JPH03503526A - Drying of biological materials in oily suspensions - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] ``Going straight υ moving view'' Saishin cod pasted.

■ Dwarf The present invention is particularly useful for insects using biopharmaceuticals for agricultural, garden, and household insects. Regarding pest control. In particular, the present invention aims to improve the ability of insect parasitic nematodes to improve their viability and disease. Concerning how to dry large quantities and small quantities for long-term storage while maintaining their original properties. It is something.

Trade 1) Company [passed away] Nematodes represent a group of unsegmented, round insects.

The anatomy is simple, with a simple intestine that is elongated and spindle-shaped. so many departments Some are roundworms, while others are parasitic on plants or animals. do. Those that parasitize insects are called entomoparasitism or entomopathogenic nematodes. It will be revealed.

The order of greatest commercial interest for insect control is the Rhabdttida. , encompassing several families, many members of which are parasitic on insects. Steinern ematids and Heterorhabditids are prominent among the above families. It is. A general discussion of the classification of nematodes and the entomoparasitic families within them, Potnar, G.O., Prent 1ce-Hal, 2nd St. “The Natural Hf5tory of Nematoda” published by I Company Newatodes J. (1983).

Nematodes have a standard life cycle with five stages, the five stages being It is described by a molting stage in which new epidermis is formed and old epidermis is shed. easy In other words, stage 5 adults lay eggs, and the eggs develop stage 1 larvae, which under suitable conditions Below, move to stage 2. Normally, stage 2 larvae simply develop into stage 3 larvae. From there, it develops into a stage 4 larva, which then completes the cycle and reaches the adult stage. Ru. However, what is interesting about the use of nematodes for insect control is that When surrounding conditions are relatively unfavorable for continued growth and reproduction, Stejn ernematid and 1 (eterorhabditid nematode stage 2 larvae are instead “stage 3 infeetiva juven” larvae. iles) J, that is, rlJJ. Under the above circumstances, the second The epidermis, characteristic of the stage, is retained and is called the sheath. The epidermis is completely nematode Surround da. IJ is susceptible to insect infection and passes through stage 4 and adulthood at the expense of parasitism. to complete the life cycle.

Stetnernematid and Heterorhabditid IJ Matoda is an effective means of insect control. The nematodes are morphologically identical and usually live in surface water films around soil particles.

Although it requires oxygen and moisture to survive, it does not eat food and uses itself as an energy source. Make use of their food stores. If the pods are removed, they are infectious.

Steinernematid and Heterorhabditid nematodes Another aspect of da ecology is important. Nematodes within these families disease the insect body. coexist with a species of bacteria that is primarily, but not entirely, responsible for the properties that cause .

Stefnernematid and Heterororbabditid nematodes Many challenges have been posed to the commercial production of Da and its use in pest control. However, this has only recently begun. Mass production of IJS begins in various places. Various techniques have been tried. For example, Sobi dated April 8, 1980. Eto Patent No. 726.164, PCT Application No. 8, promulgated on February 27, 1986. 6701074, U.S. Patent No. 4.334.498 and U.S. Patent No. 4.17 8. There is issue 356.

Formulations for applying infective bitter insects to soil have also been devised. For example, Sobie U.S. Patent Application No. 378.222 and U.S. Patent Application No. 4.178.366. Ru. One method is to use a suspension in light mineral oil. Furthermore, patent application No. 607260.67 No. 8 proposes a fermentation bed material support for nematode applications.

Steinernemattd and Heterorhabditldnematot Another significant problem in the commercialization of insect control using insects is that the infective nymphs Transport and storage on a large scale while maintaining viability and pathogenicity. Ru. Until now, relatively impractical methods have been used that only marginally reduce metabolism. It's here. These include hydrogen peroxide in water (Dutky, S, R 1° et al., J In5eet Patbol (1964) 6:417-42 2) in distilled water or o, i% formalin (Poinar, G, O, , r Nematoda J (1975) CRCPres for the biological control of insects. S, Boca Raton, Fiorida) or damp polyurethane in 0.1% formalin in a tongue sponge or moist filter paper (Bedd i ng, R, A, Phantom” - May 11fed 1o1 (1984) 10:11 7-120;) lara, A, H, et al. 1-nric Techno l W-16 (IHI); Hovell, J, F,, J Invert Pathol (1979) 33:155-156 and LLnder gren, J, E, et al. Punishment Technol W- 3 (1979)). Other transportation and storage Kura's technology involves the use of wood chips and activated charcoal.

Other methods have recently been disclosed. U.S. Patent No. 4°417,545 Transporting and/or storing nematodes and/or their eggs in a dormant state I'm talking about containers.

This container basically consists of a nematode and Sandwich the eggs, thus maintaining high humidity levels. However, such The method is for non-infectious stages of the worm and is not related to the transport of infectious nymphs. It's not bad. PCT application WO35103412 proposes a method of transport and storage. However, this is due to the maintenance of a presumed anaerobic state and the use of antibacterial agents. . Highly osmotic solutions are also used to inhibit bacterial growth. storage by suggestion Conditions also include adsorbents such as charcoal or synthetic resins, but these reagents It is not clear what it absorbs. This disclosure describes the use of formalin as an antibacterial agent. illustrates the use of aldehydes and proposes storage vessels containing nematodes and adsorbed carbon. .

The present approach is based on 5teinerne+aatid and Heteror Infectious nymphs of habditid nematodes are kept dormant to avoid depleting their reserves. Maintain it in a dormant state, return it to appropriate conditions and let it spawn, and maintain pathogenicity against insect hosts. It's about doing what you're doing.

