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WO2002013608A1 - Enrobages hydrosolubles de protection contre les uv, pour pesticides biologiques, et procede de fabrication correspondant - Google Patents

Enrobages hydrosolubles de protection contre les uv, pour pesticides biologiques, et procede de fabrication correspondant Download PDF

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Publication number
WO2002013608A1
WO2002013608A1 PCT/US2001/025193 US0125193W WO0213608A1 WO 2002013608 A1 WO2002013608 A1 WO 2002013608A1 US 0125193 W US0125193 W US 0125193W WO 0213608 A1 WO0213608 A1 WO 0213608A1
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Prior art keywords
formulation
spores
bioactive agent
group
water
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PCT/US2001/025193
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English (en)
Inventor
Jarrod Ethan Leland
Donald E. Mullins
Larry Jay Vaughan
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Virginia Tech Intellectual Properties, Inc.
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Application filed by Virginia Tech Intellectual Properties, Inc. filed Critical Virginia Tech Intellectual Properties, Inc.
Priority to US10/344,374 priority Critical patent/US20040038825A1/en
Priority to AU2001284832A priority patent/AU2001284832A1/en
Publication of WO2002013608A1 publication Critical patent/WO2002013608A1/fr

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Classifications

    • 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
    • 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
    • A01N25/22—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 containing ingredients stabilising the active ingredients
    • 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
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom

Definitions

  • the invention generally relates to methods and formulations for the protection of biologically active agents.
  • the invention provides methods and formulations for the solar and thermal protection of biologically active pest control agents.
  • Oil- based formulations of microbial biopesticides provide some advantages over water-based formulations.
  • oil based formulations are compatible with ultra-low volume (ULN) application, which is particularly important in pest control situations where delivering a minimum amount of material to the target site is an advantage.
  • UPN ultra-low volume
  • oil based formulations allow a microbial biopesticide to remain dehydrated, which can help protect the active agent, as in the case of increased thermal stress tolerance of entomopathogenic fungi (McClatchie et al., 1994).
  • Oil based formulations may also increase the ability of the biologically active agent to adhere to and disperse over hydrophobic surfaces (Bateman et al., 1993; Prior et al., 1988). They may also disrupt the waxy layer of insect cuticle, enhancing the infectivity of contact biopesticides such as entomopathogenic fungi.
  • UV light Ultraviolet (UV) light is known to be a major source of damage to bioactive agents that are exposed to sunlight.
  • U light particularly high energy UNB wavelengths (280-320 nm).
  • Sun screens and sun Mockers have been used to protect Metarhizium fungal spores in oil based formulations, but with only mixed success (Hunt et al., 1994). This may be due to the fact that when oil- based formulations are sprayed, they tend to spread out over the typically hydrophobic surface of the target substrate (such as plant leaves, or the exoskeleton of an insect). This property is good in that the formulation tends to become distributed over and adhere to the target surface.
  • the protective layer of oil that surrounds the bioactive agent thins and the bioactive agent becomes exposed.
  • the effect is particularly pronounced on hydrophobic surfaces, which discourage beading of oil droplets. This phenomenon may help explain why some sun screens perform well in published laboratory assays that are carried out on glass (which is relatively hydrophilic and causes "beading" of the oil droplets) but do not perform as well in field trials on natural hydrophobic surfaces (Burgess, 1998).
  • aqueous-based formulations have the disadvantages of not spreading over hydrophobic surfaces, non-adherence to hydrophobic surfaces, and non- disruption of cuticular waxy layers of insect targets. Also lost with aqueous formulations are possible advantages of thermohydric stress reduction (McClatchie et al, 1994) and reduced formation of reactive oxygen intermediates (Cooper and Zika, 1983; Ignoffo and Garcia, 1978).
  • a biologically active agent is first mixed with a water-soluble protective coating and dried.
  • the dried, coated agent is then suspended in a hydrophobic carrier, such as an oil for application to a target substrate.
  • a hydrophobic carrier such as an oil for application to a target substrate.
  • the oil component facilitates dispersal of the formulation over the substrate and adherence of the formulation to the substrate.
  • the water-soluble protective outer coating carried by the particles remains in contact with the active agent intact both while suspended in the oil (the coating material is not soluble in oil), and later on the surface of the substrate as the oil disperses. Because the protective outer coating is water-soluble, the bioactive agent is eventually released from the coating upon exposure to water in the environment, e.g. from water that is present on the target substrate. However, until such water is available, the coated bioactive agent remains protected from environmental insults (e.g. heat, solar radiation, and the like) by the protective coating. It is an object of the instant invention to provide novel formulations comprising a bioactive agent coated with water-soluble protective material in a hydrophobic carrier.
  • the coated bioactive agent is produced by being combined in an aqueous mixture with the water- soluble protective material (and optionally with stabilizers). The mixture is then dried, and the dried mixture is suspended in the hydrophobic carrier, which may be an oil or a mixture of several oils.
