AP293A

AP293A - Method and means for selective pest control.

Info

Publication number: AP293A
Application number: APAP/P/1992/000415A
Authority: AP
Inventors: Roelof Marthinus Horak; Sarel Johannes Pretorius; Ilana Mouton
Original assignee: Csir; South African Medical Res Council
Priority date: 1991-08-09
Filing date: 1992-08-07
Publication date: 1993-12-23
Also published as: AP9200415A0; OA10048A

Abstract

The invention relates to a method for selectively combating or

Description

THIS INVENTION relates to a method and means for selective pest control. More particularly the invention relates to a method for selectively combating snails, particularly freshwater snails, and to molluscicidal agents and formulations effective to be used in accordance with the invention.

According to the invention there is provided a method for selectively combating or controlling molluscs, which includes the step of applying to an infested environment a mollusc attractant selected to attract target snails to a toxic agent, the mollusc attractant comprising a fungal amylase as an attractive ingredient.

According to another aspect of the invention there is provided a method for selectively combating or controlling molluscs, which includes the step of applying to an infested environment a molluscicidal agent comprising a toxic ingredient selected from the group comprising gliotoxin and similarly effective epipolythiodioxo-piperazine analogues thereof.

Gliotoxin is an epipolythiodioxo-piperazine the chemical name (according to IUPAC) of which is 2,3,5a, 6 · tetrahydro - 6 - hydroxy - 3 - (hydroxymethyl) - 2 - methyl - 10H 3,10a - epidithiopyrazino [1,2 - a] - indole - 1,4 - dione (C^H^NjO^j) and having the formula

(hereinafter compound I);

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AP Ο Ο Ο 2 9 3 and the similarly active epipolythiodioxo-piperazine analogues may be compounds of the formula

(hereinafter compound II) υ» in which n = 1 - 4; R. R¹ = H, OH, alkyl, alkoxy, acyloxy·. R², R^J = H, alkyl; cr R²R² = a fused ring system; or compounds of the formula.

Π (hereinafter compound III) in which n = 1 - 4; and R^!, R² = H, alkyl or acyl.

Tne Applicants have found that gliotoxin is particularly effective for combating or controlling freshwater snails, and specifically the snails capable of carrying

Schistosome parasites. The moiluscicidal agent incorporating gliotoxin as the toxic ingredient, is preferably applied to the infested water environment in the form of a solid formulation.

The mollusc attractant may advantageously be applied to an infested environment together with or in combination with the moiluscicidal agent according to the invention.

Tne mollusc attractant and the moiluscicidal agent may be incorporated in a moiluscicidal formulation, eg in solid form to be placed in water infested with water snails.

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AP Ο Ο Ο 2 9 3 ©

The toxic ingredient gliotoxin and its similarly effective analogues as defined herein may be produced by appropriate chemical synthetic processes. Alternatively and preferably gliotoxin may be produced by a microbiological process, conveniently by way of a fermentation process with a suitable fungal species. The mollusc attractant may likewise be produced by a microbiological process, conveniently also by way of a fermentation process with a suitable fungal species. As mentioned above, the mollusc attractant may be an enzyme, namely a fungal amylase.

(£7 By selecting, by way of trial and experiment, suitable fungal species and suitable culture media, and by optimising fermentation conditions, gliotoxin may be produced in adequate quantities for use in accordance with the invention. Similarly, by selecting suitable fungal species and suitable culture media, enzymes, eg fungal amylases may be produced which are attractive to a particular target snail species.

The Applicants have found that, surprisingly, certain fungal species can produce gliotoxin as well as an enzyme, eg a fungal amylase, which is attractive to molluscs. For example, when the fungus Aspergillus fumigatus is grown on a particular culture medium it will produce a substance which is very attractive to snails, but grown on a different culture medium it will produce gliotoxin. This aspect will be reverted to in more detail below. Because Aspergillus fumigatus has pathogenic properties, it is generally preferred to produce the fungal amylase by fermentation of Aspergillus oryzae or Aspergillus niger.

and the gliotoxin by fermentation of Trichoderma viride.

