Note: Descriptions are shown in the official language in which they were submitted.
W O 94/05150 P(~r/AU93/00465
~ 2144172
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
conrPrns the storage and transport of the third stage
infective juveniles of nematodes belonging to the genera
St~ine~nema (synonym Neoaplectana) and Heterorhabditis
(synonym Ch~ ) using highly absorptive substances,
such as polyacrylamide gels, at specific water activities.
RA.-L.J.o~.d
It is well known that entomopathogenic nematodes in the
families St~ine~nematidae 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 f~e~ng) 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
Nematoloqica, Volume 27, pages 109 to 114, 1981 (entitled
"Low cost in-vitro mass production of Neoaplectana and
WO94/05150 PCT/AU93/00465
2 ~ 44~ 2 - 2 -
Heterorhabditis species (Nematoda) for field control of
insect pests") and in Annals of Applied Bioloqy, 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 WIP0 Publication
No W0 91/15569. Nematodes have also been mass produced in
liquid culture as detailed by G W Pace et al (see WIP0
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 WIP0
Publication No W0 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 Bioloqy,
R A BP.~ ~ ng 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 disadvantage
that to store the nematodes for long periods, forced
aeration of the carrier is ~PP~e~. 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
W094/OS150 PCT/AU93/00465
2 1 q ~
mixing 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 importance to the highly
adsorptive (as distinct from absorptive) properties of the
activated charcoal. Their method, however, has a number of
disadvantages, namely:
(a) this method works satisfactorily only with the species
St~;n~nema carpocapsae;
(b) activated charcoal is extremely unpleasant to handle;
(c) activated charcoal is expensive:
(d) the nematodes die within a few days if the package in
which they are contained is exposed to a temperature
higher than about 15C; and
(e) there are constraints on the ~x;mll~ size of the
package in which the stored nematodes can be held, and
on the number of nematodes that can be contAlne~ in
the package.
I Popiel, K D Holt~nn, 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 pheno~enon 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
W O 94/05150 PC~r/AU93/00465
2~4~
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 ret~;~e~ 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 Ext~e~ 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 St~;n~nema 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 WIP0 Publication No W0 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:
(1) 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 re~in~g 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
WO94/05150 PCT/AU93/00465
~ 2I~41~2
weight (this weight varies according to the species of
entomopathogenic nematode being treated); and
(2) 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 maint~ n~
(i) 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 cont~;ner 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 cont~iner with sufficient air space to
accommodate the needs of the stored infective juveniles.
WO94/05150 PCT/AU93/00465
L~
~4~- 6 -
Popiel et al claim that, using this technique, nematodes of
the species St~in~nema carpocapsae, St~;n~nema feltiae
(synonym bibionis) and Heterorhabditis bacteriophora
(synonym heliothidis) survived for several months with
5 ~ r~n~h~ 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 sub~ected 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, exp~n~ive enviLu -ntal chambers would have to
be used.
3. The mainten~nce 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
provisions for maintaining the relative humidity near,
but below, 100 per cent.
5. The technique does not work well with nematodes of the
Heterorhabditis species.
WO94/05150 PCT/AU93/00465
0 ~4417~
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 polysa~ch~ride (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-cont~; n; ng medium. The relative humidity of the
air surrounding the nematode-contA;n;ng medium is
maint~ n~ 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 state
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
WO94/0~150 PCT/AU93/00465
~ 8 -
a 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 ma~or
factors, namely (i) 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
milled, calcined attapulgite clay.
Although it has been c- ercially successful, this method
also has some disadvantages, as follows:
1. There is a marked A~.l in~ in the survival of all
nematode species after storage for 2 months at a
temperature in the range of from 23C to 28C.
2. When suspP~Aing 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 ~uantities of
W094/05150 PCT/AU93/00465
~ 172
nematodes 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 ob~ect 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 ~or commercial purposes.
This ob~ective is achieved by using highly absorptive (but
not necessarily adsorptive) gels, or other highly
absorptive subst~n~es~ 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/lOO, where RH
would be the relative humidity of the surro~ln~i ng
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
W O 94/05150 P~r/AU93/00465
10 -
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 to
mixing the nematode cream and absorbent together.
