AU713375B2 - Process for producing entomopathogenic nematode preparation and entomopathogenic nematode insecticide - Google Patents

Description

1 Specification Process for Producing Entomopathogenic Nematode Preparation and Entomopathogenic Nematode Insecticide Technical Field The present invention relates to a biological control agent used for exterminating pest insects. More specifically, this invention relates to an entomopathogenic nematode preparation used for exterminating pest insects.

Background Art In general, nematodes are classified, depending on their parasitising subjects, mainly into the free-living nematode, plant-parasitic nematode, and insect-parasitic nematode, or entomopathogenic nematode. Although the crop-damaging Meloidogyne is plant-parasitic, bacterium-predating nematodes which phagocytise pathogenic microorganisms of crops Aphelenchus avenae) and entomopathogenic nematodes which parasitise and kill pest insects (genus Steinerema, genus Heterorhabditis) are highly demanded for their development as biological control pesticides, or insecticides, taking advantage of their properties.

Especially, certain species of entomopathogenic nematodes have already been put to practical use and may be promising biological control materials.

Under the conditions that entomopathogenic nematodes are present along with host insect, they intrude into the insect hemocoel through insect's mouth parts, anal pit, leg joints, etc., where they discharge symbiotic bacteria preserved within their bodies, proliferate after demolishing the insect's immune system, and lead to the death of host insect. Only the nematode at the stage of the "infectious thirdinstar larva" is infectious to insects in their life cycle. Infectious third-instar larvae is covered with the exuvium of the second-instar larva (sheath) having durability in various adverse environments.

There have hitherto been known several methods for preventing and terminating the plant-damaging pest insects utilising this infectious third-instar larva and its culture in a large scale has already been carried out with the artificial nutrition. In general, after manufacturing the preparation of cultured nematodes in a form durable for the long-term preservation, they are applied to the field contaminated with subject pest insects.

In several conventional embodiments for preserving entomopathogenic nematodes exemplified by those described below, nematodes are preserved by: 1. a method of preserving nematodes suspended in distilled water under an aerobic condition R. Ducky, JV. Thompson, George E. Cantwell, J. Insect Pathology, No. 6, p417, 1964), T r

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Libc/03538 2. a method of penetrating nematodes suspended in distilled water into a carrier such as polyurethane foam and placing said carrier in an aerated bag (Bedding, R.

Ann. Appl. Biol., Vol. 104, No. 1, p117, 1084), 3. a method of mixing nematodes with absorbent such as active charcoal (Japanese Patent Laid-Open Publication No. Sho 61-501392, Specification of International Patent Laid-Open Publication No. 85/03412), 4. a method of homogeneously mixing clay with nematodes and adjusting the water content (Japanese Patent Laid-Open Publication No. Sho 2-503913, Specification of International Patent Laid-Open Publication No. 88/08668), 5. a method of enclosing nematodes in beads or film of alginic acid gel (Kaya, HK., Nelson, GE., Env. Entomol., Vol. 14, No. 5, p.572, 1985; Japanese Patent Laid- Open Publication No. Sho 62-116501; Specification of United States Patent No.

4615883; Japanese Patent Laid-Open Publication No. Sho 4-505701; International Patent Laid-Open Publication No. 90/10063), 6. a method of drying nematodes (Japanese Patent Laid-Open Publication No. Hei 2-501300; Specification of International Patent Laid-Open Publication No.

88/01134; Japanese Patent Laid-Open Publication No. Sho 3-503526; Specification of International Patent Laid-Open Publication No. 89/00460; Specification of International Patent Laid-Open Publication No. 89/08704), or 7. a method of preventing the water-evaporation with oil (Japanese Patent Laid- Open Publication No. Sho 52-41225).

However, in Embodiment 1, the use of an aeration equipment is necessary and this method is not suitable for the transportation of a large quantity.

