Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
  • A01N25/28—Microcapsules or nanocapsules
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
  • A01N63/22—Bacillus
  • A01N63/23—B. thuringiensis
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
  • A01N63/27—Pseudomonas
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/40—Viruses, e.g. bacteriophages

Definitions

• This invention relates to microbial insecticides. More par ticularly, the invention relates to a novel microbial insecticide composition and to the production and utilization thereof.
• Microbial insecticides of viral, bacterial, or fungal origin offer significant advantages over conventional chemical insec ticides.
• Microbial insect pathogens are generally nontoxic and harmless to other forms of life.
• microbial insecticides demonstrate a relatively high degree of specificity, and hence do not endanger beneficial insects.
• a susceptible insect host is quite slow to develop resistance to microbial pathogens.
• Microbial insecticides may be used in relatively low dosages, may be effectively applied as dusts or sprays, and may be used in combination with chemical insecticides.
• the Douglas fir tussock moth nuclear polyhedro sis virus is a microbial insect pathogen useful for con trolling the tussock moth.
• Bacillus thur inqiensis is a microbial insect pathogen useful for con trolling the tussock moth.
• spore-forming bacterium a spore-forming bacterium
• a microbial insect pathogen useful against numerous leaf-chewing insects in their larval stages, including, for example, alfalfa caterpillars, tomato hornworms, tobacco hornworms, cabbage loopers, cab bage web worms, army worms, gypsy moths, walnut caterpillars, diamondback moths, cosmopolitan green beetles, European corn borers, and other members of the order Lepidoptera.
• UV radiation with a wavelength of 253.7 nm induce a marked, extraord inary inactivation of B.t. spores, so that they are unable to germinate and grow out.
• a dosage of ,18 m W sec/cm 2 of such 253.7 nm wavelength radiation will inactivate 99.9% of the B.t. spores.
• UV radiation of wavelengths shorter than about 285 nm do not reach the earth's surface, such inactivation at 253.7 nm is of little practical concern in the field.
• nucleic acids show a maximum of extinction near a wavelength of 260 nm
• 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.
• B.t. spores could be protected from inactivation by such UV radiation (253.7nm) by physically mixing the B.t. spores with DNA, or a comparable nucleic acid which would absorb the UV rays.
• a comparable nucleic acid would be RNA, Ribonucleic Acid, which has a maximum of extinction near 260 nm.
• the present invention comprises a microbial insecticide composition and methods for the production and utilization of such composition.
• the composition comprises a microbial insect pathogen of viral, bacterial, or fungal origin which is susceptible to sunlight-induced inactivation embedded in a coacervate microbead which is comprised of a nucleic acid, typically RNA, and a proteinaceous material, whereby the microbead structure itself effectively shields the pathogen from sunlight-induced inactivation.
• the microbead is typically stabilized by chemical crosslinking.
• One typical method for preparing the microbial insecticide composition comprises: (a) preparing a paste-like mixture comprising (i) nucleic acid particles, (ii) proteinaceous material particles, (iii) microbial insect pathogens of viral, bacterial, or fungal origin, and (iv) an amount of water sufficient to wet (i.e. hydrate) substantially the entire mixture; and (b) agitating the paste-like mixture in a manner adapted to break up the mixture into discrete microbeads, whereby the microbial insect pathogens are embedded in the microbeads.
• the discrete microbeads are stabilized by treatment with a chemical crosslinking agent such as tannic acid, glutaraldehyde or a similar agent.
• a chemical crosslinking agent such as tannic acid, glutaraldehyde or a similar agent.
• the agitation of the paste-like mixture takes place in a solution containing the chemical crosslinking agent.
• Another typical method for preparing the composition comprises: (a) preparing an aqueous solution containing a nucleic acid; (b) preparing an aqueous solution containing a proteinaceous material; (c) preparing an aqueous suspension of strongly positively or negatively surface-charged microbial insect pathogens; and (d) mixing the aqueous solutions and suspension prepared in steps (a), (b), and (c) together, thereby spontaneously forming microbeads having the insect pathogens embedded therein.
