AU664644B2 - Method for the storage of entomopathogenic nematodes - Google Patents

Description

OPI DATE 29/03/94 APPLN. ID 48097/93 I IIII IIIIlI I AOJP DATE 23/06/94 PCT NUMBER PCT/AU93/00465 11111111111111111 111111 111 1 1111111 AU9348097 Or'CT) (51) International Patent Classification A01K 67/033, B65D 85/50 A01N 1/00, 1/02 (11) International Publication Number: Al (43) International Publication Date: WO 94/05150 17 March 1994 (17.03.94) (21) International Application Number: PCT/AU93/00465 (22) International Filing Date: 10 September 1993 (10.09.93) Priority data: PL 4791 10 September 1992 (10.09.92) AU (71) Applicant (for all designated States except US): COMMON- WEALTH SCIENTIFIC AND INDUSTRIAL RE- SEARCH ORGANISATION [AU/AU]; Limestone Avenue, Campbell, ACT 2601 (AU).

(72) Inventors; and Inventors/Applicants (for US onlv): BEDDING, Robin, Anthony [AU/AU]; 13 Welch Place, Flynn, ACT 2615 BUTLER, Karen, Louise [AU/AU]; 21 Blackman Crescent, Macquarie, ACT 2614 (AU).

(74) Agents: DUNCAN. Alan, David et al.; Davies Collison Cave, I Little Collins Street, Melbourne, VIC 3000

(AU).

(81) Designated States: AT, AU, BB, BG, BR, BY, CA, CH, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ, LK, LU, LV, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, UZ, VN, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published With international search report.

664,644 (54)Title: METHOD FOR THE STORAGE OF ENTOMOPATHOGENIC NEMATODES (57) Abstract Long-term storage of infective third stage juveniles of nematodes belonging to the genera Steinernema or Heterorhabditis is effected by mixing together an aqueous cream of the nematodes and particles of a highly water-absorptive material. The water present in the mixture is such that the mixture, after equilibrating, has a water activity in the range of from 0.80 to 0.995 (preferably 0.95 to 0.99). The preferred absorbent material is anhydrous polyacrylamide gel in the form of particles having an average mass of 0.01 gm. For optimal survival of the nematodes, the equilibrated mixture should be stored at a temperature in the range of from I °C to 30 in an atmosphere which maintains the water activity of the mixture in the range of from 0.80 to 0.995.

I I i WO 94/05150 PCT/AU93/00465 1 TITLE: "METHOD FOR THE STORAGE OF ENTOMOPATHOGENIC NEMATODES" Technical Field This invention concerns the storage for transport or future use of entomopathogenic nematodes. More particularly it concerns the storage and transport of the third stage infective juveniles of nematodes belonging to the genera Steinernema (synonym Neoaplectana) and Heterorhabditis (synonym Chromonema) using highly absorptive substances, such as polyacrylamide gels, at specific water activities.

Background It is well known that entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have considerable potential for the biological control of a number of insect pests. Infective third stage juveniles (J3) of these nematodes (which can survive many weeks in the environment without feeding) are able to seek out an insect, penetrate into the insect's haemocoel and there release specific symbiotic bacteria (Xenorhabdus species).

The bacteria kill the insect within a day or so and provide suitable conditions for nematode reproduction.

Methods for the large scale in-vitro mass rearing of these nematodes in solid culture have been described by R A Bedding in, for example, the specifications of his US patents Nos 4,178,366 and 4,334,498, the specification of Australian patent No 509,879, and his papers in Nematologica, Volume 27, pages 109 to 114, 1981 (entitled "Low cost in-vitro mass production of Neoaplectana and I I WO 94/05150 PCT/AU93/00465 -2 Heterorhabditis species (Nematoda) for field control of insect pests") and in Annals of Applied Biology, Volume 104, pages 117 to 120, 1984 (entitled "Large scale production, storage and transport of the insect-parasitic nematodes Neoaplectana spp. and Heterorhabditis spp.").

Further developments of these techniques are described in the specification of International patent application No PCT/AU91/00136, which is WIPO Publication No WO 91/15569. Nematodes have also been mass produced in liquid culture as detailed by G W Pace et al (see WIPO Publication No 86/01074), and by M J Friedman, S L Langston and S Pollitt (see the specification of International patent application No PCT/US88/04124, which is WIPO Publication No WO 89/04602). Thus large quantities of nematodes can be produced in a variety of ways and to use these nematodes commercially, it is necessary to have effective ways of storing them and transporting them to the end user.

In his aforementioned paper in Annals of Applied Biology, R A Bedding describes the use of crumbed polyether polyurethane foam as a carrier for stored nematodes in polyethylene bags. Although this is an effective means of storage at low temperatures, it suffers the cisadvantage that to store the nematodes for long periods, forced aeration of the carrier is needed. In addition, extraction of the nematodes from the foam takes from one to two hours.

