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WO1995010597A1 - Formulations de champignons entomopathogenes destinees a etre utilisees comme insecticides biologiques - Google Patents

Formulations de champignons entomopathogenes destinees a etre utilisees comme insecticides biologiques Download PDF

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Publication number
WO1995010597A1
WO1995010597A1 PCT/US1994/011542 US9411542W WO9510597A1 WO 1995010597 A1 WO1995010597 A1 WO 1995010597A1 US 9411542 W US9411542 W US 9411542W WO 9510597 A1 WO9510597 A1 WO 9510597A1
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Prior art keywords
entomopathogenic
formulation
conidia
oil
fungus
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Clifford A. Bradley
James H. Britton
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Mycotech Corp
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Mycotech Corp
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Priority to EP94931342A priority Critical patent/EP0738317A4/fr
Priority to AU80155/94A priority patent/AU8015594A/en
Priority to JP7512004A priority patent/JPH09506592A/ja
Publication of WO1995010597A1 publication Critical patent/WO1995010597A1/fr
Anticipated expiration legal-status Critical
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    • 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

  • Entomopathogenic fungi or insect pathogenic fungi have the potential to be a new class of bioinsecticides suitable for wide spread applications in agriculture.
  • the mode of action of the fungi, penetration through the insect cuticle, makes fungi effective against a wide range of insect pests that cannot be controlled effectively by bacterial, viral, or protozoan pathogens that act through ingestion.
  • Insect pathogenicity occurs in a broad range of fungi including the genera Beauveria, Metarhizium, Nomuraea, Verticilium, Aschersonia, Hirsutella, and Paecilomyces. Common traits of all these fungi are that they all produce conidia, conidia are the infective form of the organism and all have been shown to produce conidia in vitro.
  • Daoust et al. J. Invert. Path, 41: 151 - 160 ( 1983) discuss the effect of formulation on the viability of Metarhizium anisopliae conidia.
  • fourteen oils were tested including mineral oil, cod liver oil and twelve vegetable oils.
  • Daoust et al. teach that the fourteen oils were highly detrimental to the viability of the conidia after two months of storage. They also teach that organic acids and water were lethal to the conidia.
  • Prior et al. Prior et al.
  • vegetable oils have been used as carriers for entomopathogenic fungi.
  • vegetable oils do not confer long term shelf life and do not have good spray characteristics.
  • Conidia of Beauveria bassiana and other species of entomopathogenic fungi are strongly hydrophobic and difficult to suspend in water. Many applications of fungi use water, detergent suspensions, or wettable powders that use selected clays as suspension aids or dry powder. However, in water suspensions, conidia can germinate and lose their infectivity after only twenty-four hours.
  • fungal insect pathogens as mycopesticides requires that fungal conidia remain viable and infective during and after storage and transport and that fungal conidia be effectively delivered to the target insect in a viable form. It also is important that fungal conidia be stored and shipped economically, and applied effectively in a wide range of conditions with a variety of application equipment.
  • the present invention provides entomopathogenic formulations that include conidia of an entomopathogenic fungus suspended in a carrier.
  • Carriers include oils, emulsions, and suspensions.
  • the fungus-oil suspension can be emulsified with water and can contain other additives.
  • the fungus-oil suspension and formulations prepared from the fungus-oil suspension have the advantages of increased shelf life and temperature stability compared with dry conidia preparations or water suspensions, increased protection of conidia from ultraviolet radiation and greater infectivity than conidia suspended in water. Also provided are methods of killing insects using the formulations of the present invention.
  • Figures IA and IB are graphs depicting mortality results of bioassays involving oil and clay/water formulations of Beauveria bassiana against nymphs of Oedaleus senegalensis.
  • Figure 2 is a bar graph which shows the grasshopper species composition in treated and untreated plots prior to application of Beauveria bassiana.
  • Figure 3 is a bar graph which shows mortality results of six grasshopper species collected from field test plots that have been treated with Beauveria bassiana.
  • Figure 4 is a bar graph which shows mortality results of grasshoppers treated aerially with BbGHA1991 and held on native rangeland in cages.
  • Figure 5 is a graph depicting changes in grasshopper population as determined from ring counts after application of Beauveria bassiana.
  • Figure 6 is a graph depicting reduction in grasshopper population density after a ground application test of BbGHA1991, an isolate of Beauveria bassiana, and controls.
  • Figure 7 is a graph depicting cumulative mortality of grasshoppers collected one hour post-treatment in a test where BbGHA1991 and the control were applied from the ground.
  • the present invention provides an entomopathogenic formulation that includes conidia of an entomopathogenic fungus and a carrier oil.
  • a small volume of the formulation is capable of being spread over a large geographical area or water emulsions can be prepared from the oil formulations for high volume applications.
  • entomopathogenic formulation is intended to include a mixture of conidia of an entomopathogenic fungus and a carrier.