In summary, the methods and containers disclosed in connection with the present invention are effective in preventing infective nymphs from It is designed to maintain a state of "hidden life", a dormant state in which metabolism is suppressed. ing. There are several ways organisms can do this, each with varying degrees of success. generally known. most commonly proposed and probably most universally applicable The method involves induction of cryobiosis, i.e. low temperature, general It is to suppress metabolism at sub-zero temperatures. Another option, even more difficult to achieve, is evaporative dehydration. anhydrous induced by osmotic dehydration, and closely related by osmotic dehydration. There is osmobiosis bK allo.

In general, there are a large number of papers on the induction of anhydrous life in nematodes. Ru. However, direct induction of anhydrous life in nematodes associated with insect control is To the applicant's knowledge, the only published report (Simons, W.R., and Po1nar, G, O,, J Inve t Pathol (19 73) ii:228-230) and Yolob promulgated on February 24, 1988. There is only the disclosure of Patent No. 256.873. This application ('660) is an anhydrous A method of inducing a living condition is disclosed. This method removes large amounts of water from the surface. After drying, carefully (necessitate controlled drying) the nematode spread in a thin layer. do. This method requires large areas to dry commercial quantities of nematodes. You have to be careful about one thing. For example, 2.5 x 10' IJ square At an inch density, a 2.5x 10-hole IJ would require approximately 10' square inches. On the other hand, the same number of IJs can be easily controlled with a 500a tank. and dried. Co-pending U.S. patent application filed April 6, 1987 Application no. Discloses a method using transparency.

Furthermore, there are reports of natural drying of Neoa Iaetana under unspecified conditions. Summary of the posium (Kamionek, M. et al., “Eleyenth Int. 'l5yrap Nematol, Eur Soe Nematol-(19 ]2)).

Other types of nematodes, including free-living nematodes and plant-parasitic nematodes Nematodes are known to be able to survive naturally under dry conditions (Ev ans, A, A, A, F, et al., “Nematoma as a biological model. -da (-Ne Tatsumiatodes as Biol.

gial Models-) J (1980) Acade+*ic P ress, New York, pp.

193-211: Demeure, Y., et al. [Plant-parasitic nematodes ( -Plant Parasitic Ne++atodes-) J (19 81) Acada+mie Press, Nev York). By drying Significant changes in chemical composition may occur in preparation for anhydrous living caused by It has been shown that the plant-parasitic nematodes that are the subject of these studies have a low Pre-conditioned at 97-9H relative humidity for 48-72 hours before exposure to relative humidity. It is known that Evans et al. (cited above); Womer sley, C,, Con Bioehem Phvsiol (IHI) 68Δ:249-252+ Madin, K, A, C,, ra, LヱKyoko Z ool (1975) 143:335-342; Crove, J, H ,, et al. (ibid.) 323-334).

Freekman, D.W., et al. “New trends in soil ecology (- New Trends in 5 oil Biology”) J (Leb run, P. (ed.) (1983) University Catho Sand that lives in an anhydrous state in lique de Louvain Press It describes the abilities of the Nematoda of the Desert.

Womersley, C, Cow ar Bioche + m Ph 5iol (1981) 70B: 669-678 on anhydrous life in nematodes. The survival mechanism has been investigated in detail (similar research is by Crave, J. H., et al. L Eri Bao Hong (1979) 20 Uni 431-437; Demeura, Y ,, et al., L■lL (1979) n:189-195 and Crove , J, H,, et al., Ann Meet A+wer In5t BiolSc i, East Lansing, Michigan, 21-26 Aug. UST 1977.

For species related to insect control, N, ear oea sae is One report attempted drying using a series of humid chambers containing a liquid solution. It has been promulgated (Si+mons, W, R, and Po1nar, G, Ol, anal dust). However, relative humidity (RH) is not measured directly and the temperature at which the experiment was performed is also reported. It has not been. The IJ was kept at 96% RH for 12 hours and then moved to 93% RH. for 12 hours and then at 10-79% RH for periods of up to 28 days.

Only 79.5% RH had a survival rate greater than 40% after 12 days. In 20 days Even under these conditions, the survival potential dropped to 30%.

In other words, some of the published studies on drying nematodes include 5teinerne+ Effective against natid and heterorhabditis insect parasitic nematodes Measuring indicators that enable long-term, commercially viable, long-term, bulk storage and transportation None were provided by a possible process.

However, the method of the present invention provides a means to induce fully anhydrous life of infectious nymphs in suspension. The infective nymphs can be stored for long periods of time while retaining their ability to kill the insects they infect. The tδ0 method does not require separate equipment and can be used to control large quantities of insects. applicable to the processing and use of The method can also be applied to other biological materials, especially yeasts. Applied to the dehydration of biological materials that can ``come back to life'' after a certain amount of time. can.

The method of the present invention provides a more convenient and economical process expansion. The present invention is based on Stefnernematfd and [1terororhabdHi This method provides a method for bringing infectious nymphs of D. nematoda into an anhydrous state. This facilitates long-term storage and transportation while retaining viability and pathogenicity. Ru. The present invention also provides anhydrous-living Nema-da infectious bitter insects as such. It is. The methods and materials provided by the present invention can be used to treat insects in agricultural applications. Stefnernemat for pest control! cl and Heterorhabd It is important to make itid imitations economical and commercially viable. be. Without such methods and materials, expensive packaging and refrigeration would be used. or the infective nymphs will be cultured in close proximity to the site where they will be used. However, this approach to pest control is rarely practical, resulting in The use of chemical pesticides, which have been shown to be phylogenetically harmful, continues.