  • the bioactive agent may be any agent that is capable of acting on a biological entity and producing a desired effect in the biological entity.
  • the bioactive agent may be a biopesticidal microbe (such as a fungus, a virus, a protozoan, or a bacterium), an antagonist to a disease-causing organism, or a microbe that otherwise promotes plant growth or health.
  • the bioactive agent may be a nematode.
  • the bioactive agent may be a toxin and/or insect feeding inhibitor, such as known substances produced by the bacterium Bacillus thuringiensis, substances produced by plants such as Azadirachta indica or Melia volkensii, or synthetic chemicals.
  • the bioactive agent may be natural insect pheromones or their synthetic analogues.
  • the bioactive agent may be an insect growth regulator - a group of chemicals generally recognized by practitioners of the art having a mode of action that inhibits normal developmental processes in immature insects without killing the insect through direct toxicity.
  • the water-soluble protective material that coats the bioactive agent may be any material that is useful for protection of the bioactive agent.
  • protective materials are those that protect against damage from UN light (UV protectants such as sun screens and sun blockers), and materials that provide thermal protection, or the stabilizing of biological materials in a desiccated state. In some cases, a single material may provide more than one protection.
  • the invention provides a method of exposing a target substrate to a bioactive agent by contacting the target substrate with the formulation of the instant invention.
  • target substrates include insects and plants.
  • the target substrate is an insect pest and the bioactive agent is a biopesticidal microbe.
  • FIG. 1 Colony-forming units (log 10 CFU's) for four formulations after exposure to UNB. Each data point represents four replicate plates from one replicate of each treatment.
  • Figure 2 A-D Average adult desert locust Schistocerca gregaria mortality over time for four formulations of Metarhizium anisopliae var. acridum fungal spores. Mortality is corrected for control mortality by Abbott's formula. Each data point represents two replicate cages often insects.
  • 3A 1 x 10 3 spores/insect
  • 3B 1 x 10 4 spores/insect
  • 3C 1 x 10 5 spores/insect
  • 3D 1 x 10 6 spores/insect.
  • Figure 7 Percent germination of spores following different periods of UV exposure (48 hr incubation on 2% malt agar + Benlate).
  • ⁇ AC non-coated aerial culture
  • CAC coated aerial culture
  • CLC coated liquid culture.
  • Figure 8. Average adult Schistocerca americana mortality over time for three formulations of Metarhizium anisopliae var. acridum fungal spores. Mortality is corrected for control mortality by Abbott's formula. Each data point represents ten individually caged insects. 3A: 1 x 10 3 spores/insect; 3B: 1 x 10 4 spores/insect; 3C: 1 x 10 5 spores/insect; 3D: 1 x 10 6 spores/insect.
  • the present invention provides formulations comprising a bioactive agent.
  • bioactive agent we mean that the agent is capable of exerting an effect on a living, biological entity.
  • living, biological entities include but are not limited to plants, insects, protozoa, fungi, algae, bacteria, nematodes, molluscs, and the like.
  • Those of skill in the art will recognize that many biological entities exist for which it would be beneficial have the capability to target and effect using the formulations and methods of the instant invention, and all such biological entities may be targeted and treated by the formulations and methods of the instant invention, i.e. they are "target substrates" for the bioactive agent.
  • these biological entities may be targeted at any suitable stage of their life cycle, e.g. adult or immature stages such as caterpillars, larvae, nymphs, eggs, pupae, and the like.
  • a "target substrate” may include either the living entity itself, or its habitat.
  • the formulations and methods of the instant invention are capable of exerting a desired effect on or eliciting an effect from a targeted biological entity.
  • the types of effects that may be elicited include but are not limited to death, cessation of feeding, delayed development, inability to reproduce, inhibition of growth, increase in growth, and the like.
  • Those of skill in the art will recognize that many different types of effects exist which it may be desirable to elicit by utilizing the formulations and methods of the present invention, and all such are intended to be encompassed by the instant invention.
  • the biological entity that is targeted by the formulations and methods of the instant invention is an insect pest or a noxious or undesirable plant (e.g.. a "weed"), and the effect that is elicited is death of the insect pest or weed.
  • the formulation may an insecticide and the bioactive agent may be specific for destroying insects.
  • the biological entity that is targeted is a disease producing organism, or a host or vector of a disease producing organism (e.g. mosquito, ticks, flies and the like).
  • the formulations of the instant invention comprise a bioactive agent for which it would be beneficial to provide a water-soluble protective coating.
  • the reason for providing such a protective coating may be to protect the bioactive agent from environmental insults such as heat, solar radiation, exposure to chemicals, and the like.
  • bioactive agents include but are not limited to microbiocidal agents, biopesticidal microbes, viruses, nematodes, toxins, insect feeding inhibitors, pheromones, growth regulators, and the like.