• the suspension prepared in step (c) is first mixed with the solution prepared in step (a), and then this mixture is mixed with the solution prepared in step (b).
• the suspension prepared in step (c) is first mixed with the solution prepared in step (b), and then this mixture is mixed with the solution prepared in step (a).
• the surface charge of the pathogens is made strongly negative or strongly positive by the addition of a protein-modifying agent to a buffered aqueous suspension of the pathogens.
• the microbeads are typically crosslinked.
• the present invention also comprises a method for controlling insect pests in insect infested areas which typically comprises applying an effective amount of the insecticide composition described above to the insect infested areas.
• the present invention further comprises the insecticide composition made by the processes described above.
• Fig. 1 is a graph showing the optical density, over the solar UV and visible range, of two typical types of microbeads suitable for use in the present invention.
• Figs. 2-12 are graphs showing the comparative experimental data from Examples 1, 2 and 4-12, below, respectively.
• 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.
• Figs. 5, 6, and 8 the percentage of microbes remaining as survivors is shown as a function of the exposure time.
• Figs. 7 and 10 the number of viable spores per filter is shown as a function of the exposure time.
• Figs. 9, 11, and 12 show LD 50 data as a function of the exposure time.
• microbial insect pathogens of viral, bacterial or fungal origin which are susceptible to sunlight-induced inactivation are embedded in coacervate microbeads comprised of a nucleic acid, typically RNA, and a proteinaceous material.
• RNA nucleic acid
• proteinaceous material typically RNA
• the microbeads act as protective shields, serving to intercept and block the harmful radiation wavelengths (i.e., those wavelengths of sunlight which tend to inactivate the pathogen) before they reach the light-sensitive material of the insect pathogen.
• any insect pathogen embedded in such microbeads will be protected against sunlight-induced inactivation.
• Various proteinaceous materials may be used in combination with the nucleic acid to form the microbeads, depending on the specific insect pathogen to be protected and the microbead system to be used, including, but not limited to: protamine, cytochrome c, soy protein, hemoglobin, gelatin, synthetic amino acid poly mers," etc.
• protamine protamine
• cytochrome c soy protein
• hemoglobin gelatin
• synthetic amino acid poly mers etc.
• any proteinaceous material can be used if the conditions are adjusted so as to facilitate formation of the microbeads. Such conditions may include charge modification techniques, adjustments in pH, component concentrations, etc.
• microbeads in which the microbial insect pathogens are embedded may be advantageously produced using known techniques 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).
• an aqueous solution containing a nucleic acid preferably ribonucleic acid, RNA
• a buffering agent e.g.
• the microbeads in which the microbial insect pathogens are embedded may be produced using a new technique which we have developed.
• this new technique will be referred to as the "paste formulation” technique, and microbeads formed according to this technique will be referred to as "paste formulation” microbeads.
• a paste-like mixture is prepared comprising nucleic acid (preferably RNA) particles, proteinaceous material particles, and an amount of water sufficient to wet substantially the entire mixture, and then this paste-like mixture is agitated in a manner designed to break up the mixture into discrete microbeads.
• Such agitation may be accomplished by conventional techniques such as, for example, rapid stirring or blending (in a conventional blender), sonification, shaking, pressure extrusion, etc.
• rapid stirring or blending in a conventional blender
• sonification shaking
• pressure extrusion etc.
• a chemical crosslinking agent e.g. tannic acid, etc,
• crosslinking may be unnecessary where stabilization may be effected by other means, such as, for example, freeze drying.
• the paste formulation technique With respect to the solution formulation technique, and to a certain extent the paste formulation technique, it should be noted that while all of the above-named proteinaceous 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. For example, when using protamine, the pH should be maintained below about 11, and when using hemoglobin the pH should be maintained below about 7. Furthermore, if a protein is used that is insoluble at a given pH, the pH may have to be put in a range in which the protein is soluble, or other steps may have to be taken to make the protein soluble.
• RNA is being used as the nucleic acid in the solution formulation technique
• the pH of the mixture of solu tions 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.
• the materials to be utilized to intercept and absorb the harmful radiation form the microbead structure, thereby producing a highly protective coating.
• the bimolecular structure of the microbeads creates a ther- modynamically 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.