Therefore, in one aspect, the present invention provides St, sfnernematid and Het. Regarding the process that causes the anhydrous life/hidden life of erorhaMitid nematodes. As a first step, the infective nymphs are placed in a suitable oil, such as light mineral oil or vegetable oil. This includes suspending. The oil is preferably sufficient to prevent nematode agglomeration. Contains a small amount of non-toxic surfactant. Next, apply the resulting suspension to Use a suitable drying agent and mix until a sufficient amount of water is removed from the nematode. dry. Suitable desiccants include solids (e.g. Na25O4), liquids (e.g. cleaning agents or surfactants), gases (for example, air, 027N2, etc.). current A preferred desiccant is air having a relative humidity of less than 100%. Nematoda scraper Mix and suspend and expose to air for a sufficient period of time to render the IJ anhydrous. Noriyoshi Typically longer times than 24 hours are effective, with at least 72 hours being optimal.

After anhydrosis is induced, the dried nematodes are filtered and reduced to an oily paste. Alternatively, an oil suspension may be provided and stored in a suitable moisture-resistant container for an extended period of time. Can be stored. Storage over a period of several months is achieved with this method. case In some cases, the nematodes are suspended in a hypertonic solution of the type described above. Good too.

In another aspect, the present invention relates to anhydrous IJ obtained by the method of the present invention. do.

Transport and/or storage of IJ living in an anhydrous or hidden state Containers useful for storage are covered by U.S. Patent Application No. 10, filed September 28, 1987. No. 1.530, incorporated herein by reference. Simply put, The characteristics of these containers are to maintain the relative humidity experienced by the contained nematodes, Provides oxygen to meet minimal metabolic demands at the low metabolic levels characteristic of this condition It is possible. The container does not allow moisture to pass through and satisfies the oxygen requirements of the IJ during negative drawing life. have enough head space to allow water and air to pass through, or have RH Either it has a means for controlling it. Alternatively, drying in the oil suspension of the invention Nematodes may also be provided.

Preferably, the dried nematodes have the necessary humidity and surface exposure to air. while protecting the TJ from large amounts of water (and premature rehydration). , in a suitable non-toxic gel or matrix. Pack IJ well To do this, a suitable stock solution with a water activity of 0.97 is first prepared. one In general, non-hazardous osmolytes in water at subsaturating concentrations Solutions are suitable, such as salts (e.g. NaCl, K2SO4, potassium acetate, etc.). ), sugar (e.g. IM fructose, 1M maltose, etc.), sugar alcohol (e.g. (e.g., 1M sorbitol, 14% glycerol, etc.) and other low molecular weight polymers ( For example, polyethylene glycol °PEG"600).

The stock solution is then transferred to Terr 1-sorb"" (industrial 5e rvices International, Inc.) ~5anvet” (Celanese, starch grafted polyacrylate), Dr. ytech” (Dov. cross-linked polypropenoic acid) Used to prepare paste using Trix. suitable matrices When the gas material is prepared to diffuse oxygen sufficiently, the particles are separated. The paste should be soluble in water at higher concentrations. oily The IJ in suspension is centrifuged and the oil is decanted and silane. Next I J is stirred in a fixed amount of stock solution (e.g., 35+mL solution per 10” IJ). The matrix paste (e.g. 10” 450g matrix per JI).

The resulting nematode paste is then placed into a container. preferably The paste is [of a size that allows J to pass through but does not allow the matrix particles to pass through]. It is often placed in a mesh bag, and in this way, for example, matrices can be removed using running water. An effective method for releasing IJ from a box is provided. What material is this bag made of? You may be able to do it. Nylon is preferred because it is easy to heat seal. stitch size The size ranges from about 15 to 150 holes per inch, and the size depends on the matrix. Depends on the material. Instead, the container contains hose fittings and sieve/filter retainers. It may have a fitting or be used in a spray device. Next The antenna is sealed to allow oxygen in while limiting humidity. A suitable film (e.g. (e.g., polysulfone, polystyrene, Tyvek, etc.) A tarcap may be advantageously used. Also, make sure that there is enough head space in the container. Oxygen may be provided to completely seal the container. Alternatively, open a suitable hole. Use tall or loose caps to protect the area during storage and transportation. The container may contain extra water to compensate for water loss. here is preferably sealed in a polysulfone film container.

spend the day ゛ Superintendent L As used herein, "biological material" refers to living tissue, cell cultures, microorganisms, (yeast, bacteria, protozoa, etc.), small multicellular organisms (nematodes, insect larvae, etc.) (such as insects, etc.), and when some of the water naturally contained in the material is removed, the metabolism By entering a state of reduced demand and adding water or a suitable solution, normal It is something that can be restored.

"Insect parasitic nematode" is a nematode that parasitizes one or more insect species. Instruct Toda. The most important order of insect parasitic nematodes is Rhabditl. da, and the present invention primarily focuses on two Rhabditid families of this group, viz. 5teinerne+oatidae and Heterorbabd it 1 relating to the storage and/or transportation of dae.

However, other insect parasitic families are also targets to which the method of the invention may be applied. It is suitable for enehoidaeSEEntaphelenehidae, Mar+++ 1thidae, Neotylenchidae, 5phaerulari non-Rhab including Tetradone+natidae, and Tetradone+natidae. ditjd family, as well as Diplogasteridae.

Panagrolaimidae, Phabditidae and 5yrop Includes hone+mat 1 dae.