  • the carrier is a substance capable of dispensing the fungus appropriately without adversely affecting the fungus' ability to perform its intended function.
  • Carriers of the present invention include oils, emulsions, and suspensions.
  • Conidia is art-recognized and is intended to include asexual spores characteristic of fungi. Conidia of a fungus can be counted and used as units of measure of the fungus, for example, with respect to viability and LD50.
  • entomopathogenic fungus means a fungus which is capable of killing an insect. Such a fungus is considered a mycopesticide. Entomopathogenic fungi include those strains or isolates of fungal species in the class Hyphomycetes which possess characteristics allowing them to be virulent against insects. These characteristics include formation of stable infective conidia.
  • An effective entomopathogenic fungus preferably is lethal for target insects but less harmful for non-target insects. Also, the entomopathogenic fungus preferably does not harm vegetation or animals who might come in contact with it.
  • non-phytotoxic is intended to include a substance that is not significantly inhibitory to the growth of or poisonous to plants at application rates used in insect control or at conidia levels necessary to kill the target insect.
  • a non-phytotoxic substance such as a horticultural oil cannot adversely affect any vegetation with which the substance comes in contact.
  • Non-phytotoxic substances include horticultural oils such as Sunspray® 6N and Sunspray® 6E and agricultural oils such as Sunspray® 7E and Sunspray® 7N.
  • Oil is art-recognized and is intended to include a substance which is an unctuous, viscous liquid at ordinary temperatures.
  • Oils can be derived from either petroleum or from vegetables. Oils include light paraffmic oils such as Sunspray® 6N, Sunspray® 6E, or Sunspray® 7E as well as other petroleum-based oils and vegetable oils such as those derived from corn, cottonseed, soy beans, palm or coconut, rape seed, and sunflower seed.
  • conidia are killed by exposure to sunlight, particularly ulraviolet wavelengths. Oils preferably include those that can protect entomopathogenic fungal conidia from harmful ultraviolet radiation. Formulations which protect conidia from sunlight damage are advantageous in increasing persistence of conidia in the field after spraying. Conidia also can be killed by exposure to elevated temperatures. Oils included are those that do not adversely affect, or preferably those which enhance, conidia stability.
  • petroleum based oil includes oils that are derived from petroleum including light paraffmic oils. Petroleum based oils typically have distillation midpoints in the range of 404-435°F. In addition, light paraffmic petroleum oils can have an unsulfonated residue of at least 90 and a CAS number 64741-89-5. Light paraffmic petroleum oils include Sunspray® 6N, Sunspray® 6E, and Sunspray® 7E. Petroleum based oils preferably include those which do not adversely affect the viability and/or virulence of the conidia derived from the fungus.
  • Petroleum based oil for purposes of this invention is not intended to include mineral oil which adversely affects the viability and/or virulence of the conidia derived from the fungus.
  • the language "fungus" is art-recognized.
  • the fungi of the invention can be entomopathogenic or can produce commercially useful enzymes such as amylase and/or ligninase.
  • Examples of fungus include fungi of the subdivision Deuteromycotina (or Deuteromycetes) and fungi of the class Hyphomycetes.
  • fungi of the class are examples of the class of the class of the class
  • Hyphomycetes can produce conidia.
  • Examples of entomopathogenic fungus genera include Beauveria, Metarhizium, Paecilomyces, Tolypocladium, Aspergillus, Culicinonyces, Nomuraea, Sorosporella, Verticillium, and Hirsutella.
  • Examples of fungus genera that are not entomopathogenic include Trichoderma and Alternaria.
  • fungus examples include Beauveria bassiana, Metarhizium flavoviride, Metarhizium anisopliae, Paecilomyces fumusoroseus, Paecilomyces farinosus, Nomuraea rileyii, Aspergillus niger, Aspergillus awamori, Trichoderma riride, and Trichoderma harzianium.
  • Fungus includes those strains or isolates of fungal species that possess characteristics allowing them to be virulent against insects. These characteristics include formation of stable, infective conidia.
  • the present invention also provides an entomopathogenic formulation that includes conidia of an entomopathogenic fungus and an emulsion.
  • emulsion is intended to include mixtures of two liquids not mutually soluble which are capable of suspending the conidia of an entomopathogenic fungus.
  • Emulsions include mixtures of oil and water. In an oil and water emulsion, the oil can aid in suspending the hydrophobic conidia and allow for high volume dispersion of the fungus. In water suspension, conidia of the entomopathogenic fungus typically germinate and are noninfective within twenty-four hours. However, the oil in the emulsion surrounds the conidia and can retard germination for several days, thus, allowing storage of the formulation after addition of water for several days prior to application.
  • the present invention further provides an entomopathogenic formulation that includes conidia of an entomopathogenic fungus and a non-phytotoxic suspension as well as an entomopathogenic formulation that includes conidia of an entomopathogenic fungus, a non-phytotoxic suspension and petroleum jelly. Also included are oil or oil water emulsions with spreaders, stickers, or other additives used for purposes such as aiding conidia suspension or dispersal or enhancing ultraviolet protection.
