WO1979000155A1

WO1979000155A1 - Microbial insecticides

Info

Publication number: WO1979000155A1
Application number: PCT/US1978/000080
Authority: WO
Inventors: R Andrews; K Spence
Original assignee: Battelle Development Corp
Priority date: 1977-09-22
Filing date: 1978-09-12
Publication date: 1979-04-05
Also published as: IT7827813A0; IE47370B1; NL7809563A; FR2403744A1; JPS5455722A; GB2020976B; CA1109785A; IE781892L; IT1099109B; BE870651A; GB2020976A

Abstract

An improved insecticidal composition is disclosed comprising a microbial agent of viral or bacterial origin embedded in a microbead, wherein the microbead contains sufficient radiation-absorbing material to effectively shield the agent from sunlight-induced inactivation. Typically the microbead is comprised of a nucleic acid and a protein-containing material, and is stabilized by chemical crosslinking. A method for preparing a microbial insecticide composition is also disclosed comprising: mixing a buffered aqueous solution containing a microbial insect pathogen with an aqueous solution containing a nucleic acid, and mixing the said mixture with an aqueous solution containing a proteinaceous material, thereby spontaneously forming microbeads having the pathogen embedded therein. Typically the microbeads are subsequently stabilized by treatment with chemical crosslinking agents, and typically, prior to the two mixing steps, the microbial insect pathogen is cleaned by washing and its surface charge is modified by the addition of a protein-modifying agent adapted to provide a more uniform surface charge.

Description

DESCRIPTION;

MICROBIAL INSECTICIDES

Technical Field

This invention relates to microbial insecticides. More particularly, the invention relates to a novel microbial insecticide composition and to the production and utilization thereof.

Background Art

Microbial insecticides of viral or bacterial origin offer significant advantages over conventional chemical insecticides. Microbial insect pathogens are generally nontoxic and harmless to other forms of life. In addition, microbial insecticides demonstrate a relatively high degree of specificity, and hence do not endanger beneficial insects: Moreover, a suscep¬ tible insect host is quite slow to develop resistance to microbial pathogens. Microbial insecticides may be used in relatively low dosages, may be effectivel - applied as dusts or sprays, and may be used in combination with chemical insecticides.

For example, Bacillus thuringiensis _________ a spore-forming bacterium, is well-known as a micro¬ bial insect pathogen useful against numerous leaf- chewing insects in their larval stages, including, for example, alfalfa caterpillars, tomato hornworms.

tobacco hornworms, cabbage loopers, cabbage web worms, army worms, gypsy moths, walnut caterpillars, diamond- back moths, cosmopolitan green beetles, European corn borers, and other members of the order Lepidoptera. Unfortunately, the effectiveness and usefulness in the field of many microbial insect pathogens as insecticides is severly limited by their extreme sensitivity to sunlight. It is known that nonionizing radiation having a high photon energy (e.g. ultraviolet rays of sunlight) exerts an inactivating effect on various microbial insect pathogens. See, e.g., "Photoprotection Against Inactivation of Bacillus thuringiensis Spores by Ultraviolet Rays," Aloysius Krieg, Journal of Invertebrate Pathology, Vol. 25, pp. 267-268 (1975) . For example, it is known that ultraviolet (UV) rays with a wavelength of 253.7 nm induce a marked, extra¬ ordinary inactivation of Bacillus thuringiensis (B.t.) spores, so that they are unable to germinate and grow out. A dosage of 18 m W sec/cm^ of such UV radiation will inactivate 99.9% of the B.t. spores. We have determined that the half life of B.t. subjected to UV radiation of sunlight in the field is approximately six minutes. Likewise, it has been determined by others that the half lives of certain occluded viruses subjected to UV radiation of sunlight in the field is one and one-half hours. Thus, the effectiveness of a typical spray application of such microbial insecticides is rapidly lost in the field. Since nucleic acids show a maximum of extinction near a wavelength of 260 nm, it is believed that the UV induced death of B.t. at 253.7 nm, and of certain occluded viruses at comparable wavelengths, may be caused by a photoreaction of the genetic material, especially DNA. Thus, it has been suggested.

