WO2024254558A2 - Formulations d'huiles épaississantes d'entomopathogènes fongiques - Google Patents

Formulations d'huiles épaississantes d'entomopathogènes fongiques Download PDF

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Publication number
WO2024254558A2
WO2024254558A2 PCT/US2024/033150 US2024033150W WO2024254558A2 WO 2024254558 A2 WO2024254558 A2 WO 2024254558A2 US 2024033150 W US2024033150 W US 2024033150W WO 2024254558 A2 WO2024254558 A2 WO 2024254558A2
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composition
formulation
flies
oil
thickening agent
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WO2024254558A3 (fr
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Ikkei SHIKANO
Christian Streit
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University of Hawaii at Manoa
University of Hawaii at Hilo
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University of Hawaii at Manoa
University of Hawaii at Hilo
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • 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
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
    • A01N31/16—Oxygen or sulfur directly attached to an aromatic ring system with two or more oxygen or sulfur atoms directly attached to the same aromatic ring system
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • 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

  • Tephritidae fruit flies
  • This family Tephritidae is moderately large, with over 4000 documented species and are endemic to Africa, Asia, Australia, the Pacific, and Central and South America. Tephritidae are primarily distributed throughout temperate, subtropical, and tropical parts of the world. Tephritid flies are also known to be excellent colonizers due to their strong ability to adapt to new regions, climates, and host species. Of the known tephritid flies, there are about 250 species that are economically significant due to their deleterious impacts on production and trade of agricultural commodities.
  • Citrus, Citrus spp. coffee, Coffea arabica L.; eggplant, Solanum melongena L.; guava, Psidium guajava L.; mango, Mangifera indica L.; melons, Cucumis melon L.; papaya, Carica papaya L.; passion fruit, Passiflora edulis Sims.; persimmon, Diospyros kaki L.; tomato, Solanum lycopersicum L.; cucurbits, Cucurbita pepo L. Aside from the hurdles of producing these susceptible crops, exporting them from Hawaii requires postharvest treatment, which further exacerbates an already difficult agricultural economy.
  • the first three phases are the immature stages and visual species identification can be difficult. During the adult phase most tephritid’s are easily identified by their unique body marking and wing patterns. Proper identification is critical to ensuring the proper management, quarantine, and control programs are implemented.
  • Eggs are elongated, glistening white, rounded at one end, approximately 1mm in length (FIG. 22). They are laid into young fruits and deposited in clusters. Some will lay eggs in batches or individually, depositing eggs in as many fruits as possible over the course of the female’s lifetime. The incubation time of the eggs is impacted primarily by temperature and substrate. The substrate type impacts the time it takes for eggs to hatch in melon flies.
  • the pupal period ranges by species, host fruits, and multiple external factors like temperature, moisture content of the soil, and soil type. As flies eclose from their puparium, they make their way to the surface of the soil. Here the teneral adults seek out a safe place to dry their bodies and wings before becoming active, searching for a food source. Each adult fly is going to exhibit unique behaviors, body coloration, and wing patterns that are characteristic of that species (FIG.21). [0012] Mediterranean flies (C. capitata) are smaller than other tephritids. With an average body length of 3.5-5 mm and wingspan of 8-10 mm. Their bodies are short and stubby with black, yellow, and brown markings.
  • cucurbitae are smaller than an average house fly with their average body size between 8-10 mm long and a wingspan of 14-17 mm.
  • Males are typically smaller than the females and females are identified by their ovipositor.
  • Their bodies are a reddish- brown with three distinct lateral yellow vitta and a yellow scutellum.
  • the abdomen is reddish- brown with 5 tergites.
  • a “T” pattern is formed by a transverse dark band across the T3 tergite is intersected by a thin medial line connecting the T3-T5 tergite. This “T” marking is sometimes faint.
  • the T3-T5 tergites have black medial stripes and a transverse dark line on T3, forming a “T” on the abdomen.
  • Oriental flies wings are mostly hyaline with a dark costal banding along the anterior margin of the wings.
  • Most tephritids become sexually mature within a few days, with some taking up to two- weeks to mature, after eclosion and finding a food source. In some species, sexually mature flies will aggregate together forming a lek. Leks are formed before sunset and the light intensity is most intense. In these leks, each territory within the lek will be occupied by one male who is actively defending his site against other males.
  • a key mechanism that makes MAT effective against tephritids is the use of synthesized male-specific chemicals like methyl eugenol (ME; 4-allyl-1, 2-dimethoxybenzene- carboxylate), cuelure (C-L; 4-(p-acetoxyphenyl)-2-butanone), and raspberry ketone (RK; 4-(4- Hydroxyphenyl)butan-2-one). Combining these attractants with a toxicant or fumigant forms the basis for MAT. Pestiferous tephritids have been eradicated successfully using MAT on multiple islands throughout the pacific and Indian ocean.
  • ME methyl eugenol
  • C-L 4-(p-acetoxyphenyl)-2-butanone
  • RK raspberry ketone
  • MAT can be used to eradicate invasive tephritids, but financial restrictions and geographic distribution of pest species can determine the success of a MAT program. Multiple MAT programs that were initially set out to eradicate a specific tephritid species later became suppression programs. Sterile Insect Technique (SIT) was first proposed with its principles by E.F. Knipling and Raymond Bushland in the 1950’s as a method to suppress or eradicate pest species by the release of sterilized insects.