The following fungi have been found to produce gliotoxin when grown on suitable culture media:

Aspergillus chevalieri [(Mangin) Thom & Church]

Aspergillus fumigatus [Fresenius]

Aspergillus niger [van Tieghem]

Gliocladium deliquescens [Sopp = £. viride Matr]

Gliocladium virens [Miller, Giddens & Foster]

Penicillium cinerascens [Biourge]

Penicillium restrictum [Gilman & Abbot]

Penicillium terlikowski [Zaleski]

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Trichoderma hamatum [Bonard (Bain)]

Trichoderma viride [Pers. ex Gray]

The invention accordingly extends also to a method of producing the active toxic ingredient for a molluscicidal agent, and a method of producing a mollusc attractant, which methods include the steps of allowing a suitably selected fungus species to grow on a suitably selected culture medium, and recovering the product formed. More specifically the method for producing the toxic ingredient gliotoxin includes the steps of preparing a suitable culture medium, inoculating the culture medium with a strain of a fungus selected from the following species:

.0 Aspergillus chevalier! [(Mangin) Thom & Church]

Aspergillus fumigatus [Fresenius]

Aspergillus niger [van Tieghem]

Gliocladium deliquescens [Sopp = G. viride Matr]

Gliocladium virens [Miller, Giddens & Foster]

Penicillium cinerascens [Biourge]

Penicillium restrictum [Gilman & Abbot]

Penicillium terlikowski [Zaleski]

Trichoderma hamatum [Bonard (Bain)]

Trichoderma viride [Pers. ex Gray] allowing the fungus to grow, and recovering the product.

In selecting a suitable fungus, the following aspects should be taken into account: As a first consideration, a fungal species should preferably be selected which would produce the desired toxic chemical in commercially viable yields. Secondly, it is preferred to select a species which has no or little human pathogenic characteristics. The culture medium should be selected and adapted so as to enable the selected fungal species to produce optimally, and preferably also to facilitate the extraction of the desired product.

The Applicants have found that the species Trichoderma viride gives particularly

AP Ο Ο Ο 2 9 3

The following fungi have been found to produce fungal amylases when grown on suitable culture media:

Aspergillus orvzae Aspergillus niger [Van Tieghem]

According to another aspect of the invention there is provided a molluscicidal agent in which the toxic ingredient is selected from the group comprising gliotoxin and similarly effective epipolythiodioxo-piperazine analogues thereof as defined herein.

As already mentioned above, the gliotoxin may be the product of a fermentation process with a suitable fungal species. Furthermore, the molluscicidal agent may preferably be incorporated in a formulation which includes also a mollusc attractant which is attractive to the target snails. The attractive ingredient in the mollusc attractant may also be the product of a fermentation process with a suitable fungal species, eg a fungal amylase, which is an enzyme.

The molluscicidal agent according to the invention is particularly effective to combat or control freshwater snails, and in particular the snails capable of carrying Schistosome parasites. It will be appreciated that a molluscicidal agent which is effective to control or eradicate these Schistosome host snails would have important implications for the control or eradication of schistosomiasis as the disease caused by these parasites is known.

Schistosomiasis is a chronic disease which affects humans and various animal particularly in tropical and sub-tropical countries, and which is caused by the presence in the body of parasitic flat-worms of the genus Schistosoma. Two species of Schistosoma parasites are mainly responsible for schistosomiasis, namely Schistosoma haematobium which is carried by the Bulinids, eg Bulinus africanus (also known as

Bulinus globosus) and which causes urinary bilharziasis; and Schistosoma mansoni which is carried by the Biomphalaria host snails, eg Biomphalaria pfeifferi. and which causes alimentary bilharziasis.