^"BU

_ ^~ ibid. , at p. 267, that^' B.t.- spores could be protected from inactivation by UV radiation by physically mixing the B.t. spores with DNA, or a comparable nucleic acid which would absorb the UV rays. Such a comparable nucleic acid would be RNA, Ribonucleic Acid, which has a maximum of extinction near 260 nm. However, this technique proved to be ineffective.

Disclosure of Invention

In the present invention, materials capable of absorbing radiation which would otherwise inactivate the microbial insect pathogen of concern are incorporated in a protective coating which surrounds the pathogen in the form of a microbead. These materials serve to intercept and block the harmful radiation before it reaches the light- sensitive material of the insect pathogen. In a typical embodiment of the invention the material selected to intercept the harmful radiation comprises a nucleic acid; and in a preferred embodiment the material comprises RNA. Various other materials and combinations of materials may be used in combination with the nucleic acid depending on the specific insect pathogen to be protected and the microbead coating system to be used; including, but not limited to the following materials: protamine, cytochrome c, soy protein, hemoglobin, gelatin, etc. In general, any protein can be used if the conditions are adjusted as to facilitate formation of the microbeads. Such conditions may include charge modification techniques, adjustments in pH, component concentrations, etc.

The microbeads in which the microbial insect pathogen are embedded may be advantageously produced using any known technique for forming what have been called coacervate droplets or microbeads. One such technique was developed in conjunction with the study of the origin of life on earth, and has been used to construct precellular models. See, for example, Evreinova, et al.. Journal of Colloid and Interface Science, Vol. 36.' No. 1 (1971).

Brief Description of Drawings

Fig. 1 is a graph showing the optical density (i.e., absorption) over the solar UV range of typical microbeads suitable for use in the present invention.

Figs. 2-6 are graphs showing the comparative experimental data from Examples 1, 2, 4, 5, and 6, below, respectively. In Figs. 2-4 the number of viable spores, extrapolated to 1 ml of original sample, is shown as a function of the length of time of exposure to the UV radiation. In Figs. 5 and 6 the percentage of B.t. remaining as survivors is shown as a function of the exposure time.

Best Mode for Carrying Ou the Invention In one preferred embodiment of the invention the microbeads are produced using the above-mentioned technique as follows. An aqueous solution containing a nucleic acid such- s, for example, RNA, and a buffering agent such as, for example, phosphate, designed to maintain a pH at the position which optimizes the charge on the RNA, the protein and the microbe (where the microbe is sensitive to pH, this should be taken into account as well) , is mixed with an aqueous solution containing an appropriate protein material, such as, for example, protamine, gelatin, soy isolate, hemoglobin, etc. or synthetic a ino acid polymers. Protein-nucleic acid microbeads which are essentially solid and roughly spherical form spontaneously upon the mixing of these two solutions.

It is important to note that while all of the above-named protein materials, and others, can be used satisfactorily in forming the microbeads, care must be taken to maintain the pH of the mixture of solutions on the acid side of the isoelectric point of the particular protein being used since this is required for formation of the microbeads. If a protein is used that is insoluble at a given pH, the pH will have to be put in a range in which the protein is soluble, or other steps will have to be taken to make the protein soluble. These steps could include partial degradation, charge modification or adding other components in the buffers (e.g. detergents, alcohols, surfactants, etc.). If foaming of one or more components is a problem, simethicone type agents (U.S. patent No. 2,441,098) can be included. When RNA is being used as the nucleic acid, the pH of the mix- ture of solutions must be maintained at or above about 4.3 to prevent the RNA from precipitating out of the microbead, with the protein necessarily leaving the microbead and going back into solution.

As can be seen, in the above-described method for forming microbeads (i.e., coacervate droplets) the material to be utilized to intercept and absorb the harmful radiation, i.e.,- the nucleic acid, is incor¬ porated directly in the microbead structure, thereby producing a highly protective coating. The bimolecular structure of the microbeads creates a thermodynamically stable cooperation between the components, so that even without subsequent chemical crosslinking, as described below, the components will not individually diffuse out of the microbeads. The bimolecular structure also causes the microbeads to be highly charged. These charges should aid the microbeads in sticking to plant surfaces. These charges-can be controlled by selecting the appropriate protein to be used in forming the microbead.