Embodiment 2 is troublesome since nematodes must be taken out from sponge prior to the treatment in the field. Embodiment 3 is applicable only for specific nematodes, and also problems imposed on the processability of active carbon have been indicated. In fact, the present inventors attempted to preserve S. kushidai in accordance with this Embodiment, resulting in a very unsatisfactory preservability.

In Embodiment 5, the preparation must be suspended in a solution containing a univalent ion such as sodium citrate to dissolve gels prior to its application, making its manufacture in a large quantity difficult. Embodiment 6 requires to set up strict environmental conditions such as humidity, temperature, etc. for appropriately drying nematode. Embodiment 7 is to temporarily prevent drying up nematodes applied on plant leaves, not useable for a long-term preservation over several months.

In Embodiment 4, clay (attapulgite, etc.) inexpensive as compared with active charcoal is utilised, providing a better processability. However, since the use of clay with a small particle diameter causes a rapid migration of water from >,ematodes to clay, damaging them, this method may be effective only when clay ith relatively large particle diameter is used. That is, in the case of clay of large Libc03538 particle size, because of their smaller surface area per unit volume, the contact area between nematodes and clay is restricted so as to suppress the waterabsorbency of clay to a certain extent. In contrast, in the case of the use of clay of small particle size, clay exerts its potent water absorbency to rapidly absorb the surface water of nematodes, thereby damaging them. Furthermore, once clay of small particle size absorbs water, the water molecule penetrates into spaces of the particle structure in such a manner that clay cannot maintain the original particle structure any longer, forming large clods with no internal spaces. Since the inside of such clods is tightly closed up with clay and water, it imposes problems to cause anoxia to nematodes within the clods as the surface water is excessively removed.

On the other hand, in the case of the use of clay with large particle diameter for the treatment in the practical field such as farm and lawn, these preparations have to be first diluted with water and then sprayed. If not, the original suspension tends to plug the spray nozzle pore of ordinary drug sprayer or sprinkler with clay.

For example, in the case of the use of baked chips of attapulgite, these large particles of clay must be removed using sieve, etc. prior to use. Thus, the process becomes complicated.

The above-described "Japanese Patent Laid-open Publication No. Sho 2- 503913" discloses a method in which, after baked chips of attapulgite are deprived of their large particles with a sieve (40 mesh, 350gm opening), the clay is mixed with nematodes, layered, and spread on a filter paper (Examples In this method, although the risk of plugged nozzle at the time of treatment can be slightly avoided, there still remains the risk of plugging nozzle with the particle with 350gtm diameter (in the case of the usual use of clay as the hydrate, the particle diameter of not more than 100pgm is preferred.), and also the use of particles of small diameter resulted in the decrease in the survival rate of nematodes as compared with the case of using large particles (see Tables 1 and 2 in the above-described Publication). Therefore, this method cannot be said to be suitable for the practical use.

Disclosure of the Invention An object of this invention is to provide a preparation of entomopathogenic nematode which has good preservability and is easy to handle in the field.

The present inventors have intensively studied to develop a process for producing an insecticide preparation, which comprises steps of bringing a mass of entomopathogenic nematodes into contact with clay and allowing them to move into clay. Also, this method enabled to produce a preparation of entomopathogenic ,,--,,niematodes which has good preservability and is easy to handle in the field, thereby *4,4pmplishing the present invention.

Libc03538 That is, the present invention relates to a process for producing an insecticide preparation which comprises steps of bringing a mass of entomopathogenic nematodes retaining the surface water into contact with clay and allowing them to move into clay. Also, this invention relates to an insecticide preparation manufactured by this process.

