lasting toxin (insecticidal) action of the environment of the target pest.

Biological Analysis Method

Cell dilutions or untreated surviving HD-1 cells eradicated with Lugol iodine were mixed in a cup containing a certain amount of larvae. T. ni larvae were added to each cup for 5 days. 18 larvae were tested for each dilution. Six days later, the larvae were examined and the total number of larvae killed was recorded. The results are shown in Table (1). This is the percentage of dead larvae.

TABLE 1

Example 2

Stability test

B. containing spores t. HD-1 undigested cells were treated with 1% Lugol's solution for 4 hours at room temperature, then deionized water was washed and stored in the refrigerator for 52 days. After the end of this period, the cells were examined to show no degradation (separation of spores and crystals).

B. containing spores t. The undigested cells of HD-1 were collected by centrifugation, and the resulting pellets were suspended in sterile deionized water (10E ¹⁰ cells / ml), heated at 70 ° C. for 30 minutes, and stored in a refrigerator for 9 days. After 9 days all cells were broken down with spores and crystals separated.

Example 3

Soil test method

B. t. HD-1 recipe:

B. t. Containing undissolved spores Cultures of HD-1 were collected by centrifugation and cell suspension was resuspended in 400 ml of 1% Lugol iodine (4 × 10 E ⁸ cells / ml). The iodine cell suspension was stirred for 3 hours at room temperature, washed three times and resuspended in 400 ml of sterile 0.1 mM sodium phosphate buffer, pH 6.9. Live B. t. Detected on nutrient agar (10E-1) after iodine treatment. There were no HD-1 cells and all cells were complete (undigested) by microscopic examination.

Dipel Method:

B. t. Difel containing HD-1 cells (0.1 g, Abbott Laboratories, 16,000 International Units of Potency / mg. List # 5188) was measured in 400 ml of sterile 0.1 M sodium phosphate buffer.

Experiment method

1) Preparation of Non-Sterile Soil: 40 g of soil was placed in a 500 ml sterile flask and 200 ml of experimental solution was added.

2) Preparation of Sterile Soil: 40 g of soil was placed in a sterile 500 ml flask and autoclaved for 1 hour before adding 200 ml of experimental solution. The flask containing the soil suspension was incubated using a stirrer (200 RPM) at room temperature. Each soil suspension sample (30-40 ml) was filtered through four layers of cotton cloth and then sprayed onto the leaves of the lettuce to determine the toxicity to T. ni larvae.

B. t. Measurement of toxins

Spray the freshly prepared leaves of standard concentrations of the standard concentrations of DIPEL (1x0.19g / 100ml, 1 / 10x, 1 / 20x and l / 100x) and the experimental solution just 24 hours before use.

Leaves treated with standard or experimental solutions are removed from the plants and weighed separately and placed in a petri dish. Ten T. ni larvae starved for 7 days were applied to each leaf and fed with leaves at 25 ° C. for 24 hours. At the end of feeding, the leaves are reweighed and the average weight loss is measured for each treatment.

Under the above-mentioned preload, leaf weight loss is a linear logarithmic function of toxin concentration in excess of toxin concentrations of 16,000 Intenntional units of potency per mg. Therefore, after spraying freshly prepared difel concentrates on the lettuce plants, each assay was performed on the leaves sprayed with experimental solution B. t. The concentration of HD-1 toxin is calculated and used as a standard. The data presented in Table (2) show the percentage of toxicity remaining over time after culture in soil (%, 1 day) and subsequent analysis of leaf inhibition.

Leaves other than lettuce plants can also be used to analyze B. t toxin efficacy.

TABLE 2

In soil B. t. Inactivation of protein crystals of HD-1 is manifested by the action of soil microorganisms (West, 1984). The results in Table (II) are shown in B. t. When the HD-1 preparation was incubated under similar conditions, the toxicity of the Diffel (sporide) preparation was drastically reduced while the toxicity of cells treated with Lugol iodine was little changed.

From the above results, chemical treatment of the entire microbial cell is carried out in a manner that retains polypeptide toxin action, while making the cell stable in storage. This increases the residual activity of toxin action under field conditions.