  • Suspensions are intended to include a solid substance which, in a particulate form, is mixed with a fluid but which remains undissolved.
  • Suspensions include those where clay or diatomaceous earth is suspended in water or in oil/water emulsions. In clay/water suspensions or clay/water/oil emulsions, the clay helps the hydrophobic conidia to be suspended in water for greater dispersion and longer shelf life.
  • clay is art-recognized and is intended to include a natural earthy material that is plastic when wet and can act as a carrier in suspension of conidia of a fungus. In an emulsifiable suspension, the clay can stabilize the oil and water emulsion and allow the liquids to remain emulsified even in unagitated spray equipment.
  • the amount of clay needed depends on the final water volume to be added and can be determined by routine experimentation.
  • examples of clays within the scope of this invention include attapulgite clay, kaolin, and bentonite.
  • Petroleum jelly is art-recognized and is intended to include a gelatinous mass obtained from petroleum.
  • Petroleum jelly includes a smooth, semisolid blend of mineral oil with waxes crystallized from the residual type of petroleum lubricating oil. Petroleum jelly can make the droplets of conidia/oil suspensions and emulsions more uniform and denser. Addition of petroleum jelly reduces spray drift in aerial application and provides additional protection of the fungal conidia from ultraviolet radiation. Petroleum jelly also increases viscosity and aids in maintaining conidia in suspension, e.g., during storage.
  • conidia of an entomopathogenic fungus are hydrophobic.
  • Oils, emulsions, and suspensions of clay in oil or clay in an oil/water emulsion can keep the conidia suspended in liquid form for greater ease of delivery.
  • the advantages of these formulations are ease of delivery, temperature stability, and longer shelf life.
  • Conidia disperse uniformly in oil and disperse uniformly upon application in small volumes. Water suspensions could be applied at equally low volumes but the conidia would not be as uniformly dispersed.
  • conidia are more stable under exposure to elevated temperatures when suspended in oils.
  • Selected carrier oils include light paraffmic oils.
  • a suspension of conidia in water generally has to be used within a few days. However, a suspension of conidia in oil has a much longer shelf life, e.g., one of months instead of days.
  • small volume is intended to include volumes recognized by those of skill in the art as being small volumes for suspending entomopathogenic fungal conidia.
  • Small volume includes the amount of liquid necessary or sufficient to suspend the conidia of the entomopathogenic fungus.
  • Small volume also includes the minimum amount of a liquid such as a carrier oil needed to suspend the conidia.
  • small volume includes that volume of formulation used in ultra low volume applications, e.g. aerial applications. Examples of small volume amounts and applications are described in the examples below.
  • high volume is intended to include volumes recognized by those of skill in the art as being high volumes for suspending entomopathogenic fungal conidia.
  • High volume includes the amount of oil necessary to suspend the conidia to which water has been added to form an emulsion.
  • High volumes of conidia suspension can be used to apply the conidia to an affected area using ground equipment such as a tractor pulling a spray apparatus. Typically, ground equipment move across a field at a much slower rate than an airplane and high volumes are needed to ensure that the correct amount of conidia per unit area is applied. Examples of high volume amounts and applications are described in the examples below.
  • the language "being capable of spreading” is intended to include the ability to be applied over an area either evenly or unevenly.
  • the language includes substances such as oils or emulsions that can be evenly or unevenly distributed over a geographical area by land or by air.
  • large area is intended to include an extent of space or surface that would be recognized by those of skill in the art as being a large area. Large areas include parcels or portions of land on which insects are present.
  • viscosity that allows the entomopathogenic formulation to be sprayed through a nozzle is intended to include liquids whose ability to resist a force that causes the liquid to flow is such that the liquid can be forced through a nozzle to form a spray. The viscosity cannot be so high that it clogs the nozzle when it is forced through the opening.
  • nozzle is intended to include a spout or terminal discharging pipe through which a substance flows.
  • Nozzle includes a spout that causes a liquid to become a spray.
  • a nozzle can be used on airplane spray booms or ground sprayers to finely divide the flow of entomopathogenic formulation into a spray for efficient application.
  • the present invention provides methods of killing insects using the aforementioned formulations.
  • the formulations can be administered to an affected geographical area by either land or air.
  • an affected geographical area is intended to include a parcel or portion of land which has insects present on that land.
  • Affected geographical areas include those areas which contain large numbers of insects that cause damage to vegetation or crops.
  • applying is intended to include a method of bringing the conidia of an entomopathogenic fungus in contact, either externally or internally, with an insect, for example, by a droplet directly contacting an insect or the insect's body coming in contact with the conidia as the insect travels across a leaf.
  • Application includes direct application of the conidia of an entomopathogenic fungus to the insect as in topical application.
