Note: Descriptions are shown in the official language in which they were submitted.
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MICROBES FOR CONTROLLING PESTS
The present invention relates to the control of pests
and to biocontrol agents.
Members of the phylum Mollusca are the most abundant
animals, in terms of biomass and species numbers, after the
Arthropoda. Many species are major agricultural and
horticultural pests and their control in modern agriculture
is of extreme importance. Within the Class Gastropoda is a
Sub-Class Pulmonata which contains almost all the
terrestrial molluscs known as the pulmonate slugs. Slugs
damage or destroy a wide range of crops, especially winter
wheat and potato and are a significant problem in
commmercial horticulture and amateur gardening.
Government agencies recognize the economic importance
of slugs as pests, although there is a paucity of detailed
quantitative information on the scale of the slug damage in
agriculture and horticulture. It is a worldwide problem,
and one of the problems is predicting the degree of damage.
For example, in some locations in the U.K. as much as 35% of
potato tubers may be damaged by slug grazing. Slug damage
may be controlled by chemicals but it is a costly process.
~owever, as farming practices move towards fewer
cultivations in restricted crop rotations, stimulation of
slug populations with favoured crops becomes an increasing
problem.
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For wheat, and also barley and oats, two types of
slug damage are recorded: (i) grain hollowing where the germ
is eaten away prior to germination; and (ii) grazing of
emergent shoots. The latter is a problem also for sugar
beet. In horticulture total damage to crops such as
lettuce, cabbage, Brussels sprouts, as well as flower crops,
can be extensive. In addition, for these crops excellent
condition is a prerequisite for the best market prices.
Accordingly damaged crops have signifcantly reduced value
and indeed may be unsaleable. For the amateur gardener slug
damage is a frustrating problem.
Accordingly slug control is of major economic
importance, especially if it could be accomplished in a
cost-effective fashion in agriculture. Normally at present
in agriculture the most effective control is to introduce
appropriate crop management practices to minimize slug
population development. Biological control using bird
populations has been considered but is largely
impractical.
Chemical control using compounds such as metaldehyde
and various carbonates is the most effective method of slug
control. Methods have been developed to mix toxicants with
an attractant such as bran or wheat meal in order to form a
lethal bait. This is the basis of many pellet products
available for home gardeners and horticulturists. In
addition, pellets may contain other chemicals such as
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surfactants which improve the rate of toxicant assimilation
by the slugs, and enable lower concentrations of toxicant to
be used to obtain an effective kill. A major difficulty
with these baits is that the chemicals are frequently toxic
to other non-target animals which accidentally consume slug
pellets.
In addition to slugs, other molluscs may be of
significant economic importance and their control would be
valuable. For example, another pulmonate gastropod,
lo Biophalaria glabrata, has been identified as the freshwater
snail responsible for the transmission, as the intermediate
host, of the trematode, Schistosoma mansoni, which causes
the disease schistosomiasis. The control of snail
populations by compounds such as copper sulphate, calcium
oxide, calcium cyanamide and ammonium sulphate is currently
used to combat schistosomiasis. Thus in many situations
control of slugs and snails, and indeed other mollusca, is
important.
As alternatives to chemical control, one form of
biocontrol which has been considered is the use of selected
microorganisms. In effect microbial biocontrol is the
selected, targetted use of one or more microorganisms,
acting singly or as a consortium, which cause damage or
disease to the target organism. For example, the use of
Bacillus thuringiensis to control selectively gipsy moths by
killing their feeding caterpillar stage is a major
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mechanism. Different strains of B. thuringiensis have now
been identified which attack and kill different target
insect populations.
In many cases microbial biocontrol represents the
controlled use of opportunistic pathogens or pathogens which
normally attack the target organism. They may indeed be
part of the natural microflora, either external or internal
te.g. the gut) of the target pest. Normally potential
microbial biocontrol agents are identified by isolating
microorganisms obtained from dead or dying target organisms,
it being a reasonable assumption that the dominant microbial
population could be the causative agent in the death of the
target organism. Examinat$on of the bodies of dead slugs
and snails reveals, as expected, an extensive community of
populations of bacteria, fungi, nematodes and protozoa. As
many as 106 bacteria g~1 snail have been detected. However,
despite extensive searching, no suitable candidate
biocontrol microorganisms have been reliably (i.e.
repeatedly and confirmed) identified.