T Yukawa and J M Pitt, in the specification of International patent application No PCT/AU85/00020, have described a method of storing nematodes which involves WO 94/05150 PCT/AU93/00465 -3mixing a cream of infective juvenile nematodes with powdered activated charcoal. The nematodes are then able to survive at high densities under anaerobic or substantially anaerobic conditions for long periods of time provided they are kept at low temperatures. Yukawa and Pitt attach considerable impcrtance to the highly adsorptive (as distinct from absorptive) properties of the activated charcoal. Their method, however, has a number of disadvantages, namely: this method works satisfactorily only with the species Steinernema carpocapsae; activated charcoal is extremely unpleasant to handle; activated charcoal is expensive; the nematodes die within a few days if the package in which they are contained is exposed to a temperature higher than about 15°C; and there are constraints on the maximum size of the package in which the stored nematodes can be held, and on the number of nematodes that can be contained in the package.

I Popiel, K D Holtemann, I Glazer and C Womersley, in the specification of International patent application No PCT/US87/02043 (which is WIPO Publication No WO 88/01134), have described a storage technique which utilises a phenomenon previously associated with many other species of nematodes. That phenomenon is that when the nematodes are exposed to relative humidities of around 97 per cent for several days, they change their biochemical composition significantly and enter a state of dormancy, usually termed cryptobiosis (sometimes loosely called WO 94/05150 PCT/AU93/00465 4 anhydrobiosis when the surface water is removed from the nematodes). When the nematodes are in such a state, they use up their food reserves much more slowly than otherwise, and thus they can survive for much longer periods. Popiel et al found that entomopathogenic nematodes, like many other nematodes, can be retained in the "apparent anhydrobiotic" state if they are held at a relative humidity of about 97 per cent. In fact, W R Simons and G 0 Poinar (in a paper entitled "The Ability of Neoaplectana carpocapsae (Steinernematidae: Nematodea) to Survive Extended Periods of Desiccation", which was published in the Journal of Invertebrate Pathology, Volume 22, pages 228-230, 1973) had earlier shown that if infective juveniles of Steinernema carpocapsae (one species of entomopathogenic nematode) are kept at 96 per cent humidity for 12 hours, and then at 94 per cent for 12 hours, they survive at low humidities much better than nematodes which have not been "conditioned" in this manner.

In the aforementioned WIPO Publication No WO 88/01134, I Popiel et al describe a method for putting an aqueous suspension of infective juvenile entomopathogenic nematodes into a state of anhydrobiosis which has two steps, namely: the removal of most of the surface water from the suspension by vacuum filtering a high-density layer of nematodes only 1 to 4 mm thick, then evaporating off the remaining surface water by holding the nematodes at a relative humidity of about 97 per cent; the evaporation is effected until the nematodes form a characteristic network or foam, or until a given number of organisms have reached a predetermined WO 94/05150 PCT/AU93/00465 weight (this weight varies according to the species of entomopathogenic nematode being treated); and the induction of cryptobiosis by exposing the nematodes to a relative humidity of 97 per cent 2 per cent for a minimum of 2 days (this range of relative humidity is achieved either by placing the nematode network in a desiccator where the relative humidity is controlled by sulphuric acid/water mixes, or by using environmental chambers through which air having the prescribed relative humidity is circulated).

After induction of cryptobiosis, Popiel et al store their nematodes at relative humidities in the range of from 50 to 94 per cent, or from 95 to 99 per cent, while allowing for adequate oxygen supply. These humidities are maintained with a hydrogel or fibrous matrix that has been impregnated with a saturated solution of potassium sulphate (which maintains a relative humidity of 97 per cent), (ii) using a saturated solution of potassium nitrate (which has a relative humidity of 94 per cent), or (iii) using solutions of sulphuric acid. The preferred technique involves storing the desiccated nematodes in a container in which there is also a saturated salt solution in a package made from a hydrophobic, vapour permeable membrane, such as GORETEX (trade mark) material. Alternatively, the cryptobiotic nematodes are stored in an airtight and moisture tight container with sufficient air space to M accommodate the needs of the stored infective juveniles.

WO 94/05150 PCT/AU93/00465 6 Popiel et al claim that, using this technique, nematodes of the species Steinernema carpocapsae, Steinernema feltiae (synonym bibionis) and Heterorhabditis bacteriophora (synonym heliothidis) survived for several months with undiminished infectivity.