As mentioned above, Rhabditida, the most commercially important family. is Steinernematidae and Heterorhabditid It is ae. References in the literature to rNeoa 1actanaJ include Ste. refers to a specific genus of Jnernematidae. life for a particular species Neoa as a noun 1 actana and the word Steinernema -One example, t ha, L' upper υ mutual wa, or LIL! j--- sometimes , are used interchangeably.

While the classification of various groups of nematodes can be confusing, the present invention Insects are contagious and can occur as stages in their life cycle, as described in the background section above. Targeted for genera with stage 3 infective nymphs (IJs) with defined characteristics is clear. by the intended agricultural application, i.e. by the target insect. One or more species are particularly effective.

"Negative life state" means, according to the content of the present invention, in particular the negative life state of infectious nymphs. point to The state is a state in which the metabolism is suppressed, physiological It is in a dormant state.

In fact, if relatively insensitive methods are used to ascertain metabolism, metabolism may not be ascertained. stomach. Under these conditions, oxygen uptake is greatly reduced, making traditional methods relatively short. It cannot be detected if it is used for a long time.

"Anhydrous life" refers to a negative state of life caused by loss of water2. An “apparently anhydrous lifestyle” or “apparently hidden lifestyle” is defined by the following criteria: Refers to a fixed state: i.e., no movement, shrinkage and wrinkles. As a result, oxygen consumption decreases. (Obviously living an anhydrous life or in a state of concealment) Nematodes are determined to be still viable by rehydration and testing for viability and virulence. It can be shown that the ) In particular, the anhydrous life or negative life state of the IJ of the present invention is determined according to the following criteria. It can be proven: (1) Out of a group of at least 1,000 infective nymphs, more than 60% are rehydrated. After successful suspension in 70% methanol, viability can be verified. here The suspension is then placed in liquid nitrogen for 24 hours and then rapidly returned to room temperature. .

(2) Few (more than 70% of a group consisting of 1,000 infective nymphs are i06/ Survives when stored in an airtight container at a nematode/volume ratio of 30-.

Here, air initially fills the volume of the container. Holding time depends on temperature Ru. The required rate is greater than 15 days at 25CO and greater than 8 days at 30CO; Can be maintained at 35C' for longer than 6 days.

(3) Free liquid water is present in fruit clusters consisting of more than 1.000 IJ. In the absence of oxygen, the oxygen demand at 25CO is 1 centigrade per 9 hg of dry IJ per day. Fewer. In contrast to non-dried nematodes, "ordinary" nematodes This is easily seen as the oxygen requirement exceeds 6 children per 10' organisms.

4) Less (more than 90% of the group consisting of 1,0000 Survives exposure for 2 hours.

"Dehydrated nematodes" also refer to nematodes that are clearly in an anhydrous state of life.

"Infectious nymphs" or "IJs" are able to invade and infect insect hosts; Refers to those in the immature stage. In the family that is the subject of this invention, these are stage 3 It is IJ.

"Relative humidity J (rRHJ) is the water vapor pressure in the air relative to the saturated vapor pressure at the same temperature. It is defined in the standard way as a percentage of , and is generally expressed as a percentage.

L 2nd ship Xingcheng Tsui The infective nymphs that are the subject of the procedure here are borers (borers) that bore holes in trees and fruit. ), root weevils, caterpillars, beetle g rubs), beetle larvae, chafer beetle, and mole cri It is useful for controlling various pests, including pests (e.g. ckets). Infectious nymphs like this The main crops protected are corn, strawberries, almonds, greenhouse grains, and wheat. Includes tushinirum, sugar cane, and potatoes. Poultry breeding equipment and Other animal enclosures are also kept free of flies. In typical agricultural use, Infectious nymphs are used in many targeted environments. For example, pine mushrooms For control of 5 ciariids in insects, approximately 5 x 10' insects are placed in each house. Applies to

A small number of IJs, for example about 10', may be useful for home use.

Such a number is suitable for protecting potted plants.

Using this technique, approximately 1011 infective nymphs or 25. kg wet product can grow to about 152 kg of medium in one week. Such a large amount of IJ is stored must be collected, transported and stored.

In the method of the present invention, large quantities of IJ are stored for long periods in anhydrous living conditions for such purposes. Saved for a while. Anhydrous living conditions remove most of the surface water (e.g. (by washing or filtration), place the Jl in an oil suspension and add a suitable desiccant. Dry the mixture using the is achieved. Suitable desiccants include solids (e.g. Na25O4), liquids (e.g. (e.g., cleaning agent or surfactant), or gas (e.g., air, 02/N2, etc.) It is. Here, the desiccant is preferably at a relative humidity of less than 100% (preferably as low as possible). The atmosphere is better. The nematode has enough time for the IJ to become anhydrous. , stirred and aerated to maintain suspension. typically 24 hours Longer times are effective, with at least 48 hours being optimal. Anhydrous life is the best After drying, the dried nematodes are filtered to form an oil-containing paste or suspension. The liquid is provided and stored in suitable moisture-resistant containers for long periods of time.

With this method preservation over a period of several months is achieved. In some cases, dry Dried nematodes may be suspended in a hypertonic solution, such as concentrated sucrose. . The IJ thus dried maintains its viability and pathogenicity.1. ,’ There is.

Viability is determined by microscopic observation. Here, based on the criteria for living individuals, A clear esophageal segment, absence of the typical site of death, and the presence of a dental probe Includes motility when This kind of survival ability test is the IJ of the hidden life. If carried out at a later date, it is typically carried out after a 24 hour re-aqueous phase. difficult conditions Other tests to measure resistance to occult life include IJ rehydration and viability assessment. 55% RH for 72 hours.

