Technical Field

The invention relates to insect control employing biological agents especially for the benefit of agriculture, garden and household insects. In par¬ ticular, it relates to a method to desiccate insect par¬ asitic nematodes in both large and small quantities for long term storage while maintaining their viability and pathogenicity to insects.

Background Art

Nematodes represent a group of unsegmented round worms. They are simple in anatomy, having a sim¬ ple gut and elongated fusiform shape. They are divided into numerous Families, some of which are free living' while others are parasitic to plants or animals. Those which are parasitic to insects are called entomogenous or entomopathogenic nematodes.

The Order of greatest commercial interest for insect control is the Order Rhabditida, which contains several Families, many of whose members are parasitic to insects. Prominent among these Families are the Steinernematids and Heterorhabditids. A general discus¬ sion of the classification of nematodes, and the

entomogenous Families thereof is found in Poinar, G.O., "The Natural History of Nematodes" (1983), Prentice- Hall, Inc., N.J.

Nematodes have a standard life cycle compris- ing five stages which are delineated by a molting pro¬ cess in which a new cuticle is formed and the old one shed. Briefly, the adults of stage 5 reproduce, and the eggs generate stage _. 1 larvae which, under appropriate conditions, transit co stage 2. Normally, the stage 2 larvae simply develop to stage 3 larvae and thence to stage 4 larvae, which then complete the cycle to the adult stage. However, and of interest to the use of nematodes for insect control, when conditions are rela¬ tively unfavorable for continued growth and reproduc- tion, the stage 2 larvae of Steinernematid and Hetero- rhabditid nematodes develop instead into "stage 3 infec¬ tive juveniles" or "Us". Under these conditions, the cuticle characteristic of the second stage is retained and is called the sheath. It completely encloses the nematode. Us are infective to insects and complete their life cycle through stage 4 and adult at the expense of the host.

Steinernematid and Heterorhabditid IJ nema¬ todes are an effective means of insect control. They are identifiable morphologically and normally live in surface water films around soil particles. They require oxygen and moisture for survival, but do not feed; they utilize their own food reserves as an energy source. They remain infective if the sheath is removed.

One other aspect of Steinernematid and Hetero¬ rhabditid nematode biology is significant: nematodes within these families are symbiotic with species of bac¬ teria which are primarily but not totally responsible for their entomopathogenic properties.

The commercial production of Steinernematid and Heterorhabditid nematodes and their use in insect pest control presents a number of challenges which have only recently begun to be met. Large scale production of Us has been developed at a number of locations, and a number of techniques have been tried. See, for exam¬ ple, Soviet Patent 726,164; 8 April 1980; PCT Patent Application No. 86/01074 published 27 February 1986; U.S. Patent 4,334,49b and U.S. Patent 4,178,366. Formulations have also been devised for the application of infective juveniles to the soil. See, for example, Soviet Patent Application No. 378,222 and U.S. Patent Application 4,178,366. One approach uti¬ lizes a suspension in light mineral oil. In addition, Japanese Patent Application No. 60/260,678 proposes a fermented compost support for the application of the nematodes.

An additional and serious problem in commerc¬ ialization of insect control using Steinernematid and Heterorhabditid nematodes arises in the large scale shipment and storage of the infective juveniles in a state which maintains their viability and pathogenicity. Heretofore, relatively impractical methods, which only minimally reduce nematode metabolism have been used. These include storage and transportation in oxygenated water (Dutky, S.R., et al, J Insect Pathol (1964) 6:417- 422) in sterile water or 0.1% formalin in flasks (Poinar, G.O., "Nematodes for Biological Control of Insects" (1975) CRC Press, Boca Raton, Florida) or in 0.1% formalin on moist polyurethane sponge or saturated filter paper (Bedding, R.A. Ann Applied Biol (1984) 104:117-120; Hara, A.H. et al, USDA Adv Aqric Techno1 W-16 (1981); Howell, J.F., J Invert Pathol (1979) 3_3:155-156 and Lindergren, J.E. et al, USDA Adv Aqric

Technol W-3 (1979)). Other shipment and storage tech¬ niques have included the use of wood chips and activated charcoal.

Recently, additional approaches have been dis- closed. U.S. 4,417,545 describes a shipping and/or storage container for nematodes and/or their eggs in their dormant state. This container basically sand¬ wiches the nematodes and eggs between two pieces of foam which are saturated -.-rith water and thus maintain a high level of humidity. This approach is however directed to the noninfective stages of the worm and does not relate to the shipment of infective juveniles. PCT Application WO85/03412 suggests methods of transport and storage which depend on maintaining putative anaerobic condi- tions and the presence of an antimicrobial agent. High osmotic strength solutions are also used to prevent bac¬ terial growth. The proposed storage conditions also include an adsorbent such as charcoal or synthetic res¬ ins, although it is not clear what these agents are expected to adsorb. The disclosure exemplifies the use of formaldehyde as an antimicrobial, and proposes stor¬ age containers which contain both the nematodes and adsorbent charcoal.

