CA2049458A1 - Method and medium for packaging entomogenous nematodes - Google Patents
Method and medium for packaging entomogenous nematodesInfo
- Publication number
- CA2049458A1 CA2049458A1 CA002049458A CA2049458A CA2049458A1 CA 2049458 A1 CA2049458 A1 CA 2049458A1 CA 002049458 A CA002049458 A CA 002049458A CA 2049458 A CA2049458 A CA 2049458A CA 2049458 A1 CA2049458 A1 CA 2049458A1
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- Prior art keywords
- film
- nematodes
- matrix
- infective
- entomogenous
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/30—Rearing or breeding invertebrates
-
- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
- A01N63/12—Nematodes
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- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Entomogenous infective juvenile (IJ) nema-todes are prepared for storage and shipment by encas-ing viable IJs in a thin film which is permeable to oxygen, and which contains sufficient water to main-tain the IJs in a fully hydrated state. The film is rigid enough to substantially immobilize the IJs, resulting in a reduction in the amount of oxygen and food reserves required, thus extending the shelf life of IJs.
Entomogenous infective juvenile (IJ) nema-todes are prepared for storage and shipment by encas-ing viable IJs in a thin film which is permeable to oxygen, and which contains sufficient water to main-tain the IJs in a fully hydrated state. The film is rigid enough to substantially immobilize the IJs, resulting in a reduction in the amount of oxygen and food reserves required, thus extending the shelf life of IJs.
Description
,:~s ~ f '~ j ~
METHOD AND MEDIUM FOR PACKAGING
ENTOMOGENOUS NEMATODES
Description Technical Field This application relates to the fields of packaging living organisms for storage and/or ship-ment, and to the use of entomogenous nematodes for insect control.
Backqround of the Invention The hazards of residual toxicity and the relative lack of specificity ha~e rendered chemical insecticides unable to meet the requirements of modern agriculture and gardening. Lack of specificity results in destruction of beneficial insect species (e.g., honeybees) along with the destruction of target species. Residual toxicity affects organisms which encounter insecticide by ingesting dead insects, and so pas~ the chemical agents and their metabolites into the food chain. These problems and others have caused new interest in biological methods for pest control.
Perhaps the oldest form of biological insect control is the use of ladybugs to combat infestation by aphids. More recently, fly populations have been reduced by release of sterilized male flies, which -2~ 58 compet~ with fertile males and thus redu~e the number of fertile eggs produced by females. Othe~s have atte~pted to control insect populations through use of viruse~ or entomogenous fungi.
Relatively recently, interest has turned to entomogenous nematodes. Nematodes make up a diverse phylum of unsegmented round worms which may be free-living or parasitic. Entomogenous nematodes parasi-tize insects. Typically, an entomogenous nematode in a particular developmental stage termed an "infective juvenile" or IJ enters a host insect through the alimentary canal or spiracles. Once in the host, the IJ emerges from its protective sheath and penetrates into the host's haemocel. In the host insect's haemo-cel, the nematode releases symbiotic bacteria whichinduce septicemia in the host, and render the host corpse suitable for nematode foraging and reproduc-tion. The nematodes may spend several generations within the insect host, until food consumption and crowding trigger production of another IJ stage gen-eration. The new IJs lea~e the host corpse in search of fresh hosts.
R.W. Glaser, J Exp Zool (1940) 84:1-12 reported ~he culturing of Neoaplectana qlaseri or use in controlling Japanese beetles. N._g~aseri was applied to test fields in several states, and in some cases resulted in reduction of beetle and moth grub populations (G.O. Poinar, "Nematodes for Biological Control of Insects", CRC Press, 1975).
R. Gaugler, J Nematol (1981) 13:241-49 dis-cussed the potential uses of entomogenous nematodes for control of insect populations.
However, the use of entomogenous nematodes presents several obstacles to successful commercial development. Entomogenous nematodes are highly sensi-tive to drying, and will eventually desiccate if held at relative humidities of less than 100%. They are sensitiYe to direct sunlight, and are also somewhat prone to infection, although care mus~ be taken that the nematodes' symbiotic bacteria is not eliminated along with any infecting agents. Accordingly, par-ticular requirements must be met when storing or ship-ping entomogenous nematodes. The packaging must be able to maintain the nematodes' moisture content (e.g., by maintaining relative humidity at 100%), must provide sufficient food and oxygen to each nematode in the package (allowing for the tendency of nematodes to clump or sett~e), and preferably should protect the nematodes from infection by exogenous agents. IJs depend only on internal stores for food, but may metabolize their stores too quickly for long storage.
Economics requires that the packaging material be inexpensive, lightweight, durable, and free from aeration restraints.
Finney, U.S. Pat. No. 4,417,545 disclosed a water-saturated foam packing material for shipping nematodes.
Bedding, U.S. Pat. No. 4,178,366 disclosed the use of entomogenous nematodes (particularly N.
carpocapsae) for biological con~rol of insects, and an anhydrous oil suspension formulation for application of nematodes to vegetation by spraying. Ths formula-tion also contained a wax component to retard wa~er loss by nematodes in the sprayed droplets. Nema~odes applied to foliage in oil suspension survived longer, as the formulation retarded desiccation.
S.R. Dutky et al, J Insect Pathol (1964) 6:417 22 disclosed the storage of a nematode desig-nated DD-136 (possibly N. carpocapsae) in 0.1~ aqueous formaldehyde at 7.1C. The nematodes were suspended at a concentration of 50,000/mL, and 1 liter of sus-pen3ion wa~ stored in an insulated gallon ~ug. The suspQn~ions were oxygenated periodically.
