CA1341052C - Cellular encapsulation of biological pesticides - Google Patents

Abstract

Methods and compositions are provided for preparing and using pesticides, where the pesticides are encapsulated in non-proliferating cells. In one aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagents than prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). In another aspect of the invention a heterologous gene is introduced into a cellular host, where expression of the heterologous gene results, directly or indirectly, in production of the pesticide.
These cells are then killed under conditions which prolong the pesticidal activity when said cells are applied to the environment of a target pest. The thus treated cells can be used directly or after formulation for treatment of an agricultural host or environment of the host with the pesticide.

Description

,, , 1 54~ X52 DESCRIPTION
CELLULAR ENCAPSULATION
OF BIOLOGICAL PESTICIDES
Background of the Invention The extraordinary increase in agricultural pro-ductivity has been a result of many factors, including significantly better understanding of the methods involved caitr, agriculture, improved equipment, availa-bility of fertilizers, and improved pesticides. The latter factor has not been without detrimental aspects, hocaever, due to the negative effect on the environment.
There is, therefore, a substantial interest in developing effective and. environmentally acceptable pesticides.
Among ecologically acceptable pesticides are the protein toxir,.s produced by various microorganisms, such as Bacillus thurinli_g ensis. However, the use of B. thurin-:ZO giensis lysate or spores as a pesticide has significant drawbacks. The lifetime of the pesticide is relatively short in the environment, requiring multiple applications to give adeqL:ate protection. Consequently, these pesti-cides are not. economical in comparison to more traditional ;)_5 chemical proc;ucts having long residual activities.
Improvements in field longevity would greatly aid in expanding the: application of biological, or protein toxin-based pesticides.
As indicated above, there are many requirements for 30 pesticides associated with their particular application.
For example, in many cases it is desirable to have pesti-cides which have long residual activity in the field while not accumulating in the environment. In addition, because of economic, considerations, it is preferable ~34~ 452

-2-to have pesticides which have a reasonably broad spectrum of biocidal activity. Also, the pesticide should degrade to degradation products which are environmenta:Lly acceptable. Other considerations include ease of formulation, pesticidal activity, stability to environmental effects, such as light, water, organ:~sms, and the like, and effect on beneficial or innocuous organisms in the environment.
West, Soil Biol. Biochem. (1984) 16:357-360 reports the results of a study on the persistence of _B._t. toxin in soil. SeE~ also, West et al., J. of Invertebrate Pathology (1~~84) 43:150-155. U.S. Patent No. 4,265,880 describes emt>edding live insecticidal pathogens in a coacervate mi.crobead. Japanese Patent No. 51-5047 describes physical-chemical methods for killing _B.
thuringiensis: spores, while retaining toxicity.
Brief SWrimary of the Invention Methods and compositions are disclosed for protecting a~,ricult:ural crops and products from pests. In one aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing or-ganism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).
In another aspect of the invention, the vesticides are produced by introducing a heterologous gene into a cellular host. Expression of the hetero-:30 logous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated, as dis-closed above, under conditions that prolong the activity :35 X34, 052-

- 3-of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin.
These naturally encapsulated pesticides may then be formulated in. accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants.
Detailed Disclosure of the Invention .L 0 In accordance with the subject invention, improved pesticides are provided, having among their other ad-vantages an extended residual life, by modifying naturally-occurring pesticide-producing microorganisms, .l5 or pesticide-producing microorganisms hosting a hetero-logous gene capable of expression in the host, where expression of the gene results, directly or indirectly, in the production of a pesticide. The subject invention involves treating t:he organisms with reagents that ~'_0 prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin.
A wide variety of pesticides can be produced which ~5 will be characterized by being capable of being pro-duced intracellularly, particularly in a unicellular microorganism host, such as prokaryotes, e.g., bacteria;
or eukaryotes, e.g., fungi, exemplified by yeast and filamentous fungi, such as Neurosnora and As~ergillus;
.f0 or protists, such as amoebas, protozoa, algae, and the like.
.15

-4-The pesticidE~ can be any toxin produced by a microbe.
For example, it can be a polypeptide which has toxic activity toward a eukaryotic multicellular pest, such as insects, e.g., coleoptera, lepidoptera, diptera, hemiptera, dermaptera, and orthoptera; or arachnids; gastropods;
or worms, such as nematodes and platyhelminths. Various susceptible insects include beetles, moths, flies, grasshoppers, lice, and earwigs.
The pesticide which is produced in the host cell may be a polypeptide produced in active form or a pre-cursor or proform which requires further processing for toxin activity, e.g., by the pest, as with the crystal toxin of B. thurin~iensis var. kurstaki. Thus, the gene may encode an enzyme which modifies a metabolite to produce a pesticidal composition.
Among naturally-occurring toxins are the polypeptide crystal toxins of B. thuringiensis var. kurstaki, active against lepidoptera; B.t. var. israelensis, active against mosquitoes; :B.t. M-7, active against coleoptera; _B.
thurin~iensis var. aizawai, active against spodoptera;
and B. s hae:ricus, active against mosquito larvae. Other toxins include those of entomopathogenic fungi, such as beauverin of Beauveria bassiana and destruxins of I,~etarhizium spp.; or the broad spectrum insecticidal compounds, such as the avermectins of Streptomyces avermitilus. Cultures exemplifying the above are as follows:
Bacillus thurinpiensis var. kurstaki HD-1--NRRL B-3792; disclosed in U.S. Patent 4,448,885 Bacillus thuringiensis var. israelensis--ATCC 35646 Bacillus thuringiensis M-7--NRRL B-15939 Bacillus thurin~iensis var. tenebrionis--DSM 2803 The following B. thurin~iensis cultures are avail-able from th~~ United States Department of Agriculture