The above-mentioned methods of Popiel et al also have a number of drawbacks, as follows: 1. In order to remove surplus water, a thin layer of nematodes is subjected to vacuum filtration followed by air drying. This takes much space and time. In addition, it is possible that nematodes may be excessively dried, particularly under commercial production conditions.

2. Only thin layers of nematodes can be exposed to a relative humidity of around 97 per cent. Thus it is impractical or labour intensive to treat hundreds of kilograms of nematodes in aqueous suspension at one time. To treat large quantities of nematodes in this manner, expensive environmental chambers would have to be used.

3. The maintenance of very precise relative humidities, varying by less than 2 per cent, using the described laboratory techniques, is not commercially feasible.

4. After cryptobiosis has been induced, the thin layer of nematodes must be removed from the treatment site and placed in containers for storage, with special I provisions for maintaining the relative humidity near, but below, 100 per cent.

The technique does not work well with nematodes of the Heterorhabditis species.

I i -e c; i WO 94/05150 PCT/AU93/00465 7 Storage of entomopathogenic J3 nematodes at a relative humidity of at least 95 per cent is also a feature of the insect traps (designed for cockroaches) that are described in the specification of US patent No 5,172,514 (to T Weber, R Georgis, P Pruitt and J Wren; assignors to Biosys Corporation). In these cockroach traps, a hydrogel structure, made from a polysaccharide (such as agar, carrageenan or tragacanth), or a porous matrix of sponge, polyurethane foam or polyether foam, contains the nematodes, which "nictate" or "stand up and wiggle" on a relatively dry surface provided on the surface of the nematode-containing medium. The relative humidity of the air surrounding the nematode-containing medium is maintained at a value of at least 95 per cent by enclosing, with the medium, a quantity of "a water-liberating gel, such as swelled polyacrylamide". The effective life of the traps is not stated in the specification of US patent No 5,172,514, and although there is an implication that the effective life is significantly more than seven days, the baits in the traps are not really examples of "stored" nematodes, for the nematodes are maintained in their active state to parasitise cockroaches (in contrast to the stete of reduced metabolism that is induced when nematodes are stored for future use).

In the specification of United States Patent No 5,042,427 (which corresponds to the specification of International patent application No PCT/AU88/00127), R A Bedding showed how clay (particularly attapulgite clay) can be used to store entomopathogenic nematodes, either by forming a homogenous mixture of nematodes with the clay (which is not WO 94/05150 PCT/AU93/00465 -8a highly absorbent material) or by making a sandwich consisting of a layer of nematode cream between two layers of clay. This method (which is currently used commercially) is believed to owe its success to three major factors, namely the nematodes are constrained from moving (thus conserving food reserves), (ii) perhaps, the clay adsorbs potentially toxic nematode excretory products, and (iii) by exposing nematodes to humidities of less than 100 per cent, preferably near to a water activity of 0.97, they are induced into the cryptobiotic state. A range of entomopathogenic nematode species can be stored in this way using either chips of calcined attapulgite clay or coarsely iiilled, calcined attapulgite clay.

Although 1t has been commercially successful, this method also has some disadvantages, as follows: 1. There is a marked decline in the survival of all nematode species after storage for 2 months at a temperature in the range of from 23 0 C to 28°C.

2. When suspending the nematodes in water after storage, in preparation for their distribution by spraying, it is difficult to remove all the larger particles of clay from the suspension. These clay particles tend to block spray nozzles. And smaller clay particles cause the nematode suspension to have the appearance of muddy water.

3. More than half the weight of the stored nematode product is clay, which adds to transport costs when air freight is used.

4. There is a limit to the permissible thickness of the stored nematode product when very large quantities of WO 94/05150 PCT/AU93/00465 9 nentatodes have to be "stored" because, although the nematodes are largely cryptobiotic, and thus have a significantly reduced metabolism, it is necessary for some oxygen to be present in the stored product.

Disclosure of the Present Invention It is an object of the present invention to provide a method of storing entomopathogenic nematodes for transport or future use which avoids the major disadvantages, noted above, of the methods previously utilised or proposed for these purposes, and which can be applied to both small quantities of nematodes and large volumes of nematodes produced for commercial purposes.

This objective is achieved by using highly absorptive (but not necessarily adsorptive) gels, or other highly absorptive substances, to absorb surface water from an aqueous cream of nematodes and, having done this, to accurately provide a water activity within a narrow range suitable for inducing cryptobiosis of the nematodes without adversely affecting their survival.

"Water activity" is defined as the ratio RH/100, where RH would be the relative humidity of the surrounding atmosphere in a sealed system.