Nelsen et al, U.S. Pat. No. 4,615,883 dis-closed encapsulation of entomogenous nematodes in hydrogels, where the hydrogel capsule contains 50-98%
free water in its interior, and allows diffusion of gases sufficient for respiration. The capsules may optionally be provided with a wax membrane to retard water loss. The hydrogel capsules must be suffi-ciently tough to resist abrasion, but pliable enoughto allow release of the nematodes upon ingestion by an insect host. The capsules, having an average diameter range of 0.4-5 mm, are sprayed over an area to be treated. Hydrogels have also been employed to en-capsulate microorganisms. See, e.g., Mimura et al,U.S. Pat. No. 4,450,233; Jung, U.S. Pat. No.
4,434,231; Lim, U.S. Pat. No. 4,352,883; Asai et al, U.S. Pat. No. 4,202,905; Guttag, U.S. Pat. No.
METHOD AND MEDIUM FOR PACKAGING
ENTOMOGENOUS NEMATODES
Description Technical Field This application relates to the fields of packaging living organisms for storage and/or ship-ment, and to the use of entomogenous nematodes for insect control.
Backqround of the Invention The hazards of residual toxicity and the relative lack of specificity ha~e rendered chemical insecticides unable to meet the requirements of modern agriculture and gardening. Lack of specificity results in destruction of beneficial insect species (e.g., honeybees) along with the destruction of target species. Residual toxicity affects organisms which encounter insecticide by ingesting dead insects, and so pas~ the chemical agents and their metabolites into the food chain. These problems and others have caused new interest in biological methods for pest control.
Perhaps the oldest form of biological insect control is the use of ladybugs to combat infestation by aphids. More recently, fly populations have been reduced by release of sterilized male flies, which -2~ 58 compet~ with fertile males and thus redu~e the number of fertile eggs produced by females. Othe~s have atte~pted to control insect populations through use of viruse~ or entomogenous fungi.
Relatively recently, interest has turned to entomogenous nematodes. Nematodes make up a diverse phylum of unsegmented round worms which may be free-living or parasitic. Entomogenous nematodes parasi-tize insects. Typically, an entomogenous nematode in a particular developmental stage termed an "infective juvenile" or IJ enters a host insect through the alimentary canal or spiracles. Once in the host, the IJ emerges from its protective sheath and penetrates into the host's haemocel. In the host insect's haemo-cel, the nematode releases symbiotic bacteria whichinduce septicemia in the host, and render the host corpse suitable for nematode foraging and reproduc-tion. The nematodes may spend several generations within the insect host, until food consumption and crowding trigger production of another IJ stage gen-eration. The new IJs lea~e the host corpse in search of fresh hosts.
R.W. Glaser, J Exp Zool (1940) 84:1-12 reported ~he culturing of Neoaplectana qlaseri or use in controlling Japanese beetles. N._g~aseri was applied to test fields in several states, and in some cases resulted in reduction of beetle and moth grub populations (G.O. Poinar, "Nematodes for Biological Control of Insects", CRC Press, 1975).
R. Gaugler, J Nematol (1981) 13:241-49 dis-cussed the potential uses of entomogenous nematodes for control of insect populations.
However, the use of entomogenous nematodes presents several obstacles to successful commercial development. Entomogenous nematodes are highly sensi-tive to drying, and will eventually desiccate if held at relative humidities of less than 100%. They are sensitiYe to direct sunlight, and are also somewhat prone to infection, although care mus~ be taken that the nematodes' symbiotic bacteria is not eliminated along with any infecting agents. Accordingly, par-ticular requirements must be met when storing or ship-ping entomogenous nematodes. The packaging must be able to maintain the nematodes' moisture content (e.g., by maintaining relative humidity at 100%), must provide sufficient food and oxygen to each nematode in the package (allowing for the tendency of nematodes to clump or sett~e), and preferably should protect the nematodes from infection by exogenous agents. IJs depend only on internal stores for food, but may metabolize their stores too quickly for long storage.
Economics requires that the packaging material be inexpensive, lightweight, durable, and free from aeration restraints.
Finney, U.S. Pat. No. 4,417,545 disclosed a water-saturated foam packing material for shipping nematodes.
Bedding, U.S. Pat. No. 4,178,366 disclosed the use of entomogenous nematodes (particularly N.
carpocapsae) for biological con~rol of insects, and an anhydrous oil suspension formulation for application of nematodes to vegetation by spraying. Ths formula-tion also contained a wax component to retard wa~er loss by nematodes in the sprayed droplets. Nema~odes applied to foliage in oil suspension survived longer, as the formulation retarded desiccation.
S.R. Dutky et al, J Insect Pathol (1964) 6:417 22 disclosed the storage of a nematode desig-nated DD-136 (possibly N. carpocapsae) in 0.1~ aqueous formaldehyde at 7.1C. The nematodes were suspended at a concentration of 50,000/mL, and 1 liter of sus-pen3ion wa~ stored in an insulated gallon ~ug. The suspQn~ions were oxygenated periodically.