  • the bioactive agent itself is a biological entity.
  • bioactive biological entities examples include but are not limited to microbes such as fungi, bacteria, viruses, protozoa, nematodes, and the like.
  • the bioactive agent is a biopesticidal microbe, for example, a bacterium, fungus, protozoan, or virus.
  • a bioactive biological entity may be at any suitable stage of its life cycle when utilized in the practice of the present invention.
  • the bioactive agent is a fungal spore.
  • the bioactive biological entities may be genetically engineered, as in, for example, a microbe that has been genetically engineered to produce a protein that is either beneficial to or harmful to the targeted entity.
  • the bioactive agent is coated with a water-soluble protective coating.
  • the protective, water-soluble coating is comprised of UV-protective material.
  • the water-soluble coating may protect the biologically active agent by absorbing, blocking, or reflecting UV radiation, or by negating active oxygen radicals.
  • the coating material must also have the property of being highly water-soluble, such as Kraft lignins or lignosulfonates.
  • the water soluble protective coating comprises lignin or a lignin derivative.
  • a lignin or “a lignin derivative” we mean any polymers commonly understood by those practiced in the art to have the general chemical characteristics of lignin and any molecules modified from lignin.
  • Lignins are extracted from plant material by physical or chemical degradation of the plant material such as by steam explosion or enzymatic degradation.
  • the lignins can be rendered water-soluble by chemical modification such as by the Kraft process, reaction with a strong base followed by a strong acid to form a salt, or by the addition of a strong base followed by carbon dioxide to reduce the lignin pH.
  • the UV protective qualities of lignin have been known for many years.
  • the aforementioned patent provides a method for applying microbial pesticides in aqueous formulations with a lignin derivative that is linked with a multivalent salt.
  • the water-soluble lignin likely cross-links with cationic sights on a multivalent salt via the anionic cinnamyl alcohol residues of the lignin molecules.
  • the cross-linked lignin forms a protective coating on the microbial pesticide particles.
  • Lignins possess excellent and well-known UV-protective qualities, they may be rendered water soluble by methods which are well-known to those of skill in the art, and they are relatively inexpensive and readily available since they are waste products of the paper pulp industry.
  • Those of skill in the ait will recognize that many means to render lignin water soluble exist, including but not limited to the Kraft process, extraction with a strong base followed by a strong acid, and extraction with a strong base followed by neutralization with carbon dioxide. Water-soluble derivatives produced by such means may be utilized in the practice of the present invention, so long as they have a useful pH range.
  • Useful pH range we mean a pH range at which the lignin derivatives remain water soluble and which is tolerated by the bioactive agent, i.e.
  • the lignin derivatives that are utilized are a cationic lignin (Curan 100, a Kraft alkali lignin, Aldrich # 37-095-9) and an anionic lignin (Ultrazine, a sodium salt of a lignosulfonic acid, Aldrich #37 -097-5).
  • a cationic lignin Curan 100, a Kraft alkali lignin, Aldrich # 37-095-9
  • an anionic lignin Ultrazine, a sodium salt of a lignosulfonic acid, Aldrich #37 -097-5.
  • bioactive agents of the instant invention are coated with a protective, water- soluble coating.
  • Particles of the bioactive agent may be coated with the protective material by any of several means that are well-known to those of skill in the art.
  • the bioactive particles are mixed with the protective, water-soluble material in an aqueous-based medium and then dried.
  • various other compounds may be added to the bioactive agent-protective material mixture prior to drying.
  • additional stabilizers such as skim milk; various biologically compatible solutes (e.g. polyols) that function in osmoregulation and can protect macromolecules such as proteins (Brown, 1976; Crowe et al., 1990); non- reducing sugars (e.g. disaccharides such as trehalose) which have the ability to stabilize membranes (Crowe et al., 1984, 1990) and enzymes (Carpenter and Crowe, 1998, a, b); antioxidants (e.g.
  • formulations of the present invention may contain more than one type of bioactive agent in a single formulation.
  • Drying of the aqueous bioactive agent-protective material mixture may be carried out by any of many methods which are well-known to those of skill in the art, including but not limited to air drying, counterflow towers, freeze drying, spray diying, fluidized bed drying, and the like. Any method of drying may be utilized in the practice of the instant invention, so long as the bioactive agent remains viable and the protective coating is adequately adhered to the bioactive agent.
  • the mixture is air dried. After drying, the particle size of the dry material may be reduced and/or standardized to form a dispersible mixture, or the particle size of the final product may be determined by parameters in the drying process, particularly in the case of spray drying.
  • Preferable methods of size reduction and standardization include grinding , sieving, and pulverizing.
  • the desired size of the particles is dependent on the application requirements. For example, for ultra-low volume application, the particle size must be less than 100 micrometers in diameter.