The benefits of using highly absorbent subst~nc~ are
believed to be five-fold. Firstly, they remove surface
water rapidly so that the nematodes form a "foam" within 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
swollen particles. These spaces provide room for nematode
foA~i ng 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.
Secon~1y, 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 filter discs or the like.
Thirdly, by mixing the appropriate combinations of
nematodes and absorbent, the establishment of the required
WO94/05150 PCT/AU93/00465
~ 417~
11 --
water 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
continu~l 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
subst~nc~c 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 ~xc~c 0.995 but
re--inc below l.O0; 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
WO94/05150 PCT/AU93/00~65
~ ~ - 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.
Dep~n~ing on the particular absorbent, the absorbent may
comprise from about lS 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 ~on~e~tion, the water content
of the nematode cream must first be standardised. This can
be achieved by making the m~h~r of clean infective
~uvenile nematodes per gram constant for any particular
species of nematode or by st~nA~rdising 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
WO94/05150 PCT/AU93/00465
~ 2144~72
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 lC, or
at temperatures up to 30C. 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 obtA~ne~ 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 foAm~ ng and
gaseous diffusion.
A detailed description of the way in which nematodes are
prepared for storage in a nematode and absorbent
W094/05150 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 ~c~o~panying
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 St~;n~nema carpocapsae.
Figure 3 is a graphical presentation of the average dry
weight of nematodes (S. carpocapsae) in stored samples
after different storage times.
Preparation of the Nematodes Prior to Storing
The ~nn~r 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 Bioloqy paper by
R A R~ ng, 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 tox~n~, or mech~n~cal or
chemical damage, their longevity may be reduced. In
addition, the quality of the media on which the nematodes
have been reared and the r~n~er in which they have been
W094/05150 PCT/AU93/00465
~ 2l~ 2
reared affect their subsequent longevity. For example,
entomopathogenic J3 nematodes that have been reared in a
J liquid culture frequently store less well than those reared
in a solid culture (this is particularly true of
5 HeL~ o~habditis 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 (i) microbial
10 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).
15 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
methods described by R A Bedding, M S Stanfield and
G W C~ Oil in the specification of International patent
application No PCT/AU91/00136 (WIP0 Publication
No W0 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 rPm~;n;ng 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 technigue described in the
=
WO94/05150 PCT/AU93/00465
~ 16 -
specification of International patent application
No PCT/AU88/00127, were s~;~ented after w~h;n~ and the
excess water was drained off. The sP~;-?nt 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 L~ :ved by stirring the nematode cream while draining.
In some trials, still further water was L~-~ ved by
gathering up the cloth edges to enclose the nematode mass
in the cloth before squeezing out some of the r~; n ing
water. The resulting cream of nematodes contained from 0.5
to 3.5 million J3 nematodes per gram, depending upon the
species involved and the amount of inter-nematode water
rem~i n i ng . For some experiments, one or more of various
antifungal and antibiotic agents were added to and mixed
with the se~ ted 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
rcr-ine~ 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.
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 ap~Lu~Liate
weight of nematodes. (The appropriate weights were
WO94/05150 PCT/AU93/00465
2I~ 72
- 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 O.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 attAine~
immediately. The absorbent particles ~uickly 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
for the first four hours after the ~;x;ng 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 ;x; ng of the nematode cream and
the absorbent particles.
WO94/05150 PCT/AU93/00465
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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
15C to 23C 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 cont~; nerS, each with provision for
gaseous exchange between the interior and the exterior of
the contAlner (while minimising water loss), so that
anaerobic conditions cannot develop within the cont~i n~.r .
Various cont~; nerS 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
ext~Aefl storage temperature of the experiment immediately
after receiving a sample of the mixture of nematodes and
absorbent material. In other experiments, the contA~ners
were stored firstly at 15C for three days and then a~ the
exten~fl storage temperature. In all experiments, the
ext~ndefl storage was effected in a ~ner such that the
water activity of the a~sorbent/nematodes combination was
maint~ine.fl at a value in the range of from 0.80 to 0.99.