Nelsen et al, U.S. Pat. No. 4,615,883 dis-closed encapsulation of entomogenous nematodes in hydrogels, where the hydrogel capsule contains 50-98%
free water in its interior, and allows diffusion of gases sufficient for respiration. The capsules may optionally be provided with a wax membrane to retard water loss. The hydrogel capsules must be suffi-ciently tough to resist abrasion, but pliable enoughto allow release of the nematodes upon ingestion by an insect host. The capsules, having an average diameter range of 0.4-5 mm, are sprayed over an area to be treated. Hydrogels have also been employed to en-capsulate microorganisms. See, e.g., Mimura et al,U.S. Pat. No. 4,450,233; Jung, U.S. Pat. No.
4,434,231; Lim, U.S. Pat. No. 4,352,883; Asai et al, U.S. Pat. No. 4,202,905; Guttag, U.S. Pat. No.
3,767,790; and Fogle et al, U.S. Pat. No. 3,541,203.
In the above formulations, the nematodes are typically formulated when in the IJ stage. This essentially eliminates the requirement for food during storage, as IJs do not feed until they unsheath, but rely on stored food. However, IJs continue to require oxygen and moisture, which must be provided by the packaging or formulation.
An alternative approach is to exploit the ability of IJs to enter a cryptobiotic state, in which meta~olism is greatly reduced or halted. For example, Popiel et al, EPO 256,873 disclosed the induction of an apparent anhydrobiotic state in IJs, using care-fully controlled d~siccation. Upon slow desiccation, the IJs adapt and are able to survive reduced moisture levels in a state of reduced metabolic activity (anhydrobiosis). The anhydrobiotic IJs still require oxy~en and moisture, ~ut at a much lower rate than normal "biotic~ IJs. The reduction of metabolism also result~ in conservation of food stores. Th~ net re~ult is a method for storing and shipping IJs which is much less sensitive to moisture and oxygen require-ments than traditional methods.
Yukawa et al, PCT wo 85/03412 disclosed anematode formulation comprising a -cream~ of IJs in a solution containing an antibiotic such as formalde-hyde, optionally an agent to provide a high osmotic potential such as 30~ sucrose, and optionally an absorbent such as activated charcoal. The formulation is sto~ed under anaerobic conditions, and is asserted to be resistant to temperatures up to 40 C. The IJs in this formulation are presumably in an anaerobiotic state.
A disadvantage of some of the above formula-tions is that IJs may require a recovery period for their metabolism to revert to normal, before full infectivity is resumed. During this period, the nema-todes may be subject to predation, and may fail toparasitize insects upon ingestion when an otherwise successful infec~ion would normally occur. Also, the process of inducing the anhydrobiotic or cryptobiotic state can be very time consuming ~e.g., 2-6 days for anhydrobiosis), and has an associated mortality rate.
Disclosure of the I_vention We have now invented a medium for convenient storage and shipping of entomogenous nematodes. The medium comprises a film formed from a hydrated, oxygen-permeable, reversibly cross-linked matrix con-taining entomogenous IJs. The film has a thickness of between about 0.5 and S mm, and allows oxygen to pen-etrate to each nematodes in sufficient quantity to assure respiration. The nematodes are restrained in an immobilized state, although cryptobiosis is not -6~ jJ~
induced. As a result of the restraint, metabolic dem~nd~ for food and oxygen are reduced, which permits longer periods of storage and reduce~ the head space needed for oxygen supply in a sealed pacXage.
Another aspect of the invention is a method for preparing the nematode film. The method comprises suspending the IJs in an aqueous solution with a suf-ficient amount of cross-linkable matrix material, casting the suspension into a thin sheet (about 0.5-5 mm thick), and cross-linking the matrix to form a sheet.
Another aspect of the invention is the method of controlling an insect population, by revers-ing the cross-linking of a nematode film, freeing the nematodes, and applying the freed nematodes to an area having insects to be controlled. Another aspect of the invention is the method wherein the nematode film is applied to an area having insects to be controlled, and is then uncross-linked to free the nematodes. In the practice of the latter method, the film may advan-tageously include photoprotective agents to protect nematodes from direct sunlight, and may be designed to maintain a high level of moisture in the application area for an extended period of time.
Mod~s of Carryinq Out The Invention A. Definitions The term 'entomogenous nematode" refers to nematodes which parasitize and kill insects. Pres-ently preferred entomoyenous nematodes are derived from the Family Steinernematid and Heterorhabditid nematodes, particularly Neoaplectana carpocapsae, N.
bibionls, N. alaseri, and H. heliothidis.
The term "infective juvenile" or "IJ" refers to an entomogenous nematode in the infective third ! i ' ''' ~ il ',; ~
larval stage. IJs are characterized by retention ofthe s~cond stage cuticle or sheath after molting to thir~ ~tage. IJs do not eat, but depend on internal food sto~es. They are capable of substantial vertical and horizontal migration, and are generally the only nematode stage capable of establishing a productive infection in insects.
The term cryptobiosis'- refers to a state of dormancy in which metabolism essentially ceases. In this state, the IJ fails to respond to physical manipulation, and appears inert upon inspection.
Cryptobiotic IJs may be stored for long periods with~
out air or food, but generally require a recovery period prior to reestablishment of full infectivity.
"Anhydrobiosis" refers to a cryptobiotic or semi-cryptobiotic state which is induced by gradual desic-cation of IJs. In the anhydrobiotic state, IJs gener-ally coil and cease movement, and may survive removal of most of their body water content. Anhydrobiotic IJs may still require oxygen, but at a rate greatly reduced from motile IJs.