  • SIT Sterile Insect Technique
  • Pathogens and biological pesticides that utilize nematodes, bacteria, fungi, and viruses are being brought to the forefront in the biological control of fruit flies. Some pathogens are more effective at controlling the flies while they are in the soil (nematodes, fungi) while others may be able to be applied to adults (fungi, bacteria, viruses). Research into gut symbiosis and compatible bacterial pathogens, like Wolbachia, are being tested and applied to tephritids as another option for biological control.
  • EPN entomopathogenic nematodes
  • EPF could be employed in a similar fashion to these attract and kill stations. Horizontal transmission of B. bassiana anisopliae in multiple tephritid species has been confirmed to induce significant reductions in female numbers after exposure to infected males. [0031] To effectively utilize EPF in an attract-and-kill fashion to control tephritid fruit flies, multiple requirements must be met. First, the development of a carrier agent that prolongs the viability of EPF spores by protecting them from water exposure and ultraviolet radiation. Second, the development of an auto-dissemination station that can disperse the EPF product to the target members of a population.
  • a pesticide composition comprising entomopathogenic fungus spores or conidia in a thickened oil formulation.
  • the composition comprises the entomopathogenic fungus spores or conidia, a carrier oil, and a thickening agent.
  • the pesticide composition further comprises an abrasive material.
  • the abrasive material is in an amount of 10% by weight/volume of the composition, for example, where the abrasive material is diatomaceous earth.
  • the pesticide composition further comprises a lure for a target insect.
  • entomopathogenic fungus spores or conidia is a genus selected from the group consisting of: Beauveria, Metarhizium, Hirsutella, Isaria, Lecanicillium, Paecilomyces, Entomopthora, and Nomuraea.
  • the entomopathogenic fungus is a species selected from the group consisting of: Beauveria bassiana, Metarhizium anisopliae, Agent Reference: 11157.179WO-PCT Metarhizium brunneum, Hirsutella thompsonii, Isaria fumosorosea, Lecanicillium lecanii, Lecanicillium longisporum, Paecilomyces lilacinus, Entomopthora muscae, and Nomuraea riley.
  • the entomopathogenic fungus is B. bassiana or Metarhizium anisopliae.
  • the carrier oil is selected from the group consisting of mineral oil, petroleum distillates, an oil isolated from a botanical source, or mixtures thereof.
  • the carrier oil comprises canola oil or mineral oil.
  • the composition comprises petroleum oil.
  • the thickening agent in the pesticide composition is in an amount sufficient to result in the composition having a viscosity of 1,500 to 2,000,000 cps.
  • the pesticide composition has a consistency resembling lotion, petroleum jelly, or a paste.
  • the thickening agent is in an amount of at least 10% by weight/volume of the composition.
  • the thickening agent is selected from the group consisting of: glyceride flakes, Dermofeel® Viscolid, cornstarch, diatomaceous earth.
  • the composition comprises cornstarch is in an amount of at least 1 g/ml of the composition.
  • the composition comprises glyceride flakes or Dermofeel® Viscolid in an amount of at least 10% by weight/volume of the composition.
  • the lure for the target insect is in an amount of 0.01-10% by volume of the composition.
  • the pesticide composition has widespread efficacy against multiple species of fruit flies.
  • the composition comprises Beauveria bassiana at a concentration of at least 2.0 x 10 10 conidia per ml, the carrier oil, and the thickening agent.
  • the composition further comprises an abrasive material and/or a lure for fruit flies.
  • the carrier oil is selected from mineral oil and an oil from a botanical source (for example, canola oil).
  • the composition further comprises a thickening agent and/or a lure for fruit flies.
  • the thickening agent is in an amount of sufficient to results in the composition having a consistency resembling petroleum jelly. Accordingly in some implementation, the thickening agent is in an amount of at least 10% by weight/volume of the composition.
  • the thickening agent is selected from the group consisting of: glyceride flakes, Dermofeel® Viscolid, and cornstarch. Where the thickening agent is glyceride flake or Dermofeel® Viscolid, the composition comprises the thickening agent is in an amount of 10% by weight/volume of the composition. Where the thickening agent is cornstarch, the composition comprises the thickening agent is in an amount of at least 1 g/ml of the composition or in an amount of about 1.4 g/ml of the composition. In Agent Reference: 11157.179WO-PCT some aspects, the abrasive material is in an amount of 10% by volume of the composition. In particular embodiments, the abrasive material is diatomaceous earth.
  • the lure is selected from the group consisting of: cuelure, trimedlure, methyl eugenol, and raspberry ketone.
  • the lure for fruit flies is in an amount of 10% by volume of the composition.
  • a bait station comprising the pesticide composition disclosed herein is also described.
  • the bait station comprises a chamber and a reservoir for retaining the above described pesticide compositions.
  • the reservoir for retaining a pesticide composition is a fabric saturated with the pesticide composition.
  • the reservoir is a surface within the chamber onto which the pesticide composition is provided.
  • the method comprises providing the above described pesticide composition to the designated geographical area.