Large numbers of bacteria have been identified in the
crop contents of slugs. The crop is part of the slug's
alimentary canal and is the region where substantial
macromolecule degradation occurs as a result of a complex
array of appropriate enzymes. None appears to be a suitable
pathogen or opportunistic pathogen candidate.
For snails one report (Ducklow et al., Can J.
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Microbiol. 26, 503-506, 1980) showed that the bacterium
Vibrio parahaemolyticus was pathogenic to Biomphalaria
glabrata at adminsitered concentrations of 6.8 x 10'
bacteria per snail. Other examples involving Mycobacterium
species, Bacillus species and Vibrio species are quoted.
Another report (Ducklow et al., Microb. Ecol . 7, 253-274,
1981) showed that healthy specimens of ~. glabrata had about
10 times lower bacterial levels compared with moribund
snails. The posibility of obtaining a bacterium for the
biological control of certain species of molluscs has been
discussed (Cheng, J. Invert. Pathol. 47, 219-224, 1986).
According to the present invention there is provided
a method of controlling at a locus a pest having an oxygen-
dependent respiratory system, which method comprises
applying to the locus a cyanide-producing microorganism.
The invention also provides a biocontrol agent comprising a
cyanide-producing microorganism, an agriculturally or
horticulturally acceptable carrier or diluent and,
optionally, a substrate for cyanide generation.
The locus at which the pest is controlled may be any
habitat where the pest is to be found, for example on the
ground on agricultural or horticultural land, or in gardens
and small holdings. Depending upon the pest to be
controlled, the site within these habitats can be further
selected, e.g. under rocks, or under leaves or the fruit of
plants.
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The invention is particularly concerned with the
control of molluscs, especially slugs. However, it may be
used to control any other animals with an oxygen-dependent
respiratory system. It may therefore be applied to aphids,
for example. The pests ingest the cyanide-producing
microorganism and die as a result of cyanide release. The
introduction of cyanide is therefore highly targetted. It
is specifically targetted to the pest animal so there is no
risk of indiscriminate release of cyanide. The
cyanide-producing microorganism is applied to a locus to
kill the pests.
A number of microorganisms synthesize and release
cyanide and may therefore be employed. Cyanide is formed by
the decarboxylation of glycine:
H2NCH2COOH~HCN+CO2+4H'
In general, the microorganism may be any suitable
cyanide-producing bacteria that occurs naturally. Since
cyanide-producing microbes are themselves susceptible to the
toxic influences of cyanide (produced by their own
metabolism) it may be appropriate to select
naturally-occuring strains or mutants which are able to
tolerate higher levels of cyanide, for example 0.2 to 2.0 M
and preferably from 0.8 to 1.2 M cyanide. Cyanide-producing
bacteria able to resist higher levels of cyanide are capable
of generating higher concentrations of cyanide as a result
of continued metabolism before their own sensitivity
terminates cyanide production.
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Bacteria may be used including pseudomonads, such as
_eudomonas aeruginosa, and strains of Chromobacterium
vlolaceum. It is well established that infection of burn
wounds by Pseudomonas aeruginosa leading to general
septicaemia results in death, probably as a result of
cyanide toxicity caused by the bacterium. Chromobacterium
violaceum regularly infects animals, probably as a result of
ingesting water or soil containing the organism. It is an
opportunistic pathogen inducing septicaemic disease, and
many examples have been given (Moss and Ryall, The Genus
Chromobacterium In: The Prokaryotes: A Handbook on Habitats,
Isolation, and Identification of Bacteria, Edited by
M.P. Starr, H. Stolp, H.G. Truper, A. Balows and H.G.
Schlegel, pp 1355-1364, 1981).
A preferred naturally-occurring strain is
Chromobacterium violaceum NCIMB 9131 (Rodgers and Knowles,
J. Gen. Microbiol. 108, 261-267, 197B; sunch and Knowles, J.
Gen. Microbiol. 128, 2675-2680, 1982). Other suitable
strains of Chromobacterium violaceum can be isolated
according to the procedures described by Moss and Ryall
(19B1).