• 100m diameter solution formulation microbeads can be made by mixing an equal volume of 5% RNA and 10% protamine sulfate. Microbeads will form and settle to the bottom of the vessel. Most of these will be in the 100m range.
• the size of the micro- beads can be controlled, to some extent, by the degree of agitation during formation, with greater agitation producing smaller microbeads.
• microbeads having an effective diameter within the range of from about 10 to about 200 microns should be suitable for use in the present invention, it will generally be preferred to utilize microbeads having an effective diameter within the range of from about 40 to about 100 microns.
• the type of vegetation i.e. crops, trees, etc.
• the method of application will determine the desired microbead size.
• nucleic acid and proteinaceous material 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.
• th.e microbial insect pathogens may be embedded (i.e., entrapped) in the microbeads by simply placing them in suspension in water (a buffering agent, e.g. phosphate, acetate, etc. may optionally be added if necessary to control pH) 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 proteinaceous material-nucleic acid microbeads which form.
• a buffering agent e.g. phosphate, acetate, etc. may optionally be added if necessary to control pH
• the buffered suspension of microbes may be first mixed with the proteinaceous material solution and then the resulting suspension 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.
• the microbial insect pathogens may be embedded in the microbeads by simply placing them in suspension in water (as above, a buffering agent may be added as necessary) and then mixing this suspension with the mixture of nucleic acid particles and proteinaceous material particles (care should be taken to use only an amount of water sufficient to wet the mixture and give it a paste-like consistency). The resulting paste-like mixture is then agitated as described above so as to break it up into discrete microbeads.
• nucleic acid particles and proteinaceous material particles may be mixed with an amount of water sufficient to wet the mixture and give it a paste-like consistency, and then the microbial insect pathogens may be mixed with this paste-like mixture and embedded in discrete microbeads by agitation of the mixture as described above.
• microbeads produced according to the above- described solution formulation and paste formulation techniques possess a certain degree of stability, it will generally be advantageous to increase their stability to facilitate separation of embedded pathogens from non-embedded pathogens and to further facilitate handling. This is particularly so with respect to paste formulation microbeads.
• such stabilization is accomplished by chemically crosslinking the microbead molecules by treating them with crosslinking agents such as, for example, tannic acid, glutaraldehyde, imidoester agents, dithiobissuccimidyl propionate, etc. using conventional crosslinking techniques.
• 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.
• buffered tannic acid is non-toxic to bacterial spores at a concentration of 10% (w/v), and we believe that it will be non-toxic to most microbial insect pathogens at concentrations of 1% or less (w/v). While buffered tannic acid having a concentration within the range of from about 0.001% to 10% should be suitable for use, concentrations within the range of from about 0.5% to 1.5% will generally be preferred.
• the depth of crosslinking can be controlled rather easily by controlling the time, concentration, temperature, and other conditions of crosslinking.
• the depth of crosslinking may be controlled by stopping thecrosslinking reaction by adding a small molecule which reacts with the crosslinking reagent (e.g. lysine added to glutaraldehyde) or by using low crosslinking reagent concentrations.
• the crosslinking reagent e.g. lysine added to glutaraldehyde
• Such chemical crosslinking of the microbeads yields several advantages, including: (1) stabilization 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 use of tannic acid as the crosslinking agent increases the optical density of the resulting crosslinked microbeads (see Fig. 1), thereby providing improved shielding of the embedded microbes against sunlight-induced inactivation.
• the microbial insect pathogen may be embedded (i.e. entrapped) in the above-described solution formulation microbeads much more readily and in much greater numbers if its net surface charge is first modified so as to be made nearly totally (i.e. strongly) negative or nearly totally positive. We believe this will also be the case with regard to paste formulation microbeads.
• This surface charge modification may be accomplished, for example, by the controlled addition of a protein modifying agent such as, far example, succinic anhydride (to make strongly negative) and similar compounds (see e.g., Gary E. Means and Robert E. Feeney, Chemical Modifications of Proteins, Holden Day, Inc., 1971).
• succinic anhydride is not suitable for use with vegetative bacterial cells (e.g. Serratia marsescens, etc.), since it tends to inactivate these cells.