  • the pesticide composition is applied on a surface within the designated geographical area.
  • the pesticide composition has a consistency resembling petroleum jelly or a paste
  • the pesticide composition is provided to the designated geographical area in a bait trap.
  • FIG.1 is a graph of the lethal concentration survivorship curves from probit analysis of Bactrocera dorsalis, Ceratitis capitata, and Schlodacus cucurbitae exposed to multiple concentrations of Botanigard® ES in aqueous suspension. The data is from Trial 1. Gray shading indicates 95% confidence intervals. Data from the control and highest concentration (2.0e+08 spores per/ml), which mainly produced 0 and 100% mortality, were excluded from the analysis.
  • FIG.2 is a graph of the lethal concentration survivorship curves from probit analysis of B. dorsalis, and Z. cucurbitae exposed to multiple concentrations of Botanigard® ES in aqueous suspension. The data is from Trial 2.
  • FIG.3 shows the Kaplan-Meier survival curves of Aprehend® and the BotaniGard® in mineral oil formulation (BGM) on three substrates: filter paper, cotton cloth fabric, and PIG® Agent Reference: 11157.179WO-PCT Oil-Only Absorbent Mat. Survival curves are shaded by 95% confidence intervals with individual p-values between each control and treatment.
  • Z. cucurbitae flies were exposed to each treatment for five-minute periods before release and monitoring for mortality. After 14 days exposure, all escaped and surviving flies were censored from the analysis.
  • FIG.4 is a graph of the Kaplan-Meier survival curves of the BotaniGard® in mineral oil formulation (BGM) with diatomaceous earth incorporated at 2.5, 5.0, and 10.0%. Survival curves of Z. cucurbitae are shaded by 95% confidence intervals. Flies were passed through 4- inch treated tubes and monitored for mortality for 14 days after exposure, all escaped and surviving flies were censored from the analysis. [0045] FIG.
  • FIG. 5 is a representation of Kaplan-Meier survival curves of the BotaniGard® in mineral oil formulation with diatomaceous earth incorporated at a concentration of 10.0% (BMD). Survival curves of B. dorsalis, C. capitata, and Z. cucurbitae are shaded by 95% confidence intervals.
  • FIGs.6A-6F show representative Kaplan-Meier survival curves of the BotaniGard® in mineral oil formulation with diatomaceous earth incorporated at 10.0% (BMD) with 95% confidence intervals. Horizontal transmission trials for forced and passive transmission for B. dorsalis are shown on FIG. 6A and 6B respectively, C. capitata forced and passive transmission are represented in FIG. 6C and 6D respectively, and Z.
  • FIG.6E and 6F cucurbitae forced and passive transmission are shown in FIG.6E and 6F, respectively. Flies were censored from the data if they escaped out of cages and after 14 days for forced trials and 18 days for passive trials.
  • FIG.7 shows a graph of the average germination rates ( ⁇ SE) of the BotaniGard® in mineral oil formulation, with 10% diatomaceous earth incorporated (BMD), during 12 weeks of exposure in control (lab), direct sun (sun), and indirect sun (shade) conditions.
  • FIG. 8 is a representation of the Kaplan-Meier survival curves with 95% confidence intervals of Z. cucurbitae that were forced to contact the weathered BMD formulation.
  • FIG. 9A shows a boxplot of the number of visits by Z. cucurbitae to potential carrier oils.
  • FIG.9B shows the preferred oil (canola) was then compared in a two choice test against mineral oil. Significant differences (Tukey HSD) are indicated by different lower case letters.
  • FIGs. 10A. and 10B depict boxplot of the number of visits by Z. cucurbitae flies to canola oil with or without sugar (FIG. 10A) or molasses (FIG. 10B0. Least-squares means showed no significant difference between oil with and without a carbohydrate source incorporated.
  • FIG. 11 graph of the average germination rates ( ⁇ SE) BMD formulation thickened with cornstarch (CORN), Dermofeel® Viscolid (DMV), and mono- and di-glyceride flakes (GF) during 8 weeks of exposure in control (lab), direct sun (sun), and indirect sun (shade) conditions.
  • FIG. 12A shows the mean amount ( ⁇ SE) of cornstarch, glyceride flake and non- thickened BMD formulations that dripped out of an inverted plastic cup when exposed to constant temperatures of 25, 30, 35, and 40 ⁇ C. The formulations were applied on a cloth fabric lining (fabric) or directly to the plastic surface (none).
  • FIG. 12B is a close up of FIG. 12A (reduced y-axis values), to compare only the cornstarch and glyceride flake-thickened formulations.
  • FIGs.13A-13C depict Kaplan-Meier survival curves with 95% confidence intervals of Z. cucurbitae adults exposed to weathered thickened BMD formulation.
  • FIGs.14A-14C show the Kaplan-Meier survival curves with 95% confidence intervals of Z.
  • FIGs.15A and 15B depict boxplot of the numbers of visits by Z. cucurbitae (FIG.15A) and B.
  • FIG. 15B shows the average germination rates ( ⁇ SE) of the canola oil formulation containing three concentrations of liquid cuelure and methyl eugenol.