In the process of the present invention, when a mass of entomopathogenic nematodes (hereinafter sometimes referred to simply as "nematodes") that retain the surface water are brought into contact with clay, the water first migrates towards clay due to its water-absorbability, forming a concentration gradient of water from the mass of nematodes towards the outside of clay. Under these conditions, by suppressing drying up of the preparation and allowing it to stand at the temperature optimal for the nematode activity, usually at 25-28 0 C, nematodes start to migrate from the mass to clay in the opposite direction to that of the concentration gradient of water seeking the oxygen. These conditions can be attained, for example, by placing a mixture of nematodes and clay in a bag made of aerated film or a container partially made of aerated film, and allowing it to stand in a chamber with the relative humidity of 95% or higher, or by gently stirring the mixture in an atmosphere of the relative humidity of 95% or higher. Also, if it is for a short while, the conditions can be attained by a gentle intermittent stirring of a mixture of nematodes and clay placed in a container with a lid in a room. In this occasion, the surface water of migrating nematodes is gradually absorbed into clay, and nematodes cease to migrate when the water concentration reaches the level that stops the nematode migration. Nematodes under these conditions are inactivated (referring to the conditions where nematodes become inactive in their motions, and suppressed in their oxygen demand and energy metabolism), resulting in the enhancement of the preservability in the preparation. Although the time required to reach this state varies depending on the species of nematode, type of water absorbent used, or mass size and concentration of nematodes, it is about min-24h, usually 30 min-12h. Also, in this invention, the particle diameter of clay is not particularly limited, but it is preferably not more than 100m, and more preferably not more than 1 O0m.

According to the present invention, it has become possible to retain the activity of nematodes in the preparation for a long period of time. Since the particle diameter of clay can be reduced in the preparation of the present invention, the preparation is well hydrated, can readily be suspended, and conveniently sprayed, after diluted with a large quantity of water, over a whole area of the actual site of pest insect prevention and elimination such as the field and lawn. In addition, the preparation of the present invention can be ploughed into the field soil as such as a ,iNdered preparation.

Libc103538 In one embodiment of the present invention is exemplified a method comprising the steps of, prior to bringing clay into contact with nematodes, retaining them once in a water-absorbent, and contacting said nematodes retained in the water-absorbent and water with clay. Preferable examples of the water-absorbent include polyurethane foam, cellulose sponge, paper chip, wood chip, chip, fibre, macromolecular absorbent, etc. Although the concentration of nematode suspension to be retained in the water-absorbent is varied depending on the type of water-absorbent, nematode species to be used, and the concentration of nematodes in the desired final preparation, the suspension is preferably condensed to about 20-2 000 000 nematodes/mL (in liquid).

This method features that, when nematodes retained in the water-absorbent and water are brought into contact with clay, water first migrates from the absorbent to clay due to the clay's absorbency, and then the water movement is equilibrated at a certain level due to the water-retaining capability of the absorbent, forming a stable concentration gradient of water from the water-absorbent towards the outside of clay. Clay may be either dried or slightly moisturised (about Nematodes migrating from the water-absorbent into clay ultimately stop to move at the water concentration which can bring them to a stop. Since nematodes which have stopped the movement are now surrounded with clay which has been equilibrated with water, they will be exposed to no further dryness, and preserved under moisturised conditions optimal for their inactivation.

In addition, after the migration of nematodes, the water-absorbent is preferably separated from the clay using a sieve with 1-5mm mesh. At this time, not less than 90% nematodes are recovered in the clay, and those remaining in the water-absorbent can be recovered as a suspension by soaking them again in water. Also, water-absorbent such as polyurethane foam, cellulose sponge, etc.

can be reused by drying them in dryer after washing. Thus, a powdered preparation with clay, moisture and nematodes being homogeneously distributed therein can be obtained by separating it using a sieve. Since, in such a preparation, small-particle clay and nematodes mutually form a steric structure, they can provide spaces to supply enough oxygen to nematodes.

Although a certain percentage of nematodes tend to undergo ecdysis as they migrate in clay, this method is advantageous in that the preparation is not susceptible to the mould contamination during the long-term storage, because exuviae, contaminants, etc. from nematodes are retained within water-absorbent and hardly transferred into clay. This method is further advantageous in that the insecticidal activity of nematodes recovered in clay and contained in the preparation is elevated as compared with that of nematodes prior to the migration, because atodes with a high motility preferentially migrate into clay leaving those with low Libc-.3538 motility in the water-absorbent (in general, nematodes with a high motility also have a high insecticidal activity).