The approach of the present invention is to maintain the infective juveniles of the Steinernematid and Heterorhabditid nematodes in a state of dormancy so that their food reserves are not used up, and so that upon return to suitable conditions they revive and remain pathogenic to the insect host. In short , the methods and containers disclosed in connection with the present invention are designed to maintain the infective juveniles in a "cryptobiotic" state - a state of dor¬ mancy in which metabolism is suppressed. Several ways of doing this, with varying degrees of success, are

known for organisms in general. The most generally sug¬ gested method and perhaps the most universally applica¬ ble is the induction of cryobiosis, i.e., reduced metab¬ olism at low, usually freezing temperatures. In addi¬ tion, and more difficult to achieve, is anhydrobiosis, which is induced by evaporative desiccation, and the closely related osmobiosis induced by osmotic desicca¬ tion.

There is an extensive literature on induction of anhydrobiosis in nematodes in general. However, direct induction of anhydrobiosis in the nematodes of interest in insect control is, to applicants' knowledge, limited to a single published report (Simons, W.R., and Poinar, G.O., J Invert Pathol (1973) 22:228-230), and the disclosure of European Publication No. 256,873, published 24 February 1988. This application ('660) discloses a method for inducing an anhydrobiotic state which requires removal of the bulk surface water, followed by carefully controlled drying of the nematodes spread into a thin layer. Note that this method requires vast areas for drying commercial quantities of nematodes. For example, at a density of 2.5 x 10^ Us/in , 2.5 x 10 ¹¹ Us would require about 10^ in ² , whereas the same number of s can be desiccated with greater control under the instant method in a 500 liter tank. Copending U.S. Serial No. 034,883, filed 6 April 1987, discloses an osmotic method for inducing reduced metabolism in entomogenous infective juveniles. An additional report that Neoaplactana desiccate in nature under unspecified conditions appears in a symposium abstract (Kamionek, M. et al, "Eleventh Int'l Symp Nematol, Eur Soc Nematol" (1972)).

Other types of nematodes, including free liv¬ ing and plant parasitic nematodes, are known to survive

naturally under dry conditions (Evans, A.A.A.F. et al, in "Nematodes as Biological Models" (1980) Academic Press, New York, pp. 193-211; Demeure, Y. et al, in "Plant Parasitic Nematodes" (1981) Academic Press, New York). It has been shown that significant changes in chemical composition occur in preparation for the an¬ hydrobiosis caused by desiccation, and it is known that the plant parasitic nematodes which form the subjects of these studies, must e preconditioned at 97-98% relative humidity for 48-72 hours before being subjected to lower relative humidity (Evans et al (supra) ; Womersley, C. , Comp Biochem Phvsiol (1981) 68A:249-252; Madin, K.A.C., et al, J Expl Zool (1975) l£2:335-342; Crowe, J.H., et al, (ibid) 323-334). Freckman, D. . et al, in "New Trends in Soil

Biology" (Lebrun, P. ed.) (1983) Universitie Catholique de Louvain Press, discuss the ability of nematodes in desert soils to survive anhydrobiosis. Womersley, C. Compar Biochem Phvsiol (1981) 70B: 669-678 reviews the mechanisms of anhydrobiotic survival in nematodes; simi¬ lar studies are reported by Crowe, J.H. , et al, J Exp Zool (1979) 2j07:431-437; Demeure, Y. , et al, J Nematol (1979) 11:189-195 and Crowe, J.H., et al, Ann Meet Amer Inst Biol Sci, East Lansing, Michigan, 21-26 August 1977.

However, with respect to species of interest in insect control, the one published report of an attempt to desiccate N. carpocapsae (Simons, W.R., and Poinar, G.O., supra) utilized a series of humidity cham- _. bers containing glycerol solutions. Relative humidity (RH) was not measured directly, nor was the temperature at which the experiment was conducted reported. Us were held at 96% RH for 12 hr, transferred to 93% for a further 12 hr, and then to RHs ranging from 10-79% for

periods up to 28 days. Only at 79.5% RH was survival greater than 40% after 12 days; even under these condi¬ tions viability fell to 30% after 20 days.

In short, none of the published studies of nematode desiccation provide guidance for effecting anhydrobiosis in Steinernematid and Heterorhabditid entomogenous nematodes in a scalable process to ensure effective, commercially practical, long-term mass stor¬ age and shipment. The method of the present invention, however, provides a means to induce sufficient anhydrobiosis in suspensions of infective juveniles to permit their long term storage, with retention of ability to kill the insects they infect. The method requires no specialized equipment, and is adaptable to the treatment and use of large numbers of the worms for insect control. The method is also applicable to* the desiccation of other biological materials, particularly biological materials which are to be "revived" at some later time, such as yeasts.

The method of the invention also provides for convenient and economical process scale-up, making com¬ mercial practice of the invention feasible.