  • FIG. 15D Boxplot of average spore pick-up rates by Z. cucurbitae from cuelure-incorporated formulation is shown in FIG. 15D and by B. dorsalis from methyl eugenol-incorporated formulation is shown in FIG.15E.
  • FIGs. 16A and 16B depict boxplots of average spore pick-up rates of Z. cucurbitae (FIG. 16A) and B. dorsalis (FIG.16B) with each improvement: canola oil, thickening agent, and liquid lure. Tukey HSD test with normal distribution showed that formulations with all the additional components significantly increased the number of spores picked up by the flies.
  • FIGs.17A and 17B show Kaplan-Meier survival times for B. dorsalis (FIG.17A) and Z. cucurbitae (FIG.17B) that were passively exposed to the BMD formulation and to the final formulation (BotaniGard® with diatomaceous earth, canola oil, cornstarch, and liquid lure). Accompanying log-rank tests confirmed that the additions to the formula decreased the time to death in both sexes and species significantly. Flies that survived beyond 20 days were censored out of the analysis.
  • FIG.18 graph is a representation of at what temperature(s) (in Celsius) the thickened formulation will hold its state.
  • FIGs.19A-19D show an exemplary implementation of the formulation in a bait station.
  • Male pheromone lure traps used for monitoring populations can be modified into baiting stations as shown in FIG. 19A.
  • the bait station inside may be lined with BMD formulation- soaked fabric as shown in FIGs. 19C and 19D.
  • FIG. 19B shows the passive horizontal transmission where sexually mature unmated males and females were placed into the cage with a cup lined with fabric soaked in BMD formulation hung in the cage and inside each cup was a pheromone lure.
  • FIG. 20 shows the forced horizontal transmission.
  • FIG.21 shows the three species of tephritid fruit flies used (Z. curcurbitae on the left, B. dorsalis on the center and C. capitata on the right).
  • FIG. 22 shows eggs that are white, elongated and elliptical, approximately 1mm long. Incubation period is 1-2 days. One female can lay hundreds to thousands of eggs over her lifetime.
  • FIG. 23 illustrates that all instar phases are apodal and shift in color as they mature. Third instars have a “popping” behavior that they exhibit to leave the fruit when they are ready to pupate.
  • FIG.24 shows the pupae stage. Pupa are 5-6mm long, barrel-shaped, with a black dot on the posterior end. Pupal cases vary in color from light yellow to dark brown/coppery reddish brown. Pupal period lasts approximately one week. After eclosion, teneral adults are less active and pale in color.
  • FIG.25 illustrates some of the current and historical controls for Tephritid flies. [0066] FIG.
  • FIG. 26 shows the three material or substrates the two commercial products, BotaniGard® and Aprehend®, were tested on.
  • FIG.27 shows the crude formulation (BMD) created.
  • FIG.28 illustrates the design for the carrier oil testing.
  • FIG.29 is a representation of the B. bassiana life cycle.
  • FIG.30 shows the mineral oil formulation trials results.
  • FIG. 31 shows the Melon flies (Z. cucurbitae) killed after contacting the thickened fungal formulation. The insert shows the fungal spores growing at the initial site of infection (mouth) and a cadaver completely covered in fungus (bottom), which could lead to the spread of infection to other pests.
  • Tephritidae (Diptera) is a globally distributed fruit fly family, containing many species that are considered to be major economical pests. Tephritid flies cause severe damage through the oviposition activities of females. Females puncture the fruits which become scarred and have discoloration at the puncture site as eggs hatch and larvae (maggots) feed and bore into the fruits. This life cycle can cause up to 100% loss of fruit crops and remaining produce is subject to strict quarantine regulation being imposed on exporting countries.
  • tephritid species all of which harm Hawaii’s agricultural sector and limit their ability to export produce to the continental United States.
  • the first tephritid was Schlodacus cucurbitae (Coquillett) (melon fly) 1895; followed by Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) 1910; Bactrocera dorsalis (Hendel) (Oriental fruit fly) 1944; Bactrocera latifrons (Hendel) (Malaysian fruit fly) 1983; Bactrocera oleae (Gmelin) (olive fruit fly) 2019.
  • This disclosure discusses the three older introductions of Melon fly, Med fly, and Oriental fruit fly.
  • Management of these invasive fruit flies have shifted from broadcast sprays of crops with chemical insecticides to methods that target specific life stages and behaviors. These include field sanitization to kill eggs and larvae, insecticidal soil drenches to kill late-stage larvae and pupae, releases of biological control agents (i.e., parasitoids) that target larvae, Agent Reference: 11157.179WO-PCT sterile insect technique (SIT), and insecticide-laced bait sprays and attract-and-kill bait stations.
  • SIT sterile insect technique
  • Bait stations contain an insecticide with either male lures to target mainly male flies or a protein source to mainly target reproductively immature females, who need protein to develop their ovaries.
  • none of the available eradication tools, including bait sprays and bait stations target reproductively mature females. This is important because mature females are significantly less attracted to protein baits than immature females.
  • two established fruit fly species in the U.S. olive fly, Bactrocera oleae, in California and melon fly, Schlodacus cucurbitae, in Hawaii
  • have exhibited resistance to Spinosad which is the active ingredient in the most widely used protein bait GF-120.