Example 4

Generation of Heterologous Genes and Transformation into A-Compliant

Heterogeneous constructs are constructed from the Pseudomonas aeruginosa clones available from Northern Regional Research Laboratories (NRRL B-12127), which contain a large host-wide shuttle plasmid pRO 1614 (J. Bact. [1982] 150). : 60; United States Patent No. 4,374,200. Plasmids have unique Hind III, Bam HI, Sal I, Pvu II restriction sites, and Pst I inserts, which include carbenicillin resistance genes and the P. aeruginosa replication system, where Hind III, Bam HI and The Sal I restriction site is in the tetracycline resistance gene. The remaining plasmids were derived from pBR 322. Second plasmid pSM 1-17 has been deposited as a clone of E. coli (NRRL B-15976). Deposits will be made at 61604, Illinois, USA, Peoria, USDA, and the NRRL Research Institute. Plasmid pSM-17 confers ampicillin resistance to E. coli, which contains a 6.8 kbp Hind III DNA fragment containing the ∂-endotoxin gene from the 50 md plasmid of Bacillus thuringiensis HD 73. Sufficient amounts of toxins are expressed from the gene in E. coli and render undigested cells toxic to cabbage worms.

Modification of DNA fragments enhances the transformation of toxins and allows for toxin expression in another host, Pseudomonas fluorescens. To remove unnecessary DNA from the 6.8 kbp fragment, pSM l-1 was cleaved with Bam HI to open a cyclic plasmid near the 5 'end of the toxin gene, and treated with exonuclease Ba1 31 to 6.8 kbp Hind II insert. Is reduced to 2.9 kbp.

The (free) end of the DNA is filled with Klenow polymerase, a Bam H I binder is added and the linear DNA is ligated to reform the circular plasmid. The resulting vector, pFG 270, is cleaved with Xho I to open the cyclic plasmid at the site closest to the 3 'end of the toxin gene. Sal I binder is added, the plasmid is ligated in cyclic form and the resulting vector pFG l0.6 is amplified in E. coli. pFG 10.6 was completely cleaved into Bal I and Bam H I, and the resulting 2.lkbp fragment containing the toxin gene was purified by gel electrophoresis and ligated to the Bam H I and Sal I sites of plasmid pRO 1614. The resulting plasmid pCH 2.1 is amplified in E. coli to transform Pseudomonas fluorescens to have the ability to synthesize ∂-toxin and carbenicillin resistance. Pseudomonas fluorescens (pCH 2.1) is deposited in NRRL and accession number NRRL B-15977. Plasmids pSM 1-l7 and pCH 2.l were deposited in NRRL on July 2, 1985.

In the experiments below, P. fluorescens (pCH 2.1) is used with the control, which is an untransformed cell.

Culture deposits E. coli (pSM 1-17) -NRRL B-15976 and P. fluorescens (pCH 2.l) -NRRL B-15977 will be deposited at the NRRL site for at least 30 years. The deposit may also be sold to the public upon patenting using the deposit. It may also be sold, if necessary, in accordance with the foreign patent law of that country when a part or a copy of the application is filed. It should be understood, however, that the sale of such deposits does not permit the practice of the present invention while lowering the patent rights granted by administrative measures.

The deposits E. coli (pSM 1-17) and P. fluorescens (pCH 2.1) were deposited on March 25, 1986 as KFCC-1010 and KFCC-10194, respectively, to the Korean spawn association.

Example 5

Microbial treatment and testing with heterologous genes

Pseudomonas is killed by treatment at ambient temperature using either 1% Lugol iodine (100 g KI, 50 g I ₂ , 100 ml glacial acetic acid in lL sterile deionized water) or 2% formalin solution, respectively.

B. t. Divide the cell pellet in 2 liter broth cultures of P. fluorescens with toxin and P. fluorescens without. Half-divided cells are treated with 1% Lugol iodine for 4 hours and the other half is kept in ice.

The washed golgol treated and untreated cells are pelleted with 10 ml of sterile deionized water. Suspension (10E ⁸ -10E ¹² cells / ml) 9 ml Spray three young lettuce plants. Three sprayed plants are placed in a single closed chamber and 50-75 Trichoplusia ni larvae are applied to the plants. If no leaves are lost during the observation period, the plant is considered protected.

TABLE 3

25 larvae applied to plants for 1-2 days.

50 larvae applied to plants for 3-5 days.

Observation record after 10 days.