The pathogenicity is Galleria + ae11. onel-infectivity for fallen larvae It is measured by analyzing the nymphs. Infectious nymphs at a density of 5o per analyzer Wbatman Jllo placed on the edge of the bedding dish of m, 0.0 on the filter paper of 1. He is transferred to the water after 5 kicks with a bebe throat. 1 o of larvae are placed on the filter paper, and the ``'' is closed. and left at 22CO 80% RH. 1Iiii 30 to 50 hours after dew Mortality is recorded every two hours every day during time (LTse) compared to the control.

(Margin below) R-ho and L-ta A critical aspect of the method of the invention is control of the rate and extent of drying. Main departure The drying speed can be precisely controlled. In the practice of this invention, cultured The collected IJ is first filtered to remove most of the surface water. large number of tables Surface water is most easily removed by filtration. Quantitative suspension of water in oil Removal is important in this method, in contrast to processes that involve evaporation from thin films of nematodes. isn't it.

The nematodes are then homogeneously suspended in a suitable oil. Oil is not harmful to living organisms. Vegetable, mineral or synthetic oils may be used as long as they are suitable. The right oil will ensure uniform mixing and In particular, it should not be viscous or volatile for the safety of the fluid and handlers. this In this regard, preferred oils have a viscosity of less than about 60 centivoise and a vapor pressure of Approximately 0. Should be below OO1+amHg. Apart from these elements, The selection of the particular oil used may generally be a consideration of availability and cost.

Specific oils include soybean oil, corn oil, light mineral oil, and the like. Typically, Ltd. About 20 to 100 + og, preferably about 45 mg of nematoda (dry) per oil Dry form j1) is suspended in oil: approximately 500,000 IJ per l .

Preferably, a non-toxic surfactant aids in uniform spreading and suspension of the nematodes. It is added to oil for this purpose. In the absence of surfactant, nematodes aggregate This may result in uneven drying. If agglomeration occurs, A large amount of water is removed from the nematodes outside the flocs, while the flocculated nematodes Water is not removed sufficiently from the da. Agglomerated nematodes therefore have an anhydrous lifestyle. It is unable to enter the state and becomes anhydrous during the subsequent water removal stage. The final result is The mortality rate is unacceptably high. The addition of surfactants can also be done under less agitation conditions. Promotes proper mixing. Therefore, the nematode is subjected to low mechanical pressure by mixing. exposed. Suitable surfactants have low hydrophilicity/lipophilicity such as 5pan'80 Night of balance (HLB).

The drying step is performed at a temperature ranging from about 0°C to about 50°C, preferably from about 5°C to 30°C, most preferably from about 5°C to about 30°C. Preferably, it may be carried out at a temperature in the range of about 20°C to 25°C.

The desiccant is then added to the nematode suspension and uniformly dispersed. The desiccant is chloride solids such as calcium or sodium sulfate, detergents or other surfactants. or preferably a gas such as air or a nitrogen/oxygen mixture. can be obtained. No matter which desiccant or desiccant combination you choose, the nematode will Airage the suspension to preserve the viability of organisms with limited oxygen requirements. You should keep in mind what you need to do. Air flow velocity, temperature, The drying speed can be easily controlled by changing the air flow and the RH of the incoming air. Preferably, air is used as the agent.

The water content of the suspension should be carefully monitored during the drying process. For example, ArlFisher titration can be used to determine the water content of the suspension. gas If a desiccant is used, water loss can be determined by measuring the humidity of the escaping gas. loss can be monitored. For example, passing the effluent gas through a drying trap and adding 1iffi of desiccant may be measured. The exact rate of water removal will, of course, depend on the specific material being dried. 11 of the excess water present in the initial preparation (and present dissolved in the oil) amount of excess water) as well as the desired final water content. However, one Generally, about 1 at a rate of about 0.225 g H2O per hour for about 64 hours. Drying 0” Neoa 1ectana ear oea se IJ and obtained acceptable results.

Dry the organism to a water content that corresponds to the water content at which the organism enters an anhydrous state. In most nematodes, anhydrous living conditions are induced at approximately 97% RH.

The water content at the RH can be determined as follows. I tared io'lJ. It was placed in an aluminum dish and dried in an oven at 150° C. for 24 hours. These o Weight of IJ dried in a bun and f of IJ dried at 97% RH using prior art method ! By comparing j1, the water content of IJ required to induce anhydrous living conditions can be determined. can be determined. Therefore, carefully reduce the water content of the organism to that level, Anhydrous conditions can be induced and the RH reduced for further preservation. for example, Co-pending U.S. patent application Ser. No. 897.660 to POpiel et al. 10'N, 10'N, parallel IJ exposed to H was about 140 to about 168 vag. Discloses that it has a final weight. Same amount of oven dried N, ear ocase IJ has a dry weight of approximately 90 yaH. Therefore, 97%RH A 106L group of two U = IJ dried in mg). This figure expressed as a ratio of 11gIJ20/mg dry weight is approximately 0.7. Similar ratios are established for each type and species of desired organism. obtain. Therefore, determining the number of organisms present and the desired final water content 1 Thus, a drying endpoint can be determined based on the amount of water removed.

Following drying, remove oil by decantation or filtration if desired. It is possible. The resulting dried organism can be preserved as an oil suspension or paste. It can be done. Most of the oil and surfactants are recovered and recycled for further use. It can be done. Desiccated organisms should be dried in a suitable manner to maintain appropriate humidity and oxygen supply levels. may be stored in the container provided.