Disclosure of the Invention

The invention provides processes for placing Steinernematid and Heterorhabditid nematode infective juveniles into a state of anhydrobiosis, thus facilitat¬ ing long-term storage and shipment while maintaining their viability and pathogenicity. The invention also provides the anhydrobiotic nematode infective juveniles per se. The methods and materials provided by the invention are significant in enabling the economically sound and commercially practical use of Steinernematid

and Heterorhabditid nematodes for insect control in agricultural applications. Absent such methods and materials, high cost packaging and refrigeration would have to be used, or the infective juveniles would have to be cultivated very close to the site of application rendering this approach to pest control for the most part impractical, and resulting in the continued use of chemical insecticides some of which have been shown to be ecologically harmful. Therefore, in one aspect, the invention is directed to a process for inducing an anhydrobiotic/ cryptobiotic state in Steinernematid and Heterorhabditid nematodes, which comprises, as a first step, placing the infective juveniles into suspension in a suitable oil, for example, light mineral oil or vegetable oil. The oil preferably contains a small amount of a non-toxic surfactant, in an amount sufficient to prevent the nema¬ todes from clumping together. The resulting suspension is then dried using a suitable drying agent, with ix- ing, until a sufficient amount of water has been removed from the nematodes. Suitable drying agents may be solid (e.g., Na2≤θ4), liquid (e.g., detergents or surfact¬ ants), or gaseous (e.g., air, O2/N2, etc.). The pres¬ ently preferred drying agent is air at a relative humid- ity < 100%. The nematodes are maintained in suspension with stirring and aeration for a suitable period of time sufficient for the Us to become anhydrobiotic. Typical effective periods are of greater than 24 hours, optimally at least 72 hours. After anhydrobiosis is induced; the desiccated nematodes can be filtered to provide an oil-containing paste or oil suspension, and can be stored for extended periods in suitable moisture- proof containers. Storage for periods of several months has been achieved by this method. Optionally, one may

suspend the nematodes in a hyperosmotic solution of the type disclosed above.

In another aspect, the invention is directed to the anhydrobiotic Us obtained by the method of the invention.

Containers useful in shipping and/or storage of the Us which have been thus placed in an anhydro¬ biotic or cryptobiotic state are disclosed in Serial No. 101,530, filed 28 September 1987, and incorporated herein by reference. Briefly, these containers are characterized by their capacity to maintain the relative humidity experienced by the contained nematodes and to provide sufficient oxygen to supply minimal metabolic requirements in the low level of metabolism character- izing this state. The containers are either impermeable to moisture but have sufficient headspace to meet the oxygen requirements of the cryptobiotic Us, or are per¬ meable to water and air, but provide a means to control RH. Alternatively, one may provide the desiccated nema- todes of the invention in an oil suspension.

Preferably, the desiccated U are provided in a suitable non-toxic gel or matrix which maintains the necessary humidity and surface exposure to air, while simultaneously protecting the Us from bulk water (and premature rehydration) . To package the IJs properly, one first prepares a suitable storage solution with a water activity of about 0.97. Suitable solutions are generally aqueous solutions of non-toxic osmolytes at concentrations below saturation, for example salts (e.g., NaCl, K2SO , potassium acetate, etc.), sugars (e.g., 1M fructose, 1M maltose, etc.), sugar alcohols (e.g., 1M sorbitol, 14% glycerol, etc.), and other low molecular weight polymers (e.g., polyethylene glycol "PEG" 600). The storage solution is then used to

prepare a paste using an absorbent particulate matrix material, such as Terri-sorb ^tM (Industrial Services International, Inc.), Sanwet™ (Celanese, a starch- grafted polyacrylate) , Drytech™ (Dow, a cross-linked polypropenoic acid). Suitable matrix materials must remain in discrete particles when the paste is prepared in order to provide for sufficient oxygen diffusion, but may be water soluble at higher concentrations. The Us in oil suspension are. centrifuged, and the oil decanted. Then the Us are stirred into a paste in a quantity of storage solution (e.g., 35 mL solution per 10 ⁸ Us), then stirred into the matrix paste (e.g., 450 g matrix per 10 ⁸ Us). The resulting nematode paste is then inserted into containers. Preferably, the paste is placed into mesh bags having a mesh size large enough to admit the Us, but small enough to retain the matrix particles, thus providing an efficient means for releas¬ ing the Us from the matrix, e.g., using a stream of water. The bags may be of any material: nylon is pre- ferred because it may easily be heat-sealed. The mesh size may range from about 15 to 150 openings per inch, depending upon the matrix material. Alternatively, the container may have hose fittings and sieve/strainer attachments, or may be-adapted for use in a spraying device. The container is then sealed in such a manner that oxygen may enter while moisture is confined. One may advantageously use a sifter cap (for mechanical strength) covered with a suitable film (e.g., poly- sulfone, polystyrene, Tyvek™, etc.). Alternatively, one may seal the container completely, providing sufficient oxygen in the headspace of the container. Or, one may use a cap with a suitably sized aperture, or a loosely fitting cap, and provide sufficient excess moisture in the container to compensate for the expected loss of

oisture during storage and transport. It is presently preferred to seal the container with polysulfone film.