To isolate a strain of bacteria which is tolerant to
cyanide, typically sodium cyanide, a lawn of a parent strain
of bacteria which are slightly resistant to cyanide may be
allowed to form over the surface of a solid growth medium,
e.g. nutrient agar. The medium either contains cyanide, or
cyanide is then added. Preferably the surface contains more
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than one concetration of cyanide, most preferably a gradient
of cyanide concentrations, e.g. a continuous gradient. This
may be achieved, for example, by cutting a small well in the
nutrient agar and filling the well with a suitable
concentration of a cyanide solution. Cyanide diffuses into
the solid growth medium away from the well establishing a
concentration gradient. At least one region of the surface
contains a concentration of cyanide, e.g. 0.2 to 2.0 M,
preferably 0.8 to 1.2 M in which the parent strain would not
be expected to grow. This is seen as a clear zone in which
no parent organisms can grow surrounded by a region of
growth of the lawn. By selecting colonies of bacteria which
do grow in these regions of high concentrations of cyanide,
strains of bacteria which are tolerant to higher levels of
cyanide than the parent strain may be selected. This
procedure may be repeated any number of times to improve
gradually the cyanide resistance properties of the mutants
derived in each round of screening.
Alternatively, a microbe may be isolated from the
alimentary canal of a pest, for example from the crop of a
mollusc, and altered so that it incorporates an expressible
gene coding for cyanide synthase. Such a microbe is
therefore naturally adapted for colonisation of and growth
in the alimentary canal of the pest it is wished to control.
For this purpose, a dominant member of the bacterial
population of the alimentary canal of the pest is isolated
and manipulated to produce cyanide.
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This may be achieved by natural selection of a
cyanide-producing mutant of the natural microbe. It is well
established that, for many functions in many natural
isolates, the expression of a particular gene to yield a
particular phenotypic property is restricted, if not
entirely nil. That is, genes are present, capable of
expressing a given protein to produce a specific phenotypic
property, but for unexplained reasons the genes are normally
silent. These are known as cryptic genes. Cryptic genes
can be made to express properly to reveal their function.
In the present case mutants of a naturally-occurring
microbe, isolated from the alimentary canal of a pest such
as a slug and containing a cryptic gene for cyanide
synthase, may be selected which express this gene, thereby
producing a cyanide-generating mutant of a natural gut
microbe.
It may also be achieved by genetic manipulation
through recombinant DNA techniques. In C. violaceum the
cyanide-generating system from glycine depends on a single
enzyme called cyanide synthase ~Bunch and Knowles, J. Gen.
Microbiol. 128, 2675-2680, 1982). A gene library can be
prepared from the DNA of C. violaceum. The fragment
encoding the cyanide synthase gene may then be identified.
Standard procedures can be used. This gene is then cloned
into a plasmid which is then used to transform the
appropriate, selected natural microbe.
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Viable cyanide-producing microbes are formulated for
use with an agriculturally or horticulturally acceptable
carrier or diluent. It may be provided as a dry powder
either by spray-drying or freeze-drying, typically
containing about 109 viable microbes g~~. For solid
formulations, they may be in the form of a powder, pellets
or capsules for example. The microbes may be dispersed in
water for liquid formulations. A growing culture of the
cyanide-producing microbe may be employed.
The microbes are preferably administered with a
substrate for cyanide generation such as an amino acid from
which cyanide can be generated. Preferably this is glycine
although methionine, glutamic acid or sodium glutamate may
be used. However, sources of thefie amino acids do occur in
natural habitats, for example as a result of the breakdown
of all proteins.
The formulations may be provided with an attractant
for the pest. For slugs, this can be a farinaceous material
such as bran or wheat meal. Vitamin B~s are also suitable
for this purpose. Other formulation additives such as a
surfactant or a binder may also be present. Suitable
surfactants include non-ionic agents, such as condensation
products of polyalkylene oxide and alkylphenols and fatty
acid esters of polyoxyalkylenes, cationic agents such as
quaternary ammonium salts, e.g. cetyltrimethylammonium
chloride, cetylpyridinium chloride and anionic agents such
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as sodium salts of secondary long chain alkyl sulphates e.g.
sodium lauryl sulphate, salts of alkyl aryl sulphates,
sodium deoxycholate, sodium taurocholate and sodium
tauroglycocholate (TGC). Suitable binders include gelatine,
starch, synthetic or natural resins or gums, e.g.
carboxymethyl cellulose and tragacanth, or clay.
The proportion of attractant such as a farinaceous
material, if present, in formulations containing
cyanide-providing microbes will vary according to the
lO required properties of the final composition, e.g. whether
it is to be liquid or solid. Generally the farinaceous
material may be present in a proportion of 5 to 95%,
preferably 20 to 60 % and most preferably from 45 to 53 % by
weight of the final composition.