The Applicants have found that the toxic ingredient gliotoxin is effective to eradicate or at least control the host snail Bulinus africanus (also known as Bulinus globosus). Furthermore the Applicants have found that the fungal amylase produced by the fermentation of for example the fungi Aspergillus orvzae or Aspergillus niger is an attractant substance attractive to the host snail Bulinus africanus (also known as Bulinus globosus).

Fungal amylase products are commercially available, and such commercial products may conveniently be used in accordance with the invention. Thus the Applicants have found that the fungal amylase commercially available under the trade name Biozyme F from Amano Pharmaceutical Company of Japan gives favourable results as an attractant in respect of host snails of the species Bulinus africanus. A similarly effective fungal amylase is commercially available under the trade name Biobridge supplied by Enzy me SA of South Africa.

By incorporating a mollusc attractant in a solid molluscicidal formulation together with the molluscicidal agent including the toxic ingredient in accordance with the invention, the target snails are attracted to the formulation and will proceed to eat the substance. When the gliotoxin is ingested, the snails will die. An advantage is that a fungal amylase is not generally attractive to other animals, including water animals such as fish, so that such other animals will not be tempted to eat the toxic substance.

The Applicants have found that the concentration of the fungal amylase attractant in the molluscicidal formulation should be carefully controlled, since if the amylase is present in too high a concentration it causes repulsion of the snails. On the other hand, if the concentration is too low, the target snails will not be attracted. It is necessary, therefore, empirically to determine a suitable effective concentration of the fungal amylase attractant in a solid formulation for a specific target snail. The Applicants have found that an effective concentration of fungal amylase in a solid formulation according to the invention may vary between about 0,0025% and 0,01% for the Bulinus africanus snail. For example, in a 350 mg molluscicidal tablet produced in accordance with the invention, between about 8/zg and 34/zg of enzyme powder may be included.

AP Ο Ο Ο 2 9 3

Presently commercially available molluscicides are usually applied as aqueous solutions to dams and rivers inhabited inter alia by the schistosomiasis host snails. Such application has several disadvantages: Tn the first place, because most existing commercial molluscicides are inorganic substances, they are regarded by some as 5 undesirable in their application to a natural environment. Furthermore, at molluscicidal^; concentration, these substances are generally not only toxic to the target snails but also to other freshwater snails and to other water organisms. At higher concentrations such¹existing commercial molluscicides may also be toxic to fish, an aspect which precludes their use in dams where a build-up of toxins can occur.

Existing commercial molluscicides are usually applied as liquid formulations by means of pumps or spraying apparatus, methods of application which are clumsy and expensive and with which overdosage may occur. Repeated, regular applications are necessary in running water because there is no sustained activity. Because conventional molluscicides are generally water miscible, all users of the treated water, humans as well as animals, are exposed to the chemicals concerned.

Gliotoxin, and the manner in which it may be applied in accordance with the invention, does not entail these disadvantages. Gliotoxin is active against the schistosomiasis host snails at low concentrations. For example, it has been found in laboratory trials that concentrations of gliotoxin in water from as low as 0,1 mg/l are lethal to snails, and in particular the schistosomiasis host snails. Laboratory trials^ indicated that a molluscicidal agent according to the invention containing gliotoxin as the toxic active toxic ingredient does not affect fish, tadpoles and water insects, even when applied in concentrations significantly higher than molluscicidal concentrations.

In practice, a molluscicidal agent according to the invention may be applied as an aqueous solution to the water habitat of the'snails. Preferably, however, the toxic ingredient of the invention may be incorporated, advantageously with a mollusc attractant as set out above, in a solid environmentally acceptable matrix selected to allow the slow release of the attractant, while preventing the dissolution of the toxin, to make up a solid molluscicidal formulation. The invention accordingly extends to a solid formulation

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Μι effective for combating or controlling molluscs, particularly freshwater snails, which comprises gliotoxin or a similarly effective epipolythiodioxo-piperazine analogue thereof as herein defined, and a suitable moTlusc attractant incorporated in a solid environmentally acceptable matrix. As already indicated above, the mollusc attractant mav be a fungal amviase.