-5-(USDA) at Texas.
Brownsville, Requests should be made Co Joe Garcia, otton Insects Research Unit, USDA, ARS, C

P.O. Box 1033, Brownsville, Texas 78520 USA.

B. thuringiensis HD2 B. thwringiensis var.finitimus HD3 B. thu~ring_iensisvar.alesti HD4 B. thu:rin~3.ensisvar.kurstaki B. thu:ring'~ensisvar.sotto HD770 B. thu:cin iensis var.dendrolimus B. thu:ringiensis var.kekenyae B. thu:ringiensis var.galleriae B. thuringiensis var.canadensis B. thuni_ ngiensisvar.entomocidus B. thuringiensis var.subtoxicus HD109 B. thuringiensis var.aizawai HD11 B. thuringiensis var.morrisoni B. thuringiensis var.ostriniae HDSO1 B. thuringiensis var.tolworthi HD537 B. thuringiensis var.darmstadiensis HD146 B. thunin~ietzsis var.toumanoffi HD201 B. thuringiensis var.kyushuensi:s HD541 B. thuringiensis var.thom~soni HD542 B. thuningiensis var.Pakistani HD395 B. thuz~in~iensis var.israelensis HD567 ;05 B. thunin~iensis var.Indiana HD521 B. thurin iensis var.dakota B. thuringiensis var.tohokuensis HD866 B.. thuringiensis var,kumanotoensis HD867 B. thurin iensis var.tochigiensis HD868 _30 B. thuningiensis var.colmeri HD847 B. thuz~in~iensis var. wuhanensis HD525 :35 ~~4; X52_

