EP0444024A1 - Dessiccation de substances biologiques dans une suspension d'huile - Google Patents

Dessiccation de substances biologiques dans une suspension d'huile

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Publication number
EP0444024A1
EP0444024A1 EP89902874A EP89902874A EP0444024A1 EP 0444024 A1 EP0444024 A1 EP 0444024A1 EP 89902874 A EP89902874 A EP 89902874A EP 89902874 A EP89902874 A EP 89902874A EP 0444024 A1 EP0444024 A1 EP 0444024A1
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EP
European Patent Office
Prior art keywords
composition
infective
nematodes
nematode
water
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP89902874A
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German (de)
English (en)
Other versions
EP0444024A4 (en
Inventor
Milton J. Friedman
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BIOSYS
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BIOSYS
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Publication of EP0444024A1 publication Critical patent/EP0444024A1/fr
Publication of EP0444024A4 publication Critical patent/EP0444024A4/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/30—Rearing or breeding invertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A01N63/12—Nematodes

Definitions

  • the invention relates to insect control employing biological agents especially for the benefit of agriculture, garden and household insects.
  • 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.
  • 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.
  • stage 2 larvae of Steinernematid and Hetero- rhabditid nematodes develop instead into "stage 3 infec ⁇ tive juveniles" or "Us".
  • stage 3 infec ⁇ tive juveniles or "Us”.
  • 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.
  • Steinernematid and Hetero ⁇ rhabditid nematode biology are 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.
  • 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.
  • 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.
  • 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.
  • cryobiosis i.e., reduced metab ⁇ olism at low, usually freezing temperatures.
  • anhydrobiosis which is induced by evaporative desiccation, and the closely related osmobiosis induced by osmotic desicca ⁇ tion.
  • 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).
  • the method of the invention also provides for convenient and economical process scale-up, making com ⁇ pitchal practice of the invention feasible.
  • 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.
  • 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.
  • 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 ⁇ 4), 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.
  • 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.
  • 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.
  • 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) .
  • 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).
  • 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.
  • 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.), SanwetTM (Celanese, a starch- grafted polyacrylate) , DrytechTM (Dow, a cross-linked polypropenoic acid).
  • an absorbent particulate matrix material such as Terri-sorb tM (Industrial Services International, Inc.), SanwetTM (Celanese, a starch- grafted polyacrylate) , DrytechTM (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.
  • 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.
  • 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.
  • 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.
  • a sifter cap for mechanical strength
  • a suitable film e.g., poly- sulfone, polystyrene, TyvekTM, etc.
  • one may seal the container completely, providing sufficient oxygen in the headspace of the container.
  • 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.
  • Bio materials 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.
  • a state of reduced metabolic demand e.g., an anhydrobiotic state
  • 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.
  • 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, ⁇ ntaphelenchidae, Mermithidae, Neotylenchidae, Sphaerulariidae, and Tetradonematidae.
  • the most important families for commercial use are the Steinernematidae and the Heterorhabditidae.
  • Neoaplactana refers 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.
  • “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. )
  • anhydrobiotic or cryptobiotic state of the Us of the invention can be evidenced by any of the following criteria: ⁇ l 3-
  • 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.
  • RH relative humidity
  • 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.
  • 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.
  • infective juveniles or about 25 kg of wet product, can be grown per week in about 152 kg of culture medium.
  • 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.
  • 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 ha ⁇ been achieved by this method.
  • 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.
  • the critical aspect of the process of the invention is the control over the rate and extent of drying.
  • the instant invention provides extraordinar con- trol over the drying rate.
  • 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.
  • the nem ⁇ atodes may clump, resulting in uneven desiccation.
  • 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.
  • HLB hydrophile/lipophile balance
  • the desiccation process may be conducted over
  • -I a range of temperatures, from about 0°C to about 50°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
  • a liquid such as a detergent or other sur ⁇ factant, or, preferably, a gas, such as air or nitrogen/ oxygen mixtures.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • This figure, expressed as a ratio of mg H2 ⁇ /mg dry weight, is about 0.7.
  • a similar ratio may be estab ⁇ lished for each type and species of organism desired.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • anhydrobiotic nematodes Us 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.”
  • 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.
  • rehydration may advantageously be less abrupt.
  • 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.
  • 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.
  • the anhydrobiotic nematodes may be placed in the environment directly, and soil moisture or normal or deliberate irrigation can furnish the necessary water.
  • 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 H2 ⁇ /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 ⁇ 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.
  • 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 ⁇ C in air-tight vials containing humidifying agents to maintain an RH of 97%.
  • a sample of Us is rehydrated and assayed for viability and infectivity.
  • 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-sorbTM (1.0 Kg, Industrial Services International, Inc.). The Terri- - sorbTM, 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.
  • 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-sorbTM (800 g), covered, and set asic'e to swell to prepare solution "B".
  • 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.
  • 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.
  • 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 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.
  • solutions with high osmotic pressure for example, 2.5% NaCl, 10% myoinositol, and the like.
  • the Us are immersed in water or isotonic aqueous solution and rehydrated by the direct process (part A) .
  • Pathogenicity is determined by assaying the infective juveniles against Galleria mellonella larvae.
  • the Us in suspension, at 50 Us per assay dish
  • the Us 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 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 suitable delivery device e.g., hose-end sprayer, tank sprayer, watering can, etc.
  • the IJ liquid is preferably diluted to 2 gallons.
  • the liquid is diluted to 1 gallon.
  • the resulting solution is then applied to the soil around the affected area (i.e
  • 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.

