CA2064021A1 - Microbes for controlling pests - Google Patents

Abstract

ABSTRACT

MICROBES FOR CONTROLLING PESTS

The present invention provides a method of controlling at a locus a pest having an oxygen dependent respiratory system, which comprises applying to the locus a cyanide producing microorganism. The invention also provides a biocontrol agent for controlling pests comprising a cyanide producing microorganism, and agriculturally or horticulturally acceptable carrier or diluent, and a substrate for cyanide production.

Description

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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.

2 ~ 2 1 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 2 ~ 2 1 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 2 ~ 2 ~

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.

2~g~21 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 % ~

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 2 ~ 2 ~
-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.

2~021 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 2~6~

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 2 ~

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 2 ~ 2 ~

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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_ 16 -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

2 ~ 2 1 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 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:-2 ~

-Treatment Time for slug dPath (h). Cumulative Number and %

_ 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.

Claims (13)

1. A method of controlling at a locus a pest having an oxygen dependent respiratory system, which comprises applying to the locus a cyanide-producing bacterium.

2. A method of claim 1 wherein the locus is agricultural or horticultural land, or a garden.

3. The method of claim 1 or 2 wherein the pest is of the phylum Mollusca.

4. The method of any one of the preceding claims wherein the microorganism is a strain of Pseudomonas or of Chromobacterium.

5. The method of claim 4 wherein the microorganism is Chromobacterium violaceum.

6. A biocontrol agent for controlling pests comprising a cyanide producing bacterium, and an agriculturally or horticulturally acceptable carrier or diluent.

7. The agent of claim 6, which further comprises a substrate for cyanide production.

8. The agent of claim 6 or 7 wherein the pest is of the phylum Mollusca.

9. The agent of any one of claims 6 to 8 wherein the microorganism is a strain of Pseudomonas or of Chromobacterium.

10. The agent of claim 9 wherein the microorganism is Chromobacterium violaceum.

11. The agent of any one of claims 7 to 10 wherein the substrate is glycine.

12. A method of controlling at a locus a pest having an oxygen-dependent respiratory system, said method being substantially as hereinbefore described in Example 5.

13. A biocontrol agent for controlling pests, said agent being substantially as hereinbefore described in Example 4 or 5.