The newly hatched cabbage worms are placed in a Petri dish containing droplets of the material to be biopsied. Once the larvae absorbed the liquid, they were removed with a small cup containing the larvae diet. After seven days, the larvae are examined and the total number of dead animals recorded. Table (4) below shows the results.

TABLE 4

Another method of erasing cells is to determine the stability effects of the cells on sound waves.

P. fluorescens strain 33 is an untransformed cell containing no Bt toxin, while strain pCH contains a 2.1 kb toxin gene. As a first task, stationary phase cultures of strain 33 and strain pCH are collected by centrifugation and cell pellets are suspended in sterile deionized water at concentrations of 10E ¹⁰ and 10E ⁹ . Cell aliquots are exposed to a 70 ° C. water bath with varying time and cell viability is determined by plate count of king's B agar. (King, EO et al. (1954) J. Lab. Clin.Med. 44: 304). The medium had the following components: Proteose peptone 3, 2.0 percent; Bactoagar, 1.5 percent; Glycerol, CP, 1.0 percent; K ₂ HPO ₄ [anhydrous] 0.15 percent; MgSO 4 _˙ 7H ₂ O, 0.15 percent; pH 7.2 strain 33 died completely within 5 whereas strain pCH had no viable cells for 10 minutes.

Strain 33 cells (treated by heat at 70 ° C., untreated or treated with Lugol [treated for 2 hours at room temperature with 1% Lugol's iodine]) were suspended in 10 ml of deionized water at 10E ⁹ per ml. Cells) concentration was formed. The cell suspension is subjected to sonication for 5 minutes on a Bronson 200 Sonifier (microbial emissions 10, 50% duty cycle, pulsed mode).

The optimum density of the cell suspension (575 nm) is measured before and after sonication. Optical density reduction indicates cell division.

Tables (5) and (6) below show the results.

TABLE 5

Cells of strain pCH (5 × ^{10 E 10} / ml) were starched in a water bath at 70 ° C. and then analyzed for virulence against T. ni larvae. The biological assay procedure uses cabbage worms to place them in a petri dish and drop them off for analysis. Once the larva absorbs the liquid, the larva is removed with a small cup containing the larvae diet. After six days, the larvae are examined and the total number of dead animals recorded.

TABLE 6

In the greenhouse (1) spraying Romaine lettuce leaves with insecticidal plants, (2) growing the treated plants in the greenhouse for up to 19 days (greenhouse covered with UV transparent roof), and (3) retaining insecticidal activity in the leaves Leaf inhibition analysis of cabbage worms was carried out using P. fluorescens + B. The persistence of t toxin (strain pCH) cells was investigated.

Determination of B. t toxin supplying inhibition is as described above and the method of spraying plants is described in Example (5).

In this experiment, the toxin activity of 1.0 is the same as the insecticidal force consisting of Dipel 0.19 for 100 ml sprayed at a rate of 10 ml per plant (ie, before the end of the discharge).

As shown in table (7), after 11 days Pseudomonas showed full activity when the diepel lost 2/3 of its initial activity.

It can be seen from the data in Table 7 that the immobilized Pseudomonas cells are in a way to protect the insecticidal toxin from inert action on the leaf surface.

TABLE 7

From the above results it can be seen that while all the pesticidal action of the living bacteria is retained, the toxin containing exterminated bacteria offers many advantages. It can be seen that the maintenance of dead bacteria during storage, transport and application is more convenient and economical than using live ones. At the same time, the microorganisms encapsulate and protect the toxin to maintain the biological action of the toxin for a long time. In addition, denaturation of the protease prevents protein denaturation of the polypeptide pesticide.

Various methods of killing microorganisms without denaturing protein structures or destroying their structure can be used to provide the desired pesticidal action, as well as to provide pesticidal compositions used or applied as agents in plants and plant products. Can be.

Although the foregoing embodiments and embodiments of the invention have been described for a better understanding, variations and modifications are possible within the scope of the appended claims.

Claims (3)

A method for producing a single-celled microorganism close to undegraded intracellular toxin, characterized by culturing a microorganism containing a gene encoding a polypeptide toxin and subjecting microbial cells under protease inactivation conditions or cell wall strengthening conditions. The method of claim 1, wherein the gene encoding the polypeptide toxin is a heterologous gene. The method of claim 1, wherein the gene encoding the polypeptide toxin is a homologous gene.