Another embodiment of the present invention is a method comprising the steps of dehydrating a suspension of cultured nematodes to a clod and embedding this nematode clod in clay. The suspension is preferably concentrated and dehydrated to about 40-4 000 000 nematodes/mL (in the form of clod), though the content may vary depending on the species used. When this nematode clod is placed in clay the water content of which has previously been adjusted to 10-30% to prevent dehydration and allowed to stand, water migrates from the nematode clod to clay, forming a concentration gradient of water. Nematodes which migrate from the clod to clay ultimately cease to move at the water concentration capable of stopping the nematode movement. This method is advantageous in that the production process is simple because of using no water-absorbent.

For a long-term preservation of the nematode preparation in accordance with the present invention, it is preferable to preserve it after acclimatising, under the aerobic conditions to prevent drying, the nematode preparation thus manufactured to the temperature to suspend the activity of 1 nematode (varied depending on the nematode species, 5°C in the case of S. carpocapsae or S.

.glaseri, and 10-15°C in the case of S. kushidai or H. bacteriphora) for a predetermined period of time sufficiently to induce nematodes to diapause. Nematodes induced into diapause become extremely less oxygen-demanding, and can be stored even in a tightly closed container for a long period of time.

In order to prevent the putrefaction during the storage, the preparation of the present invention may 20 properly be mixed with agents such as antifungal agents, etc.

Thus, according to an embodiment of the present invention there is provided a process for producing a pesticide preparation, which comprises steps of bringing a mass of entomopathogenic nematodes retaining the surface water into contact with clay, and maintaining said mass of 'entomopathogenic nematodes retaining the surface water and clay in contact with one another as discrete contiguous entities forming part of a non-homogeneous composition, whereby, while said mass of entomopathogenic nematodes retaining the surface water are in contact with one another, said nematodes migrate into said clay.

Best Mode for Carrying Out the Invention The present invention will be further described below with reference to the following Examples, which are not to be construed to limit the scope of the invention.

[I:\DayLib\LIBZZ]03538adoc:gcc Example 1 Production process using cellulose sponge Eight mL of nematode cream containing 10 000 000 nematodes was thoroughly absorbed into 0.65g of cellulose sponge (5mm dice), and mixed with 17g of dried clay (containing an antifungal agent). After storing this mixture under these conditions for 24h (at 25°C, in an atmosphere of relative humidity of 95% or higher), the clay containing nematodes was separated from sponge on a sieve (1-mm mesh). The nematode preparation thus manufactured was stored under the conditions of 15°C and relative humidity of 95% or higher. After storing the preparation for a predetermined period of time, it was suspended in a suitable amount of water, and a few drops of the suspension i applied on a slide glass were 9* 0e 4* a 9

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7 examined under microscope to obtain the ratio of viable nematodes. The results are shown in Table 1.

Example 2 Production process using wet clay To dried clay (containing an antifungal agent) was dropwise added water in an amount of 15% (weight ratio), and wet clay was first prepared by homogeneously pulverising the above mixture in a blender. About 5g of nematode clods (comprising 10 000 000 dehydrated and sieved nematodes) was placed in 27g of the wet clay thus prepared and they were briefly mixed. After the mixture in this state was placed in an aerated bag for 24h (at 250C, in an atmosphere of relative humidity of 95% or higher), the particle diameter of the preparation was unified using a sieve (1mm mesh). Nematode-clay preparations thus manufactured were stored at different temperatures in an atmosphere of relative humidity of 95% or higher. After storing for a predetermined period of time, preparations were suspended in a appropriate amount of water. A few drops of the suspension were applied to a slide glass, and examined under a microscope to obtain the ratio of viable nematodes. The results are shown in Table 1.