Modes of Carrying Out the Invention

A. Definitions

"Biological materials" as used herein refers to living tissue, cell cultures, microorganisms (such as yeasts, bacteria, protozoa, etc.), small multicellular organisms (such as nematodes, insect larvae, and the like), which are capable of entering a state of reduced metabolic demand (e.g., an anhydrobiotic state) upon removal of a portion of the material's natural water content, and which may be returned to its normal state upon addition of water or a suitable solution.

"Entomogenous nematodes" refers to nematodes which .are parasitic to one or more species of insect. The most important Order of entomogenous nematodes is the Rhabditida, and the invention is directed chiefly to storage and/or shipment of two Rhabditid families in this group: the Steinernematidae and the.Hetero- rhabditidae. However, other entomogenous families may also be suitable as subjects to which the methods of the invention may be applied, and include Diplogasteridae, Panagrolaimidae, Rhabditidae, and Syrophonematidae, together with non-Rhabditid families including the Allantonematidae, Aphelenchoididae, Ξntaphelenchidae, Mermithidae, Neotylenchidae, Sphaerulariidae, and Tetradonematidae. As set forth above, for the Rhabditida, the most important families for commercial use are the Steinernematidae and the Heterorhabditidae. References in the literature to "Neoaplactana" refer to a particu¬ lar genus of the Steinernematidae, and the terms

Neoaplactana and Steinernema as designators for specific species—e.g., N. qlaseri or S. qlaseri—which are used interchangeably.

While the classification of the various groups of nematodes may be confusing, it is clear that the invention is directed to those genera which have the characteristics of being infective to insects, and which have as a stage in their life cycles, stage 3 infective juveniles (Us) with the characteristics described in the background section above. Depending on the agricul¬ tural application intended, i.e., the insect targeted, one or more of the species may be particularly advanta¬ geous.

"Cryptobiotic state" refers, in the context of the present invention, specifically to a cryptobiotic state of the infective juvenile. It is a reversible physiological state of dormancy in which metabolism is suppressed. If relatively insensitive methods are employed to ascertain metabolism, the metabolism may, in fact, go undetected. In this state, oxygen uptake is greatly reduced and may be undetectable using certain conventional means for relatively short times.

"Anhydrobiosis" refers to a cryptobiotic state induced by water loss. "Apparent anhydrobiosis" or "apparent cryptobiosis" refers to such a state as deter¬ mined by the following criteria: lack of movement, shrunken and shriveled appearance, and reduced oxygen consumption. (The apparently anhydrobiotic or crypto¬ biotic nematodes can be shown still viable by rehydrating them and testing for viability and pathoge¬ nicity. )

In particular, the anhydrobiotic or cryptobiotic state of the Us of the invention can be evidenced by any of the following criteria:

τl 3-

(1) In a population of at least 1,000 infec¬ tive juveniles, more than 60% can successfully be rehydrated and found viable after being suspended in 70% methanol wherein the suspension is then plunged into liquid nitrogen for 24 hours and thereafter rapidly thawed to room temperature.

(2) In a population of at least 1,000 infec¬ tive juveniles, more than 70% survive maintenance in an airtight container at a nematode/volume ratio of 10°/30 mi _f wherein air occupies the volume of the container initially. The maintenance time is temperature depen¬ dent. The required percentage can be maintained for more than 15 days at 25°C, for more than 8 days at 30°C, or for more than 6 days at 35°C. (3) in an aggregation of more than 1,000 Us, with no free liquid water present, the oxygen demand is less than 1 ml oxygen per 90 mg dry weight of Us per day at 25°C. The contrast with nonanhydrobiotic nema¬ todes can easily be seen, in that for "normal" nema- todes, the oxygen demand would be in excess of 6 ml of oxygen per 10° organisms.

(4) In a population of at least 1,000 Us, more than 90% survive exposure to 45°C for 2 hours.

"Desiccated nematodes" also refers to nema- todes in an apparent anhydrobiotic state.

"Infective juvenile" or "U" refers to a nonadult stage capable of invading and infecting an insect host. For the families which are the subject of the present invention, these are stage 3 Us.

"Relative humidity" ("RH") is defined in a standard manner as the ratio of water vapor pressure in the air to the saturation vapor pressure at the same temperature, and is normally expressed as a percent.

B. General Description

The infective juveniles which are the subjects of the procedures herein are useful in controlling a variety of insect pests, including borers, root weevils, caterpillars, beetle grubs, corn root worms, Japanese beetles, and mole crickets. Major agricultural products which are protected by such infective juveniles include corn, strawberries, almonds, greenhouse crops, mush- ,. rooms, sugar cane, a~.d potatoes. Poultry raising facil- ities and other animal housing may be kept free of flies. In a typical agricultural application, infective juveniles are applied to the target environment in large numbers. For example, for control of sciarid flies in mushroom houses, approximately 5 x 10° worms are sprayed in each house. Smaller numbers of Us, e.g., about might be useful for home applications; this number would be suitable for protection of a single potted plant.

Using present technology, approximately 10^ infective juveniles, or about 25 kg of wet product, can be grown per week in about 152 kg of culture medium.