The term "reversibly cross-linkable matrix material" refers to a substance which may be cross-linked to form a relatively rigid gel. The matrix material must be capable of permitting diffusion of gases sufficient for nematode respiration while immobilized, must retain sufficient water to prevent desiccation, and must be substantially non-toxic to the nematodes employed in the film. The oxygen perme-ability will of course vary with the species o~ nema-tode selected, the degree of immobilization, the con-centration of IJs in the film, and the thickness o~
the film cast. However, by immobilizing the IJs within the film, we have found that oxygen diffusion rates may be used which are far lower than the oxygen diffusion rates required for non-immobilized, biotic ~ ~ r ~
nematodes. The degree of hydration should be about 50-90~ in order to assure non-desiccation. The matrix material must be capable of being cross-lis~ked to a degree sufficient tc substantially immobilize the IJ5.
The degree of cross-linking may be estimated by the rigidity of the resulting film, e.g., using a duro-meter. The films of the invention are rigid enough that ~2~ of the entrained nematodes are able to migrate out of the gel within 72 hours. The cross-linked film must be capable of being unlinked or dis-solved in a non-toxic solvent to release the nema-todes. Finally, the cross-linking and unlinking con-ditions must be mild enough for the IJs to tolerate.
Reversibly cross-linkable matrix materials useful in the present invention include sodium alginate, cara-geenan, gelatins, xanthan gums, and the like. The presently preferred matrix material is alginic acid.
Alginic acid suspensions are cross-linked by the addi-tion of Ca++, and are unlinked by removal of Ca+~, e.g. r by addition of citrate and/or EDTA.
~ he phrase "limits water loss refers ~o the reduction in water loss by evaporation. In general, the water loss should be low enough that the film is not desiccated in the container during storage or shipment. For a 1 ft2 film, a water loss of about 0.15 g/day at ambient temperatures is acceptable.
B. General Method Infective juvenile nematodes are prepared by any acceptable means, such as the methods disclosed by Glaser, supra, or Bedding, U.S. Pat. No. 4,334,498.
~ suspension of IJs in buffered aqueous solution is prepared with a suitable concentration of matrix material. The concentration of IJs may range from about 1 x 105/mL to about 6 x 105/mL, preferably about 3 x 105/mL. The concentration of matrix ,/ L~ 5 ~
g material depends upon the particular material selectad~ and may be determined by routine experimen-tation Where alginic acid is employed, the preferred concentration is about 0.75% to about 10~, preferably about 2% when cast. In general, the suspension is prepared to provide sufficient viscosity that it may be cast on a screen of opening size <1-2 mm or on a solid support. The suspension is mixed well and cast as a film of thickness 4 to about 6 mm, and cross-linking is initiated. Where a cross-linking agent is required, it may be added after casting, or immedi-ately prior to ca~ting. In the case of alginic acid, the preferred cross-linking agent is a divalent metal cation, preferably Ca++. About 0.5 M to 2.0 M CaC12 is added to the cast film, and the gel allowed to harden to provide a nematode film of the invention.
The film may then be removed from the support and cut to an appropriate size for packaging. The film may be prepared embedded in the screen, and ~he entire screen and film composition may be cut to appropriate size.
The film may be stored in a polymer bag or bottle which is capable of transmitting o~ygen while limiting water 10s5 by evaporation. Water loss should be limited to < 0.15 g/day per square foot of matrix.
Oxygen permeability should provide about 70 cc of 2/
day per square foot of matrix.
Unlinking is accomplished in a manner dependent upon the particular matrix material selected. In the case of alginic acid, the divalent metal cation is removed, typically by complexation with EDTA and/or citric acid. The film may be immersed in a suitable solution, and stirred until the film has substantially disintegrated. The resulting suspension contains viable, biotic ~Js, and non-toxic matrix materials, and may be applied directly to an area having insects to be controlled.
-10- ~ -3~
Viability of nematodes may be assessed micro~copically, by observing the reaction to prodding with a dis~ec~ion needle. Infectivity is tradition-ally assessed by applying the IJs to Galleria mellonella larvae and noting the rate of mortality.
Normally, at least 40~ of the G. mellonella larvae will be dead within 48 hours of application.
C. Examples The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.
Example 1 (Preparation of Nematode Film) (A) NeoaPlectana carpocapsae IJs were cleaned and disinfected by washing with a suitable disinfectant, such as dilute hypochlorite.
Deionized water (7.5 Kg), Proxel~ (a bio-cide, 8 g), Keltone~ HV9 (an alginic acid analog, 300 g), Min-U-Gel 9 ( a montmorillonite clay extender, 225 g), and Waterlock~ G400~ (a modified starch humectant, 15 g) were blended in a mixing vessel with strong agitation to form a uniform slurry of high viscosity.
To this slurry was added an aqueous suspension of dis-infected IJs (6.3 x 10 IJ/mL, 8048 g), and the mix-ture blended slowly under very mild agitation.
The slurry (80 g) was then applied to one square foot of fiberglass window screening having about 15 strands/inch, and was spread to a uniform layer 3-6 mm thick. The entire sheet was then immersed in an aqueous solution of CaC12 (1.6 M: cal-cium phosphate is also acceptable), and held un~il the gel had set (about 30-60 seconds). The sheet was then transferred to a pure water bath for about 10-30 sec-ond3 to remove excess calcium.
The sheet is then allowed to drip dry, and is packaged in bags of semipermeable polymer film, or in bottles having loose caps or semipermeable film lids. The package need only supply about 70 cc of oxygen per day to support the IJs immobilized in one square foot of film- In contrast, an equal number of free-swimming IJs in suspension would require about 180 cc of oxygen per day.