The size of the microbeads can be controlled by controlling the concentration of the nucleic acid and the protein in the formation vessel. For example, 100μ beads can be made by mixing 5% RNA with 10% protamine sulfate. Beads will form and settle to the bottom of the vessel. Most of these will be in the 100y range. While a relatively wide range of nucleic acid and protein concentrations can be used to make these microbeads generally, in preparing microbeads for use in the present invention (i.e. for entrapping microbial insect pathogens) it is preferred to use a nucleic acid : protein ratio in the range from about 1:5 to 5:1. If the insect pathogen Bacillus thuringiensis is to be embedded in the microbeads, it is preferred to use RNA as the nucleic acid and protamine sulfate as the protein, and to use an RNA: protamine sulfate ratio of approximately 1:2.

In those embodiments of the invention wherein it is desired to utilize the microbeads described above, the microbial insect pathogen may be embedded (i.e., entrapped) in the microbeads by simply mixing it with an aqueous solution of the desired buffering agent, e.g. phosphate, and then mixing this suspension with an aqueous solution containing the desired nucleic acid. The resulting suspension is then mixed with the aqueous protein solution as described above and the pathogen is spontaneously embedded in the protein-nucleic acid microbeads which form. As an alternative, the microbe may be carried in the protein solution and then mixed with an aqueous solution containing the nucleic acid.

It has been found that subsequent shaking of the vessel in which the solutions have been mixed will cause the microbeads to coalesce and spontaneously reform, usually resulting in additional pathogens being embedded in the microbeads.

While the microbeads produced according to the above-described technique possess a certain degree of stability, (i.e., resistance to breakage and coalescence) , it may be advantageous to increase their stability to facilitate separation of embedded pathogens from non-embedded pathogens and to further facilitate handling. In one preferred embodiment of the invention, this stabilization is accomplished by chemically crosslinking the microbead protein molecules by treating them with crosslinking agents such as, for example, glutaraldehyde, imidoester agents, dithiobissuccimidyl propionate, etc. If it is desired to use glutaraldehyde, an aqueous solution of 0.25%, or less, (by weight) should be used, since we have found that as the glutaraldehyde concentration is increased, certain pathogens, in particular. Bacillus thuringiensis, will tend to become inactivated. It should be noted that the depth of cross- linking can be controlled rather easily by controlling the time, concentration, temperature, and other conditions of crosslinking. For example, the depth of crosslinking may be controlled by stopping the cross- linking reaction by adding a small molecule which reacts with the crosslinking reagent (e.g. lysine added to glutaraldehyde) or by using small crosslinking reagent concentrations.

Such chemical crosslinking of the microbeads yields several advantages, including: (1) stabili- zation against the shear forces created by spray application of the insecticide? (2) maintenance, if desired, of fluid centers within the microbeads; (3) maintenance, if desired, of a pH level inside the microbead which is lower than that of the environment surrounding the microbead (i.e., alkaline digestive juices of the insect gut) so that the interior of the microbead may be kept at a pH value near the optimum pH value for viability, storage, etc.- of the microbial pathogen; (4) control of the position in the insect gut where the pathogen is released (i.e. the greater the crosslinking, the further along in the gut release will occur and vice-versa) , thereby increasing the infectivity of the pathogen. We have also found that the microbial insect pathogen may be embedded (i.e. entrapped) in the above-described microbeads much more readily and in much greater numbers if its net surface charge is first modified so as to be made nearly totally negative or nearly totally positive. This surface charge modification may be accomplished, for example, by the controlled addition of a protein modifying agent such as, for example, succinic anhydride, imidoesters, and similar compounds (see e.g., Gary E. Means and Robert E. Feeney, Chemical Modifications of Proteins, Holden Day, Inc. , 1971) . The effectiveness of this charge modification technique will generally be increased by first washing the microbial pathogen composition in separate organic (e.g. 60% ethanol solution, by weight) and inorganic (e.g. 1M sodium chloride solution) washes.