Maintain anhydrous living conditions for subsequent storage, transportation and handling if required Organisms may be suspended in hypertonic solutions (e.g., concentrated sucrose) that can . The oxygen demand of the IJ is typically 9 hg dry once anhydrous conditions are achieved. Less than 1 − of oxygen per day by dry weight.

At the end of the induction period, anhydrosis can be confirmed with reference to any of the criteria listed above.

Existence After the induction period, the nematodes can be stored in a suitable container.

The container maintains an appropriate range of R1'l and low IJ in anhydrous/concealed living conditions. Adequate oxygen supply must be provided to accommodate high levels of metabolism. this state The IJ of is approximately 0.6 to 1-1 of oxygen per 90 mg dry weight per day at 25°C. request. Storage methods should not be designed to provide this amount of available oxygen. Must be.

Oxygen demand is 90, corresponding to approximately 106 organisms (N, car oca 5ae) Based on tested species of vag IJ (referenced by weight; for other species , the relative amount of oxygen per organism varies with size, but 1 mg of IJ The required volume of oxygen per insect is relatively constant throughout the parasitic rod.

For storage containers that provide adequate oxygen in a small package, the package is Semi-permeable materials that allow oxygen to pass through as long as they are made to maintain the correct RH It can be made from.

For example, a saturated solution of potassium sulfate maintains 97% R11, and this solution It can also be mixed into a matrix, e.g. a hydrogel such as Terasorb. or within a fibrous matrix, such as cellulose or other fibers. .

The simplest implementation provides sufficient air space to meet the requirements of the storage IJ for the desired period of time. It is an airtight (and moisture-proof) container. In this embodiment, the exact Maintaining a good RH is automatic and does not require any special precautions. This is a small number Although practical for large numbers of nematodes, it is difficult to maintain large numbers of nematodes, e.g. If it is desired that the head The amount of space may be calculated based on the oxygen demand values above.

one person tax After storage, anhydrous nematode IJ must be rehydrated before use. . Upon rehydration, the anhydrous state is lost and the IJ regains metabolic activity. Survival ability and pathogenicity can be confirmed as explained above. Two common approaches to rehydration Approaches are suitable: direct hydration and "slow" hydration.

Direct hydration involves placing dried IJ in water or an isotonic aqueous solution, or containing an IJ preparation. Fill the compartment with water or an isotonic aqueous solution. After 2-3 hours of rehydration, the IJ is rehydrated. and can be tested for viability/virulence. Preferably used within 24 hours after rehydration should.

In some cases, it may be advantageous to rehydrate slowly. This kind of “relaxation” In rehydration, insects are kept in a controlled environment for a predetermined period of time, e.g. 100% RH air. It is placed in air or a relatively high osmotic solution for 20 hours. A suitable solution is For example, it contains 2.5% NaCl or 10% myo-inositol. This acclimatization After the period, soak the nematodes in water and rehydrate directly as described above. The above Although the procedure was suggested as slow rehydration of the preserved nematode preparation, rehydration It should be noted that it is also possible to rely on environmental conditions to provide moisture for hydration. Ru. Therefore, other methods involve placing anhydrous nematodes directly in the surrounding environment and Soil moisture or regular or slow irrigation to provide the necessary water I. But J.

train! L The following examples are illustrative and do not limit the invention.

five mice Koen Gong 111 Yukai - Kesari.

K bar 1. ＜ is other insect parasitic species grown under 1.1 standard culture conditions. te, Beddiy+g, R, A, Ann 104:117-120, infective nymphs were obtained. This document is , is incorporated herein for reference. Hyamin (hyamir+e) washed The IJ is suspended in sterile water at approximately 10' IJ/-.

Support 15-1 i kandogyu and tana±...p9'', qsu, 曵GenKma, fierce proverb~guidance) - explained in Preparation A Approximately 10"N, group-sized IJ prepared as above was added to 2.3 (g/g H2 Vacuum filtered in a Buch+ funnel to a water content of >0/dry weight. With a round bottom mixer IJ was mixed with soybean oil (20nd) in a round bottom flask equipped with a water jacket. . The suspension was maintained at a 20" C1 mixing speed of 200 pm.

Pressurized air was passed through a drying column (Drierite) to reduce R11 to approximately Q%. Feed it through the 0.7a1cm polymer spa... through the jar and into the flask. It bubbled. The effluent air is directed into a tared drying tube (Drierite). The rate of water loss was calculated from the weight increase at the outlet of the drying tube. air flow velocity was controlled using a rotameter and adjusted to a water loss rate of 0.225 g/11. . After 64 hours, the water content of the nematode was determined by Kart Fisher titration. As it turned out, it decreased to 0□7.

Decant the final dry IJ and add a humidifier to maintain 97% R1. ti Airtight bottle;: Store at 25℃ and save %. hill yo 2 (A) Approximately 10” N.eaz52-q,c-ppH prepared using liquid culture method ae IJ was vacuum filtered to a water content of 2.3 (g/g 20/dry weight) . IJ at 0.3% 5pan in a temperature-controlled reactor with a motor-driven mixer Mineral oil (2Off) containing 80 (ICI America's Inc.) ) mixed with The suspension was maintained at 20° C. and a mixing speed of 200 rpm.

Dry pressurized air (RIN = 5%) was bubbled into the tank through a sparger. . The rate of water loss (approximately 22 , 5g/h) was determined. Air flow velocity is controlled using a rotameter. Approximately 64 hours After 1 hour, the water content of the nematode decreased to 0.7 and was subjected to arm Fisher titration. It was approved.