  • the coated, bioactive agent is suspended in a hydrophobic carrier such as oil.
  • a hydrophobic carrier such as oil.
  • suitable hydrophobic carriers include but not limited to the many types of refined and unrefined vegetable and mineral oils.
  • Any suitable type of oil may be utilized in the practice of the present invention, so long as the oil does not dissolve the protective coating, is not toxic to the bioactive agent, and has viscosity suitable for the application method.
  • the carrier may be comprised of a mixture of such hydrophobic components, e.g. a mixture of oils which may include, for example, a petroleum distillate, mineral oils, or vegetable oils.
  • a low viscosity oil such as diesel fuel or kerosene may be mixed in adequate proportions with higher viscosity oils such as peanut oil, sunflower oil, corn oil, or any other suitable naturally occurring or synthetic oil.
  • the hydrophobic carrier may contain various additives such as stabilizers, detergents, and the like.
  • the oil formulation is then applied to a target substrate.
  • the formulation may be applied directly to the target (e.g. an insect pest, or the leaf of a plant) or indirectly by applying the formulation to an area to which the targeted entity will be exposed (e.g. a crop, or the habitat of an insect pest).
  • target e.g. an insect pest, or the leaf of a plant
  • area to which the targeted entity will be exposed e.g. a crop, or the habitat of an insect pest.
  • potential areas for application include but are not limited to seeds, crops, fields, soil, bare ground, dwellings, building structures, bodies of water, marshes, forests, brush, and the like.
  • formulation of the present invention may be applied to any substrate so long as the targeted biological entity is eventually contacted by the formulation (either by direct application of the formulation, or indirectly by contact with an area to which the formulation has been applied.)
  • Formulations of this type are typically applied by spraying, although those of skill in the art will recognize that any suitable method may be utilized. The exact type and manner of spraying may vary from situation to situation, depending on factors such as the type of target, general environmental conditions, amount of formulation to be applied, cost, and the like. Any suitable means of spraying or otherwise applying the formulation of the instant invention may be utilized in the practice of the instant invention.
  • the concentration of bioactive agent in the formulation will vary depending on several factors, including but not limited to the nature of the bioactive agent itself, and the type and means of administration of the formulation. For example, for ULV application of M. anisopliae var. acridum spores, an application rate of 2 x 10 12 - 2 x 10 13 spores/ha has been recommended (Kpindou et al., 1997 ; Lensen, 1999). Spores are applied in a volume of 0.5 - 2 L/ha depending on the particular sprayer used (Lomer et al., 1997). Thus, the concentration of spores would need to be between 1 x 10 9 and 4 x 10 10 spores / L of total liquid formulation. Those of skill in the ait are well acquainted with such determinations.
  • Curan 100 and Ultrazine are two commercially available lignin derivatives (Aldrich Chemical Co. catalog numbers # 37-095-9 and # 37-097-5, respectively). Both of these products are extracted from wood by steam-explosion followed by chemical extraction. Curan 100 is a Kraft alkali lignin and Ultrazine is a sodium salt of a lignosulfonic acid .
  • Submerged spores were produced in a 1-L BioFlo III System (New Brunswick Scientific).
  • the liquid media consisted of 40 g/L waste brewers' yeast and 40 g/L fructose (Sigma).
  • Waste brewers' yeast was harvested from a batch of Stroh's beer by NPC incorporated (Eden, NC).
  • the pH of the media was adjusted to 7.0 with sterile 1 N, NaOH before autoclaving.
  • Aerial conidia of Metarhizium anisopliae var. acridum (IMI 330189) from Sabouraud dextrose agar was used as an inoculum. Conidia were harvested by scraping 2-3 week old cultures and suspending in 0.05% Tween 80 in sterile distilled water.
  • Conidia densities were determined by hemacytometer and adjusted to 6 x 10 ⁇ conidia/mL. Viability of the inoculum was determined as percent germination after 24 hr at 24 °C on 2% malt agar. Media was inoculated to produce an initial concentration of 1.2 x 10 5 spores/mL of media. Temperature in the 1-L fermenter was maintained at 24 °C. The air-flow rate was 0.5 L/min (0.5 VVM). The minimum dissolved oxygen was set at 0.5% of saturation and was maintained by allowing the agitation rate to automatically adjust between 130 RPM and 500 RPM. Antifoam 289 was delivered by automatic delivery system that monitored foaming.
  • the contents of the bioreactor was sampled approximately every 5 hours, at which time the dissolved oxygen, temperature, and agitation rate were recorded.
  • the final spore concentration after 5 days was determined by hemacytometer to be 8.2 x 10 8 spores/mL.
  • each prelyophihzation solution added to the washed spores was 20 mL, which was approximately equal to the volume of each pellet, for a total volume of 40 mL.
  • the spores were suspended in the prelyophihzation solutions and then immediately frozen at -80 °C, and lyophilized.