Example 3 Production process using wet clay To dried clay (containing an antifungal agent) was dropwise added water in an amount of 12% (weight ratio) and wet clay was first prepared by homogeneously pulverising the above mixture in a blender. Fifty-seven g of clods of S. kushidai (comprising 114 000 000 dehydrated and sieved nematodes) was placed in 160g of the wet clay thus prepared and they were briefly mixed in a beaker using a spatula. The top of the beaker was covered with a piece of aluminium foil, and stored at 250C for lh with a brief mixing using a spatula once every 15 min. Then, the particle diameter of the preparation was unified using a sieve (1mm mesh). The nematode-clay preparation thus manufactured was placed in polyfilm bag (slightly aerated), and stored under the conditions at maintaining temperatures shown in Table 1 for a period of each indicated time in an atmosphere of relative humidity of or higher. Then, a few drops of the preparation suspended in a suitable amount of water were applied to a slide glass, and examined under a microscope to find the ratio of viable nematodes. The results are shown in Table 1.

Comparative Example 1 Production process solely using dried clay Eight mL of nematode cream containing 10 000 000 nematodes was added to 17g of dried clay and they were thoroughly mixed. The nematode-clay preparation us manufactured was stored under the conditions at each temperature and in the ilative humidity of 95% or higher. After storing the preparation for an indicated Libc/03538 4 8 period of time, it was suspended in an appropriate amount of water, and a few drops of the suspension were placed on a slide glass, and examined under a microscope to find the ratio of viable nematodes. The results are shown in Table 1.

Table 1 Nematode Preparation Storage temperature nematode Storag Species method conc. e days (nematodes/g) 20 30 40 60 90 120 days days days days days days days k Ex. 1 15°C 292,000 99.3 97.5 93.7 91.4 86.2 80.6 76.5 k Ex. 2 15°C 307,000 99.9 95.1 94.8 88.0 78.0 75.0 k Comp. Ex. 1 15°C 298,000 80.0 70.8 42.5 26.5 7.8 c Ex. 2 5°C 200,000 92.0 93.0 89.2 85.6 84.9 c Comp. Ex. 1 5 0 C 224,000 87.0 83.0 78.3 67.4 48.8 37.6 k Ex. 3 15°C (10days)->5C 456,000 93.3 89.4 89.5 k: S. kushidai c: S. carpocapsae The results in Table I clearly indicate that the nematode preparations manufactured by the process of Examples 1, 2 or 3 have remarkably better preservability than those manufactured by the process of comparative Example.

Industrial Applicability The present invention has enabled the improvement of preservability of nematode preparations. Since the preparation of this invention can make the particle diameter small, it can be conveniently diluted with a large quantity of water and spread over a whole area.

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Claims (10)

1. A process for producing a pesticide preparation, which comprises steps of bringing a mass of entomopathogenic nematodes retaining the surface water into contact with clay, and maintaining said mass of entomopathogenic nematodes retaining the surface water and clay in contact with one another as discrete contiguous entities forming part of a non-homogeneous composition, whereby, while said mass of entomopathogenic nematodes retaining the surface water are in contact with one another, said nematodes migrate into said clay.

2. The process according to claim 1, wherein said mass of entomopathogenic nematodes retaining the surface water is held in a water-absorbent. o i

3. The process according to claim 2, wherein said absorbent is any one of sponge, paper chips, wood chips, or fibre, or a mixture thereof.

4. The process according to any one of claims 1 to 3, wherein said entomopathogenic nematodes retaining the surface water form nematode clods.

5. The process according to claim 4, wherein said clay which has previously retained water 5 is used.

6. The process according to any one of claims 1 to 5, wherein the particle diameter of said S* clay is not more than 100pm.

7. A process for producing a pesticide preparation, substantially as hereinbefore described with reference to any one of the examples but excluding the comparative examples. •:20

8. A pesticide preparation manufactured by the process according to any one of claims 1 to 7.

9. The preparation according to claim 8, further comprising an antifungal agent.

,10. A method of controlling pests at a locus, which method includes or consists of applying to said locus, an effective amount of a pesticide preparation according to claim 8 or claim 9. Dated 7 October, 1999 Kubota Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [I:\DayLib\LIBZZ]03 53 8a.doc:gc