These large numbers of Us must be preserved, shipped, and stored.

In the process of the present invention, large numbers of Us are maintained for long periods for these purposes in an anhydrobiotic state which is attained by removing bulk surface water (e.g. by decanting or fil¬ tering), placing the Us into oil suspension, and drying using a suitable drying agent, with mixing, until a suf¬ ficient amount of water has been removed from the nema- todes. Suitable drying agents may be solid (e.g.,

Na2Sθ4), liquid (e.g., detergents, surfactants), or gas¬ eous (e.g., air, O2/ 2, etc.). The presently preferred drying agent is air at a relative humidity < 100% (pref¬ erably as low as practical). The nematodes are main-

tained in suspension with stirring and aeration for a suitable period of time sufficient for the Us to become anhydrobiotic. Typical effective periods are of greater than 24 hours, optimally at least 48 hours. After anhydrobiosis is induced, the desiccated nematodes can be filtered to provide an oil-containing paste or suspension, and can be stored for extended periods in suitable moisture-proof containers. Storage for periods of several months haπ been achieved by this method. Optionally, one may suspend the desiccated nematodes in a hyperosmotic solution, e.g., concentrated sucrose. s desiccated in this way retain their viability and pathogenicity.

Viability is determined by microscopic obser- vation wherein the criteria for viable individuals include: a transparent esophageal region, the absence of the typical death position, and motility when rolled with a dental probe. These tests for viability, when performed on cryptobiotic Us, are preceded typically by _a 24 hour rehydration period. In an additional test which also measures resistance to hardship conditions, the cryptobiotic Us are exposed to a 55% RH condition for 72 hours prior to rehydration and viability evalua¬ tion. Pathogenicity is determined by assaying the infective juveniles against Galleria mellonella larvae. The infective juveniles in concentrations of 50 per assay dish are pipetted in 0.5 ml water onto a single Whatman No. 1 filter placed in the lid of a 45 mm petri dish. Ten insect larvae are placed on the filter, and the dish is closed and placed at 22°C and 80% RH. Mor¬ tality is recorded at daily 2 hr intervals between 30 and 50 hr post-exposure, and the time required to effect 50% mortality (LT50) is compared to controls.

The Invention Process

The critical aspect of the process of the invention is the control over the rate and extent of drying. The instant invention provides exquisite con- trol over the drying rate. In the practice of the invention, cultured Us are first filtered to remove most of the surface water. Bulk surface water removal is most easily effected by filtration. Quantitative removal of water pri'-.r to oil suspension is not critical in this process, in contrast to processes involving evaporation from thin layers of nematodes.

The nematodes are then uniformly suspended in a suitable oil. The oil may be vegetable, mineral, or synthetic, so long as it is non-toxic to the organisms. Suitable oils should not be especially viscous or vola¬ tile, as these properties create difficulties in uniform mixing and worker safety. In this regard, preferred oils should have a viscosity less than about 60 centipoise, and a vapor pressure less than about 0.001 mmHg at ambient temperature. Apart from these factors, selection of the particular oil used may be generally left to considerations of availability and price. Exem¬ plary oils include soy oil, corn oil, light mineral oil, and the like. Typically about 20-100 mg of nematodes (dry weight), preferably about 45 mg, are suspended per ml of oil: roughly 500,000 Us per ml.

A non-toxic surfactant is preferably added to the oil to assist in uniformly dispersing and suspending the nematodes. In the absence of a surfactant, the nem¬ atodes may clump, resulting in uneven desiccation. When clumping occurs, too much water is removed from nema¬ todes outside the clumps, while not enough water is removed from the clumped nematodes. The clumped nema¬ todes would thus fail to enter an anhydrobiotic state,

and would become dehydrated during later water-removal steps. The net result is an unacceptably high mortality rate. The addition of a surfactant also facilitates adequate mixing under less agitating conditions. Thus, 5 the nematodes are subject to reduced mechanical stress from mixing. Suitable surfactants will have a low hydrophile/lipophile balance (HLB) , such as Span® 80.

The desiccation process may be conducted over

-I a range of temperatures, from about 0°C to about 50°C,

10. preferably about 5°-30°C, and most preferably about 20-25°C.

The drying agent is then added and uniformly dispersed in the nematode suspension. The drying agent may be a solid, such as calcium chloride or sodium

15 sulfate, a liquid, such as a detergent or other sur¬ factant, or, preferably, a gas, such as air or nitrogen/ oxygen mixtures. Whichever drying agent or combination of drying agents is selected, it should be kept in mind that the nematodes have a finite oxygen demand, and that

20 the suspension must be aerated to preserve viability of the organisms. It is preferred to use air as a drying agent, as the rate of drying can be easily controlled by varying the airflow rate, temperature, and RH of inflow¬ ing air.