-6-Bacillus cereus--ATCC 21281 Bacillus mori.tai--ATCC 21282 Bacillus popi.lliae--ATCC 14706 Bacillus lentimorbus--ATCC 14707 Bacillus sph_aericus--ATCC 33203 Beauveria bassiana--ATCC 9835 Metarhizium anisopliae--ATCC 24398 Metarhizium flavoviride--ATCC 32969 Streptomyces avermitilus--ATCC 31267 The torn need not be the same as a naturally occurring to}cin. Polypeptide toxins may be fragments of a naturally-occurring toxin; expression products of deletion, transver;sion or transition mutations, where two or fewer number percent of the amino acids may be changed; or a repetitive sequence capable of processing by the intended pest host. In addition, fusion products may be prepared where one, five or more amino acids are provided at t:he N-terminus to provide, for example, reduced prote.olytic degradation of the toxin(s). in some instances, a plurality of the same or different toxins may be encoded and expressed, where processing sites may be introduced between each toxin moiety in the polytoxin.
Illustrative host cells may include either prokary-otes or eukaryotes" normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possi-bility of toxicity to a mammalian host. As -_,_ hosts, of particular interest will be the prokaryotes and lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteri-aceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacil.laceae; Rhizobiaceae, such as Rhizo-bium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactob acillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.
Among eukary~~tes are fungi, such as Phycomycetes and.
Ascomycetes, which. includes yeast, such~as Saccharomyc_es and Schizosaccharomyces; and Basidiomycetes yeast, such as r Rhodotorula, Aureobasidium, S~orobolomyces, and the like.
Characteristics of particular interest in selecting a host cell :Eor purposes of production. include ease of introducing the heterologous gene into the host, availability of expression systems, efficiency of expres-sion, stabil:Lty of the pesticide in the host, and the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide micro-capsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracel-lular packag~_ng or formation of inclusion bodies; leaf affinity; la<:k of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to thE~ toxin; and the like. Other considera-tions includE: ease of formulation and handling,- eco-nomics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharornyce~~ sp., and Sporobolomyces sp.; phylloplane organisms such Pseudomonas sp., Erwinia sp. and Flavo-bacterium sp.; or such other organisms as Escherichia, _$_ Lactobacillus sp., Bacillus sp., and the like. Speci-fic organisms include Pseudomonas aeru~inosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thurin-~iensis, Escherichia coli, Bacillus subtilis, and the like.
The cell will usually be intact and be substantially in the proliferative form when killed, rather than in a spore form, ,although in some instances spores may be employed.
The cel:Ls may be inhibited from proliferation in a variety of ways, so long as the technique does not deleteriousl~~ affect the properties of the pesticide, riot diminish the cellular capability in protecting the pesticide. '.:he techniques may involve physical treat-ment., chemic<~1 treatment, changing the physical charac-ter of the cE:ll or leaving the physical character of the cell substantially intact, or the like.
' Various techniques for inactivating the host cells includ~s heat, usually 50°C to 70°C; freezing; UV
irradiation; lyophilization; toxins, e.g., antibiotics;
phenols; ani:Lides, e.g., carbanilide and salicylanilide;
hydroxyurea; quaternaries; alcohols; antibacterial dyes;
EDTA and ami<iines; non-specific organic and inorganic 2S chemicals, such as halogenating agents, e.g., chlorinating, brominating c>r iod:inating agents; aldehydes, e.g., glutaraldehyde or :formaldehyde; toxic gases, such as ozone and ethylene oxide; peroxide; psoralens; desiccating agents; or the like,which may be used individually or in combination. The choice of agent will depend upon the particular pesi:icide, the nature of the host cell, the nature of the modification of the cellular structure, :35 such as fixing and preserving the cell wall with cross-linking agents, or the like.
The cells generally will have enhanced structural stability which will enhance resistance to environmental degradation in the field. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bioavailability or bioactivity of the toxin.
The hetE~rologous genes) may be introduced into the host in any ~~onvenient manner, either providing for extrachromosomal maintenance or integration into the host genome. (By heterologous is intended that the gene is not present in the host into which it is introduced, nor would the gene normally be found in such host. That is, even if t:he host organism and the source of the heterologous gene exchange information, the heterologous gene would nc>rmall:y not be found in the wild-type host cells in nature. 'Usually, the term heterologous will involve species of different genera as host and gene source.) Various constructs may be used, which include replication systems from plasmids, viruses, or eentro-meres in comf~ination with an autonomous replicating segment (ors) for stable maintenance. Where only integration i.s des:ired, constructs can be used which may provide for replication, and are either transposons or have transposon~-like insertion activity or provide for homology with the genome of the host. Frequently, DNA sequences are employed having the heterologous 1 3~1 X52 -lo-gene between sequences which are homologous with se-quences in the genome of the host, either chromosomal or plasmid. Desirably, the heterologous genes) will be present in multiple conies. See for example, U.S.
Patent No. 4;,399,216. Thus, conjugation, transduction, transfection and transformation may be employed for introduction of the heterolojous gene.
A large number of vectors are presently available which depend upon eukaryotic and prokaryotic replication systems, such as ColEl, P-1 incompatibility plasmids, e.g., pRK290, yeast 2m a plasmid, lambda, and the like.
Where are extrachromosomal element is employed, the DNA construct will desirably include a marker which allows for a selection of those host cells containing :15 the construct. The marker is commonly one which provides for biocide resistance, e.g., antibiotic resistance or heavy metal resistance, complementation providing prototrophy to an auxotrophic host, or the like. The replication systems can provide special properties, such as runaway replication, can involve cos cells, or other special feature.
Where the heterologous genes) has transcriptional and translational initiation and termination regulatory signals recognized by the host cell, it will frequently be satisfactory to employ those regulatory features in conjunction with the heterologous gene. However, in those situations where the heterologous gene is modified, as for example, removing a leader sequence or providing a sequence which codes for the mature form of the pesticide, where the entire gene encodes far a precursor ~.t will freqmantly be necessary to manipulate the DNA
sequence, so that a transcriptional initiation regulatory sequence may he provided which is different from the ?~5 ~ 34 ~ ~5 2 natural one.
A caide variety of transcriptional initiation sequences exist for a wide variety of hosts. The sequence can provide for constitutive expression of the pesticide or regulated expression, where the regulation may be induc:ible by a chemical, e.g., a metabolite, by temperature, or by a regulatable repressor. See for .
example, U.S" Patent No. 4,374,927. The particular choice of thE~ promoter will depend on a number of factors, the strength of the promoter, the interfer-ence of the F>romater with the viability of the cells, the effect of regulatory mechanisms endogenous to the cell on t:he promoter, and the like. A large number of promoters are available from a variety of sources, :15 including con~inercial sources .
The cellular host containing the heterologous pesticidal gene may be grown in any convenient nutri-ent medium, where t:he DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the hetero-logous gene. These cells may then be harvested in accordance with conventional ways and modified in the various manners described above. Alternatively, the cells can be fixed prior to harvesting.
The meth~~d of treating the host organism containing the toxin can fulfill a number of functions. First, it may enhance snructural integrity. Second, it may provide for enhanced i~rotE~olytic stability of the toxin, by modi-fying the to:cin so as to reduce its susceptibility to pro-teolytic degradation and/or by reducing the proteolytic activity of proteases naturally present in the cell. The cells are pref=erably modified at an intact stage and when there has been a substantial build-up of the toxin protein. The:;e modifications can be achieved in a ..