  • dorsalis has shown a propensity to develop resistance to Spinosad in selection experiments in the lab and resistance alleles have been found in field-populations of Mediterranean fruit fly, Ceratitis capitata, in Spain.
  • an over-reliance on GF-120 to target female fruit flies may be an unsustainable strategy.
  • Many tephritids exhibit unique mating behaviors where males will aggregate together to form leks. This behavior gives an opportunity for the formulation to be dispersed amongst both sexes of the flies through mating.
  • Z. cucurbitae are known to gather at dusk on non-host plants, which surround crop fields. They release pheromones to attract females while defending small territories from competing males.
  • Fipronil is relatively slow- acting and provides the males with enough time to horizontally transmit the fipronil to reproductively mature females via contact during courtship or through their regurgitant during food-sharing.
  • Field experiments in Hawaii demonstrated that the Amulet C-L bait stations significantly reduced the numbers of female Z. cucurbitae.
  • Fungal biopesticides have been shown to be effective against tephritid fruit flies but the optimal method of application has not been determined. So far, fungal pathogens have been tested as soil drenches to target late-stage larvae and pupae, mixed into protein baits to target adults, and directly applied to adults. Fungal pathogens take at least several days to kill their hosts.
  • bassiana formulation could be used in a bait station.
  • the formulation maximizes spore longevity and pickup of spores by male flies.
  • a pesticide composition targeting fruit flies comprising Beauveria bassiana in a concentration of at least 2.0 x 10 10 conidia per ml is described.
  • BotaniGard® is the source of B. bassiana in the disclosed pesticide composition, for example the BotaniGard® ES formulation.
  • the pesticide composition comprises dilutions of the BotaniGard® ES formulation such that the concentration of B. bassiana in the composition is about 2.0 x 10 10 conidia per ml.
  • the pesticide composition further comprises a carrier oil and an abrasive material.
  • a general pesticide composition comprising the entomopathogenic fungus spores or conidia, a carrier oil, and a thickening agent is described herein.
  • This composition is the first Agent Reference: 11157.179WO-PCT entomopathogenic fungus formulation that not only extends the shelf life of such a pesticide composition while also enhancing the distribution (or spreadability and adhesion) of the entomopathogenic fungus to insect to effect pest control properties.
  • the thickened oil-based formulation allows the entomopathogenic fungus spore or conidia to remain in a dormant stage while awaiting contact with target insects.
  • the general pesticide composition further comprises an abrasive material and/or a lure for a target insect.
  • the thickening agent is not abrasive
  • the inclusion of the abrasive material may be preferred to enhance the efficacy of the pesticide composition, as abrasion of the insect exoskeleton improves adhesion of the entomopathogenic fungus spore or conidia to the insect.
  • the carrier oil is mineral oil, petroleum distillate, or an oil isolated from a botanical source, for example, commercially available vegetable oil, canola oil, soybean oil, peanut oil, or castor oil.
  • the carrier oil is mineral oil or canola oil.
  • the carrier oil is mineral oil.
  • the composition comprises petroleum jelly.
  • the pesticide composition comprises the thickening agent in an amount sufficient result in the composition having a viscosity of 1,500 to 2,000,000 cps.
  • the composition has a viscosity of 1,500 to 2,000,000 cps in the temperature in which the pesticide composition would be administered, for example, 2oC-40oC, 10oC to 30oC, 18oC to 25oC, 20oC to 22oC, or about 21oC.
  • the pesticide composition comprises an amount of the thickening agent sufficient to result in the composition having a consistency resembling lotion, petroleum jelly, or a paste.
  • the pesticide composition comprises the thickening agent is in an amount of at least 5%, at least 10%, or about 10% by weight/volume of the composition.
  • suitable thickening agents may be found in hydrocarbons, gelatines, starches, gelatins, silica, or polymerized or hydrogenated oils.
  • the thickening agent is selected from glyceride flakes (for example, monoglyceride and/or diglyceride flakes), hydrogenated oil from a botanical source (for example, Dermofeel® Viscolid or hydrogenated vegetable oil), or cornstarch.
  • the pesticide composition comprises mono- and di-glyceride flakes or Dermofeel® Viscolid at an amount of about 10% by weight/volume.
  • the pesticide composition comprises a starch (for example, cornstarch) at an about of at least 1 g/ml, for example, about 1.4 g/ml.
  • the thickening agent includes an abrasive material.
  • the pesticide composition comprises two sources of thickening agent, once of which is the abrasive material.
  • Options for the abrasive material include calcium silicate, Agent Reference: 11157.179WO-PCT calcium carbonate, silicon dioxide, talcum powder, bentonite clay powder, and other anticaking agents and abrasive minerals, such as diatomaceous earth.
  • the abrasive material is diatomaceous earth.
  • the amount of abrasive material in the pesticide composition is at least 2.5%, at least 5%, 2.5-10%, or about 10% by weight/volume.
  • the pesticide composition comprises diatomaceous earth as the abrasive material in an amount of 10% by volume.
  • the pesticide composition further comprises a lure for a target insect in an amount of at least 0.01%, at least 0.1%, at least 1%, 0.01-10%, 0.01-1%, 0.1-1%, 0.1-10%, about 0.01%, about 0.1%, about 1%, or about 10% by volume of the composition.