  • Application also includes spraying an affected geographical area with the conidia in liquid form. The suspended conidia can be sprayed on the affected geographical area from the air such as from a low flying plane or from the ground such as by an individual with a tank sprayer.
  • Application also includes spraying vegetation or soil and subsequent contact between an insect and conidia on sprayed vegetation or soil.
  • insects are art-recognized. Examples of insects include grasshopper, locust, white fly, gypsy moth, Colorado potato beetle, corn borer, citrus root weevil, corn root worm, and thrip.
  • BbGHA1991 were produced in a series of solid cultures using 1.5 or 3 kg dry weight culture substrate as follows:
  • the strain was maintained as a dried laboratory solid culture containing viable conidia stored at 4°C.
  • Broth cultures of composition described below were inoculated with conidia from this maintenance culture and incubated at 25°C on a rotary shaking water bath for three to six days.
  • Broth culture medium results in production of high numbers of single-celled blastospores.
  • Typical broth cultures o contain in excess of 1x10 blastospores per ml.
  • 100ml of broth culture were transferred to 1.5 liters broth in 2800ml flasks incubated at 25°C with approximately 500cc/minute sparged air flow.
  • Solid culture substrate was prepared by mixing equal parts by weight of dry barley flakes and the same inoculum culture medium described above except that glucose was omitted.
  • Wetted barley was autoclaved in polypropylene bags for 20 minutes to one hour (depending on volume) at 15psi at 121°C, cooled, and inoculated by transferring broth cultures directly to bags of substrate which were mixed externally by hand.
  • 1.5 kg dry weight barley flakes were mixed with 1500ml nutrient solution and autoclaved in one bag, cooled and inoculated with 300ml of broth culture.
  • Inoculated solid substrate was transferred to an autoclave-sterilized polycarbonate box 27cm x 48cm x 15cm fitted with a screen bottom and connectors for air inlet and outlet.
  • the substrate formed a bed about eight to ten centimeters deep on the screen.
  • 3kg dry weight flakes were processed and incubated in 18" diameter x 24" deep round steel vessels fitted with screens.
  • the culture beds were about 20 to 30cm deep. Cultures were incubated at 20-30°C for 10 days with an air flow of about 0.5 to 2 liters/minute. Air flow varied to maintain culture temperature. After eight to twelve days incubation, cultures were transferred to a dryer consisting of screens and equipped with a fan. Cultures were spread on screens and dried to a final moisture content of less than 10% with a flow of dry air at 20-25°C.
  • Conidia suspension was homogenized for two minutes and diluted as appropriate (generally diluted to certain or estimated 1x10° to 1x10' conidia/ml in the final dilution.) Conidia concentration was determined by microscopic count at 400x magnification using a hemocytometer. (Neubauer-Levy or Petroff-Hauser Chamber or equivalent). Viability was determined by placing a drop of diluted conidia suspension on Sabaroud's Dextrose Agar Yeast Extract (SDAY, Difco) plate. The drop was covered with a sterile microscope cover slip and plates were incubated 16-20 hours at 25°C. Plates were examined at 400x and germinated and ungerminated conidia were each counted.
  • Conidia were considered germinated if swollen or if a hypha was emerging from the conidia. Conidia suspensions were sampled in duplicate. For each sample, a total of at least 100 conidia were counted in at least three microscope fields of view.
  • Beauveria bassiana conidia powder prepared according to 5 Example 1 was suspended at the rate of 75 g per liter of Sunspray® 6N Horticultural Oil (CAS #64741-89-85). Sunspray® 6N oil has a distillation midpoint of 404-424°F and unsulfonated residue of 90. The final preparation contained 5x1012 conidia per liter.
  • Formulations were sprayed by injecting a measured volume of formulation into the air stream of an air brush set at a height of 1.8m above a 14" diameter target area. Volume injected was 0.09 ml and was calculated to be equivalent to an application rate of 1x10 3 conidia in 3,785 ml (one gallon) per acre. Each formulation was sprayed on 3rd and 4th instar Melanoplus sanguinipies. For each formulation, ten separate, replicated sets of five grasshoppers (50 grasshoppers per formulation) were sprayed. An untreated control group consisting of 50 grasshoppers was handled as the test grasshoppers except that they were not sprayed. A "treated control" group consisting of 50 grasshoppers was sprayed with formulation oil without conidia.
  • Grasshoppers were held individually in screen cap, four ounce cups and fed daily with a diet of romaine lettuce and oat cereal. Holding room conditions were 26-33°C, 25-42% rh, 14 hour light, 10 hour dark photo period. Table 1 shows mortality over 12 days in the treated and control groups.