25 The water content of the suspension should be carefully monitored during the drying process. For example, one may employ Karl Fisher titration to deter¬ mine the water content of the suspension. When a gase¬ ous drying agent is used, one may monitor water loss by

30 measuring the humidity of the outflowing gas. For exam¬ ple, one may pass the outflow through a desiccant trap, and measure the weight gain of the desiccant. The exact rate of water removal will of course vary with the par¬ ticular organism to be desiccated, the amount of excess

water present in the initial preparation (and dissolved in the oil), and the final water content desired. In general, however, we have obtained acceptable results by drying about 10° Neoaplectana carpocapse Us at a rate of about 0.225 g H2θ/hour for about 64 hours.

The organisms are desiccated to a water con¬ tent corresponding to the water content at which they enter an anhydrobiotic state: for most nematode Us, an anhydrobiotic state ^; s induced at about 97% RH. The water content at that RH may be determined as follows:

10° Us are placed in a tared aluminum dish and dried in an oven at 150°C for 24 hours. By comparing the weight of these oven-dried Us with the weight of s desic¬ cated at 97% RH under prior art practices, one can determine the water content of the Us necessary to induce anhydrobiosis. Thus, one may carefully reduce the organisms' water content to that level, allow anhydrobiosis to occur, and then reduce the RH still further for storage. For example, Popiel et al. in copending U.S. patent application 897,660 discloses that 10° N. carpocapse Us subjected to 97% RH for 72 hours have a final weight of about 140 to 168 mg. The same amount of oven-dried N. carpocapse Us have a dry weight of about 90 mg. Thus, 10° N. carpocapse Us dried at 97% RH contain about 50 to 78 mg of water (average = 64 mg). This figure, expressed as a ratio of mg H2θ/mg dry weight, is about 0.7. A similar ratio may be estab¬ lished for each type and species of organism desired. Thus, by determining the number of organisms present and the desired final water content, one may establish -a desiccation endpoint based on the amount of water removed.

Following desiccation, the oil may be removed if desired by decanting or filtering, and the resulting

desiccated organisms stored as an oil suspension or paste. Most of the oil and surfactant may be recovered and recycled for further use. The desiccated organisms may be stored in containers suitably provided to main¬ tain appropriate humidity and oxygenation levels. Optionally, the organisms may be suspended in a hyper¬ osmotic solution (e.g., concentrated sucrose) capable of maintaining an anhydrobiotic state for subsequent stor¬ age, shipping and handling. The oxygen demand for Us is generally less than 1 ml of oxygen per day per 90 mg of dry weight, once anhydrobiosis has been achieved.

At the end of the induction period, assurance of anhydrobiosis can be had by reference to any of the criteria set forth above.

Storage

After the induction period, ¹ the nematodes may be stored in suitable containers.

The containers must maintain the RH in a suit¬ able range, and provide sufficient oxygen supply to accommodate the low level of metabolism of the Us in their anhydrobiotic/cryptobiotic state. Us in this state require on the order of 0.6-1 ml of oxygen per 90 mg dry weight per day at 25°C. The manner of storage must be designed so as to to provide this amount of available oxygen.

The oxygen demand has been referenced against weight, based on a tested species in which 90 mg of s corresponds to approximately 10° organisms (N. carpocapsae) . For other species, the relative amount of oxygen per organism will vary according to the size, while the needed oxygen volume per mg of Us remains relatively constant across the entomogenous spe¬ cies.

For the storage containers to provide adequate oxygen in a small package, the packaging can be made of semipermeable materials which allow the passage of oxy¬ gen, as long as provision is also made for maintenance of the correct RH. For example, a saturated solution of potassium sulfate will maintain a 97% RH, and this solu¬ tion could be incorporated in a gel matrix, e.g., a hydrogel such as Terasorb, or can be placed in a fibrous matrix, e.g., cellulose or other fiber. The simplest embodiment is an airtight (and moisture-tight) container with sufficient air space to accommodate the needs of the stored Us for the desired time period. In this embodiment, maintenance of correct RH is automatic; no special precautions need be taken. This is practical for smaller numbers of nematodes, but may be troublesome if large numbers of nematodes, e.g., 10 , are desired to be stored over long time periods since considerable volume may be required. The amount of head space can be calculated on the basis of the value of the oxygen requirement, as set forth above.

Rehydration

After storage, anhydrobiotic nematodes Us must be rehydrated for use. Upon rehydration, the anhydrobiotic state is lost, and the Us regain their metabolic activity. Viability and pathogenicity can be confirmed as described above. Two general approaches to rehydration are appropriate: direct and "slow."

For direct rehydration, the desiccated. Us are simply placed in water or an isotonic aqueous solution, or the compartment containing the U preparation is filled with water or an isotonic solution. After 2-3 hr of rehydration, the Us are rehydrated and may be tested

for viability/pathogenicity. They should preferably be used within 24 hours of rehydration.

In some cases, rehydration may advantageously be less abrupt. For such "slow" rehydration, the worms are placed in controlled environments for a preliminary period, such as in 100% RH air or relatively high osmotic pressure solutions for 20 hr. Appropriate solu¬ tions may contain 2.5% NaCl or 10% myoinositol, for example. After this period of acclimation, the nema- todes are immersed in water and directly rehydrated as described above.