The surfactant, if present, may be used in a
proportion of 0.05 to 1%, preferably 0.1 to 0.7% and most
preferably from 0.1 to 0.4% by weight of the final
composition.
The binder, if present, may be used in a proportion
20 of 0.05 to 5.0 %, preferably 0.05 to 2.5 % and most
preferably 1.0 to 2.0 % by weight of the final camposition.
The substrate for cyanide generation, when used, may
be present in an amount of 10 to 60, preferably 15 to 35 and
most preferably 20 to 30 by weight of the final composition.
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Application of the cyanide-producing microbes can be
in any appropriate fashion, depending upon the type of
formulation. Liquid formulations may be sprayed onto an
affected area. Solid formulations may be dispersed wherever
damage due to a pest is prevalent or feared. The liquid or
solid formulations may be applied as a coating on an
absorbent substrate, for example paper, e.g. filter paper or
paper board, or on a porous ceramic tile or a shallow dish
of similar material. Alternatively, the formulations may be
made into pellet form, and either used in moist condition or
treated further,-e.g. freeze-dried.
An amount of the microbes is applied sufficient to
ensure that the pest is eradicated or at least kept under
control. Typically from 105 to 107 viable microbes are
needed per gram of animal. From 105 to 107 viable microbes
are required per slug. The viable microbes grow in the gut
of the pest animal and amplify the lethal effect.
The following Examples illustrate the invention.
EXAMP~E 1: General procedure for preparinq slug pellet
A high cyanide-yielding strain of C. violaceum or
other suitable microbe, such as an appropriate strain of P.
aeruginosa, is cultured and formulated as a dry powder
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eit:her by spray-drying or freeze-drying techniques in such a
way that the powder retains viability. An appropriate cell
density to ensure lethal infection of slugs is qreater than
109 viable cells g~l administered powder. This ensures
rapid colonization of the slug's crop to establish a
cyanide-producing population with the ability to generate
sufficient cyanide to kill the slug. The dry viable cell
powder is formulated with a suitable slug attractant, such
as bran, with or without other additives, such as
surfactants, to form the bait pellet. Glycine is included
in the bait pellet to provide the best substrate for cyanide
generation.
EXAMPLE 2: General procedure for preparinq a bacterium,
isolated from a slug's crop, containing a plasmid
incorporating a cvanide synthase gene
The DNA from one of the chosen high-yielding strains
of C. violaceum and containing the gene(s) for cyanide
synthase ~cys gene) is digested with a restriction
endonuclease, e.g. PstI or HindIII, to produce a random
mixture of smaller DNA fragments, one of which carries the
cys gene. The fraqments are inserted into a suitable
plasmid vector, e.g. pKT231, pGS58 or pHG327, using standard
procedures of opening the plasmid DNA and using the same
restriction endonuclease as for the genomic DNA fragment
generation stage. The plasmid DNA and the C. violaceum
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fragments are ligated together to form a chimaeric (or
recombinant) DNA vector based on the plasmid and containing
part of the C. violaceum DNA. The randomly generated
recombinant DNA plasmids are transformed into suitable
recipient strains of E. coli or P. putida and transformants
screened for their capacity to generate cyanide from
glycine. Suitable transformants now carrying a plasmid
containing the cyanide synthase gene are assessed for
genetic stablity, i.e. to ensure, through repeated
sub-culturing, that the required trait is not spontaneously
lost. A selected recombinant plasmid is transferred to a
selected, natural isolate from the crop of the slug. This
organism is used as the slug-specific cyanide-producing
molluscicide in the pellet form as described in Example 1.
EXAMPLE 3: Isolation of a cyanide resistant strain of C.
violaceum
A mutant of C. violaceum NCIMB 9131, termed strain
Lig a8-1, was isolated as a cyanide-resistant mutant. A
lawn of C. violaceum NCIMB 9131 was spread uniformly on the
surface of a nutrient agar plate containing a basic growth
medium (see details in Example 4). A central well was cut
in the agar into which a solution of l.OM sodium cyanide was
placed. The plate was incubated at 0C for 1.0 hour in
order to prevent growth and allow the added cyanide to
diffuse from the well into the agar. The plate was then
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incubated at 30C for two to three days to allow the
bacterium to grow. A zone of clearing round the well,
corresponding to regions of high cyanide concentration, was
produced because the cyanide concentration was high enough
to prevent bacterial growth (it is important to note that
although C. violaceum may produce cyanide, at high enough
concentrations the organism is itself inhibited by cyanide).