  • Conidia powder produced in a series of bench scale cultures by the method of Example 1 were pooled. Conidia in powder were 99% viable. Conidia powder was mixed in 20 gallons of Sunspray ® 6E which was loaded into a spray plane owned and operated by the U.S. Department of Agriculture. On the first spray date, the plane circled test plots for about 30 minutes. Wind was too high to spray and the plane returned to the airport. Upon return to the airport, temperature of the oil suspension was about 105°F, measured with a medical thermometer. The suspension was drained from the plane to pails which were stored overnight at about 4°C. The next morning the suspension was reloaded in the plane and a portion sprayed on a test plot. Unused suspension was drained into pails. Pails were stored at cool room temperature for eleven months. At this time, samples were taken and conidia viability assayed. Viability after use in field trial with exposure to elevated temperature and approximately eleven months of storage was greater that 90%.
  • Example 7 Storage Stability of Beauveria bassiana at 40°C in Sunspray® Horticultural Oils 6N and 6E
  • Example 8 Storage Stability of Beauveria bassiana in Sunspray® Agricultural Oil 7E at Different Temperatures
  • Strains were BbGHA1991, RS 252, originally isolated from Colorado Potato Beetle used commonly in Beauveria research, and a Mycotech strain, designated GMB6 isolated from Gypsy moth.
  • Strain RS 252 was obtained from the USDA ARS collection of entomopathogenic fungi, Ithaca, New York.
  • Conidia powder from each strain was suspended at 0.5g per 10ml Sunspray® 7E oil in a series of separate screw cap vials. Samples of dry conidia powder were also placed in vials. Vials were placed in dark incubators at 25°, 32°, and 42°C. Vials were sampled at time intervals and conidia viability was determined by the method described in Example 1. Results are shown in Table 3. Table 3
  • Conidia powder prepared according to Example 1 was used to prepare an ES.
  • the purpose of the ES was to minimize the amount of oil and allow high volume application using water as a diluent.
  • the ES was mixed in the following ratios:
  • Conidia mixed in water typically germinate within 24 hours. As a result, water suspensions must be used immediately after mixing. In the ES formulation described in Example 9, conidia do not germinate for several days after water addition. This allows use of formulation diluted to final volume over several days.
  • Conidia powder was formulated as an ES as described in
  • Example 9 with 1.5g conidia powder in 10ml Sunspray® 6E oil, 3.5g Attaclay RVM and 25ml water. The mixture was stored at room temperature and samples were observed daily at 400x in a phase contrast microscope. After 10 days, conidia had not germinated. At seven and ten days, incubator samples were taken for viability assay. Conidia viability was 80-90% in both samples.
  • Emulsifiable concentrate was prepared as in Example 9 in the following ratio (equivalent to 1x10*3 conidia in two liters final volume per acre): 0.6g conidia powder, 4ml Sunspray® 6E oil, 3.2g Attaclay to a final volume of 10 ml with water. Prior to mixing, 5% (w/w) petroleum jelly was added to the oil/water/clay suspension.
  • Example 12 Spray Tower Test ofES Formulations
  • Duplicate ES formulations were prepared as described in Example 9 with 1.5g conidia powder in 10ml Sunspray® 6E oil, 5.25g Attaclay RVM and 27ml water. One sample was made the day prior to spray and one immediately prior to spray. Formulations were sprayed on 3rd instar Melanoplus sanguinipies using the spray tower and methods described in Example 5 except that 0.05ml of formulation was sprayed on each replicate of five grasshoppers. Mortality results are shown in Table 4 below. Results demonstrate the stability and infectivity of conidia in oil/clay/water emulsion suspension 24 hours after water addition.
  • Oil and water suspensions were compared in a topical 5 bioassay on 3rd instar Senegalese grasshoppers, Oedalius senegalensis.
  • grasshoppers were treated individually with a single 0.25 microliter droplet delivered by microsyringe to the pronotum of the insect.
  • 30 grasshoppers were treated, held individually in small plastic cups, fed daily with fresh, l o native grass and monitored for mortality.
  • Controls were sprayed cover slips held in the dark at room temperature
  • Trac-Rac is an equatorial mounted, single axis, follow-the-sun tracking device, used to increase the amount of radiation a specimen would receive in specified time period. The total amount of energy available to the test specimen is increased by maintaining near-normal incidence (noontime conditions) from sunrise 0 to sunset. The specimens were mounted unbacked with the sprayed surface toward the sun.
  • thermocouples were monitored every 30 seconds with a five minute average and maximum recorded every hour, 24 hours per 5 day utilizing two 30 AWG Type T thermocouples.
  • One thermocouple was attached with thermally conductive epoxy to the back surface of an unsprayed cover slip.
  • the other thermocouple was located in a shaded, well-ventilated area to monitor the ambient air temperature. Temperature data was recorded with a Campbell Scientific Model 21x o datalogger.
  • Table 6 shows average conidia viability for each formulation immediately prior to sunlight exposure, with 6.5 and 30 hour exposure and formulations held in the dark. Maximum temperature on the cover slips during the first 6.5 hours was 18.2°C and was 25.3°C during the second day of the test. Ambient temperature was within ⁇ 2°C of temperature on the cover slips. Minimum temperature was 8.1°C.