It should be noted that although the foregoing procedures are suggested for deliberate rehydration of the stored nematode preparations, it may be possible to rely on conditions in the environment to provide mois¬ ture for rehydration. Thus, in an alternative mode, the anhydrobiotic nematodes may be placed in the environment directly, and soil moisture or normal or deliberate irrigation can furnish the necessary water.

Examples

The following examples are illustrative but do not limit the invention.

Preparation A

(Preparation of Infective Juveniles) N. carpocapsae or other entomogenous species are grown under standard culture conditions and infec¬ tive juveniles obtained as described by Bedding, R.A. , ^• Ann Appl Biol (1984) 104:117-120. incorporated herein by reference. The hyamine-washed Us are suspended in ster¬ ile water at about 10° Us/ml.

Example 1 (Anhydrobiotic Induction in Neoaplactana carpocapsae) About 10° N. carpocapsae Us prepared as described in Preparation A were vacuum filtered on a Buchner funnel to a water content of 2.3 (g/g H2θ/dry weight). The Us were mixed into soy oil (200 ml) in a water-jacketed round bottom flask with a round bottom mixer. The suspension was maintained at 20 ^β C, with a mixing speed of 200 .pm. Pressurized air was passed through a drying column (Drierite) to reduce the RH to about 0%, and bub¬ bled into the flask through a 0.7 x 1 cm polymeric sparger. Outflowing air was directed through a tared drying tube (Drierite), and the rate of water loss cal- culated from the increasing weight of the exit drying tube. The air flow rate was controlled using a rotameter, and adjusted to a water loss rate of 0.225 g/h. After 64 hours, the water content of the nematodes had been reduced to 0.7, as confirmed by Karl Fisher titration.

The resulting desiccated Us were decanted and stored at 25°C in air-tight vials containing humidifying agents to maintain an RH of 97%. There was no detect¬ able loss in viability after 38 days of storage.

Example 2

(A) About lOlO N. carpocapsae Us prepared using a liquid culture method were vacuum filtered to a water content of 2.3 (g/g H2θ/dr weight). The Us are mixed into light mineral oil (20 1) containing 0.3% Span® 80 (ICI America's Inc.) in a temperature con¬ trolled reaction vessel with a motor-driven mixer. The suspension is maintained at 20°C, with a mixing speed of 200 rpm.

Dry pressurized air (RH = 5%) was bubbled into the tank through a sparger. The rate of water loss (about 22.5 g/h) was determined by Karl Fisher titration of the vessel contents. The air flow rate is controlled ⁵ using a rotameter. After about 64 hours, the water con¬ tent of the nematodes was reduced to 0.7, and was con¬ firmed by Karl Fisher titration.

(B) About loH N. carpocapsae s prepared as described in Preparation A are vacuum filtered to a ° water content of 2.3 (g/g H2θ/dr weight). The Us are mixed into light mineral oil (200 1) in a water-jacketed reaction vessel with a motor-driven mixer. The suspen¬ sion is maintained at 20°C, with a mixing speed of 200 rpm. ^ Pressurized air is passed through a drying column (Drierite) to reduce the RH to about 0%, and is bubbled into the tank through a sparger. The rate of water loss is determined by Karl Fisher titration of the vessel contents. The air flow rate is controlled using 0 a rotameter, and adjusted to a water loss rate of 225 g/h. After about 64 hours, the water content of the nematodes is reduced to 0.7, and is confirmed by Karl Fisher titration.

The resulting desiccated Us are decanted and stored at 25 ^β C in air-tight vials containing humidifying agents to maintain an RH of 97%. At varying periods following storage, (e.g., 24 hours, 1 week, 2 weeks, 4 weeks, 2 months, 3 months, and 4 months) a sample of Us is rehydrated and assayed for viability and infectivity.

(C) Similarly, proceeding as in parts A and B above, but substituting about 7 x lθl° N. bibionis or H. heliothidis Us for N. carpocapsae. the corresponding compositions are prepared.

(D) Similarly, proceeding as in parts A-C, but substituting 5% O2/ 2 for the drying air, similar results are obtained.

(E) Similarly, proceeding as in parts A-C above, but reducing the drying air (at 5% RH) and slowly adding sodium sulfate to the reaction vessel (monitoring the drying rate by Karl Fisher titration), similar results are obtained.

Example 3

(Formulation and Packaging) (A) Solution "A" was prepared as follows: K2SO4 (1.65 Kg) was mixed with Proxel GXL (11.0 g, ICI America's Inc.) in distilled water (11 L) . Ten liters of solution A was then mixed with Terri-sorb™ (1.0 Kg, Industrial Services International, Inc.). The Terri- - sorb™, solution was covered, and set aside to swell, with occasional stirring to prepare solution "B".

Containers were prepared from plastic bottles ^' with sifter caps (Continental Plastics) by covering the sifter caps with pressure-sensitive polysulfone film (Avery) .