The term "highly absorptive substance" will be understood by persons who work with absorptive materials and are familiar with their classification. Without limiting the general understanding of the term "highly absorptive", the preferred highly water-absorptive substances of the present i WO 94/05150 PCT/AU93/00465 invention are substances which absorb at least 75 per cent of their own weight of water to have a water activity in the range of from 0.80 to 0.995 When carrying out the technique of the present invention, anhydrous or nearly anhydrous absorbent particles are preferably mixed with a nematode cream in predetermined combinations so that both the absorption of the surface (free) water and the establishment of the desired water activity are achieved without requiring further adjustment of the resultant mixture (for example, the addition of extra water). Fungicides and/or antibiotics may be mixed with the nematode cream or with the absorbent prior mixing the nematode cream and absorbent together.

The benefits of using highly absorbent substances are believed to be five-fold. Firstly, they remove surf;. o water rapidly so that the nematodes form a "foam" withiL a few minutes. Where particles of an absorbent (such as polyacrylamide gel) that swells in the presence of water are used, large interstitial spaces are formed between the -0 swollen particles. These spaces provide room for nematode foaming and for the better diffusion of gases. Thus an aerated matrix is rapidly produced and the nematodes suffer the effects of anaerobic conditions for a short time only.

Secondly, because these absorbents remove water from a three dimensional matrix, large volumes of nematodes can be processed at the same time, without the need to prepare thin layers of nematodes on fiJ lter discs or the like.

Thirdly, by mixing the appropriate combinations of nematodes and absorbent, the establishment of the required i i i- WO 94/05150 PC/AU93/00465 i 11water activity can be easily achieved. This means that there is a prompt start of the induction of cryptobiosis, accompanied by changes in the biochemical composition of the nematodes so that, for instance, the levels of glycerol and trehalose in the nematodes rise and (particularly in the case of Heterorhabditis species) coiling may take place. Fourthly, the mixture not only induces cryptobiosis in the nematodes, but it also, thereafter, maintains the water activity at a value which is appropriate for the continual storage of the nematodes. Fifthly, the weight of absorbent used can be significantly less than that of the nematode cream.

Nematodes stored in this manner using highly absorbent substances can be reactivated by dispersing the nematodes and absorbent in water. However, they are most effectively reactivated by adding water to the nematode/absorbent combination so that the water activity exceeds 0.995 but remains below 1.00; the nematode/absorbent combination being then left for two to four hours before finally dispersing the nematodes and absorbent in a surplus of water. Where particles of the absorbent swell in the presence of water (for example, when the absorbent is polyacrylamide gel), it is then a simple matter to sieve out those particles to leave a clean suspension of infective juvenile nematodes.

Thus, according to the present invention, a method of storing third stage infective juveniles (J3) of entomopathogenic nematodes comprises forming a mixture of an aqueous concentrate (cream) of clean J3 nematodes and WO 94/05150 PCT/AU93/00465 12 particles of a highly water-absorbent (as defined above) material, the proportions of the aqueous concentrate and water-absorbent material being such that the mixture, after equilibrating, has a water activity in the range of from 0.92 to 0.995.

Preferably the water activity of the mixture is from 0.92 to 0.995, and most preferably between 0.95 and 0.99. The final value of the water activity of the mixture may take from 24 to 72 hours to establish.

Depending on the particular absorbent, the absorbent may comprise from about 15 per cent to 75 per cent of the resulting mixture. Before using a particular kind of absorbent, experiments should be conducted to determine the water activity resulting from various combinations of it with nematode cream. In this connection, the water content of the nematode cream must first be standardised. This can be achieved by making the number of clean infective juvenile nematodes per gram constant for any particular species of nemnatode or by standardising the viscosity of the nematode cream used. The more viscous the nematode cream, the lower the quantity of absorbent that is required to achieve a particular water activity of the final absorbent/nematodes mixture. The exact water activity required to give optimal induction of cryptobiosis varies a little from species to species of entomopathogenic nematodes. After a period of induction (usually 2 to 3 days), the nematodes may be stored with the mixture having the attained water activity, or the water activity

I:

WO 94/05159 PCT/AU93/00465 13 of the resulting combination may be lowered by adding further absorbent.

It has been discovered that when certain absorbents are combined with J3 entomopathogenic nematodes so that a water activity of between 0.92 and 0.995 is achieved, there is a greatly improved longevity of the nematodes after storage at a wide range of temperatures. The mixture may be stored in a refrigerator, having a temperature as low as 1 0 C, or at temperatures up to 30 0 C. Furthermore, after such storage, the nematodes can be readily suspended in water simply by mixing the absorbent/nematodes combination with water and sieving out the absorbent. The nematode suspension thus obtained can be applied directly to soil or plants, by spraying or by other means, for the control of insect pests.