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Abstract

Est décrit un procédé pour induire un état anhydrobiotique chez les sujets jeunes infectieux des nématodes entomogènes. Ces sujets jeunes infectieux anhydrobiotiques se prêtent particulièrement au stockage et au transport. Ce procédé permet une dessiccation soigneusement contrôlée desdits sujets en les mettant en suspension dans de l'huile, et en éliminant graduellement l'eau à l'aide d'un agent déshydratant, notamment en utilisant de l'air à une humidité relative inférieure à 100 %, en présence éventuelle d'un tensio-actif.
EP19890902874 1988-02-10 1989-02-06 Desiccation of biological materials in oil suspension Withdrawn EP0444024A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15485088A 1988-02-10 1988-02-10
US154850 1988-02-10

Publications (2)

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EP0444024A1 true EP0444024A1 (fr) 1991-09-04
EP0444024A4 EP0444024A4 (en) 1992-09-09

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EP19890902874 Withdrawn EP0444024A4 (en) 1988-02-10 1989-02-06 Desiccation of biological materials in oil suspension

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EP (1) EP0444024A4 (fr)
JP (1) JPH03503526A (fr)
AU (1) AU3192789A (fr)
WO (1) WO1989007446A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU665398B2 (en) * 1992-02-28 1996-01-04 Commonwealth Scientific And Industrial Research Organisation Preparations containing entomopathogenic nematodes for biological control of the banana weevil
WO1994019940A1 (fr) * 1993-03-04 1994-09-15 Commonwealth Scientific And Industrial Research Organisation Procede d'emballage de nematodes entomopathogenes pour stockage et transport
AUPP616798A0 (en) * 1998-09-28 1998-10-22 Commonwealth Scientific And Industrial Research Organisation Method and apparatus for the storage of entomapathogenic nematodes
US6399118B1 (en) * 2001-06-29 2002-06-04 Fish Biotech Ltd. Process for storing enriched nematodes
US20050266027A1 (en) 2004-05-25 2005-12-01 Watson James B Live organism product
GB0720543D0 (en) * 2007-10-19 2007-11-28 Arab Orangisation For Agricult Composition for and method of pest control
DE112019004535A5 (de) * 2018-09-11 2021-06-02 Mann+Hummel Gmbh Trocknungssystem, trocknerkartusche und verwendung einer trocknerkartusche

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Publication number Priority date Publication date Assignee Title
US2929150A (en) * 1956-05-07 1960-03-22 Standard Brands Inc Dehydration
US3034968A (en) * 1959-04-09 1962-05-15 Standard Brands Inc Process for preparing viable dry bacteria and molds
US4178366A (en) * 1975-08-26 1979-12-11 Commonwealth Scientific And Industrial Research Organization Nematode larvae as biological insecticides
SU810758A1 (ru) * 1978-11-20 1981-03-07 Государственный Союзный Трест Помонтажу Электрооборудования Электро-Станций И Подстанций "Электроюжмонтаж" Способ осушки минеральных масел
US4518696A (en) * 1983-01-11 1985-05-21 Chr. Hansen's Laboratory, Inc. Stabilized liquid bacterial suspension for oral administration to animals
FI84621C (fi) * 1983-12-29 1991-12-27 Daicel Chem Dehydraticeringsfoerfarande.

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EP0444024A4 (en) 1992-09-09
WO1989007446A1 (fr) 1989-08-24
AU3192789A (en) 1989-09-06
JPH03503526A (ja) 1991-08-08

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