Since the charge-modified pathogen apparently competes with the like-charged^'component of the microbead for positions in the bead, care must be taken to reduce the concentration of such like- charged component to a level which will permit incorporation of the pathogen into the microbead. For example, if it is desired to entrap Bacillus thuringiensis cells, spores, and toxin crystals, all of which have been modified to a strongly negative surface charge, in an RNA-protein microbead as described above, it may be necessary to reduce slightly the concentration of the RNA solution (RNA is also negatively charged) prior to mixing with the protein solution.

Likewise, in such a microbead system, if the surface charge of the pathogen has been modified to a strongly positive surface charge, then it may be necessary to reduce slightly the concentration of the protein solution (protein is positively charged) prior to mixing with the RNA solution. This latter system may be more attractive for purposes of the present invention since it does not require reduction of the amount of radiation-absorbing material (i.e. RNA). Since a primary object of the present invention is the protection of light-sensitive microbial insect pathogens against inactivation by harmful radiation from the sun, it is obviously important, in selecting the materials to be used in formulating the microbeads, to select materials which strongly absorb such harmful radiation. While other materials might be selected, the most preferred material is a nucleic acid such as RNA (Ribonucleic Acid) , and it is particularly preferred to use a combination of a nucleic acid and a protein. The absorption of" microbeads comprised of RNA and protamine, produced according to the above-described technique, is shown in Fig. 1. Fig. 1 shows the absorption of a 0.1% solution of microbeads (e.g.

"BUREAU

__..OMPI microbeads made by combining 0.1% RNA and 0.1% Protamine) over the solar TJV range.

In many cases the presence of a nucleic acid in the microbead will offer a second advantage. It has been suggested that the damage caused by wave¬ lengths of sunlight greater than 313 nm is, in the case of many microbes, primarily the result of the reaction of the microbe's nucleic acids with free radicals (it is believed that radiation damage to tyrosine produces H2O2 which, in turn, produces free radicals) . The nucleic acid present in the microbead structure will tend to react specifically with the free radicals which would otherwise react with the microbe's nucleic acids, thus preventing any damage. Since the pathogenic effect of the microbial agent cannot be realized so long as the agent remains embedded within the microbead, care must be taken in selecting the materials to be used in formulating the microbeads to select materials which will permit release of the agent after ingestion of the microbeads by the insect. While other materials might be selected, we have found that microbeads comprised of a protein and a nucleic acid (e.g. RNA) provide quite satisfactory release characteristics. After ingestion of the microbeads by the insect, the microbeads will be attacked by proteases and nucleases in the insect digestive tract (i.e., gut) , which will lead to release of the microbe. Thus, it is important to select microbead materials which are not resistant to such type of attack. We have found that if Bacillus thuringiensis (cells, spores and toxin crystals) embedded in microbeads comprised of RNA and protamine (produced according to the above-described technique) are incubated at room temperature in the presence of insect digestive juices, release of the B.t. begins within minutes, with progressive and complete release following within one half hour.

We have also found that if Bacillus thuringiensis embedded in microbeads comprised of RNA and protamine (produced according to the above- described technique) are incubated at room temperature in the presence of amino acids and/or sugars such as would be found in an insect digestive tract, the germinating spores themselves dissolve the microbeads in approximately two hours. This does not occur in water or buffer alone, so that the microbeads will remain intact on leaf surfaces.

It should be noted that, while the only microbial insect pathogen discussed in detail above is the bacterial pathogen Bacillus thuringiensis, the present invention is suitable for use with any light sensitive microbial insect pathogen, including those of viral origin. Example 6, below, shows the applicability of the present invention to a bacterial virus. The positive results shown in Example 6 indicate that the present invention should also be suitable for use in protecting insect virus.

Industrial Applicability The following Examples illustrate several different embodiments of the present invention. It is intended that all matter in these Examples and in the foregoing description of the preferred embodiments and accompanying drawings be interpreted as merely illustrative and not in a limiting sense.