WO94/05150 PCT/AU93/00465
21~41~
;.
_ 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 St~;n~nema
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 outline~ above, had as
much water squeezed out of it as possible. The sqll~e7e~
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 secon~.) 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 O.Ol gm) and this
species of nematode, and how these water activities
affected nematode mortality if the combination is stored at
23C
Based on previous pr~l;~in~y experiments, the nematode
cream was divided into seven batches and combined with the
PAG crystals in the following proportions by weight:
WO94/05150 PCT/AU93/00465
~- . ¢--
- 20 -
Nematode PAG
Batch No Cream Crystals
Proportion Proportion
1 3
2 3.5
3 4
4 4.5
6 5.5
7 6
Samples from each of these combinations of gel crystals and
nematode cream were then added to 250 ml plastic food
cont~in~rs, the upper rims of which were perforated with 5
holes, each about 2 mm in diameter. Lids were placed on
the cont~1~ers. The weight of mixture placed into each
contA~n~r was chosen so that each cont~iner 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
pol~lo~ylene cont~in~rs, 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 cont~iners were then stored at 23C.
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 cont~iners~ The total numbers of live and dead
nematodes from each small cont~iner, and from the samples
WO94/05150 PCT/AU93/00465
~ 4 1 72
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 nematodes in
the various samples of the stored nematodes are recorded in
Figure 3. It wlll 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 ~cline 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 l~ -i n 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 nematode were tested with a range of
preservatives, antifungal agents and antibiotics. In a
series of such experiments, control batches without
WO94/05150 PCT/AU93/00465
.. ~
- 22 -
preservatives were tested. The methodology used is
described below.
Nematodes were produced and processed as in Example 1, but
with lO~ 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, O.O1 gm). Surface water in the nematode cream
could not be st~nd~rdised as readily as in Example 1
because Heterorhabditis bacteriophora cream loses water
much more readily than St~in~nema carpocapsae.
Accordingly, the H. bacteriophora nematodes were
st~n~rdised 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 cont~;n~s of the same dimensions as the smaller
cont~lners used in Example 1, with about 8 gm of a sample
of a combination in each container. The cont~1n~rs were
then placed in zip sealed polyethylene bags cont~ ng 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
ascert~;ne~ 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
W094/051SO PCT/AU93/00465
2144~ 7~
- 23 -
combination act as a sort of buf~er between the nematodes
and the external atmosphere.
The polyethylene bags were then kept at 23C. The storage
contA~n~rs were PX~e~ weekly for as long as the
treatments with which they were associated were providing
useful information. (Mite infestation resulted in the
ab~n~onment 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
t Nem/gm R~tlo Water 8torege X
~ rt s 10~ Nem PAaActivltr Duratlon 8urv~vAl
d~t~
1991
1510 Oct 2.6 4:1 13 wks 79~
16 Oct 2.73.5:1 0.96 12 wks 96%
20 Nov 2.54:1 0.97 14 wks 96%
5 Dec 2.54:1 0.98 17 wks 90%
1992
2020 Mar 2.4 4:1 17 wks 90%
20 Mar 2.44:1 22 wks 93%
7 May 2.54:1 0.978 20 wks 93%
21 May 2.233.2:1 0.94418 wks* 98%
21 May 2.233.4:1 0.95818 wks* 98%
2521 May 2.23 3.6:1 0.963 18 wks* 98%
21 May 2.233.8:1 0.96518 wks* 96%
* Ongoing experiments
-
W094/05150 PCT/AU93/00465
~ - 24 -
In these examples and in other experimental work conducted
by the present inventors, the storage method of this
invention has been successfully tested with representative
species of both genera of entomopathogenic nematodes,
St~n~nema (synonym Neoaplectana) and Heterorhabditis
(synonym Ch~ ~ ema). All species of entomopathogenic
nematodes which were tested proved to be ArenAhle to
storage in this way. Thus, since these species belong to
two distinct families of nematodes (Steinernematidae and
Heterorhabditidae) and include all the spec~es 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 commerclal
production of large volumes of entomopathogenic nematodes.