  • Results show that submerged spores of Metarhizium anisopliae var. acridum were able to survive the coating process in the presence of skim milk, Curan 100TM and UltrazineTM. Survival rates ranged from 83-91%. In contrast, survival of lyophilized uncoated submerged spores was poor (13%).
  • Each of the lyophilized samples produced a solid block that was easily broken into a fine powder. The particle size of this powder was easily reduced to less than 120 ⁇ m by grinding the material against the surface of a metal sieve with a rubber spatula. The concentration of spores within the lyophilized powder is very high, ranging from 5 xlO 9 to 7xl0 9 /g of dry powder and is well within the necessary range for, for example, ULV application.
  • This example demonstrates that the formulations with coated spores in an oil carrier protect spores from damage by UV radiation.
  • Submerged spores were lyophilized with three different aqueous solutions (treatments 1-3): 10% skim milk plus 1% glycerol; 10% Curan 100TM plus 1%) glycerol; and 10% UltrazineTM plus 1% glycerol as described in Example 1.
  • Aerial conidia which were used as a reference in this experiment, were harvested from 20- day old plate cultures on Paris medium. Briefly, the procedure consisted of depositing test inocula, coated spores, or crude aerial conidia on membrane filters; exposing dried and sealed membrane filters to radiation; and recovering irradiated inocula from the filters.
  • the concentration of aerial conidia (treatment 4) was determined using direct hemacytometer counting.
  • the propagule concentration for the lyophilized material was based on mass of lyophilized material and a predetermined spore concentration (spores/g of lyophilized material).
  • 10 mL of 1 x 10 7 spores/mL were deposited onto a 0.45 ⁇ m membrane filter (ME25 Schleicher and Schuell, Dassel, Germany). Deposition onto the filter was done using a Millipore vacuum filtration system (Millipore 10-047-04, 10-047-02). The total area of the inoculum deposit using this system is 10 cm 2 .
  • Six membrane filters were prepared for each treatment.
  • Laboratory irradiation experiments consisted of exposing the dried fungal formulations on the membrane filters to broad wavelengths greater than 295nm, at increasing amounts of time. Irradiation tests were conducted in a controlled environment chamber using an artificial sunlight device.
  • the artificial sunlight device consisted of two 400 W high pressure metallic halogen lamps (HQI-TS, OSRAM, F67120, Molsheim, France), which emit a continuous spectrum from 270 to 1100 nm.
  • a long pass glass filter (WG295-Schott Glastechnike, Mainz, Germany) was used to block shorter wavelengths (under 295 nm) to simulate natural sunlight.
  • UV irradiance 0.585 W m "2 for 0, 4, 8, 12 or 16-hours. These exposures correspond to UV irradiances of 0, 4.32, 8.46, 12.78, and 17.10 kJ m "2 , respectively.
  • Shaded propagule-seeded membrane filters were used as controls for each of the different formulations.
  • One membrane was covered with a metal plate to block radiation and placed in the controlled environment chamber for 16 hr.
  • the surface temperature of the seeded surface of membrane filters was regulated at 25 + 1 °C and the air humidity ranged from 40 to 50% relative humidity (RH).
  • RH relative humidity
  • spores were removed from the filter by placing the membrane in 10 mL of sterile distilled water (also containing 15- ⁇ L Tween 80) in 25-mL flasks and then shaking the flasks for 5 minutes at 700 oscillations min "1 (10 cm vertical travel) on a mechanical shaker.
  • Percent germination was determined for a suspension of 1 x 10 6 spores/mL on Paris medium. Estimates of germination rate were based upon examination of 200 spores at 320X on duplicate plates after 24 and 48 hours at 25 °C. A single plate was scored for each treatment/exposure combination. Controls and treatments exposed for 4 hr of UV had grown enough to check germination after 24 hr. All other treatments required 48 hr to produce enough growth to distinguish germination. In addition, colony-forming units (CFUs) were determined for suspensions of irradiated spores on four replicate Paris media Petri plates using a spiral plating method.
  • CFUs colony-forming units
  • the spore suspension used for CFU counts was approximately 3.3 x 10 5 spores/mL before the spiral plating. After 4 days of incubation in the dark at 25° ⁇ 1 ° C, colonies were enumerated on three consecutive days to ensure that CFU's accounted for delayed growth.
  • the results of this experiment are presented in Tables 3 and 4 and Figure 1. Two criteria were used to examine the viability of M. anisopliae var. acridum spores after exposure to UVB, percent germination and colony forming units (CFU's). Viability estimates based on CFU's depend upon percent recovery of spores from the membrane filter, whereas viability estimates based on percent germination are not.
  • the results demonstrate greater UV stability for the lyophilized formulations than for fresh aerial conidia. Coating of the spores appears to provide significant UV protection, and the two lignin formulations appear to provide greater UV protection than skim milk coating.