Among the various highly absorbent materials that have been tested by the present inventors, satisfactory results have been achieved using methyl cellulose powder, polyacrylate starch gel powder, and a mixture of anhydrous polyacrylamide gel and starch powder. However, particulate anhydrous polyacrylamide gels (PAGs) have been found to give especially good results. A PAG having an individual dry particle weight of from 0.005 gm to 0.02 gm is preferred because, after swelling, such particles provide adequate interstitial space for nematode foaming and gaseous diffusion.

A detailed description of the way in which nematodes are prepared for storage in a nematode and absorbent WO 94/05150 PCT/AU93/00465 14 combination, and examples of realisations of the present invention, will now be described. In the following description, reference will be made to the accompanying drawings.

Brief Description of the Drawings Figure 1 is a graph showing the water activity of various mixtures of polyacrylamide gel and water.

Figure 2 is graphical presentation of mortality data for various absorbent/nematodes combinations of the nematode species Steinernema carpocapsae.

Figure 3 is a graphical presentation of the average dry weight of nematodes carpocapsae) in stored samples after different storage times.

Preparation of the Nematodes Prior to Storing The manner in which the nematodes are reared and processed prior to their combination with an absorbent has an important influence on their further longevity. The preferred method of nematode production is described in the aforementioned 1984 Annals of Applied Biology paper by R A Bedding, modified as described in the specification of the aforementioned International patent application No PCT/AU91/00136. Obviously, if nematodes are subjected to adverse conditions such as high temperature, anoxia, exposure to pathogens or bacterial toxins, or mechanical or chemical damage, their longevity may be reduced. In addition, the quality of the media on which the nematodes have been reared and the manner in which they have been

.A

WO 94/05150 PCT/AU93/00465 reared affect their subsequent longevity. For example, entomopathogenic J3 nematodes that have been reared in a liquid culture frequently store less well than those reared in a solid culture (this is particularly true of Heterorhabditis species). Apart from these factors, utilisation by the nematodes of their internal food reserves prior to storing should be reduced to a minimum.

Also, it is important that the nematodes are as clean as possible when added to the absorbent so that microbial degeneration of the whole mixture is not encouraged and (ii) the subsequent evaluation of the amount of surface water in the nematode cream can be effected more precisely (either by counting the number of nematodes per mass of cream or by measuring the viscosity of the cream).

All the nematodes used in the series of trials conducted by the present inventors to confirm the efficacy of the present invention were reared and extracted using the Smethods described by R A Bedding, M S Stanfield and G W Crompton in the specification of International patent application No PCT/AU91/00136 (WIPO Publication No WO 91/15569). However, the nematodes may be reared on insects in vivo or in liquid culture, provided the nematodes are free from appreciable amounts of extraneous matter remaining from the culture medium and are relatively free from nematode stages other than J3 (preferably no adult nematodes are present and certainly no more than 2 per cent of the nematodes should be adults).

The entomopathogenic nematodes reared for the trials by the present inventors, as in the technique described in the WO 94/05150 PCT/AU93/00465 16 specification of International patent application No PCT/AU88/00127, were sedimented after washing and the excess water was drained off. The sediment of nematodes was then pumped from settling tanks into sieves lined with cloth through which the water, but not the nematodes, could pass. Water was drained off in this way and further water was removed by stirring the nematode cream while draining.

In some trials, still further water was removed by gathering up the cloth edges to enclose the nematode mass in the cloth before squeezing out some of the remaining water. The resulting cream of nematodes contained from to 3.5 million J3 nematodes per gram, depending upon the species involved and the amount of inter-nematode water remaining. For some experiments, one or more of various antifungal and antibiotic agents were added to and mixed with the sedimented nematodes while they were still in the tanks. Hence, after removal of much of the surface water (and thus also of most of the antibiotic and/or antifungal agent), some of the antibiotic and/or antifungal agent remained to be absorbed by the absorbent in the storage process. The antifungal agents used by the present inventors were humic acid, brown coal dust (which contains up to about 50 per cent humic acid), powdered sulphur, sulphurous acid, and a mixture of sulphurous acid and powdered sulphur.

I

Combining the Nematodes and the Absorbent The technique usually adopted for combining the nematodes and the absorbent material was as follows. Particles of the absorbent were weighed and added to the appropriate weight of nematodes. (The appropriate weights were r WO 94/05150 PCr/AU93/00465 -17 determined by prior experimentation to ascertain which combinations fall within the required range of water activities). The absorbent and nematodes were then immediately stirred and mixed together so that the absorbent particles were evenly distributed in the mixture.

The water activity of the mixture of nematodes and highly water-absorbent particles has to be in the range of from 0.80 to 0.995. As noted above, the water activity is preferably in the range of from 0.92 to 0.995, and most preferably is in the range of from 0.95 to 0.99. As noted above, polyacrylamide gel is the preferred absorbent and Figure 1 shows the ratio of water to polyacrylamide gel to achieve a water activity for the mixture in the range of from 0.95 to 0.995.