variety of ways, such as by using chemical reagents having a broad spectrum of chemical reactivity. The intact cells can be combined with a liquid reagent medium containing the chemical reagents, with or without agitation at temperatures in the range of about -10 to 60°C. The reaction time may be determined empirically and will vary widely with the reagents and reaction conditions. Cell concentrations will vary from about 10E2 to 10E1~ per ml.
Of particular interest as chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80, lore particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment wi~h aldehydes, such as formaldehyde and glutaraldehy~le; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See:
Humason, Gre~_chen L., Animal Tissue Techniques, W.H.
Freeman and e~ornpany, '1967) ; or a combination of physical (heat) and chemical agents that prolong:the activity of v~he toxin produced in the cell when the cell is applied to the environment of the target pest(s).
For hal~~genation with iodine, temperatures will generally range from about 0 to 50°C, but the reaction can be conveniently carried out at room temperature.
Conveniently, the iodination may be performed using triiodide or iodine at 0.5 to S~ in an acidic aqueous medium, part:LCUlarly an aqueous carboxylic acid solution that may var;,~ from about 0.5-5M. Conveniently, acetic acid may be used, although other carboxylic acids, :35 ~34~ X52 generally of from about 1 to 4 carbon atoms, may also be employed. The time for the reaction will generally range from less than a minute to about 24 hrs, usually from about 1. to 6 hrs. Any residual iodine may be removed try reaction with a reducing agent, such as dithionite, sodium thiosulfate, or other reducing agent compatible with ultimate usage in the field.
In addition, the modified cells may be subjected to further treatment" such as washing to remove all of the reaction medium, isolation in dry form, and formulation with typical stickers, spreaders, and adjuvants generally utilized in agricultural applications, as is well kno~,rn to those skilled in t:he art.
Of particular interest are reagents capable of cross-linking the cell wall. A number of reagents are known in the art for this purpose. The treatment should result in enhanced stability of the pesticide. That is, there should be enhanced persistence or residual activity of the pesticide undE:r field conditions. Thus, under condi-'0 tions where the pE:sticidal activity of untreated cells diminishes, the activity of treated cells remains for periods of from 1 to 3 times longer.
The cel:Ls may be formulated in a variety of ways.
They may be employed as wettable powders, granules or dusts, by mi:{ing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (pow-dered corncobs, nice hulls, walnut shells, and the like).
The formulat:Lons may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or~sur-factants. L~:quid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients '34~a~2 may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.
The pesticidal concentration will vary widely depending upon they nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1%
by weight and may be 100% by weight. The dry formulations will have fr~~m about 1-95% by weight of the pesticide while the li~~uid formulations will generally be from about 1-60% by weight of the solids in the liquid phase.
The formulat:ions will generally have from about lE2 to about lE4 ceLls/mg. These formulations will be admini-stered at about :? cz (liquid or dry) to 2 or more lb/ha.
The foryulations can be applied to the environment of the pest(;s), e.g., plants, soil or water, by spraying, dusting, spr=inkling or the like.
The following examples are offered by way of illustra-tion and not by way of limitation.
Example 1 After treatment of intact spore-containing cells (prior to auoolysis) of B. thuringiensis with lugol's iodine, the c=ells are killed; however, they retain toxicity to =Crichoplusia _ni larvae.
The intact cells of Bacillus thuringiensis (HD-1) were harvested just prior to autolysis of the sporulating cells by cent=rifugation and the cell pellet suspended in deionized wat=er to give a concentration of 6.O x 10E9 cells/ml. An aliquot of the cell suspension was diluted to 1.5 x lOEFt cells/ml and exposed to 1% lugol's iodine for 4 hr at room temperature (a 1% lugol's solution contains 1.0 g potassium iodide, 0.5 g iodine and 1.0 ml glacial acetic acid per liter.) The treated cells ~ ~4 ~ °5 z were washed ,and resuspended in sterile deionized water to give a cell concentration of 6.0 x 10E9. No viable cells were detected by plate counts on nutrient agar after the 4 hr iodine treatment. Lugol's treated and untreated control cells were then bioassayed for toxicity to T, ni larvae.
Since tile cells of the subject invention are naturally-occurring cells, it would not be necessary to treat there under killing conditions in order to realize the benefits of the subject invention. Thus, treatment of the cells, as described herein, can be optimized by a person skilled in the art to achieve the highest leve~_ of prolongation of toxin (pesticidal) activity in t:he environment of the target pest(s).
B. Bioassay procedure Dilutions of lugol's killed cells or untreated live HD-1 cells were mixed with a constant volume of larval diet c:up. A single S day ald _T. _ni Iarva was then added to each cup. Eighteen larvae were tested per dilution. The larvae were examined after six days and the total number o:E larvae killed was recorded. The results are shown :in Table 1. They are given in percent larvae killed.