(B) Approximately 10"N. 3 x 11J prepared as described in Preparation A to 2.3 Vacuum filter to a water content of (g/g I(20/dry weight)).

I in a temperature-controlled reactor equipped with a motor-driven mixer and a water jacket. Mix J with light mineral oil (2005).@turbidity at 20℃1, mixing speed 200r Maintain at pm.

Apply 1 liter of pressurized air to the drying column (Drierite) to reduce the RH to approximately 0%. Bubble into the tank through the sparge. Marl of container contents The rate of water loss is determined by Fisher titration. Rotameter Air Flow Velocity The water loss rate was adjusted to 225 g/h. After about 64 hours, Nemato The water content of the powder decreases to 0.7, as confirmed by Karl Fisher titration.

Decant the final dry IJ and include a humidifier to maintain 97% R11. Store in an airtight bottle at 2 S''C for various storage periods (e.g. 1, 24 hours). , 1 week, 2 weeks, 4 weeks, 2 months, 3 months, and 4 months). Hydrate and analyze for viability and infectivity.

(C) Using the same procedure as in Sections A and B, Li! - Approximately 7x in the two-pronged position of the moon μ lO”　N. Arise 姐臘also 1, or L static υ℃, t elm anus also use IJ and correspond Prepare a composition.

(D) Follow the same procedure as in sections A-C, but use 5% 02/N2 instead of dry air. , get similar results.

(E) Following the same procedure as in sections A to C, reduce dry air (5! RH) and add sodium sulfate. 1, while monitoring the drying rate with Karl Fisher titration. ) add slowly to get similar results.

lf! J1. ,=$− (Prescription. Empress-7 Yode 122 otsu-kichi. Ho-Z-g) (A) Solution "Prepared as follows. L? :. in distilled water (XIU).

K2S0a (1.65 Kg) to Proxel GXI, (lgo.Og, IC1Δmeriea’s lne. ) mixed with 109 dissolved MA, its 1lTe! "r-day 5orb" (1.0 Kg.

Industrial 5 services International.　　 Ine. ) mixed with Cover and store the Terri-sorb solution to swell. Prepare solution "B" by stirring occasionally.

Schiff 9 - Cap 1000 pieces / 100% plastic bottle) (Plastic bottle of size! Prepare the container and attach the sifter cap with pressure-sensitive polysulfone film ( Avery).

An oil-dried immatog suspension was prepared as described above and the concentration (#/d) was determined. Hanging Then add the suspension to 1. Put it in a centrifuge chamber (450-1500-suspension chamber 1.-section). The suspension was heated at 3000 rpm for 5 minutes. Centrifuged. Then remove the tube Then, decant the oil. Next, add solution A (35d/lo" nematode) Add to each tube and mix to make a paste. The resulting paste was then mixed with solution B. (450 g/l (1" nematode) and mixed. This mixture was x5 inch nylon bag (50 g/bag, from Moss Ushito Netting) The bag was heat-sealed.

The sealed bag is then covered with a film-covered sifter-cap. 00 pieces in a plastic container1. A transportable final product was obtained.

(B) Another storage medium was prepared as follows: sucrose in running water (11 g). (3400 g) and ProxeJ GXL (11.0 g), Prepare solution by mixing until completely dissolved. ri-snrb” (800 g) 1, cover and store 7 to swell, and make a solution. ``B'' was made by A.

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

叉1ヱ1. ．． ．． ”− (Re-water tax) Anhydrous Life 1 must be rehydrated before use.

Upon rehydration, the anhydrous state is eliminated and the IJ regains its normal metabolic activity. . IJ can be rehydrated quickly ("directly") or slowly. Once, re-water Once combined, [J] should preferably be used within about 24 hours.

(A)l! J! L-water: Place dried IJ in water or isotonic aqueous solution for 2 to 3 hours. absorb water. The amount of water or solution is at least initially removed from the IJ during the drying process. It should be equal to the amount of water removed. Preferably, the amount of liquid added is Restore lost water and rehydrate! There should be enough water to suspend J. be. Use IJ immediately before rehydration finishes to finish rehydration in the environment It is also possible. Therefore, IJ may be mixed with an appropriate amount of water and used immediately.

(B) Slow: Slow rehydration is, for example, Heteror It is preferentially used for less hard species of nematodes such as habditids. Ru. The dried IJ is placed in a controlled high humidity environment for approximately 20 hours. For example, the environment is 1 00% RH air.

Alternatively, for example, 2.5% NaCl or 10% myo-inositol, etc. Hypertonic solutions can be used. After approximately 20 hours of conditioning, IJ was dissolved in water or isotonic water. immerse in liquid and rehydrate by direct step (section A).

Once standing on a cusp (Takumi Masato Kunioka) Analyzing infective nymphs against Galleria mellonella larvae to determine pathogenicity. IJ (in suspension, at 501 J per analyzer), 45 A piece of Vhatman placed on the edge of a mm Petri dish No. 1 O, 5 on filter paper Pipette into an ail of water. Place 10 insect larvae on the filter paper, close the dish, Leave at 22°C and 80% RE. Mortality was measured for 2 hours daily for 30-50 hours after exposure. The time required to reach 50% mortality (LTse) was recorded at intervals and compared to the control value. Compare.

shirutan - standing (Panri) 50 g of IJ matrix composition prepared as in Example 3 above to 8 oz. Dilute and stir the mixture in the container for 10-15 seconds to release the IJ. Then liquid Decant the body and use a suitable delivery device, e.g., hose sprayer, tank sprayer. Add to bowl, watering can, etc. If using a watering can, preferably use IJ liquid. , dilute to 2 gallons. If using other means, dilute the liquid to 1 gallon. Ru.