  • the number of colony forming units is based on four replicate plates. Each exposure time/ formulation treatment combination was performed once.
  • Example 2 demonstrates that the formulations with coated spores in an oil carrier can be highly virulent to a target pest.
  • Submerged spores were lyophilized with three different aqueous solutions: 10% skim milk and 1% glycerol; 10% Curan 100TM 100TM and 1% glycerol; and 10% UltrazineTM and 1% glycerol as described in Example 1.
  • Aerial conidia which were used as a reference in this experiment, were harvested from 20-day old plate cultures on Paris medium.
  • the inoculum concentration of submerged spores for the three coated-spore treatments was determined by weighing a mass of lyophilized material having a known concentration of spores per gram. Concentration of aerial conidia was determined by direct counting with a hemacytometer. All inoculum suspensions were made in 70%) diesel fuel : 30% peanut oil as a carrier. Controls insects were exposed to the carrier only.
  • the percent germination of each inoculum was measured by observing 200 spores at a concentration of approximately 1 x 10 6 spores/mL in 70% diesel fuel : 30% peanut oil on semi-synthetic medium after 24 hr of incubation at 24 °C.
  • stock formulated suspensions consisted of milk-coated spores titrated at 4.2 x 10 8 spores/mL, Curan 100TM -coated spores were titrated at 5.1 x 10 8 spores/mL, UltrazineTM - coated spores were titrated at 3.9 x 10 8 spores/mL, and crude aerial conidia were titrated at 4.8 x 10 s spores/mL.
  • the stock concentrations were the highest doses used for the bioassay, and 1:10 serial dilutions of the stock concentration were used for the three lower doses.
  • Each formulation was tested at four doses ranging from 0.8 x 10 3 to 0.8 x 10 6 spores/insect for both milk- and UltrazineTM -coated submerged spores, and from 1.0 x 10 3 to 1.0 x 10 6 spores/insect for both Curan 100TM -coated submerged spores and crude aerial conidia.
  • Bioassays were performed on approximately 8-day old Schistocerca gregaria (African desert locust) adults. Groups of 10 adults with a sex ratio of approximately 1:1 were treated with 2 ⁇ L of formulated inoculum using topical application with a micropipette directly under the pronotal shield. Treatment groups of 10 adults were contained in wire cages (27 cm x 19 cm x 13 cm) and maintained at 28 °C, 43% relative humidity and a ligh dark regime of 12h:12h ( L:D ) in individual controlled-humidity chambers. Relative humidity was maintained at 43%>. Relative humidity was monitored within each test chamber with a probe attached to a data logger.
  • Locusts were provided with a constant supply of fresh wheat bran and cages were replenished with fresh wheat seedlings daily. Locust mortality was determined daily. Dying locusts in this bioassay demonstrated characteristic signs of Metarhizium infection; they moved to the top of the cage, exhibited paralysis, and turned red. In addition, feeding appeared to stop approximately 24 to 48 hr before death.
  • the % of spores that germinated over time was determined for each of the liquid-culture spores and aerial-culture spores (Figure 3) in each of the 5 suspension media by observing a least 200 spores at 400 X magnification on 2% malt agar + 0.001% Benelate after incubating at 24 °C.
  • a 30-mL sample of approximately 2 x 10 6 spores/mL was spread onto a 30 mm Petri plate. Germination was stopped with 20% formalin and plates were stored at 4 °C until % germination was determined.
  • a spore was considered to be germinated if the germ tube was greater than half the diameter of the spore.
  • Dry mass ratio is based on the estimated dry mass of spores from liquid culture using an approximate 70% moisture of the pellet after washing.
  • the addition of suspension media to aerial conidia was calculated to produce a product that had the same number of spores/g of coating material but not the same dry mass ratio.
  • the concentrations of spores in the formulations of liquid-culture spores and aerial-culture spores were approximately 5 x 10 9 spores/g and 9 x 10 9 spores/g of total formulation, respectively.
  • spore suspension was delivered to a 60 mm sterile Petri plate, and each treatment was replicated 4 times.
  • 1.3 mL of spore suspension was delivered to a 30 mm sterile Petri plate, and each treatment was replicated 3 times.
  • the % moisture of the spore suspensions were determined by change in mass after heating at 100 °C for 24 hours. The mass of material delivered to each Petri plate was determined in order to determine change in %> moisture during the air drying process.
  • the suspended spore samples were dried at ambient (30 to 60%) relative humidity in a laminar flow hood for 82 hr.
  • the coated spores were then transferred to a desiccation chamber containing silica gel for an additional 56 hours of drying.
  • the %> germination of spores over time was determined for each of the treatments after the drying process. All dry coated-spore samples were rehydrated in a 100%) relative humidity environment before suspending in distilled water and spreading on 2% malt agar + 0.01 % Benelate for determining % germination. The method for determining % germination is described above. A sample of the coated spores were then ground against the surface of a 170 openings per linear inch sieve using a glass rod. The %> germination of spores over time was determined for the LCS treatments after the sieving process (Fig. 5).
  • Example 4 demonstrates that the formulations with coated spores from air drying in an oil carrier protect spores from damage by UV radiation.
  • the sieved product from the liquid-culture spore and aerial-culture spores treatments corresponding to suspension media number 5 (1:0.5:0.5:0.1 Spores : Curan 100TM : Skim Milk : Glycerol; dry mass ratios) in Example 4 were used in this example.
  • Coated spores from liquid culture (CLC) and from aerial culture (CAC) were compared to non-coated spores from aerial culture (NAC) with respect to tolerance to UV light when suspended in an oil formulation.
  • the methods for exposing spores to simulated sunlight was identical to that described in Example 2: UV Protection (Freeze-Dried Product).
  • Germination before exposure refers to spores that were resuspended from the filter membrane with water after being after being deposited with a 70% diesel fuel : 30% peanut oil carrier. Values in this table are corrected for initial viability, which was determined for the spore types suspended directly in water. EXAMPLE 6. Virulence (Air-Dried Product)
  • Example 4 demonstrates that the formulations with coated spores from air drying in an oil carrier can be highly virulent to a target pest.
  • the sieved product from the liquid- culture spore and aerial-culture spore treatments corresponding to suspension media number 5 (1:0.5:0.5:0.1 Spores : Curan 100TM : Skim Milk : Glycerol; dry mass ratios) in Example 4 were used in this example.
  • Coated spores from liquid culture (CLC) and from aerial culture (CAC) were compared to non-coated spores from aerial culture (NAC) with respect to virulence to the grasshopper Schistocerca americana when suspended in 70%) diesel fuel:30% peanut oil.
  • Aerial conidia which were used as a reference in this experiment, were harvested from 2 to 3 week old plate cultures on Sabouraud Dextrose Agar medium.
  • the inoculum concentration of submerged spores for the three coated-spore treatments was determined by weighing a mass of air dried material having a known concentration of spores per gram. Concentrations of non-coated aerial culture spores were . determined by direct counting with a hemacytometer. All inoculum suspensions were made in 70% diesel fuel : 30%) peanut oil as a carrier. Control insects were exposed to the carrier only.
  • the percent germination of each inoculum was measured by observing 200 spores at a concentration of approximately 1 x 10 6 spores/mL in 70% diesel fuel : 30%) peanut oil on semi-synthetic medium after 24 hr of incubation at 24 °C. Based on the number of viable spores, the following spore concentrations were made for each of the three spore types: 5 x l0 8 , 5 x l0 7 , 5 x l0 6 , and 5 x l0 5 .
  • Bioassays were performed on 2 to 3 week-old Schistocerca americana adults. Groupss of 10 adults with a sex ratio of approximately 1 : 1 were treated with 2 ⁇ L of formulated inoculum using topical application with a micropipette directly under the pronotal shield. Treatment groups of 10 adults were contained individually in wire-screened cages (27 cm x 19 cm x 13 cm) and maintained at 32 °C, 50 ⁇ 5% relative humidity and a ligh dark regime of 14h:10h ( L:D ). Relative humidity was maintained at 50%. Locusts were provided with a constant supply of romaine lettuce and cages were replenished daily. Locust mortality was determined daily.
  • Dying locusts in this bioassay demonstrated characteristic signs of Metarhizium infection; they exhibited paralysis and turned red.
  • the results of this experiment are provided in Figures 8 A-D.
  • the virulence of the lyophilized, protected spores is similar to that of fresh aerial conidia, especially at higher doses.
  • This example demonstrates that formulations of a bioactive agent as described in the present invention protect the agent from UV damage so that the agent retains its effectiveness.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Dentistry (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Wood Science & Technology (AREA)
  • Agronomy & Crop Science (AREA)
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Abstract

Cette invention se rapporte à une formulation pour la protection d'un agent bioactif, à un procédé de production de cette formulation et à des procédés d'utilisation de cette formulation. Dans ladite formulation, l'agent bioactif est enrobé d'un matériau protecteur hydrosoluble, il est séché et il est placé en suspension dans un excipient hydrophobe, tel qu'une huile. Cette formulation sert à la diffusion de l'agent bioactif sur un substrat cible. L'agent bioactif peut être un microbe biopesticide, le matériau protecteur peut être une lignine et le substrat cible peut être une plante ou un insecte parasite.