The product contains fully biotic entomogen-ous nematode IJs, may be stored under refrigeration if desired. If stored at ambient temperature, the prod-uct exhibits adequate viability for over 30 days. If stored under refrigeration (e.g., at 5C), the product will be useful even after six months of storage.
(B) A nematode film was prepared as in part A above, but substituting H. heliothidis ~or N.
carpocapsae, and using calcium phosphate instead of calcium chloride.
Example 2 To use the nematode film, the entire film was immersed in a 40 oz aqueous solution containing sodium citrate 10% and EDTA 1~. After about 15 min utes, the film disintegrated, and was ready for dilu-tion.
The suspension is diluted for further use.
For use with a "back-pack' type sprayer, the suspen-sion is diluted with about 1.5 gallons of water. Use with a hose-end type sprayer dilutes the suspension with about 200 gallons. Alternatively, the suspension may be added to 3 gallons of water in a watering can for use with potted plants. The suspension is suf-ficient to cover about 550 square feet.
~ :1 t. ` '., ` v ~ ; .i 8 If desired, the film container may be dimen-sioned to pro~ide sufficient volume for the solution for unlinking the film, so tha~ the film may be dis-solved without removing it from the container. Water is simply added to the container up to a fill line, a packet containing sodium citrate and EDTA is added, and the container is closed and shaken vigorously.
In the above formulations, the nematodes are typically formulated when in the IJ stage. This essentially eliminates the requirement for food during storage, as IJs do not feed until they unsheath, but rely on stored food. However, IJs continue to require oxygen and moisture, which must be provided by the packaging or formulation.
An alternative approach is to exploit the ability of IJs to enter a cryptobiotic state, in which meta~olism is greatly reduced or halted. For example, Popiel et al, EPO 256,873 disclosed the induction of an apparent anhydrobiotic state in IJs, using care-fully controlled d~siccation. Upon slow desiccation, the IJs adapt and are able to survive reduced moisture levels in a state of reduced metabolic activity (anhydrobiosis). The anhydrobiotic IJs still require oxy~en and moisture, ~ut at a much lower rate than normal "biotic~ IJs. The reduction of metabolism also result~ in conservation of food stores. Th~ net re~ult is a method for storing and shipping IJs which is much less sensitive to moisture and oxygen require-ments than traditional methods.
Yukawa et al, PCT wo 85/03412 disclosed anematode formulation comprising a -cream~ of IJs in a solution containing an antibiotic such as formalde-hyde, optionally an agent to provide a high osmotic potential such as 30~ sucrose, and optionally an absorbent such as activated charcoal. The formulation is sto~ed under anaerobic conditions, and is asserted to be resistant to temperatures up to 40 C. The IJs in this formulation are presumably in an anaerobiotic state.
A disadvantage of some of the above formula-tions is that IJs may require a recovery period for their metabolism to revert to normal, before full infectivity is resumed. During this period, the nema-todes may be subject to predation, and may fail toparasitize insects upon ingestion when an otherwise successful infec~ion would normally occur. Also, the process of inducing the anhydrobiotic or cryptobiotic state can be very time consuming ~e.g., 2-6 days for anhydrobiosis), and has an associated mortality rate.
Disclosure of the I_vention We have now invented a medium for convenient storage and shipping of entomogenous nematodes. The medium comprises a film formed from a hydrated, oxygen-permeable, reversibly cross-linked matrix con-taining entomogenous IJs. The film has a thickness of between about 0.5 and S mm, and allows oxygen to pen-etrate to each nematodes in sufficient quantity to assure respiration. The nematodes are restrained in an immobilized state, although cryptobiosis is not -6~ jJ~
induced. As a result of the restraint, metabolic dem~nd~ for food and oxygen are reduced, which permits longer periods of storage and reduce~ the head space needed for oxygen supply in a sealed pacXage.
Another aspect of the invention is a method for preparing the nematode film. The method comprises suspending the IJs in an aqueous solution with a suf-ficient amount of cross-linkable matrix material, casting the suspension into a thin sheet (about 0.5-5 mm thick), and cross-linking the matrix to form a sheet.
Another aspect of the invention is the method of controlling an insect population, by revers-ing the cross-linking of a nematode film, freeing the nematodes, and applying the freed nematodes to an area having insects to be controlled. Another aspect of the invention is the method wherein the nematode film is applied to an area having insects to be controlled, and is then uncross-linked to free the nematodes. In the practice of the latter method, the film may advan-tageously include photoprotective agents to protect nematodes from direct sunlight, and may be designed to maintain a high level of moisture in the application area for an extended period of time.
Mod~s of Carryinq Out The Invention A. Definitions The term 'entomogenous nematode" refers to nematodes which parasitize and kill insects. Pres-ently preferred entomoyenous nematodes are derived from the Family Steinernematid and Heterorhabditid nematodes, particularly Neoaplectana carpocapsae, N.
bibionls, N. alaseri, and H. heliothidis.
The term "infective juvenile" or "IJ" refers to an entomogenous nematode in the infective third ! i ' ''' ~ il ',; ~
larval stage. IJs are characterized by retention ofthe s~cond stage cuticle or sheath after molting to thir~ ~tage. IJs do not eat, but depend on internal food sto~es. They are capable of substantial vertical and horizontal migration, and are generally the only nematode stage capable of establishing a productive infection in insects.
The term cryptobiosis'- refers to a state of dormancy in which metabolism essentially ceases. In this state, the IJ fails to respond to physical manipulation, and appears inert upon inspection.
Cryptobiotic IJs may be stored for long periods with~
out air or food, but generally require a recovery period prior to reestablishment of full infectivity.
"Anhydrobiosis" refers to a cryptobiotic or semi-cryptobiotic state which is induced by gradual desic-cation of IJs. In the anhydrobiotic state, IJs gener-ally coil and cease movement, and may survive removal of most of their body water content. Anhydrobiotic IJs may still require oxygen, but at a rate greatly reduced from motile IJs.
The term "reversibly cross-linkable matrix material" refers to a substance which may be cross-linked to form a relatively rigid gel. The matrix material must be capable of permitting diffusion of gases sufficient for nematode respiration while immobilized, must retain sufficient water to prevent desiccation, and must be substantially non-toxic to the nematodes employed in the film. The oxygen perme-ability will of course vary with the species o~ nema-tode selected, the degree of immobilization, the con-centration of IJs in the film, and the thickness o~
the film cast. However, by immobilizing the IJs within the film, we have found that oxygen diffusion rates may be used which are far lower than the oxygen diffusion rates required for non-immobilized, biotic ~ ~ r ~
nematodes. The degree of hydration should be about 50-90~ in order to assure non-desiccation. The matrix material must be capable of being cross-lis~ked to a degree sufficient tc substantially immobilize the IJ5.
The degree of cross-linking may be estimated by the rigidity of the resulting film, e.g., using a duro-meter. The films of the invention are rigid enough that ~2~ of the entrained nematodes are able to migrate out of the gel within 72 hours. The cross-linked film must be capable of being unlinked or dis-solved in a non-toxic solvent to release the nema-todes. Finally, the cross-linking and unlinking con-ditions must be mild enough for the IJs to tolerate.
Reversibly cross-linkable matrix materials useful in the present invention include sodium alginate, cara-geenan, gelatins, xanthan gums, and the like. The presently preferred matrix material is alginic acid.
Alginic acid suspensions are cross-linked by the addi-tion of Ca++, and are unlinked by removal of Ca+~, e.g. r by addition of citrate and/or EDTA.
~ he phrase "limits water loss refers ~o the reduction in water loss by evaporation. In general, the water loss should be low enough that the film is not desiccated in the container during storage or shipment. For a 1 ft2 film, a water loss of about 0.15 g/day at ambient temperatures is acceptable.
B. General Method Infective juvenile nematodes are prepared by any acceptable means, such as the methods disclosed by Glaser, supra, or Bedding, U.S. Pat. No. 4,334,498.
~ suspension of IJs in buffered aqueous solution is prepared with a suitable concentration of matrix material. The concentration of IJs may range from about 1 x 105/mL to about 6 x 105/mL, preferably about 3 x 105/mL. The concentration of matrix ,/ L~ 5 ~
g material depends upon the particular material selectad~ and may be determined by routine experimen-tation Where alginic acid is employed, the preferred concentration is about 0.75% to about 10~, preferably about 2% when cast. In general, the suspension is prepared to provide sufficient viscosity that it may be cast on a screen of opening size <1-2 mm or on a solid support. The suspension is mixed well and cast as a film of thickness 4 to about 6 mm, and cross-linking is initiated. Where a cross-linking agent is required, it may be added after casting, or immedi-ately prior to ca~ting. In the case of alginic acid, the preferred cross-linking agent is a divalent metal cation, preferably Ca++. About 0.5 M to 2.0 M CaC12 is added to the cast film, and the gel allowed to harden to provide a nematode film of the invention.
The film may then be removed from the support and cut to an appropriate size for packaging. The film may be prepared embedded in the screen, and ~he entire screen and film composition may be cut to appropriate size.
The film may be stored in a polymer bag or bottle which is capable of transmitting o~ygen while limiting water 10s5 by evaporation. Water loss should be limited to < 0.15 g/day per square foot of matrix.
Oxygen permeability should provide about 70 cc of 2/
day per square foot of matrix.
Unlinking is accomplished in a manner dependent upon the particular matrix material selected. In the case of alginic acid, the divalent metal cation is removed, typically by complexation with EDTA and/or citric acid. The film may be immersed in a suitable solution, and stirred until the film has substantially disintegrated. The resulting suspension contains viable, biotic ~Js, and non-toxic matrix materials, and may be applied directly to an area having insects to be controlled.
-10- ~ -3~
Viability of nematodes may be assessed micro~copically, by observing the reaction to prodding with a dis~ec~ion needle. Infectivity is tradition-ally assessed by applying the IJs to Galleria mellonella larvae and noting the rate of mortality.
Normally, at least 40~ of the G. mellonella larvae will be dead within 48 hours of application.
C. Examples The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.
Example 1 (Preparation of Nematode Film) (A) NeoaPlectana carpocapsae IJs were cleaned and disinfected by washing with a suitable disinfectant, such as dilute hypochlorite.
Deionized water (7.5 Kg), Proxel~ (a bio-cide, 8 g), Keltone~ HV9 (an alginic acid analog, 300 g), Min-U-Gel 9 ( a montmorillonite clay extender, 225 g), and Waterlock~ G400~ (a modified starch humectant, 15 g) were blended in a mixing vessel with strong agitation to form a uniform slurry of high viscosity.
To this slurry was added an aqueous suspension of dis-infected IJs (6.3 x 10 IJ/mL, 8048 g), and the mix-ture blended slowly under very mild agitation.