  • the lure is selected from the group consisting of: cuelure, methyl eugenol, and raspberry ketone.
  • the pesticide composition comprises the lure for fruit flies in an amount of at least 0.01%, at least 0.1%, at least 1%, or about 10% by volume of the composition.
  • the pesticide composition comprises the lure for fruit flies in an amount of about 10% by volume of the composition.
  • the pesticide composition further comprises a phagostimulant, for example sugar or molasses.
  • a bait station for controlling an insect populations comprising the pesticide composition described herein.
  • the bait station comprises a chamber and a reservoir for retaining the pesticide composition described herein.
  • the pesticide composition in the bait trap comprises a lure.
  • the chamber houses the reservoir for retaining a pesticide composition, and the chamber comprises an opening, wherein insects attracted by the pesticide composition is able to enter the chamber.
  • the reservoir for retaining a pesticide composition is a fabric saturated with the pesticide composition or another form of membrane material that absorbs an oil-based formulation. In such embodiments, the consistency of the pesticide composition resembles a lotion.
  • the membrane material may be filter paper (for example Whatman 1540-125 filter paper), nylon, polysulfone, polyethersulone, polyvinylidene fluoride, or polytetrafluorethylene.
  • the reservoir for retaining a pesticide composition may be a surface within the chamber onto which the pesticide composition having a consistency resembling petroleum jelly is provided.
  • the bait station comprises a surface covered by the pesticide composition with the abrasive material and the lure. The surface is exposed to the environment Agent Reference: 11157.179WO-PCT and is not contained within a chamber or vessel.
  • the surface may be a board coated with the pesticide composition having a consistency resembling petroleum jelly or a paste. Where the pesticide composition has a consistency of a lotion, the surface may be sprayed with the pesticide composition.
  • a method of controlling an insect population in a designated geographical area is further described. The method comprising providing a pesticide composition described herein to the designated geographical area.
  • the pesticide composition is applied on a surface within the designated geographical area, for example, on or around a surface on a cropping system or on or around the surface of a potential roosting location of the targeted insect.
  • the method comprises providing a bait trap described herein to the designated geographical area.
  • Aprehend ® is sprayed on along bed frames, baseboards, walls, etc. to produce a barrier that bed bugs will walk across while searching for a blood meal and pick up fungal conidia.
  • BotaniGard ® ES BioWorks, Inc., Victor, NY, USA
  • Both Aprehend and BotaniGard ® ES contain B. bassiana strain GHA at different concentrations and different inert ingredients.
  • BotaniGard ® ES was diluted in Heavy Mineral Oil (Fisher Scientific Co., Fair Lawn, NJ) to 2.0 x 10 10 conidia per ml, while Aprehend® is a ready-to-use product and 2.2 x 10 9 conidia per ml. [0101] Each product was applied to three different materials: filter paper, fabric (97% cotton; 3% spandex), and PIG ® Oil-Only Absorbent Mat (Polypropylene) (New Pig Corp., Tipton, PA, USA) (FIG. 26). BotaniGard® in mineral oil (“BGM formulation”) and Aprehend® were Agent Reference: 11157.179WO-PCT applied to each material and left to aerate for 24 h in the dark at 25oC.
  • the lid of a 9 cm petri dish lid was lined with each treated material.
  • Petri dishes had a hole on the side for flies to be aspirated in.
  • Twenty Z. cucurbitae flies were aspirated into each treated petri dish and left for 15 min to ensure flies had sufficient time to walk around in the dish.
  • the flies were then released into 30 ⁇ 30 ⁇ 30 cm mesh cages with water, sugar, and yeast hydrolysate. Mortality was monitored daily for 14 d. Each day, dead flies were removed from the cages and placed in humidity chambers to confirm infection status as described above. Two replicates of each treated material were tested for each formulation, with reciprocal control cages for each material. B.
  • BotaniGard® ES was more effective than Aprehend®, albeit at a higher concentration of spores.
  • the BotaniGard® formulation was further customized to be more effective for fruit flies (FIG. 27).
  • the first modification to the formulation of BotaniGard® in mineral oil was the addition of an abrasive material, diatomaceous earth (DE), at three concentrations (2.5%, 5%, 10% by weight/volume). DE is commonly used in gardens to control arthropod pests.
  • DE diatomaceous earth
  • DE In addition to absorbing insects’ cuticular lipids, DE has sharp edges that scratch the surface of the arthropod’s exoskeleton and causes it to desiccate. Many studies have shown that the addition of DE can enhance the efficacy of fungal entomopathogens. They hypothesize that the scratches made by DE to the insect’s exoskeleton improve the adherence and penetration of fungal hyphae.
  • the formulation containing each concentration of DE was applied to 10 ⁇ 10 cm strips of white cotton fabric and then left to aerate for 24 hours in the dark at 25oC.
  • a 50 ml conical bottom centrifuge tube (Thermo Fisher Scientific Inc., Waltham, MA) with a 1 cm diameter hole at the bottom of the tube, which served as an exit hole for the flies, was lined with the treated fabric.
  • a group of twenty mixed-sex Z. cucurbitae were then placed into the top end of the tube and the lid was closed.