The required water activity of the mixture is not attained immediately. The absorbent particles quickly take up the free surface water of the nematode cream, and then absorb water that is released from within the nematodes. Thus, when the free surface water is first taken up, the absorbent material has a water activity lower than its final value, which is attained after 24 to 72 hours. In a typical mixing of a nematode cream and polyacrylamide gel particles, the water activity of the gel particles is 0.92 f for the first four hours after the mixing has taken place.

The water activity then increases to 0.94 eight hours after mixing, but does not attain the (intended) value of 0.97 until 24 hours after the mixing of the nematode cream and the absorbent particles.

-I r. I-m i WO 94/05150 PCT/AU93/00465 -18- The normal procedure adopted by the present inventors after mixing together a nematode cream and a quantity of anhydrous polyacrylamide gel particles is to leave the mixture overnight at a temperature in the range of from 15 0 C to 23 0 C in conditions allowing for aeration but reduced evaporation. This was sometimes achieved by keeping the mixture in a bowl covered with aluminium foil or "Gladwrap" (trade mark). After this overnight storage period, samples from the mixture of absorbent particles (now swollen with water) and nematodes are placed in a variety of storage containers, each with provision for gaseous exchange between the interior and the exterior of the container (while minimising water loss), so that anaerobic conditions cannot develop within the container.

Various containers were used in the series of experiments.

Some had positive ventilation arrangements (for example, a series of holes in the container). Most, however, were containers which were provided with a membrane, or included a panel, of a material through which air can permeate.

In some experiments, the containers were stored at the extended storage temperature of the experiment immediately after receiving a sample of the mixture of nematodes and absorbent material. In other experiments, the containers were stored firstly at 15 0 C for three days and then at the extended storage temperature. In all experiments, the extended storage was effected in a manner such that the water activity of the absorbent/nematodes combination was maintained at a value in the range of from 0.80 to 0.99.

WO 94/05150 PCT/AU93/00465 19 Having broadly described the techniques used to perform the present invention, particular examples of the experiments conducted by the present inventors will now be described, by way of illustration.

Example 1 A sample comprising 4 kilograms of a nematode cream comprising approximately 6436 million J3 Steinernema carpocapsae Agriotos strain nematodes (1.609 million/gm), produced from six culture vessel trays (of the type described in aforementioned WIPO Publication No WO 91/15569) and processed as outlined above, had as much water squeezed out of it as possible. The squeezed cream had a viscosity such that when it was placed in a 4 cm open ended tube held vertically, it did not drop out of the tube within 2 minutes. (The addition of even small quantities of water to some of this cream reduced its viscosity so that it readily dropped from the tube within a few seconds.) The squeezed cream was used to determine what water activities were achieved with various combinations of sieved polyacrylamide gel crystals (the crystals having an average mass of 0.01 gm) and this species of nematode, and how these water activities affected nematode mortality if the combination is stored at 230C.

Based on previous preliminary experiments, the nematode cream was divided into seven batches and combined with the PAG crystals in the following proportions by weight: p. WO 94/05150 PCr/AU93/00465 20 Nematode PAG Batch No Cream Crystals Proportion Proportion 1 3 1 2 3.5 1 3 4 1 4 4.5 1 5 1 6 5.5 1 7 6 1 Samples from each of these combinations of gel crystals and nematode cream were then added to 250 ml plastic food containers, the upper rims of which were perforated with holes, each about 2 mm in diameter. Lids were placed on the containers. The weight of mixture placed into each container was chosen so that each container had a sample with approximately 110 million nematodes stored within it.

Smaller samples of the mixtures of absorbent particles and nematodes were also stored in larger numbers of small polypropylene containers, each 40 mm in diameter and 20 mm high, with slip-on lids that allowed the passage of some air between the lid and the container side. Both the larger and the smaller containers were then stored at 23°C.

At weekly intervals, the total contents of three small containers of each nematode/gel combination were washed out. In addition, weighed samples were taken from the larger containers. The total numbers of live and dead nematodes from each small container, and from the samples WO 94/05150 PCT/AU93/00465 -21 from the large containers, were counted. The results of these observations are shown in graphical form in Figure 2.

As can be seen from Figure 2, at nematodes:gel combinations of 3:1, 3.5:1 and 4:1, mortality was well below 20 per cent after six weeks, whereas the nematode mortality climbed steeply with the other combinations of nematode cream and gel particles.