PCT/US2001/025193 2000-08-11 2001-08-10 Enrobages hydrosolubles de protection contre les uv, pour pesticides biologiques, et procede de fabrication correspondant WO2002013608A1 (fr)

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US10/344,374 US20040038825A1 (en) 2001-08-10 2001-08-10 Water soluble uv-protective coatings for biological pesticides and process for making same
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1764079A1 (fr) * 2005-09-16 2007-03-21 Beiersdorf AG Compositions cosmétiques autobronzantes
EP1764083A1 (fr) * 2005-09-16 2007-03-21 Beiersdorf AG Ecrans solaires comprenant lignosulfonates
WO2007034275A3 (fr) * 2005-09-19 2007-07-05 Battelle Uk Ltd Formulations pour la stabilite amelioree de pesticides
WO2007118297A3 (fr) * 2006-04-13 2007-12-06 Univ Fraser Simon Nouvelle composition attracticide visant à lutter contre les insectes nuisibles
WO2008062413A3 (fr) * 2006-11-21 2009-03-12 Mitam Ltd Formulations de champignons entomopathogènes pour la lutte contre les insectes
WO2010106314A2 (fr) 2009-03-18 2010-09-23 Syngenta Limited Formulation
JP2014508099A (ja) * 2010-09-17 2014-04-03 ダウ アグロサイエンシィズ エルエルシー 安定性が改善された液体農業用配合剤
CN106309212A (zh) * 2016-08-20 2017-01-11 张进 一种抗紫外线辐射的护肤化妆品及其制备方法
WO2019084246A1 (fr) * 2017-10-25 2019-05-02 Advanced Biological Marketing, Inc. Procédé de formulation de chimies microbienne et agricole combinées, composition dérivée de microbe, et son utilisation
WO2025040938A1 (fr) * 2023-08-23 2025-02-27 Abdalla Elfaki Osman Mohammed Procédé de production d'un produit de lutte biologique et produit pour la lutte contre les locustes et procédé de lutte contre les locustes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859377A (en) * 1987-07-10 1989-08-22 The United States Of America, As Represented By The Secretary Of Agriculture Starch encapsulation of entomopathogens
US4983390A (en) * 1987-04-01 1991-01-08 Lee County Mosquito Control District Terrestrial delivery compositions and methods for controlling insect and habitat-associated pest populations in terrestrial environments
US6001346A (en) * 1993-02-25 1999-12-14 The Regents Of The University Of California Aqueous emulsion comprising biodegradable carrier for insect pheromones and methods for controlled release thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4983390A (en) * 1987-04-01 1991-01-08 Lee County Mosquito Control District Terrestrial delivery compositions and methods for controlling insect and habitat-associated pest populations in terrestrial environments
US4859377A (en) * 1987-07-10 1989-08-22 The United States Of America, As Represented By The Secretary Of Agriculture Starch encapsulation of entomopathogens
US6001346A (en) * 1993-02-25 1999-12-14 The Regents Of The University Of California Aqueous emulsion comprising biodegradable carrier for insect pheromones and methods for controlled release thereof

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1764079A1 (fr) * 2005-09-16 2007-03-21 Beiersdorf AG Compositions cosmétiques autobronzantes
EP1764083A1 (fr) * 2005-09-16 2007-03-21 Beiersdorf AG Ecrans solaires comprenant lignosulfonates
WO2007034275A3 (fr) * 2005-09-19 2007-07-05 Battelle Uk Ltd Formulations pour la stabilite amelioree de pesticides
WO2007118297A3 (fr) * 2006-04-13 2007-12-06 Univ Fraser Simon Nouvelle composition attracticide visant à lutter contre les insectes nuisibles
WO2008062413A3 (fr) * 2006-11-21 2009-03-12 Mitam Ltd Formulations de champignons entomopathogènes pour la lutte contre les insectes
WO2010106314A2 (fr) 2009-03-18 2010-09-23 Syngenta Limited Formulation
JP2014508099A (ja) * 2010-09-17 2014-04-03 ダウ アグロサイエンシィズ エルエルシー 安定性が改善された液体農業用配合剤
EP2615912A4 (fr) * 2010-09-17 2015-04-01 Dow Agrosciences Llc Formulations agricoles liquides ayant une stabilité améliorée
AU2011301966B2 (en) * 2010-09-17 2015-06-04 Dow Agrosciences Llc Liquid agricultural formulations of improved stability
CN106309212A (zh) * 2016-08-20 2017-01-11 张进 一种抗紫外线辐射的护肤化妆品及其制备方法
WO2019084246A1 (fr) * 2017-10-25 2019-05-02 Advanced Biological Marketing, Inc. Procédé de formulation de chimies microbienne et agricole combinées, composition dérivée de microbe, et son utilisation
US11229203B2 (en) 2017-10-25 2022-01-25 Agrauxine Corp. Method of formulation of combined microbe and agricultural chemistry, microbe-derivative composition, and use of same
WO2025040938A1 (fr) * 2023-08-23 2025-02-27 Abdalla Elfaki Osman Mohammed Procédé de production d'un produit de lutte biologique et produit pour la lutte contre les locustes et procédé de lutte contre les locustes

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