  • the small opening on the bottom of the tube was inserted into a 30 ⁇ 30 ⁇ 30 cm mesh cage and the flies were allowed to exit the tube on their own (via walking) and enter the cage. It was not possible to control the amount of time spent in the tube. The flies could not be forced to exit as flies that attempted to fly in the tube became stuck on the oily treated fabric.
  • One cup without a treated liner contained a HOBO® MX2300 Series Data Logger (Onset Computer Co., Cape Cod, MA) to monitor temperature and relative humidity.
  • HOBO® MX2300 Series Data Logger Onset Computer Co., Cape Cod, MA, one location was selected that was exposed to direct sunlight for most of the day and another location that was shaded for most of the day. Cups were also hung in the laboratory as a control (25 ⁇ 1o C; 70 ⁇ 5% RH). Treated cups were tested for 12-week period during the months of May to August.
  • the vial was vortexed for one minute to release the spores from the fabric.
  • the spore suspension was then plated on Sabouraud Dextrose Agar (SDA) (10 cm petri dish) by pipetting three 10 ⁇ l drops on Agent Reference: 11157.179WO-PCT each plate and gently tilting in a circular motion to spread the droplets without the droplets touching each other. Plates were then incubated at 25°C for 18 hours. After incubation, spores were counted under a phase-contrast microscope at 400x zoom. Spores were considered germinated when the germ tube was longer than the diameter of the conidia. The first 300 conidia were counted per drop and the average of all three drops was used to estimate the germination rate for each plate.
  • SDA Sabouraud Dextrose Agar
  • Example 3 Formulation Optimization A. Changing the Carrier Oil
  • the mineral oil used in our BMD formulation may be a potential deterrent to the flies. Therefore, the attraction of Z. cucurbitae to alternative oils was compared, which included soybean oil, canola oil, peanut oil, (J.M. Smucker Co., Orrville, OH) and castor bean oil (NOW foods Inc., Bloomingdale, IL).
  • Pieces of fabric (7.5 ⁇ 7.5 cm) were soaked with each oil and let drip dry and aerate for 24 hours prior to testing.
  • One oil-soaked fabric piece was placed in a 10 cm petri dish and a cotton wick (3.75 cm) soaked in a 9:1 water-yeast hydrolysate solution was placed in the center of the fabric piece.
  • Four fabric pieces, each treated with a different oil (canola, soybean, peanut, and castor oils), with protein wicks were then placed in separate corners of a 40 ⁇ 40 ⁇ 60 cm cage containing 250 ⁇ twenty mixed-sex flies (FIG.31).
  • the petri dish lids were removed at the same time and a timelapse recording for each petri dish was taken for one hour.
  • the thickening agents were glyceride flakes (MDF) (Mono and Agent Reference: 11157.179WO-PCT Diglyceride flakes, Modernist Pantry LLC., Eliot, ME), Dermofeel® Viscolid MB (DV) (Evonik Co., Hopewell, VA), and cornstarch (ACH Food Companies Inc., Chicago, IL).
  • MDF glyceride flakes
  • DV Dermofeel® Viscolid MB
  • ACH Food Companies Inc., Chicago, IL cornstarch
  • the MDF and DV thickening agents were added to the formulation at a concentration of 10% (or 1g per 10 ml of oil) to achieve a Vaseline-like consistency.
  • the cornstarch was directly mixed into the formulation at a rate of 1.4g/ml of oil.
  • canola oil was heated to 60° C (140° F).
  • Germination rates were assessed every two weeks (0, 2, 4, 6, and 8 weeks) using the methods previously described, except instead of cutting a 1 cm 2 piece of treated fabric, a 1 cm 2 area inside the cup was swabbed. Additionally, cups containing fabric liners treated with non-thickened formulation were also hung for comparison. [0115] In addition to spore viability, the effectiveness of the weathered thickened formulations on fly mortality was tested. Mortality tests were conducted with the same thickened formulations described above, except that the DV-thickened formulation was not tested. The cornstarch and glyceride flake-thickened formulations were applied to modified 50 ml centrifuge tubes.
  • the treated tubes were then hung inside an inverted yellow plastic cup and hung in a direct sun-exposed area and a shaded area on the roof of Gilmore Hall and in the laboratory. This experiment was conducted at the same time as the spore viability tests of the weathered thickened formulations. Mortality tests were conducted every two weeks (0, 2, and 4 weeks) as described previously. Briefly, the treated tubes were removed from the yellow cups and a mixed-sex group of 25 Z. cucurbitae were passed through each tube to allow all flies to walk over the formulation. Two replicates per thickening agent and location were used, with 25 flies per replicate (50 flies total per treatment and location). Controls for each thickened formulation without BotaniGard® were also tested.
  • Modified centrifuge tubes lined with BMD formulation on fabric, or treated on the inside wall with cornstarch and glyceride flake-thickened formulations were hung inside inverted yellow cups and weathered at three locations.
  • cucurbitae were provided with zucchini, B. dorsalis with ripe papayas, and C. capitata with clementine oranges with the rind peeled back. After 24 hours, fruits were removed from the cages and placed into 1 L plastic cups. The cups were lined with a coffee filter and had holes at the bottom to allow liquid from the decomposing fruit to drain. These cups were placed into another 1 L cup, which collected the liquid. The stacked cups containing fruits were placed in a secondary container (28 L clear storage container), which was covered with a mesh cloth (FIG.20). Fresh fruits were added to the cups daily to ensure larvae had enough food to fully develop before pupation.
  • the Z. cucurbitae colony was originally collected from infested zucchini fruits from a commercial farm inEwa, HI. Experiments on this population began at generation F6. B. dorsalis and C. capitata colonies were obtained from the USDA Agricultural Research Service Pacific Basin Branch (Hilo, HI). All flies were used after they became sexually mature, which was approximately 14 d after eclosion for Z. cucurbitae and B. dorsalis and 10 d for C. capitata fruit flies.
  • All treatment formulations contained BotaniGard® ES and 10% DE with the following remaining ingredients: (1) canola oil, (2) canola oil + cornstarch, (3) canola oil + lure, (4) canola oil + cornstarch + lure, and (5) BMD (no additional components other than BotaniGard®, mineral oil, and 10% DE).
  • BMD canola oil + cornstarch + lure
  • BMD BMD (no additional components other than BotaniGard®, mineral oil, and 10% DE).
  • All non-thickened formulations were applied to fabric lining the inside of yellow plastic cups while thickened formulations were directly applied to the inside wall of the cups. Each treated cup was aerated for 24 hours. Twenty male and twenty female sexually mature virgin Z. cucurbitae were placed into each cage. A treatment cup with a C-L plug was then hung in each cage.
  • Lethal concentrations were calculated using a generalized linear model using a binomial distribution and probit link. Probit analysis assumed that the percent response (fly deaths) is related to the log concentration (concentration of spores) as the cumulative normal distribution. Lethal concentrations with 100% and 0% mortality were excluded from the data analysis. The germination of B. bassiana spores were analyzed by generalized linear model (GLM) using a binomial distribution. The survival times, mean, and median survival times were obtained by Kaplan-Meier survival estimator [230,231]. In all mortality trials, flies that survived beyond 14 days were censored from the data set.
  • GLM generalized linear model
  • the mean survival time of the Kaplan-Meier estimation becomes biased when more than 30% of the data is censored while the median survival time is minimally biased [232]. Survival differences between the entire distributions of survival curves were compared using nonparametric log-rank tests weighing each death with the Kaplan-Meier estimate of survival as a log-rank (rho 0) [233,234]. Spore germination percentages and thickening agent run-off tests were analyzed using three-way repeated measures ANOVA with multiple pairwise comparisons to determine the group mean differences with Bonferroni adjustment. Choice tests for different oils, lure concentrations, and phagostimulants were analyzed using GLM using a Poisson distribution with least-squares pairwise comparison. All analyses were performed on R version 4.1.0. Example 10.
  • Table 6 Kaplan-Meier survival times for B. dorsalis, C. capitata, and Z. cucurbitae, which were passively exposed to the BMD formulation. The results report passive horizontal transmission mortality following unexposed male and females with access to a BMD formulation treated trap.
  • BMD non-thickened formulation
  • CORN cornstarch thickened formulation
  • GF glycosylated formulation
  • Germination rates of cornstarch and glyceride flake-thickened and non-thickened BMD formulations ( ⁇ SE) over a four-week period in Trials 1 and 2.
  • Mean temperature and relative humidity ( ⁇ SE) differed in each location and may have impacted the spore viability and longevity of each formulation.
  • Agent Reference: 11157.179WO-PCT Agent Reference: 11157.179WO-PCT Table 19. Pairwise T-test grouping (lure*week) with pairwise comparisons of formulation (concentration) type effects germination percentages.

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Abstract

L'invention concerne une formulation de pesticide épaissie comprenant des spores ou conidies de champignons entomopathogènes. La composition pesticide comprend les spores ou conidies de champignons entomopathogènes, une huile support et une huile épaississante, la viscosité de la formulation pesticide étant de 1 500 à 2 000 000 cps. Dans certains modes de réalisation, la composition pesticide comprend en outre un agent abrasif et/ou un leurre pour un insecte cible. L'invention concerne également des pièges d'appât comprenant la composition pesticide et des procédés de commande d'une population d'insectes dans une zone géographique désignée.
PCT/US2024/033150 2023-06-09 2024-06-07 Formulations d'huiles épaississantes d'entomopathogènes fongiques Pending WO2024254558A2 (fr)

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MX9407876A (es) * 1993-10-12 1997-02-28 Clifford A Bradley Formulaciones de hongos entomopatogenos para uso como insecticidas biologicos.
US5983558A (en) * 1994-08-15 1999-11-16 Griffin Corporation Insect bait station and method of treating insects with toxicant
TWI629935B (zh) * 2012-05-14 2018-07-21 陶氏農業科學公司 昆蟲引誘劑調配物及昆蟲防治技術
FR3075040A1 (fr) * 2017-12-15 2019-06-21 Laboratoires Arkopharma Composition antiparasitaire a usage externe
WO2019238734A1 (fr) * 2018-06-15 2019-12-19 Bayer Aktiengesellschaft Formulations liquides et stables au stockage pour spores fongiques

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