Within this zone of clearing mutant colonies of C. violaceum
were observed because the organisms were able to tolerate
higher cyanide concentrations. One such mutant, strain Lig
88-1, was resistant to higher concentrations of cyanide and
as a result was able to produce cyanide to greater
concentrations than its parent. A sample of this strain has
been deposited at the NCIMB, Aberdeen, UK on 26th June 1989
under Accession No. NCIMB 40159
EXAMPLE 4: Growth of C. violaceum strain Lig 88-1 and
formulation of a biocontrol agent in _pellet form
C. violaceum strain Li.g 88-1 was cultured on a minimal salts
medium modified from the medium of Miller (Expts. in
Molecular Genetics, page 431, Cold Spring Harbor, New York;
Cold Spring Harbor Laboratory 1972) by omission of ammonium
salts.
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The medium contained in distilled (deionized water gl ':
Na2HPO4, 6; KH2PO4,3; and NaCl, 0.5, together with 10.0 ml
of an 0.01 M CaCl2 solution; 1.0 ml of a l.OM MgSO4.7H20
solution and l.Oml of a trace element solution based on that
described by ~auchop and Elsden (J.Gen.Microbiol. 23,
457-469 1960) with the exception that the Fe2+ concentration
was raised to 30 ~M. Glutamate at a final concentration of
10 mM was the basic carbon and energy source. This medium
was found to give satisfactory growth of the strain of C.
violaceum; however, it was not optimized for maximum
organism yield. This was not important for the subsequent
use of the cells of C. violaceum produced and, indeed, any
suitable growth medium based on other nutrient compositions
would be suitable. Tn order to promote the synthesis of
cyanide synthase, the enzyme responsible for cyanide
production, glycine and methionine at final concentrations
of 2.OmM and 0.5mM respectively were included in the growth
medium. Typically cultures were grown in 500 ml aliquots in
conical flasks placed in a rotary shaker (250 rev.min 1) at
30C.
Cultures were grown to late exponential phase and
harvested by centrifugation at 5000 rev.min for 10 minutes.
The resulting pellet of organisms was typically resuspended
to 20.0 ml of the minimal salts medium described above. A
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viable cell count on the concentrated C. violaceum cell
suspension was determined using standard microbiological
procedures.
Pellets containing the biocontrol microbe were
constructed as follows:
B ran : 12.5g
Cottonseed Flour : 5.75g
Mineral salts medium supplemented with 20 mM
glutamate; 4mM glycine and lmM methionine: 10 ml
Concentrated C. violaceum cell suspension in mineral
salts medium : 10 ml
The complete pellet mixture was mixed to a homogeneous
matrix and moulded into pellets using a microtitre tray
(Flow Laboratories, Rickmansworth, G3). The tray was placed
in a freeze-drier and freeze-dried for 15 to 20 hours. In
some cases, for control purposes (see below), pellets were
made without any microorganism. In other instances the
newly formed pellets were not freeze dried.
EXAMPLE 5: Use of C. violaceum strain Lig 88-1 as a
biocontrol agent administered as a liquid formulation
A concentrated cell suspension of C. violaceum Lig 88-1 was
pipetted onto an area of filter paper which was incorporated
as the top layer of three covering the base of an aquarium
tank (dimensions 11.5 cm x 8.0 cm x 2.5 cm). The remaining
layers of filter paper has previously been moistened with
2a~ s,l
water in order to provide a humid environment for the slug
colony. A control colony in a tank of the same dimensions
was established with the cell suspension replaced with
distilled water. Food pellets (prepared as $or the
microbe-free pellets in Example 4, were provided - 5 for
each colony). 10 newly-caught slugs were placed in each
tank and death recorded as follows:-
.
Treatment Time for slug death (h). Cumulative number & % given
48 72 96 120
Early
exponential 3 4 7 10
phase
C. violaceum 30% 40% 70% 100%
Control - 0 0 0 0
water 0% 0% 0% 0~
In another experiment of the same type the following results
were obtained:-
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Treatment Time for slug dPath (h). Cumulative Number and %
_
24 48 72 96
Early
exponential 0 1 5 9
phase
C. violaceum 0% 10% 50% 90%
Control - 0 0 0
water 0% 0% 0% 10%
In both these experiments grazing of the slug populations as
they moved about the surface resulted in ingestion of
growing C. violaceum. Compared with the control populations
not exposed to the microbe, death of the slugs was rapid.