~ 34 ~ ~5 2 Table 1 Bioassay of Lugol's-Treated Intact Spore-Containing Bacillus .thuringiensis (HD-1) Cells Cell Dilution 1.0E9 10E8 10E7 10E6 10E5 Untreated 1.00 100 68.8 0 0 Lugo l ' s Treated 94.4 61.0 0 0 0 Example 2--Stability Testing Intact, spore-containing cells of _B._t. HD-1 were treated caith 1% lugol's solution for 4 hr at room temperature, washed in dei,onized water, and stored in the refrigerator for 52 days. After this period the cells remained whole, and there was no evidence of lysis (release of spores and crystal).
Intact, spore-containing cells of _B._t. HD-1 were harvested by centrifigation and the resulting pellet suspended in sterile deionized water (10E10 cells/ml), heated to 70'C for 30 min, and stored in the refrigera-tor for 9 da:,~s. After this period, virtually alI of the cells have l:,~sed, releasing spores and crystals.
Example 3 Soil Experiment Procedure B.t. HD-1 preparation:
An intact spore-containing culture of _B._t. HD-1 was harvested by centrifugation and the cell pellet resus-pended in 400 ml of 1% lugol's iodine (4 x 10E8 cells/ml).
The iodine-cell suspension was stirred for 3 hr at room temperature, washed 3 times and resuspended in 400 ml ~ 34 1 05 2 sterile 0.1. M sodium phosphate buffer, pH 6.9. No viable B.t. fID-1 cells were detected on nutrient agar (l0E-1) after treatment with iodine, and microscopic examination revealed that all cells were intact (unlysed).
Dipe l* preparation Dipel*(0.1 ~;, Abbott Laboratories, 16,000 Interna-tional Units of fotency/mg. List x/5188), which contains B.t. HD-1 ce_Lls, wa,s measured into 400 ml of sterile 0.1 M sodium pho~;phate buffer.
Experimental Design:
1) Non--sterile soil preparation: 40 g of soil was plac=ed in a sterile 500 ml flask and 200 ml of experimental sol_ati_on added .
2) Sterile soil preparation: 40g of soil was placed in a st=erile 500m1 flask and autoclaved for 1 hr prior to adding 200m1 of experimental solution.
Flas)cs containing soil suspensions were incubated on a gyratory shaker (200RPM) at room temperature. Samples (30-40m1) of each soil suspension were filtered through 4x cheeseclcth and sprayed onto the leaves of lettuce plants for subsequent measurement of toxicity against larvae of T. ni .
Measurement _of Bst- Toxin by Feeding Inhibition on_a Leaf Inhibition Assay Leaves of Romaine lettuce seedlings are sprayed with freshly prepared standard concentrations of Dipel*
(1x 0.19g/100m1, 1/10x, 1/20x, and 1/100x), and experi-mental solutions 24 hr before use.
Le,3ves treated with standards or experimental solutions ar~~ removed from the plant, weighed individually, and placed in individual petri dishes. Ten starving,

7 day old T. ni Larvae are applied to each leaf and allowed to feed on the leaf for approximately 24 hrs at 25°C. At the end of the feeding period the leaves are re-weighed and tine average weight loss determined for each trear_ment.
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~ 3~ ~ ~5 2 Under the conditions described above, leaf weight loss is a lob linear function of toxin concentration over concentrations of toxin equivalent to 0.19 mg/ml-0.019 mg/ml ;~~ipe:L (16,000 international units of potency per mg). This, the freshly prepared Dipel concentrations sprayed onto lettuce plants and run with each assay serve as standards by which the concentration of _B._t.
HD-1 toxin on leaves sprayed with experimental solu-tions can be equated. The data in Table 2 show the percentage of the original toxicity (day I) remaining at various times after incubation in soil and subse-quently assa,~ed with the leaf inhibition assay.
Leaves other than lettuce plant leaves can be used in assaying for B.t. toxin efficacy.