A sample of the extracted nematodes was used to establish the average dry weight of the live nematodes after storage, after allowing the living nematodes to migrate through tissues. The results of measurements made, in this experiment, of the average dry weight of live nematdes in the various samples of the stored nematodes are recorded in Figure 3. It will be noted that there is an apparent increase in the average weight of the nematodes after two weeks. This is probably because the nematodes which died first were the smallest nematodes. However, the decline in dry weight was least in those nematodes stored at cream:gel ratios of 3:1, 3.5:1 and 4:1, indicating that more food reserves remain in nematodes stored at these combinations.

Example 2 After preliminary experiments to determine the range of water activity most suitable to store Heterorhabditis bacteriophora (synonym heliothidis), various batches of this species of nemlatode were tested with a range of .preservatives, antifungal agents and antibiotics. In a series of such experiments, control batches without IVWO 94/05150 PCT/AU93/00465 22 preservatives were tested. The methodology used is described below.

Nematodes were produced and processed as in Example 1, but with 10% more fat added to the rearing medium. Batches of from 200 gm -to 500 gm of an aqueous cream of nematodes were combined with particles of sieved PAG (average particle weight, 0.01 gm). Surface water in the nematode cream could not be standardised as readily as in Example 1 because Heterorhabditis bacteriophora cream loses water much more readily than Steinernema carpocapsae.

Accordingly, the H. bacteriophora nematodes were standardised by making a thick cream and then diluting it with water, so that there were about 2,5 million infective third stage juvenile nematodes per gram. This diluted cream of nematodes was combined with polyacrylamide gel particles as in Example 1.

All of the nematodes and gel combinations were then stored in containers of the same dimensions as the smaller containers used in Example 1, with about 8 gm of a sample of a combination in each container. The containers were then placed in zip sealed polyethylene bags containing a few grams of a mixture of water and polyacrylamide gel (100:1). This resulted in some free water in each bag, so that the relative humidity of the atmosphere in each bag was potentially 100 per cent. The present inventors ascertained that despite the potential relative humidity of 100 per cent within the bags, the water activity of the combinations of polyacrylamide gel and nematodes remained about 0.97. This indicates that the gel particles of the r i p 1 WO 94/05150 PCT/AU93/00465 23 combination act as a sort of buffer between the nematodes and the external atmosphere.

The polyethylene bags were then kept at 23 0 C. The storage containers were examined weekly for as long as the treatments with which they were associated were providing useful information. (Aite infestation resulted in the abandonment of some experiments although the nematodes in the infested samples were still in otherwise excellent condition.) The results of this series of experiments are summarised in Table 1, below.

Table 1 Start Nem/gm Ratio Water Storage date X 106 Nem:PAG Activity Duration Survival 1991 10 Oct 2.6 4:1 13 wks 79% 16 Oct 2.7 3.5:1 0.96 12 wks 96% Nov 2.5 4:1 0.97 14 wks 96% Dec 2.5 4:1 0.98 17 wks 1992 20 Mar 2.4 4:1 17 wks Mar 2.4 4:1 22 wks 93% 7 May 2.5 4:1 0.978 20 wks 93% 21 May 2.23 3.2:1 0.944 18 wks* 98% 21 May 2.23 3.4:1 0.958 18 wks* 98% 21 May 2.23 3.6:1 0.963 18 wks* 98% 21 May 2.23 3.8:1 0.965 18 wks* 96% Ongoing experiments WO 94/05150 PCT/AU93/00465 -24- In these examples and in other experimental work conducted by the present inventors, the storage method of this I invention has been successfully tested with representative species of both genera of entomopathogenic nematodes, Steinernema (synonym Neoaplectana) and Heterorhabditis (synonym Chromonema). All species of entomopathogenic nematodes which were tested proved to be amenable to storage in this way. Thus, since these species belong to two distinct families of nematodes (Steinernematidae and Heterorhabditidae) and include all the species found therein, it seems logical that all such nematodes can be stored in accordance with the present invention.

It will be apparent that the method of the present invention is well suited for use in the commercial production of large volumes of entomopathogenic nematodes.

k I

Claims (27)

1. A method of storing third stage infective juveniles (J3) of entomopathogenic nematodes comprising forming a mixture of an aqueous concentrate of clean J3 entomopathogenic nematodes and particles of a highly water-absorbent material, the proportions of the aqueous concentrate and water-absorbent material being such that the mixture, after equilibrating, has a water activity in the range of from 0.88 to 0.995.