The solution is then applied to the soil around the affected area (i.e. not directly to the foliage) . The amount of solution is sufficient to treat approximately 225 square feet of soil. unused Store the solution frozen.

(A) Best for vegetable gardens (e.g., root worms, click beetle larvae, onion worms, For treatment of carrot weevils, cutworms, chinensis beetles, mole crickets, flea beetles, etc.) The solution is typically applied to 225 square feet of soil in a 450-foot field row. Use in 3 inch strips on each side or in a 6 inch diameter circle around the 1100 plants. Ru.

(B) Lawn insects (e.g., bean beetles, lawn butterfly and moth larvae, insects, masked scarab beetles, grub beetles, grubs, woodlice, and click beetles. To treat larvae, fall armyworm larvae, larvae of fall armyworms, mole crickets, etc.), apply the solution at 225 ml. Simply spray up to one foot.

(C) Rhodopentron, azalea, cyclamen, chrysanthemum, tulip, iris pests of ornamental plants such as primroses, primroses (e.g. Lotsuru weevil, grub, rose-shaped scarab beetle, Hatake-seed beetle, bean beetle For the treatment of Japanese beetles, Japanese beetles, Japanese beetles, etc., the solution is generally Use in 3-inch strips on each side of a 450-foot field row in 450 feet of soil. or use in a 6 inch diameter circle around 1100 plants or use 6 inch Use directly on 1100 potted plants in containers.

(D) For the eradication of fire ants, 50 g of the product prepared in Example 3 above was used. J Matrix Dilute the composition to 20 oz., stir the mixture in a container for 10-15 seconds, and to release. Four ounces of this suspension was then added to a watering can and diluted to one gallon. do. Apply the solution to the ant mound. Continue this way until all 20 ounces are used. Repeat the process to cover up to five anthills.

Following application, water the anthill and keep it moist. Preferably once or twice a week to saturate the anthill.

Submission of translation of written amendment (Article 184-8 of the Patent Law) August 1990

Claims (25)

[Claims] 1. A method of drying biological material, the method comprising: drying biological material in a suitable oil; suspending to form an oily suspension; and including the addition of a suitable desiccant for the removal of moisture, said desiccant being It is well dispersed in the suspension. 2. The method according to claim 1, wherein the desiccant is a gas having a relative temperature of less than 100%. Law. 3. the biological material is a living virus, bacteria, or other microorganism; 3. The method of claim 2, wherein the cell is a cell of a multicellular organism. 4. 3. The method of claim 2, wherein the biological material is a nematode. 5. The oil contains a non-toxic surfactant in an amount sufficient to prevent nematode agglomeration. 5. The method according to claim 4, further comprising: 6. The nematode is Steinernematid and Heterorh. The person according to claim 4, which is an infectious nymph selected from abditid nematode. Law. 7. Infectious nymphs of the preconsort nematode are N. Carpocapsae sp. The method described in claim 6. 8. the infectious nymph of the nematode is of the genus Heterorhabditis; The method according to claim 6. 9. The infective nymphs of the nematode are N. Claim 6, which is a species of Bibionis. The method described in. 10. A composition suitable for insect control, the composition being suitable for controlling insect parasitism in anhydrous conditions. infectious nymph nematoda, and Contains an amount of suitable oil sufficient to suspend the nematodes. 11. Claims further comprising a non-hazardous surfactant in an amount sufficient to emulsify with water. The composition according to item 10. 12. The infective nymphs of the nematode are Steinernematid and H. The set according to claim 11, selected from etororhabditid infective nymphs. A product. 13. The infective nymphs of the nematode are N. carpocapsae species, The composition according to claim 12. 14. The infective nymphs of the nematode are H. is of the genus heliothidis, The composition according to claim 12. 15. The infective nymphs of the nematode are N. bibionis sp. 13. The composition according to 12. 16. An insecticide composition comprising: The composition contains insect parasitic infectious nymph nematodes in an anhydrous state; The anhydrous state in the product is caused by the dehydration of the oil phase. 17. Claim 16, wherein at least 70% of the infective nymphal nematodes are alive. The composition described in . 18. Claim 17, wherein at least 85% of the infective nymphal nematodes are alive. The composition described in . 19. Claim 18, wherein at least 95% of the infective nymphal nematodes are alive. The composition described in . 20. A composition of an insect parasitic infectious nymph with a long shelf life, the composition comprising: Insect-parasitic infectious nymph nematode in the shaded state, free from water activity of approximately 0.97. Contains a granular matrix material that absorbs harmful storage solutions and water. 21. 21. The composition of claim 20, further comprising an effective amount of an antimicrobial agent. 22. The storage solution may be an aqueous salt solution, an aqueous sugar solution, an aqueous sugar alcohol solution, or 21. The composition of claim 20, wherein the composition is selected from aqueous solutions of low molecular weight polymers. 23. The storage solution is NaCl aqueous solution, K2SO4 aqueous solution, potassium acetate aqueous solution. liquid, fructose aqueous solution, maltose aqueous solution, myo-inositol aqueous solution, Solpi an aqueous solution of PEG600, an aqueous solution of glycerol, and an aqueous solution of PEG600. A composition according to claim 22. 24. The storage liquid is selected from an aqueous NaCl solution and an aqueous myo-inositol solution. 24. The composition of claim 23. 25. A granular matrix material that absorbs water envelops Terri-sorbTM. 21. The composition of claim 20, comprising: