METHOD AND MEDIUM FOR PACKAGING ENTOMOGENOUS NEMATODES

Abstract: Entomogenous infective juvenile (IJ) nematodes are prepared for storage and shipment by encasing viable IJs in a thin film which is permeable to oxygen, and which contains sufficient water to maintain 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: 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. Background of the Invention The hazards of residual toxicity and the relative lack of specificity have 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 pass 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 compete with fertile males and thus reduce the number of fertile eggs produced by females. Others have attempted to control insect populations through use of viruses 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 which induce 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 leave the host corpse in search of fresh hosts. R. . Glaser, J Exp Zool (1940) 84_:1-12 reported the culturing of Neoaplectana qlaseri for use in controlling Japanese beetles. N. glaseri 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) L3-241-49 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 sensitive to direct sunlight, and are also somewhat prone to infection, although care must 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 settle), 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 control of insects, and an anhydrous oil suspension formulation for application of nematodes to vegetation by spraying. The formula¬ tion also contained a wax component to retard water loss by nematodes in the sprayed droplets. Nematodes 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 7.1 C. The nematodes were suspended at a concentration of 50,000/mL, and 1 liter of sus- pension was stored in an insulated gallon jug. The suspensions 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 enough to 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 metabolism 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 desiccation. 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 oxygen and moisture, but at a much lower rate than normal "biotic" IJs. The reduction of metabolism also results in conservation of food stores. The net result is a method for storing and shipping IJs which is much less sensitive to moisture and oxygen require- ents than traditional methods. Yukawa et al, PCT WO 85/03412 disclosed a nematode 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 stored 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 to parasitize insects upon ingestion when an otherwise successful infection 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 Invention 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 5 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 induced. As a result of the restraint, metabolic demands for food and oxygen are reduced, which permits longer periods of storage and reduces the head space needed for oxygen supply in a sealed package. 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. Modes of Carrying Out The Invention A. Definitions The term "entomogenous nematode" refers to nematodes which parasitize and kill insects. Pres¬ ently preferred entomogenous nematodes are derived from the Family Steinernematid and Heterorhabditid nematodes, particularly Neoaplectana carpocapsae, N. bibionis, N. qlaseri, and H. heliothidis. The term "infective juvenile" or "IJ" refers to an entomogenous nematode in the infective third larval stage. IJs are characterized by retention of the second stage cuticle or sheath after molting to third stage. IJs do not eat, but depend on internal food stores. 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 of nema¬ tode selected, the degree of immobilization, the con¬ centration of IJs in the film, and the thickness of 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 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-linked to a degree sufficient to substantially immobilize the IJs. The degree of cross-linking may be estimated by the rigidity of the resulting film, e.g., using a duro- eter. 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., by addition of citrate and/or EDTA. The phrase "limits water loss" refers to 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 2 shipment. For a 1 ft 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. A 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 10 5/mL to about 6 x 105/mL, preferably 5 , about 3 x 10 /mL. The concentration of matrix material depends upon the particular material selected, 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 <l-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 casting. 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 CaCl- 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 the 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 oxygen while limiting water loss 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 0~/ 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 IJs, and non-toxic matrix materials, and may be applied directly to an area having insects to be controlled. Viability of nematodes may be assessed microscopically, by observing the reaction to prodding with a dissection 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 10 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. 15 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- 20 cide, 8 g) , Keltone- HVS> (an alginic acid analog, 300 g) , Min-U-Gels> (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 30 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 CaCl- (1.6 M: cal¬ cium phosphate is also acceptable), and held until the gel had set (about 30-60 seconds). The sheet was then transferred to a pure water bath for about 10-30 sec¬ onds 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 5 C), 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 for 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. If desired, the film container may be dimen¬ sioned to provide sufficient volume for the solution for unlinking the film, so that 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: WHAT IS CLAIMED; 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. 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. qlaseri - - 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. 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 0- 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. 10 15. The package of claim 14 wherein said container has a volume of at least 20 oz. 15 20 25 30 35

Inventors: GROVE DONNA IRVING, PRUITT PAUL L, GROVE, DONNA, IRVING, PRUITT, PAUL, L.
Applicants: BIOSYS
Application year: 1990
Main classification: C12N5
IPC codes: A01K67, A01N1, A01N37, A01N63, C12N5