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1341 052 _ Inactivation of t:he protein-crystal of _B._t. HD-1 in soil has been shown to be due to the activity of soil microorganisms (~~est, 1984). The results of Table II
demonstrate that when Dipel and a lugol's iodine-treated pre-~.ysed B.t. HD-1 preparation were incubated under similar conditions, the toxicity of the Dipel (spore-crystal) preparation degenerated rapidly, cahile the toxicity of thE~ lugol's iodine-treated cells remained essential:Ly unchanged It is evident from the above results that chemical treatment of whole microbial cells can be performed in such a way as to retain polypeptide toxin activity, while rendering thE: cell stable to storage conditions.
This provides increased residual activity of toxin :LS activity under field conditions.
Example 4--Construction of a Heterolo~ous Gene and Transformation into A Suitable Host.
A construction began with a clone of Pseudomonas :?0 aeruginosa, available from Northern Regional Research Laboratories (NRRL B-12127), containing a broad host range shuttle plasmid pRa1614 (J. Bact. [1982) 150:60; U.S.
Patent No. 4,374,200) . The plasmid has unique HindIII, BamHI, and Sall and PvuII restriction sites, a PstI inser-~'.S tion, which ivncludes the carbenicillin resistance gene and a P. aeruginosa replication system, where the HindIII, BamHi and Sal=I restriction sites are in a tetracycline resistance gene. 'The remainder of the plasmid is derived from pBR322. A second plasmid, pSMl-17, has SO been deposited as a clone of E. coli (NRRL B-15976).
This deposit was made with the permanent collection of the Northern Regional Research Laboratory, U.S. Depart-ment of Agric:ultur~e, Peoria, Illinois 61604, USA.
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f ~'~'~~ X52 Plasmid pSMl-17 confers ampicillin resistance to E. coli and contains a 6..8 Kbp HindIII DNA fragment that includes the d-endotoain J;ene from the 50 and plasmid of Bacillus thuringiensis HD73. Sufficient toxin is expressed from this gene in E. coli to make the intact cells toxic to cabbage loopers. A further modification of the DNA
fragment was done to enhance toxin expression and to accomplish a}cpression of toxin in an alternate host, Pseudomonas f-luorescens . In order to eliminate unwanted DNA from the 6.8 Kbp fragment, pSMl-17 was digested with BamHl to open the circular plasmid at a site nearest to the 5' end of.-' the toxin gene, and was treated with the exonuclease Fia131 to reduce the 6.8 Kbp HindII insert to about 2.9 Kbp. Tha_ free ends of the DNA were filled in a5 with Klenow polymerase, BamHl linkers were added, and the linear DL'JA caas ligated to reform the circular plasmid. ThE: resultant vector, pFG270, was digested with Xhol to open the circular plasmid at a site nearest to the 3' end of the toxin gene. Sall linkers were added, ?0 the plasmid eras ligated to its circular form, and the resultant vector, pFG10.6 was amplified in _E. coli.
pFG10.6 was digested completely with Sall and BamHI and the resulting 2.1.1Zbp fragment containing the toxin gene was purified by ge~1 electrophoresis and ligated into the >5 BamHI and Sal_I sites of plasmid pR01614. The resultant plasmid, pCHa!.1, w;as amplified in E. coli and used to transform Pseudomonas fluorescens to carbenicillin resistance and the ability to synthesize d-endotoxin.
Pseudomonas fluorescens (pCH2.1) was deposited with NRRL and was given the accession number NRRL B-15977.
plasmids pSM:I-17 and pCH2.1 were deposited in the repository on July 2, 1985.
_! 5 In the following illustrative experiments P. fluorescens (pCH2.1) was used in conjunction with controls of untrans-formed cells.
The culture dleposits E. coli(pSMl-17)--NRRL B-15976 and P. fluorescens_(pCH2.1)--NRRL B-15977 were made in the permanent collection of the NRRL repository, to be main-tained for at least thirty years. These deposits are available to the public upon the grant of a patent dis-closing them. The: deposits are also available as required by foreign patent laws in countries wherein counterparts of tt~~e subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Example 5--Treatment and Testing of Microbes Hosting a Heterologous Gene.
The Pseudomonads were either killed by 1% lugol's iodine (1008, KI, _'i0 g I2, 100 ml glacial acetic acid in 1 L sterile distilled water) or by treatment with a 2%
formalin solution, each at ambient temperatures.
Cell pE:llets from two liter broth cultures of P. fluorescEm_s with and without _B._t. toxin are divided in half. Half the cells are treated with 1% lugol's iodine for ~+ hr, while the other half are held on ice.
Washed lugo:L's treated cells and untreated cells are repelleted :Ln 10 ml of sterile deionized water.. Nine ml of this suspension (10E8-10E12 cells/ml) are sprayed on three young lettuce plants. The three sprayed plants are placed :gin a single enclosed chamber and 50-75 Trichoplusia _ni larvae are applied to the plants. Plants are considered protected if there is no visible loss of foliage over the observation period.

~3~'~ ~~2 TABLE 3: Plant Assay 1 - P. fluorescens.
4t Viable Total Microorganism Treatment Cells/ml Larvae Protection __ _____________________._______________________________________ ____ P. fluorescens Live 2.5 x 10E1175 Not protected _P, fluorescens Live 3.1 x 10E1175 Protected + Iit Toxin P fluorescens 1% Lugol's 0 75 Not protected Iod ine 4 hrs P. fluorescens I% Lugol's 0 75 Protected + Bt Toxin Iodine 4 hers * Set up day 25 2-day larvaeapplied plants, 1, to day 3, 50 5-daffy applied plants, larvae to day 10, observation reported.

In the next study, newly hatched cabbage loopers are placed in a Petri dish containing droplets of the material to 'be bio~assayed. The larvae imbibe the liquid and are then removed to small cups containing larval diet. The larvae are examined after seven days and the total number of animals killed is recorded. The follow-ing Table 4 indica~.tes the results.

TABLE 4': Bioassay 1.~ fluorescens - P.

~/ Larvae Pficroorganism Treatment Killed/ % Larvae Viable Cell -_---______ Total Killed Count/ml _____.______ ___________-_____--_____r.__________ S P. fluorescens Live 0/15 0 8 x P. fluorescens Live 11/18 62 2 5 x 10E12 + Bt Toxin .

P. fluorescens 1% Lugol's 0/15 0 0 4 hrs P, fluorescens1% Lugol's 8/13 62 0 + Bt Toxin 4 hrs Bioassay 2 - P. fluorescens !l Larvae Microorganism Treatment Killed/ % Larvae Viable Cell Total Killed Count/ml P. fluorescens Live 0/15 0 3 1 7 x .

_P. fluorescens Live 3/20 1.5 4.5 x 10E11 + Bt Toxin P. fluorescens 1% LuF;ol's 0/15 0 0 4 hrs P. fluorescens 1% LuF;ol's 8/15 53 0 + Bt Toxin 4 hrs ' -25- 1 3 4 1 4 5 2 Bioassay 3 - P. fluorescens ll Larvae Microorganism Treatment Killed/ % Larvae Viable Cell Total Killed Count/ml ____________ _._____________________________________~__________ S P. fluorescens Live-frozen 9/10 90 2 5 x 10E12 + Bt Toxin, 14 days .

P. fluorescens 1% Lugol's 1/15 0 4 hrs P. fluorescens 1% Lugol's 11/15 73 0 + Bt Toxin P. fluorescens 2% formalin 0/15 0 0 4 hrs P. fluorescens 2% formalin 10/15 67 0 + Bt Toxin 4 hrs Bioassay 4 - P, fluorescens II Larvae Microorganism Treatment Killed/ % Larvae Viable Cell Total Killed Count/ml P'. fluorescens Live 6/14 30 3 x P. fluorescens Live 20/20 100 3 x 10E10 + Bt Toxin P. fluorescens 1% Lugol's 0/20 0 ~ 0 4 hrs P. fluorescens 1% Lug;ol's 18/20 90 0 2.'p + Bt Toxin 4 hrs In the next study, different methods of killing the cells were employed to determine the effect on cell stability to s;onication. _P. fluorescens strain 33 is an untransfoxmecl cell not containing the Bt toxin, while strain pCH contains the 2.1 kb toxin gene. In the first study, stationary phase cultures of strain 33 and strain pCH were harvested by centrifugation and cell pellets suspended in sterile deionized water S at concentrations of 10E10 and 10E9, respectively.
Aliquots of cells were exposed to a 70°C water bath for varying amowntsof time and cell viability measured by plate counts on King's B agar. (See King, E~:O. et al.(1954) J.
Lab. ~ Clin. Med. 44:304. The medium has the following ingredients: Proteose peptone No. 3, 2.0 percent; Bacto agar, 1.5 percent; glycerol, C.P., 1.0 percent; K2HP04 [anhydrous], 0.15 percent; MgS04~7H20, 0.15 per cent;
adjusted to ~pH 7.2.) Strain 33 was substantially completely killed within 5 min, while strain pCH showed substantially_no viable cells within 10 min.
Cells of strain 33 (untreated, heat treated at 70°C, or lugol's treated [1% lugol's iodine, 2 hr, room temperature]) were suspended in 10 ml deionized water to give a cell concentration of approximately 10E9/ml. The cell suspension was then subjected to sonication for 5 min on a Bronson 200 Sonifier (using a microprobe output 10; 50% duty cycle, pulse mode).
The optical density (575 nm) of the cell suspension was measured before and after the sonication treat-ment. A decrease in optical density indicates cell disruption. The following Tables 5 and 6 give the results.

. 1349 052 TADL~ 5: Sonication - f. fluorescens 33 Optical Density (575nm) Treatment Pre-sonication Post-sonication _ _ _ _____________________________ __________ Live 0.24 0.05 Lugol's 0.20 0.19 (1%, 2hr, room temp) Heat 70C

1 0.22 0.06 3 0.21 0.07 5 0.22 0.09 0.20 0.10 30 0.21 0.11 ' 60 0.19 0.13 90 0.22 0.13 15 Cells of strain pCH (5x10E10/ml) were heat treated in a 70°C water bath and assayed for toxicity against T. ni larvae:. The bioassay procedure involved newly hatched cabbage loopers which were placed in a petri dish containing droplets of the materials to be bioassayed. The larvae were allowed to imbibe the liquid and were them removed to small cups containing larval diet. The larvae were examined after six days and the total number of animals killed recorded.

' ~'~4 ~ X52 TABLE 6: Bioassay Heat Treated P. fluorescens Treatment (70°C) ~~ Larvae Killed/
Time (min) ~~ Viable Cells/Total Cells Total Larvae lx % Killed 3 2 x 10E5 / 5.5 c 10E10 8/8 100 5 2 x 10E6 / 5.5 x 10E10 8/8 100 0 / 5.5 x 10E10 8/8 100 l0 Analysis of the persistence of the lugol's treated cells of P. fluorescens + B.t. toxin (strain pCH) was done in the greenhouse by (1) spraying leaves of Romaine lettuce with the pesticidal cells, (2) exposing the treated plants to the greenhouse environment for up to 19 days (the greenhouse was covered with a UV transpar-ent roof), anal (3) assaying the cabbage looper pesti-cidal activity remaining on the leaves with the leaf in-hibition assay. Measurement of B.t. toxin by feeding :20 inhibition is~ as described previously, and the method for spraying the plants is described in Example 5. In this experiment a toxin activity level of 1.0 is equi-valent to thE: pesticidal activity achieved with 0.19 g of Dipel/100 ml of spray applied at a rate of a~proxi-mately 10 mlfplant (i.e., just prior to run-off).
As shown in Table 7, after 11 days the Pseudomonas preparation is st:i:l1 fully active whereas the Dipel example has lost over 2/3 of its initial activity.
We interpret these data to mean that the fixed Pseudo moms cell i~~ in some manner protecting the pesticidal toxin from inactivation on the leaf surface.