• Modification to a strongly positive surface charge may be accomplished, for example, by using tannic acid to link positively charged proteins (e.g. protamine) to the surface of the pathogen.
• tannic acid to link positively charged proteins (e.g. protamine) to the surface of the pathogen.
• B.t. spores 10-20% of the B.t. spores being embedded in the microbeads, and that using strongly positively surface charged B.t. spores (treatment with tannic acid then protamine sulfate.) results in about 20-40% of the B.t. spores being embedded in the microbeads.
• the effectiveness of these charge modification techniques may be increased by first washing the microbial insect pathogen, and it may be desirable, in certain embodiments, to wash in separate organic (e.g. 60% ethanol solution, by weight) and inorganic
• the charge-modified pathogen apparently competes with the like-charged component of the microbead for positions in the bead, it may be necessary to reduce the concentration of such like-charged component to a level which will facilitate incorporation of the pathogen into the microbead.
• concentration of such like-charged component For. example, if it is desired to entrap microbial insect pathogens 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.
• RNA radiation-absorbing material
• RNA ribonucleic acid
• proteinaceous material such as, for example, hemoglobin, protamine, or a synthetic amino acid polymer.
• the solid line in Fig. 1 shows the optical density of a solution of microbeads made by combining equal volumes of 0.33% RNA and 0.5% protamine
• Fig. 1 shows the optical density of a solution of microbeads made by combining equal volumes of 0.67% RNA and 1% protamine (crosslinked with tannic acid) over the solar UV and visible, (to 600 nm) range.
• the presence of a nucleic acid in the microbead will offer a second advantage. It has been suggested that the damage caused by wavelengths 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 H 2 O 2 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.
• microbeads comprised of a protein and a nucleic acid (e.g. RNA) provide quite satisfactory release characteristics.
• microbeads After ingestion of the microbeads by the insect, the micro- beads 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.
• Bacillus thuringiensis cells, spores and toxin crystals
• RNA and protamine produced according to the above-described solution formulation technique
• Autographa californica NPV viruses embedded in the microbeads of the present invention are, in fact, released in the insect gut, and that upon being so released they exert a killing effect as desired.
• the present invention is suitable for use with any light sensitive microbial insect pathogen, including those of viral, bacterial, or fungal origin.
• Examples 1-5, 7 and 10 below illustrate the applicability of the present invention to a typical spore-forming bacteria (i.e. B.t. cells, spores, and toxin crystals), and Example 8, below, illustrates the applicability of the invention to three species of vegetative bacterial cells.
• 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 be suitable for use in protecting non-occluded insect viruses against sunlight induced inactivation.
• Examples 9, 11 and 12 illustrate the applicability of the present invention to two species of occluded insect viruses.
• the microbial insect pathogens which are labelled and/or referred to as "unprotected” comprise pathogens which were not embedded in microbeads.
• the "unprotected" pathogens were treated, exposed, and tested for viability in a manner as nearly identical as possible to the pathogens which were embedded in microbeads (i.e. the "unprotected” pathogens constituted control experiments).
• Example 1 1 x 10 9 spores of Bacillus 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 spontaneously, 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.
• Example 2 Example 2
• 1 mg/ml dithiobissuccimidyl propionate in DMSO was added to crosslink and stabilize the microbeads.
• 0.2 ml of this solution were placed on a 0.2 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 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.
• Example 5 A concentration of 1 x 10 9 spores of Bacillus thurmgiensis (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).
• 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 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 7 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% 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 determined by the method described in the following text: Grace C. Rovozzo and Carroll N. Burk, A Manual of Basic Virological Techniques, Prentice-Hall Biological Techniques Series, 1973, page 168, using P-broth agar and E. coli BB as indicator bacteria.
• Microbeads having B.t. (cells, spores, and toxin crystals) embedded therein were prepared as in Example 3, except that dithiobissuccimidyl propionate in DMSO was not used to crosslink the microbeads.
• the microbeads were crosslinked and stabilized by adding phosphate-buffered tannic acid (1% w/v/) to the microbead suspension, and allowing it to stand at room temperature for about 30 minutes.