The slurry (80 g) was then applied to one square foot of fiberglass window screening having about 15 strands/inch, and was spread to a uniform layer 3-6 mm thick. The entire sheet was then immersed in an aqueous solution of CaC12 (1.6 M: cal-cium phosphate is also acceptable), and held un~il the gel had set (about 30-60 seconds). The sheet was then transferred to a pure water bath for about 10-30 sec-ond3 to remove excess calcium.
The sheet is then allowed to drip dry, and is packaged in bags of semipermeable polymer film, or in bottles having loose caps or semipermeable film lids. The package need only supply about 70 cc of oxygen per day to support the IJs immobilized in one square foot of film- In contrast, an equal number of free-swimming IJs in suspension would require about 180 cc of oxygen per day.
The product contains fully biotic entomogen-ous nematode IJs, may be stored under refrigeration if desired. If stored at ambient temperature, the prod-uct exhibits adequate viability for over 30 days. If stored under refrigeration (e.g., at 5C), the product will be useful even after six months of storage.
(B) A nematode film was prepared as in part A above, but substituting H. heliothidis ~or N.
carpocapsae, and using calcium phosphate instead of calcium chloride.
Example 2 To use the nematode film, the entire film was immersed in a 40 oz aqueous solution containing sodium citrate 10% and EDTA 1~. After about 15 min utes, the film disintegrated, and was ready for dilu-tion.
The suspension is diluted for further use.
For use with a "back-pack' type sprayer, the suspen-sion is diluted with about 1.5 gallons of water. Use with a hose-end type sprayer dilutes the suspension with about 200 gallons. Alternatively, the suspension may be added to 3 gallons of water in a watering can for use with potted plants. The suspension is suf-ficient to cover about 550 square feet.
~ :1 t. ` '., ` v ~ ; .i 8 If desired, the film container may be dimen-sioned to pro~ide sufficient volume for the solution for unlinking the film, so tha~ the film may be dis-solved without removing it from the container. Water is simply added to the container up to a fill line, a packet containing sodium citrate and EDTA is added, and the container is closed and shaken vigorously.
Claims (15)
1. A nematode film for storage and shipment of entomogenous infective juvenile nematodes, which film comprises:
an oxygen-permeable, reversibly cross-linked matrix having a thickness of about 0.5 to about 5 mm, having a water content sufficient to preserve infective juvenile nematodes in a non-desiccated state, and having sufficient rigidity to substantially immobilize infective juvenile nematodes; and viable entomogenous infective juvenile nematodes immobilized in said cross-linked matrix in a non-cryptobiotic state.
an oxygen-permeable, reversibly cross-linked matrix having a thickness of about 0.5 to about 5 mm, having a water content sufficient to preserve infective juvenile nematodes in a non-desiccated state, and having sufficient rigidity to substantially immobilize infective juvenile nematodes; and viable entomogenous infective juvenile nematodes immobilized in said cross-linked matrix in a non-cryptobiotic state.
2. The film of claim 1, wherein said reversibly cross-linked matrix comprises calcium alginate.
3. The film of claim 2 wherein said entomogenous nematodes are selected from Steinernematid and Heterorhabditid infective juveniles.
4. The film of claim 3 wherein the infective juveniles are of the species N. carpocapsae.
5. The film of claim 3 wherein the infective juveniles are of the species H. heliothidis.
6. The film of claim 3 wherein the infective juveniles are of the species N. bibionis.
7. The film of claim 3 wherein the infective juveniles are of the species N. glaseri.
8. A method for preparing a nematode storage film, which method comprises:
suspending entomogenous infective juvenile nematodes and reversibly cross-linkable matrix material in aqueous suspension;
forming a film from said aqueous suspension, said film having a thickness of about 0.5 to about 5 mm, wherein said nematodes are present within said film; and cross-linking said matrix material to provide a film which mechanically immobilizes said nematodes.
suspending entomogenous infective juvenile nematodes and reversibly cross-linkable matrix material in aqueous suspension;
forming a film from said aqueous suspension, said film having a thickness of about 0.5 to about 5 mm, wherein said nematodes are present within said film; and cross-linking said matrix material to provide a film which mechanically immobilizes said nematodes.
9. The method of claim 8, wherein said reversibly cross-linkable matrix material comprises alginic acid.
10. The method of claim 9, wherein said cross-linking comprises adding Ca++.
11. A package of entomogenous infective juvenile nematodes, which package comprises:
an oxygen-permeable, reversibly cross-linked matrix having a thickness of about 0.5 to about 5 mm, having a water content sufficient to preserve infective juvenile nematodes in a non-desiccated state, and having sufficient rigidity to substantially immobilize infective juvenile nematodes; and viable entomogenous infective juvenile nematodes immobilized in said cross-linked matrix in a non-cryptobiotic state;
a container dimensioned to receive said matrix, wherein said container permits entry of about 70 cc O2 per square foot of matrix, and limits water loss by evaporation.
an oxygen-permeable, reversibly cross-linked matrix having a thickness of about 0.5 to about 5 mm, having a water content sufficient to preserve infective juvenile nematodes in a non-desiccated state, and having sufficient rigidity to substantially immobilize infective juvenile nematodes; and viable entomogenous infective juvenile nematodes immobilized in said cross-linked matrix in a non-cryptobiotic state;
a container dimensioned to receive said matrix, wherein said container permits entry of about 70 cc O2 per square foot of matrix, and limits water loss by evaporation.