Example 1 q 1 x 10^ sppoorreess ooff BBacillus thuringiensis, including bacterial cells, spores and asporal (crystalline) bodies, obtained from a sporulation medium culture, were mixed in 10 ml of a .15 N phosphate buffer at pH 7.5. 1.5 ml of this solution was mixed with 1.5 ml of a buffered 1.34% aqueous solution (by weight) of yeast RNA (obtained from

Sigma as grade B) . Then 0.4 ml of this suspension was mixed with constant stirring in 1.9 ml of a buffered .36% aqueous solution (by weight) of protamine sulfate (obtained from Sigma as grade B) . RNA-protamine microbeads formed sponta¬ neously, each entrapping some of the bacterial cells and/or spores and/or asporal bodies. Shaking the mixture resulted in breakage and subsequent . spontaneous reformation of additional microbeads. The microbeads were placed in a glass petri dish and exposed to a General Electric G30T8 30 watt germicidal lamp. The petri dishes were placed on a rotary shaker 78 cm below the lamp and shaken at 40 rpm. Viability was determined by plating on brain heart infusion agar obtained from Difco.

When exposed to germicidal ultraviolet radiation (peak radiation at 254 nm) sufficient to kill 99.99% of any unprotected B.t. , the B.t. which was embedded in the microbeads (i.e., the protected bacterial cells and/or spores and/or asporal bodies) nearly all survived. This is shown in Fig. 2. The early die-off shown by the line marked "protected B.t." is thought to be due to the low percentage of B.t. actually embedded in the microbeads. Under microscopic observation the percentage of B.t. actually embedded was observed to range from 0.5% to 1.5%. Example 2 Microbeads with B.t. embedded therein were prepared as in Example 1, and then 1 mg/ml dithiobissuccimidyl propionate in DMSO was added to crosslink and stabilize the microbeads. 0.2 ml of this solution were placed in a 0.22 y Millipore filter and allowed to dry under vacuum. The filters were exposed as in Example 1 without shaking. After shaking, the filters were washed off in dilution buffer and plated as in Example 1. Results of this procedure are shown in Fig. 3.

Example 3 g

1 x 10 spores of B.t. obtained from culture in sporulation medium were first washed in a 60% ethanol solution and then washed in a 1 M NaCl solution, with the B.t. being separated from these washes by centrifugation. The washed B.t. was then suspended in 20 ml of a 1 M sodium carbonate bugger solution at a pH of 8.0. Next, dry succinic anahydride, a protein modifying agent, was added to the suspension as six separate additions of 2.5 mg/ml each. The additions were made under constant stirring and the mixture was stirred for 10 minutes between each addition. The pH was held at 8.0 +_ 0.1 by addition of NaOH. When the reaction was completed, as indicated by the pH ceasing to change, the B.t. was separated out (centrifuged) and washed.

This modified B.t. (negatively charged) was then incorporated into RNA-prota ine microbeads according to the procedures set forth in Example 1 and the microbeads were crosslinked as in Example 2. It was found that this modified B.t. >. entered the microbeads much more readily and in much higher numbers than the unmodified B.t. used in Examples 1 and 2. Presumably this was due to the modification of the surface charge on the B.t. from positive to negative.

Example 4

A solution of unprotected B.t. (cells, spores, and toxin crystals) and protected B.t. (i.e., embedded in microbeads as described in Example 3, but without crosslinking), 60% unprotected and 40% protected (determined microscopically) , was subjected to 254 nm radiation as described in Example 1.

Essentially no unprotected spores remained viable after 15 minutes of such irradiation, while 1 x lθ6 protected spores (i.e., 40% of the total original mixture) remained viable after one hour of such irradiation. Viability was determined as in Example 1. The results of this experiment are shown in Fig. 4.

Example^' 5 A concentration of 1 x 10^ spores of

Bacillus thuringiensis (including cells, spores and asporal crystals) of B.t. was suspended in 10 ml of .15 N phosphate buffer, pH 7.5 (B.t. preparation was obtained and modified as in Example 3). 0.5 grams of RNA (Calbiochem, grade B) was dissolved into this . suspension and mixed by vigorous mixing in a Vortex mixing device. The suspension was then added to a buffered 10% solution of protamine sulfate (Calbiochem, grade B, by weight) and vigorously shaken for 5 seconds. Glutaraldehyde (25%, from Sigma) was added to the solution to a final concentration of 0.15% (by volume) . After 30 minutes a pellet formed at the bottom of the tube which consisted of large microbeads (100 - 150 μ) . The supernate was drawn off and the pellet was resuspended to a final volume of 20 ml by shaking. This solution was placed in a 0.22 μ Millipore filter and dried overnight under vacuum. The filters were exposed to sunlight (1:00 pm, RH 23%, temperature 89°F) . The filters were then washed in acetate buffer (.15 N, pH 4.0) to break up the microbeads and release the B.t. , which was plated as in Example 1. The results of this experiment are shown in

Fig. 5. Unprotected spores were nearly all killed after 30 minutes.