2. A method as defined in claim 1, in which the water activity of the mixture is in the range of from 0.92 to 0.995.

3. A method as defined in claim 1, in which the water activity of the mixture is in the range of from 0.95 to 0.99.

4. A method as defined in claim 1, claim 2 or claim 3, in which said highly water-absorbent material is a material that absorbs at least 75 per cent of its own weight of water to have a water activity in the range of from 0.80 to 0.995. A method as defined in claim 1, claim 2 or claim 3, in which the highly water-absorbent material is anhydrous polyacrylamide gel. _I WO 94/05150 PCT/AU93/00465 26

6. A method as defined in claim 5, in which the particles of polyacrylamide gel have an average mass of about 0.01 gm.

7. A method as defined in claim 1, claim 2 or claim 3, in which the highly water-absorbent material is selected from the group consisting of anhydrous polyacrylamide gel, methyl cellulose powder, polyacrylate starch gel powder, and a mixture of anhydrous polyacrylamide gel and starch powder.

8. A method as defined in any preceding claim, including the addition of an antifungal agent.

9. A method as defined in claim 8, in which the antifungal agent is selected from the group consisting of: humic acid; brown coal dust; powdered sulphur; sulphurous acid; and a mixture of sulphurous acid and powdered sulphur. A method as defined in any preceding claim, including the addition of an antibiotic agent to the mixture.

11. A method as defined in any preceding claim, including the step of placing the mixture in a container, said Scontainer having ventilation means permitting the WO 94/05150 PCT/AU93/00465 27 passage of air between the interior and the exterior of the container.

12. A method as defined in claim 11, in which the ventilation means comprises perforations in said container.

13. A method as defined in claim 11, in which the ventilation means comprises a membrane or a panel of a material through which air can permeate.

14. A method as defined in any preceding claim, including the subsequent step of maintaining the water activity of said mixture at a value in the range of from 0.80 to 0.99. A method as defined in claim 14, in which, in the subsequent step, the water activity of said mixture is maintained at a value in the range of from 0.90 to 0.99.

16. A method as defined in claim 11, claim 12 or claim 13, in which said absorbent material is polyacrylamide gel, including the step of placing said container in an atmosphere in contact with free water.

17. A method as defined in claim 16, in which said atmosphere in contact with free water is the interior A of a second container, a quantity of a mixture of polyacrylamide gel particles and water, which has a M- T/AU 93 00 4 6 RECEIVED 1 9 AUG 1994 28 water activity of substantially 1.00, being also contained in said second container.

18. A method as defined in any one of claims 11 to 17, in which said mixture is stored at a temperature in the range of from 1 0 C to 30 0 C.

19. A method as defined in claim 18, in which said storage temperature is a temperature in the range of from to 28 0 C. A method as defined in any preceding claim, in which the entomopathogenic nematodes are of the Steinernema species. of the Heterorhabditis species, or otherwise belong to the families Steinernematidae and Heterorhabditidae.

21. A mixture of third stage infective juveniles (J3) of entomopathogenic nematodes and particles of a highly water-absorbent material, with water present in a quantity such that the water activity of the mixture is in the range of from 0.88 to 0.995.

22. A mixture as defined in claim 21, in which the water activity of the mixture is in the range of from 0.92 to 0.995.

23. A mixture as defined in claim 21, in which the water activity of the mixture is in the range of from 0.95 to 0.99. K 1 SRA' I- PCT/AU 93 00 4 6 RECEIVED 07 APR 1994 i 29

24. A mixture as defined in claim 21, claim 22 or claim 23, in which the water-absorbent material is selected from the group consisting of anhydrous polyacrylamide gel, methyl cellulose powder, polyacrylate starch gel powder, and a mixture of polyacrylamide gel and starch powder. A mixture as defined in claim 24, in which the particles of a water-absorbent material comprise anhydrous polyacrylamide gel particles having an average mass of about 0.01 gm.

26. A mixture as defined in any one of claims 21 to including an anti-fungal agent.

27. A mixture as defined in claim 26, in which the anti- fungal agent is selected from the group consisting of: humic acid; brown coal dust; powdered sulphur; sulphurous acid; and a mixture of sulphurous acid and powdered sulphur.

28. A mixture as defined in any one of claims 21 to 27, including an antibiotic agent.

29. A mixture as defined in any one of claims 21 to 28, in a container, said container having ventilation means permitting the passage of air between the interior and the exterior of the container. JAMENDED SHEET SPCT/AU 9 3 0 46 RECEIVED 0 7 APR199 t A mixture as defined in claim 29, in which the ventilation means comprises perforations in said container.

31. A mixture as defined in claim 29, in which the ventilation means comprises a membrane or a panel of a material through which air can permeate.

32. A mixture as defined in any one of claims 21 to 31, in which the entomopathogenic nematodes are of the Steinernema species, of the Heterorhabditis species, or otherwise belong to the families Steinernematidae and Heterorhabditidae.

33. A method as defined in claim 1, substantially as hereinbefore described. A D S AMENDED SHEET 'I f