  • Conidia sprayed in water suspension showed 13% viability after 6.5 hours of exposure. All of the oil based formulations showed 78 to 94% viability after 6.5 hours. One formulation, #10, retained 10-25% viability with 30 hours exposure. Dark controls all showed at least 68-96% viability. Formulations using light paraffmic oils in the carrier provided a significant degree of solar protection.
  • Conidia preparations of Beauveria bassiana GHA1991 were produced in a series of bench scale cultures according to the method described in Example 1. Conidia were produced and stored at room temperature for 11-13 months. A portion of the conidia were o stored as dry powder and a portion was stored in Sunspray® 6E oil.
  • Formulations were prepared as described in Example 4 in Sunspray® 6E oil to a final concentration of 4.5 x 10*2 viable conidia per liter. Formulations were transported from Butte, Montana, under ambient conditions to Bowman, North Dakota, and stored at ambient conditions. 5
  • Application was by air using an unmodified USDA Cessna "Ag Truck", equipped with centrifugal pump and standard flat fan spray nozzles (Tee-jet seize 8002).
  • Application volume was determined by pump pressure and the number of nozzles on the spray o boom. Volume was calibrated by measuring delivery volume of each nozzle tip over 30 seconds in three replicated tests at the selected pump pressure. Aircraft spray time over the plot was recorded by electronic timer. Final application volume was confirmed by the difference in reservoir volume before and after application.
  • Application volume was 5 4.6 liters/hectare (2 quarts per acre). Conidia were applied at the rate of 2x10*3 conidia per hectare (8xl0*2/acre). Application was in the early morning with plots sprayed from a height of 10-20 feet. Application coverage was monitored by inspection of oil sensitive spray cards placed throughout the plots. 0
  • Efficacy was evaluated by three methods: observation of post-treatment grasshopper population samples, grasshoppers caged in the fields, and population density estimates.
  • grasshoppers of each species were collected by sweep net from test, untreated control and oil-only treated control plots on the day of application.
  • Grasshoppers were held individually in 120cc plastic cups with screen lids, fed fresh, untreated field vegetation supplemented with romaine lettuce and oat cereal and monitored daily for mortality. Cups were held at ambient temperature for 12 days. Mycosis was confirmed by conidiation of Beauveria bassiana on grasshopper cadavers.
  • Pre-spray grasshopper populations were estimated in counts on the two days immediately prior to application and post-spray counts made at three day intervals. Both population samples ( Figure 3) and field cages ( Figure 4) showed high levels of mortality in treated plots compared with controls for all species in both trials. Beauveria bassiana mycosis was confirmed in more than 90% of dead grasshoppers in population samples from treated plots with no evidence of mycosis in dead grasshoppers from control plots.
  • Figure 5 shows ring count density estimate after treatment. Significant population increases occurred in control plots with evidence of new hatch in later population samples. Weather was probably a factor in o these trials. Through the first three days post-application, weather was clear with peak afternoon air temperatures of 27-39°C. Between the third and fourth days, a cold front brought rains and cooler temperature through day eight. During this period, peak afternoon temperatures were 9-15°C with minimums of 4.5-7°C, with intermittently heavy rain. 5 The more rapid mortality of population samples held indoors, compared with those grasshoppers in outdoor field cages indicate infection was retarded at cooler temperatures.
  • Conidia powder was prepared as described in Example 1 and transporte :dd L under ambient conditions. Powder contained 7x10 conidia per gram.
  • conidia powder was suspended in Sunspray® 7N oil to a final concentration of 5x10 2 viable conidia per o liter (75g powder per liter) for application at five liters/hectare.
  • Emulsifiable suspension contained 300g conidia powder in five liters Sunspray® 6E oil.
  • the oil suspension was mixed with 460g Attaclay RVM and water to a final volume of 20 liters for a one hectare application. 5
  • INIDA Instituto Nacional de Investigacao e Desinvolvimento Agraria
  • INIDA is located at Sao Jorge, Sao Tiago Island, Republic of Cabo Verde.
  • INIDA personnel chose a site three kilometers east of the town o of Tarrafal, on the north end of Sao Tiago island.
  • the area was planted with rows of acacia trees, the rows being three to six meters apart.
  • the ground was very rocky with sparse vegetation.
  • the grasshopper population was composed almost entirely of fifth instar larvae and adult Oedaleus senegalensis. 5
  • a total of nine treatment plots 100 x 200 meters (two hectares) each, were laid out with a compass and flagging tape. Three plots were treated with oil-formulated spores, three with emulsifiable suspension-formulated spores, and three were left untreated. 0
  • the ULV oil formulation was sprayed at a rate of 2.5 x 10 spores per hectare, in a volume of five liters per hectare, from hand-held Microulva sprayers (Micron Sprayers, Ltd.).
  • the emulsifiable suspension-treated plots also received 2.5 x 10*3 S p 0res p er hectare, but in a volume of 20 liters per hectare.
  • Application was made via gasoline-powered backpack sprayers.
  • Conidia of Metarhizium flavoviride (USDA ARSEF 2023) were produced by the method of Example 1, except that a slant culture of the Metarhizium flavoviride strain was used as the source culture.
  • Final dried conidia powder contained 8.3xl0 ⁇ viable conidia per gram.
  • Conidia powder was suspended in Sunspray® 6E oil to a
  • a conidia preparation was made directly from the infected pupae and labeled Del B. Two single colony isolates made by dilution of this preparation designated GMP1Y4 and GMP1W1 dry conidia powder were prepared by the method described in Example 1. Conidia were suspended in Sunspray® 6E oil at the rate of 1x10° conidia per ml.
  • Conidia suspensions were sprayed on three foot lengths of oak tree limbs at a rate of one ml per ten square inches of surface to a ⁇ final concentration of approximately 1x10 conidia per square inch of surface.
  • Limbs were allowed to dry for one to two hours after spraying and were placed horizontally in trays.
  • Gypsy moth larvae were released on one end of the limb and a light bulb was placed at the other end of the limb to attract the larvae. After the larvae had traversed the length of the limb or had been exposed on the limb for one to three hours, the larvae were removed and held in cups on a standard gypsy moth diet and observed daily for mortality. Results are shown in Table 7. Strain GMP1 Wl formulated in oil showed significant mortality.
  • a strain of Beauveria bassiana ARSEF 201 which is virulent to gypsy moth in laboratory bioassay was used to prepare a dry conidia powder by the method of Example 1.
  • Conidia powder was o formulated in Sunspray® 6E oil to a final concentration of 4.8x10 /ml.
  • the concentrated oil suspension was diluted 20:1 with water and shaken to form an emulsion at the time of application.
  • Field test plots were located in Delaware and composed of mixed hardwood woodlots, primarily white and red oak, sweet gum and beech. Two plots, one of three acres and one of 0.5 acre, isolated by open areas were used as test plots. Two control plots of similar size were delineated.
  • Conidia emulsion suspensions were applied to the point of runoff with a backpack sprayer to the lower six to eight feet of tree trunk of all dominant trees in the test plots.
  • the sprayed portion of trees contained high numbers of larvae.
  • Application was made when larvae were four to six instar and pupation was first observed.
  • About 15 minutes after spraying 100 larvae were removed from each test plot and held in a laboratory in standard diet cups for two weeks.
  • Table 8 shows mortality in larvae in this contact spray test.

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  • General Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Pest Control & Pesticides (AREA)
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  • Dentistry (AREA)
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  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

Formulations entomopathogènes comprenant les conidies d'un champignon entomopathogène et un support. On peut citer à titre de supports les huiles, les émulsions et les suspensions. Des procédés de destruction d'insectes tels que des sauterelles à l'aide de ces formations sont également décrits.
PCT/US1994/011542 1993-10-12 1994-10-12 Formulations de champignons entomopathogenes destinees a etre utilisees comme insecticides biologiques Ceased WO1995010597A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP94931342A EP0738317A4 (fr) 1993-10-12 1994-10-12 Formulations de champignons entomopathogenes destinees a etre utilisees comme insecticides biologiques
AU80155/94A AU8015594A (en) 1993-10-12 1994-10-12 Formulations of entomopathogenic fungi for use as biological insecticides
JP7512004A JPH09506592A (ja) 1993-10-12 1994-10-12 生物学的殺虫剤として使用するための昆虫病原性真菌類の組成物

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US08/134,563 1993-10-12

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039844A1 (fr) * 1995-06-07 1996-12-19 Mycogen Corporation Materiaux et procedes servant a inoculer des microbes dans des vegetaux
WO2002060260A1 (fr) * 2001-01-30 2002-08-08 Consejo Superior De Investigaciones Cientificas Vehicule porteur de spores d'un microorganisme entomopathogene et procede permettant de lutter contre les insectes incommodants
EP1297746A1 (fr) * 2001-09-26 2003-04-02 Sumitomo Chemical Company, Limited Champignon entomopathogène destiné à être utilisé comme insecticide
WO2010044680A1 (fr) 2008-10-14 2010-04-22 Millennium Microbes Limited Champignons entomopathogènes et leurs utilisations
EP2273873A4 (fr) * 2008-04-07 2011-09-07 Bayer Cropscience Lp Formulation aqueuse stable contenant des spores
WO2015069708A1 (fr) 2013-11-08 2015-05-14 Novozymes Bioag A/S Compositions et procédés pour traiter les parasites
AU2015201322B2 (en) * 2008-04-07 2016-08-25 Basf Corporation Stable aqueous spore-containing formulation
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
CN117617267A (zh) * 2023-11-20 2024-03-01 华南农业大学 一种玫烟色棒束孢油悬浮剂及其在防治柑橘木虱中的应用
US11993068B2 (en) 2022-04-15 2024-05-28 Spora Cayman Holdings Limited Mycotextiles including activated scaffolds and nano-particle cross-linkers and methods of making them
WO2024254558A3 (fr) * 2023-06-09 2025-04-10 University Of Hawaii Formulations d'huiles épaississantes d'entomopathogènes fongiques
US12467171B2 (en) 2023-10-13 2025-11-11 Spora Cayman Holdings Limited Large-scale production of mycelium-based textiles at mushroom farm facilities

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
JP4581410B2 (ja) * 2004-01-21 2010-11-17 住友化学株式会社 殺虫性油剤

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US4755207A (en) * 1986-07-28 1988-07-05 Mycogen Corporation Synergistic mycoherbicidal compositions
US5057316A (en) * 1989-03-15 1991-10-15 Ecoscience Laboratories, Inc. Method and device for the biological control of insects

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GB2255018B (en) * 1991-04-26 1995-03-15 C A B International Entomopathogenic sprays

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US4755207A (en) * 1986-07-28 1988-07-05 Mycogen Corporation Synergistic mycoherbicidal compositions
US5057316A (en) * 1989-03-15 1991-10-15 Ecoscience Laboratories, Inc. Method and device for the biological control of insects

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Title
ANNALS OF APPLIED BIOLOGY, Volume 122, issued 1993, R.P. BATEMAN et al., "The Enhanced Infectivity of Metarhizium Flavoviride in Oil Formulations to Desert Locusts at Low Humidities", pages 145-152. *
JOURNAL OF INVERTEBRATE PATHOLOGY, Volume 41, issued 1983, R.A. DAOUST et al., "Effect of Formulation of the Viability of Metarhizium Anisopliae Conidia", pages 151-160. *
See also references of EP0738317A4 *
WEED TECHNOLOGY, Volume 5, issued 1991, W.J. CONNICK et al., "An Improved Invert Emulsion with High Water Retention for Mycoherbicide Delivery", pages 442-444. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039844A1 (fr) * 1995-06-07 1996-12-19 Mycogen Corporation Materiaux et procedes servant a inoculer des microbes dans des vegetaux
WO2002060260A1 (fr) * 2001-01-30 2002-08-08 Consejo Superior De Investigaciones Cientificas Vehicule porteur de spores d'un microorganisme entomopathogene et procede permettant de lutter contre les insectes incommodants
ES2171149A1 (es) * 2001-01-30 2002-08-16 Univ Valencia Politecnica Vehiculo portador de esporas de un microorganismo entomopatogeno y metodo para combatir insectos dañinos.
EP1297746A1 (fr) * 2001-09-26 2003-04-02 Sumitomo Chemical Company, Limited Champignon entomopathogène destiné à être utilisé comme insecticide
US7033586B2 (en) 2001-09-26 2006-04-25 Sumitomo Chemical Company, Limited Insecticidal Paecilomyces tenuipes strain FERM BP-7861
AU2015201322B2 (en) * 2008-04-07 2016-08-25 Basf Corporation Stable aqueous spore-containing formulation
EP2273873A4 (fr) * 2008-04-07 2011-09-07 Bayer Cropscience Lp Formulation aqueuse stable contenant des spores
US10362786B2 (en) 2008-04-07 2019-07-30 Bayer Intellectual Property Gmbh Stable aqueous spore-containing formulation
WO2010044680A1 (fr) 2008-10-14 2010-04-22 Millennium Microbes Limited Champignons entomopathogènes et leurs utilisations
WO2015069708A1 (fr) 2013-11-08 2015-05-14 Novozymes Bioag A/S Compositions et procédés pour traiter les parasites
US10383339B2 (en) 2013-11-08 2019-08-20 Novozymes Bioag A/S Compositions and methods for treating pests
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
US11993068B2 (en) 2022-04-15 2024-05-28 Spora Cayman Holdings Limited Mycotextiles including activated scaffolds and nano-particle cross-linkers and methods of making them
WO2024254558A3 (fr) * 2023-06-09 2025-04-10 University Of Hawaii Formulations d'huiles épaississantes d'entomopathogènes fongiques
US12467171B2 (en) 2023-10-13 2025-11-11 Spora Cayman Holdings Limited Large-scale production of mycelium-based textiles at mushroom farm facilities
CN117617267A (zh) * 2023-11-20 2024-03-01 华南农业大学 一种玫烟色棒束孢油悬浮剂及其在防治柑橘木虱中的应用

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MX9407876A (es) 1997-02-28
AU8015594A (en) 1995-05-04
EP0738317A1 (fr) 1996-10-23
CA2173953A1 (fr) 1995-04-20
EP0738317A4 (fr) 1998-09-09
JPH09506592A (ja) 1997-06-30

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