Example 6 The purpose of this example was to demonstrate protection of a virus according to the present invention. The reactions and responses of an insect virus and a bacterial virus (called bacterial phage) should be similar since both are composed basically of a nucleic acid in a protein coat. Accordingly, we chose to model our system with the bacterial virus of E^ coli, phage T-4.

T-4 bacterial phages were grown in nutrient broth with 0.5% NaCl (P-broth) . E^_ coli BB was inoculated into 100 ml P-broth and allowed to grow overnight. In the morning a 1:100 dilution was made to fresh broth and growth was allowed to proceed for one hour. 1 x 10' phages were added to this rapidly growing E^_ coli BB culture and allowed to grow for six hours (37°C, rapid shaking) . At the end of the period, 5 drops of chloroform were added to kill all bacteria in the culture. This is the phage stock. Microbeads were prepared by mixing 0.100 grams of protamine sulfate (Calbiochem, grade B) in . 10 ml phage stock. This suspension was added to 1%

"BUREAU

_ OMPI ^■ - W1 - RNA (by weight) in P-broth. The microbeads formed spontaneously. The UV exposure was carried out as in Example 1. Timed samples were taken and dilutions were made in P-broth. The viable phages were deter- mined by the method described in the following text: Grace C. Rovozzo and Carroll N. Burk, A Manual of Basic VirologicaT Techniques, Prentice-Hall Biological Techniques Series, 1973, page 168, using P-broth agar and E__;_ coli BB as the indicator bacteria. The results of this experiment are shown in

Fig. 6. These data show that unprotected virus were all killed in approximately 5 minutes. On the other hand, after an initial drop similar to that seen in the bacterial tests, the virus which were embedded in the microbeads show strong UV light resistance

(approximately 40% are protected from inactivation) .

It should be understood that the term "nucleic acid" as used throughout this specification and in the claims is intended to include all polynucleotides. Likewise, the term "protein" is intended to include all polypeptides.

It is also to be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

CLAIMS :

1. An insecticidal composition comprising a microbial agent of viral or bacterial origin embedded in a microbead, characterized in that the microbead is comprised of a nucleic acid and a protein-containing material, whereby the microbead structure itself effectively shields the agent from sunlight-induced inactivation.

2. The insecticidal composition of Claim 1 wherein the effective diameter of the microbead is within the range of from 10 to 150 microns.

3. The insecticidal composition of Claim 1 wherein the microbead is stabilized by chemical crosslinking.

4. The insecticidal composition of Claim 1 wherein the microbial agent comprises a virus.

5. The insecticidal composition of Claim 1 wherein the microbial agent comprises Bacillus thuringiensis.

6. A method for preparing a microbial insecticide composition wherein the improvement comprises:

(a) mixing a buffered aqueous solution containing a microbial insect pathogen with an aqueous solution containing a nucleic acid; and

(b) mixing the said mixture with an aqueous solution containing a proteinaceous material, thereby spontaneously forming microbeads having the pathogen embedded therein.

7. A method as in Claim 6 wherein the microbeads are subsequently stabilized by treatment with chemical crosslinking agents.

8. A method as in Claim 6 wherein prior to the two mixing steps the microbial insect pathogen is cleaned by washing and its surface charge is.modified by the addition of a protein-modifying agent adapted to provide a more uniform surface charge.

9. The method of Claim 6 wherein the microbial insect pathogen comprises Bacillus thuringiensis, the nucleic acid comprises RNA, and the proteinaσeous material comprises protamine.

10. The insecticidal composition of Claim 1 wherein the microbial agent possesses an essentially uniform surface charge when it is embedded in the microbead.

11. A method for controlling insect pests in insect infested areas wherein the improvement comprises applying an effective amount of the insecticidal composition of Claim 1 to said insect infested areas.