~ "~4 ~ ~5 2 Table 7: Greenhouse Persistence data Activity Day pCH Dipel 1 0.75 1.0 5 1.15 0.85

8 1.1 1.2 11 1.0 0.3 19 0.13 <0.01 l0 It is evident from the above results that the killed bacter is containing toxin provide many advan-tages, while still retaining all or a substantial proportion of the mesticidal activity of the live bacteria. M<<intenance of dead bacteria for storage, transportation and application is more convenient and economical than employing the live organisms. At the same time, the organisms provide encapsulated protection of the toxins, so as to maintain the toxins' bioactivity for long periods of time. In addition, denaturation of the proteases prevents proteolytic degradation ~of the polypeptide pesticides. Various means for killing the organisms without disrupting their structure oz' denaturing the protein pesticide can be employed to provide the desired,pesticidal activity, while providing for a convenient pesticidal composition for use in formulation and application to plants and plant products.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (11)

1. A;pesticidal composition comprising pesticide-containing substantially intact treated cells having prolonged pesticidal activity when applied to the environment of a target pest, wherein said pesticide is a polypeptide, is intracellular and is produced naturally or as a result of expression of a heterologous gene in said cell.

2. A pesticidal composition according to claim 1, wherein said cells are killed under protease deactivating or cell wall strengthening conditions, while retaining pesticidal activity.

3. A pesticidal composition, according to claim 1, wherein said cells are prokaryotes selected from the group consisting of Enterobacteriaceae, Bacillaceae, Rhizobiaceae, Spirillaceae, Lactobacillaceae, Pseudomonadaceae, Azotobacteraceae, and Nitrobacteraceae; or lower eukaryotes selected from the group consisting of Phycomycetes, Ascomycetes, and Basidiomycetes.

4. A pesticide composition, according to claim 3, wherein said prokaryote is a Bacillus specie selected from a pesticide-producing strain of Bacillus thuringiensis, consisting of B. thuringiensis M-7, B.
thuringiensis var. kurstaki HD1, B. thuringiensis HD2, B.
thuringiensis var. finitimus HD3, B. thuringiensis var.
alesti HD4, B. thuringiensis var. kurstaki HD73, B.
thuringiensis var. sotto HD770, B. thuringiensis var.
dendrolimus HD7, B. thuringiensis var. kenyae HD5, B. thuringiensis var. galleriae HD29, B. thuringiensis var. canadensis HD224, B. thuringiensis var.
entomocidus HD9, B. thuringiensis var. subtoxicus HD109, B. thuringiensis var. aizawai HD11, B. thuringiensis var. morrisoni HD12, B. thuringiensis var. ostriniae HD501, B. thuringiensis var. tolworthi HD537, B.
thuringiensis var. darmstadiensis HD146, B. thuringiensis var. toumanoffi HD201, B. thuringiensis var.
kyushuensis HD541, B. thuringiensis var. thompsoni HD542, B. thuringiensis var. pakistani HD395, B.
thuringiensis var. israelensis HD567, B. thuringiensis var. Indiana HD521, B. thuringiensis var. dakota, B.
thuringiensis var. tohokuensis HD866, B. thuringiensis var. kumanotoensis HD867, B. thuringiensis var. tochigiensis HD868, B. thuringiensis var. colmeri HD847, B. thuringiensis var. wuhanensis HD525, B. thuringiensis var. tenebrionis, and other Bacillus species selected from B. cereus, B. moritai, B. popilliae, B. lentimorbus, and B. sphaericus.

5. A method of protecting plants against pests which comprises applying to said plants an effective amount of a pesticidal composition comprising pesticide-containing substantially intact unicellular micro-organisms, wherein said pesticide is intracellular and is produced naturally or as a result of expression of a heterologous gene in said microorganism, and said microorganism is treated under conditions which prolong the pesticidal activity when said composition is applied to the environment of a target pest.

6. A method according to claim 5, wherein said microorganisms are prokaryotes selected from the group consisting of Enterobacteriaceae, Bacillaceae, Rhizobiaceae, Spirillaceae, Lactobacillaceae, Pseudo-monadaceae, Azotobacteraceae, and Nitrobacteraceae; or lower eukaryotes, selected from the group consisting of Phycomycetes, Ascomycetes, and Basidiomycetes.

7. A method according to claim 5, wherein said unicellular microorganisms are killed under protease deactivating or cell wall strengthening conditions, while retaining pesticidal activity.

8. Substantially intact unicellular microorganism cells containing an intracellular toxin, which toxin is a result of expression of a heterologous gene which codes for a polypeptide toxin, wherein said cells are killed under protease deactivating or cell wall strengthening conditions, while retaining pesticidal activity when said cell is applied to the environment of a target pest.

9. Cells according to claim 8, wherein said microorganism is a Pseudomonad and said toxin is derived from a B. thuringiensis.

10. Plasmids selected from (a) plasmid pSM1-17, which confers ampicillin resistance to E. coli; and contains a 6.8 Kbp HindIII DNA fragment that includes the .delta.-endotoxin gene from the 50 md plasmid of Bacillus thuringiensis HD73; and (b) plasmid pCH2.1, which contains a 2.1 Kbp DNA
fragment containing the toxin gene from B.
thurinqiensis HD73 ligated into the BamHI
and SalI sites of plasmid pRO1614.

11. Microorganisms selected from E. coli transfected with pSM1-17 and P. fluorescens transfected with pCH2.1.