• the suspension of crosslinked microbeads was diluted with dilution buffer (phosphate) in a manner selected to produce a diluted suspension containing approxi mately 100 spores per ml, and 1 ml samples of this diluted sus ⁇ pension were pulled into separate 0.22 Millipore ® filters and permitted to dry overnight.
• the filters were exposed under a General Electric sunlamp measured at 1572 watts/m at 15 inches and providing radiation in the wavelength range of about 290nm to
• Example 8 Vegetative bacterial cells of the species Pseudomonas fluorescens, Serratia marcescens, and Escherichia coli were treated with succinic anhydride, in three separate series of experiments, by placing approximately 1 x 10 9 cells in 20 ml of 1
• the resulting suspension was mixed with 2 ml of a buffered 1% (w/v) Protamine sulfate (obtained from Cal Biochem.) solution, spontaneously forming the microbeads, and then 0.4 ml of a 10% (w/v) tannic acid solution was added to the suspension of microbeads.
• the buffer referred to above was 0.15 N phosphate buffer solution at pH 7.5.
• the suspension of microbeads in tannic acid was allowed to stand for 30 minutes at room temperature, and then was washed three times in the phosphate buffer solution described by centrifuging.
• Example 8 This data suffers the same error as described in Example 8 (i.e. microbead suspension which was exposed too heavy to permit kill off of virus which were not embedded in the microbeads).
• microscopic observation did reveal that about 30% of the virus was embedded in the microbeads, and that the microbeads offered protection against sunlight-induced inactivation (whether the virus are inside or outside, but under, the microbeads).
• Example 10 1 gram of hemoglobin (crude powder) was mixed with 0.34 gram of RNA in dry powder form. Approximately 1 x 10 9 spores of B.t. were suspended in a buffer solution (0.15 N acetate, pH 5.0), and then added to the RNA-hemoglobin mixture. Additional buffer solution was added in an amount sufficient to form a hydrated ⁇ i.e. wet) mass having a paste-like consistency. The total amount of buffer solution mixed with the RNA-hemoglobin mixture was about 1 ml. This paste was forced (i.e.
• Example 11 Autographa californica nuclear polyhedrosis virus (approximately 1 x 10 7 polyhedral inclusion bodies) was embedded RNA-Protamine microbeads using the technique described in Example 10.
• 1 gram of protamine (dry powder form) and 0.67 gram of RNA (dry powder form) were used, the buffer was 0.15 N phosphate solution at pH 7.5, and tannic acid was used to crosslink and stabilize the microbeads.
• the microbead suspensions were washed twice in distilled water and then diluted 1:100 in distilled water.
• the diluted suspensions were exposed under a General Electric sunlamp using the technique described in Example 1, and the exposed suspensions were diluted in phosphate buffer as needed for the infectivity tests, described below. Trichoplu sia ni larvae were used to measure infectivity of the protected virus (i.e. embedded in the microbeads) and the unprotected virus
• Infectivity was determined by placing 10 1 of separate dilutions (1:10 2 to 1:10 7 ) on a 0.2 gram piece of diet. The larvae were allowed to eat the entire piece of diet, and then a new piece of diet (2 grams) was placed in the vial. When all of the control larvae (not fed virus microbeads) had pupated, the dead larvae were autopsied to verify that death was caused by infection caused by the Autographa californica virus (this was found to be the case). LD 50 's for the T. ni. larvae were determined for virus which had been protected by the microbeads and for the control virus, which had not been protected by any microbeads. The general procedures for making LD 50 determinations are described in Microbiology, at p. 639, B. D. Davis,et al., Harper and Row, 1973. The LD 50 data are shown in Fig. 11.
• Example 12 Example 11 was repeated using Douglas fir tussock moth nuclear polyhedrosis virus in place of the Autographa californica virus and Douglas fir tussock moth larvae in place of the T. ni. larvae, except that the larvae were autopsied at the end of a 10 day period.
• the LD 50 data are shown in Fig. 12.
• nucleic acid as used throughout this specification and in the claims is intended to include all polynucleotides.
• protein is intended to include all polypeptides.

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