Oil-desiccated nematode suspensions were pre¬ pared as above, and the concentration (#/mL) determined. The suspension was then placed in centrifuge tubes (450 mL suspension/500 mL tube) , and the suspension centri- fuged at 3000 rpm for 5 minutes. The tubes were then _. removed and the oil decanted. Next, solution A (35 mL/10° nematodes) was added to each tube, and mixed into a paste. The resulting paste was then added to solution B (450 g/10° nematodes) and mixed. This mixture was then placed into 3" x 5" nylon bags (50 g/bag, prepared from mosquito netting), and the bags heat-sealed. The sealed bags ^' were then placed in 200 plastic containers

with film-covered sifter caps to provide a final prod¬ uct, ready for shipment.

(B) An alternate storage medium was prepared as follows:

Solution "A" was prepared by mixing sucrose (3400 g) with Proxel GXL (11.0 g) in distilled water (11 L) until the sucrose was completely dissolved. Ten liters of solution A was mixed with Terri-sorb™ (800 g), covered, and set asic'e to swell to prepare solution "B".

Solutions A and B were then used as in part (A) above.

Example 4 (Rehydration) Anhydrobiotic s must be rehydrated for use.

Upon rehydration, the anhydrobiotic state is lost, and the Us regain their normal metabolic activity. s may be rehydrated quickly ("direct") or slowly. Once rehydrated, the Us should preferably be used within about 24 hours.

(A) Direct Rehydration: Desiccated Us are placed in water or an isotonic aqueous solution and allowed to absorb water for 2-3 hours. The amount of water or solution used should be at least equal to the amount of water initially removed from the s in the desiccation process. Preferably, the amount of liquid added is sufficient to both replace the lost water and provide water to suspend the rehydrated Us. The Us may be applied immediately, before rehydration has been completed, to finish rehydration in the environment. Thus, one may mix the Us with a suitable amount of water and apply immediately.

(B) Slow Rehydration: Slow rehydration is used preferentially for less hardy strains of nematode,

e.g., Heterorhabditids. Desiccated Us are placed in a controlled high-humidity environment for about 20 hours. The environment may be, for example, air at 100% RH. Alternatively, one may use solutions with high osmotic pressure, for example, 2.5% NaCl, 10% myoinositol, and the like. After the conditioning period of about 20 hours, the Us are immersed in water or isotonic aqueous solution and rehydrated by the direct process (part A) .

Example 5

(Pathogenicity Assay) Pathogenicity is determined by assaying the infective juveniles against Galleria mellonella larvae. The Us (in suspension, at 50 Us per assay dish) are pipetted in 0.5 mL of water onto a single Whatman No. 1 filter placed in the lid of a 45 mm petri dish. Ten insect larvae are placed on the filter, and the dish is closed and placed at 22°C and 80% RH. Mortality is recorded at daily 2 hr intervals between 30 and 50 hours post exposure, and the time required to effect 50% mor¬ tality (LT50) is compared to control values.

Example 6 (Application) Fifty grams of U-matrix composition, prepared as in Example 3 above, are diluted to 8 oz, and the mix¬ ture shaken in the container for 10-15 seconds to release the Us. The liquid is then decanted, and added to a suitable delivery device, e.g., hose-end sprayer, tank sprayer, watering can, etc. If administered by watering can, the IJ liquid is preferably diluted to 2 gallons. If administered by other means, the liquid is diluted to 1 gallon.

The resulting solution is then applied to the soil around the affected area (i.e., not directly on foliage). This amount of solution is sufficient to treat about 225 square feet of soil. Unused solution is stored under refrigeration.

(A) For treatment of vegetable garden pests (e.g., root maggots, wireworms, onion maggots, carrot weevils, cutworms, cucumber beetles, mole crickets, flea beetles, and the like), the solution may be applied gen- erally to 225 ft of soil, as a 3" band on each side of 450 ft of garden row, or as a 6" diameter circle around 1100 individual plants.

(B) For treatment of lawn insects (e.g., Japanese beetles, sod webworms, cutworms, masked chafers, leatherjackets, white grubs, pillbugs, wire- worms, armyworms, June beetles, mole crickets, etc.), n the solution is simply sprayed on up to 225 ft .

(C) For treating pests on ornamental plants such as rhododendrons, azaleas, cyclamen, chrysanthe¬ mums, tulips, iris, primroses, etc., (e.g., European chafers, black vine weevils, white grubs, rose chafers, Asiatic garden beetles, Japanese beetles. Oriental bee¬ tles, cutworms, etc.), the solution may be applied gen¬ erally to 225 ft ² of soil, as a 3" band on each side of 450 ft of garden row, as a 6" diameter circle around ^" 1100 individual plants, or directly to 1100 potted plants in 6" containers.

(D) For eradicating fire ants, 50 g of IJ-matrix composition, prepared as in Example 3 above, are diluted to 20 oz, and the mixture shaken in the con¬ tainer for 10-15 seconds to release the Us. Four oz of this suspension is then added to a watering can, and then diluted to 1 gallon. The contents are applied to one fire ant mound. The procedure is repeated until all

20 oz are used, thus covering up to 5 anthills. Follow¬ ing application, the anthills should be watered to keep them moist. Preferably, the anthills are saturated with water once or twice a week.