12. The package of claim 11, wherein said matrix has a surface area of about 0.5 square feet.
13. The package of claim 11 wherein said matrix comprises alginate.
14. The package of claim 13 which further comprises an amount of sodium citrate and EDTA
sufficient to dissolve said matrix.
sufficient to dissolve said matrix.
15. The package of claim 14 wherein said container has a volume of at least 20 oz.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US31359489A | 1989-02-21 | 1989-02-21 | |
| US313,594 | 1989-02-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2049458A1 true CA2049458A1 (en) | 1990-08-22 |
Family
ID=23216348
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002049458A Abandoned CA2049458A1 (en) | 1989-02-21 | 1990-02-15 | Method and medium for packaging entomogenous nematodes |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0460057A4 (en) |
| JP (1) | JPH04505701A (en) |
| AU (1) | AU635921B2 (en) |
| CA (1) | CA2049458A1 (en) |
| WO (1) | WO1990010063A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8420379B2 (en) | 2008-01-10 | 2013-04-16 | Universal Bio Research Co., Ltd. | Material for capturing microbes, device for capturing microbes, method of capturing microbes, and method of producing material for capturing microbes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5172514A (en) * | 1990-11-19 | 1992-12-22 | Biosys Corporation | Insect trap |
| US5170744A (en) * | 1991-12-13 | 1992-12-15 | Biosys Corporation | Long-term storage of infective juvenile nematodes in pseudoplastic layers |
| WO1994005150A1 (en) * | 1992-09-10 | 1994-03-17 | Commonwealth Scientific And Industrial Research Organisation | Method for the storage of entomopathogenic nematodes |
| WO1994019940A1 (en) * | 1993-03-04 | 1994-09-15 | Commonwealth Scientific And Industrial Research Organisation | Method for packaging entomopathogenic nematodes for storage and transport |
| AU6226799A (en) * | 1998-10-13 | 2000-05-01 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Hydrocolloid coating of cells |
| PL3578043T3 (en) * | 2018-06-05 | 2022-11-21 | Bühler AG | Large-scale, high density storage of larvae |
Family Cites Families (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4409331A (en) * | 1979-03-28 | 1983-10-11 | Damon Corporation | Preparation of substances with encapsulated cells |
| US4391909A (en) * | 1979-03-28 | 1983-07-05 | Damon Corporation | Microcapsules containing viable tissue cells |
| US4352883A (en) * | 1979-03-28 | 1982-10-05 | Damon Corporation | Encapsulation of biological material |
| US4407957A (en) * | 1981-03-13 | 1983-10-04 | Damon Corporation | Reversible microencapsulation of a core material |
| GB2095558B (en) * | 1981-03-30 | 1984-10-24 | Avon Packers Ltd | Formulation of agricultural chemicals |
| CA1165694A (en) * | 1981-09-21 | 1984-04-17 | Jean R. Finney | Package for the transportation of nematodes |
| SE441009B (en) * | 1982-03-08 | 1985-09-02 | Kjell Nilsson | WAY TO IMMOBILIZE LIVING BIOMATERIAL IN PEARLY POLYMERS |
| US4798786A (en) * | 1982-05-06 | 1989-01-17 | Stolle Research And Development Corporation | Living cells encapsulated in crosslinked protein |
| US4806355A (en) * | 1983-06-06 | 1989-02-21 | Connaught Laboratories Limited | Microencapsulation of living tissue and cells |
| US4803168A (en) * | 1983-09-01 | 1989-02-07 | Damon Biotech, Inc. | Microencapsulation with polymers |
| FI853870L (en) * | 1984-02-07 | 1985-10-04 | Biotech Australia Pty Ltd | LAGRINGS- OCH TRANSPORTSYSTEM FOER NEMATODER. |
| US4663286A (en) * | 1984-02-13 | 1987-05-05 | Damon Biotech, Inc. | Encapsulation of materials |
| US4778749A (en) * | 1984-06-01 | 1988-10-18 | Karyon Technology, Inc. | Tissue culture and production in permeable gels |
| US4615883A (en) * | 1985-10-23 | 1986-10-07 | Plant Genetics, Inc. | Hydrogel encapsulated nematodes |
| JPH0628570B2 (en) * | 1986-02-13 | 1994-04-20 | 雪印乳業株式会社 | Method and device for manufacturing capsule body |
-
1990
- 1990-02-15 AU AU51670/90A patent/AU635921B2/en not_active Ceased
- 1990-02-15 EP EP19900904057 patent/EP0460057A4/en not_active Withdrawn
- 1990-02-15 JP JP2504062A patent/JPH04505701A/en active Pending
- 1990-02-15 WO PCT/US1990/000923 patent/WO1990010063A1/en not_active Application Discontinuation
- 1990-02-15 CA CA002049458A patent/CA2049458A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8420379B2 (en) | 2008-01-10 | 2013-04-16 | Universal Bio Research Co., Ltd. | Material for capturing microbes, device for capturing microbes, method of capturing microbes, and method of producing material for capturing microbes |
Also Published As
| Publication number | Publication date |
|---|---|
| AU5167090A (en) | 1990-09-26 |
| JPH04505701A (en) | 1992-10-08 |
| AU635921B2 (en) | 1993-04-08 |
| EP0460057A4 (en) | 1992-05-20 |
| EP0460057A1 (en) | 1991-12-11 |
| WO1990010063A1 (en) | 1990-09-07 |
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