Pieces or chunks of suitable size, eg in the form of tablets or briquettes, of such a solid formulation may then be placed in suitable locations in infested waters. Water snails, and specifically the schistosomiasis host snails, attracted to the preparation by the attractant, will be killed when the toxin is ingested. Because only specific snails will be .0 attracted by the preparation, the toxin concentration will be optimised in its effect on such snails, while other water animals, such as fish, tadpoles, etc will not be affected.

The active ingredients may be incorporated in the matrix by admixing or blending. Such a solid matrix incorporating the toxic ingredient according to the invention and the mollusc attractive ingredient may then be formed into tablets or briquettes. Such solid forms facilitate distribution and application, and application can be limited to specific infested areas, and can be controlled.

Where the toxic ingredient, eg gliotoxin, is incorporated in a solid matrix, overdosage will not easily take place. In the event that trace amounts of gliotoxin are released from the solid matrix, the gliotoxin will undergo a 50 per cent degradation after

100 hours in solution when exposed to sunlight, thereby eliminating or at least reducing the possibility of a toxin build-up. Human and animal users cf the water are therefore not exposed to undue health risks.

Because the fungal amylase attractant gradually leaches out of the matrix, and so becomes available to attract the target snails, the formulation will gradually loose its attraction. When this occurs, the formulation must be replaced. It has been found that the duration of the attraction of the amylase may vary between about 24 and 48 hours.

ΛΡ Ο Ο Ο 2 9 3 e

The invention and the manner in which it may be put into practice will now be further described and explained by way of the following examples, based on laboratory trials.

EXAMPLE 1

PRODUCTION OF GLIOTOXIN WITH ASPERGILLUS FUMIGATUS:

A liquid culture medium was prepared by admixing with 1( water the following c ingredients;

£. 1 g KH, PO₄

1,8 g Asparagine 0,5 g MgSO₄15 g CaCO₃20 g Brown sugar

The liquid medium was inoculated with Aspergillus fumigatus and shaken for 9 days at 25-28°C. The culture filtrate was then extracted with chloroform, and the gliotoxin was crystallised from ethyl acetate. A yield of 50-90 mg/( was obtained with different trials.

EXAMPLE 2

PRODUCTION OF GLIOTOXIN WITH TRICHODERMA VIRIDE:

A liquid culture medium was prepared by admixing with 1 ( of water the*?:

following ingredients:
Vg	(NH₄)₂SO₄
0,8g	k₂hpo₄
0,4g	MgSO₄
0,008g	FeCl₃
0,016g	Peptone
15g	Sucrose

The liquid medium was inoculated with Trichoderma viride and shaken for 7 days at 27°C. The culture filtrate was then extracted with choloroform and 63 mg/( was

EXAMPLE 3

PRODUCTION OF A MOLLUSC ATTRACTANT WITH ASPERGILLUS FUMIGATUS:

A culture medium was prepared bySoiIing a potato, drying the boiled potato in an oven, and grinding or milling the dried potato to form a granulated dried product.

2 g of this product and 10 g of glucose were added to 1? water, and the medium inoculated with Aspergillus fumigatus· The culture was allowed to stand for 7 days at 37°C, whereupon the mycelia were filtered out. 1 g of CaSO₄ was added to 80 ml of filtrate, and the product was freeze dried and pulverised. The resultant powder was found to be extremely attractive to fresh water snails.

:0 The attraction of the product for snails was clearly demonstrated in tests.

An ordinary' commercial potato dextrose broth used as a culture medium, was found to give unsatisfactory results.

EXAMPLE 4

PREPARATION OF A MOLLLSCICIDAL FORMULATION (ON A LABORATORY 15 SCALE):

250 mg of an emulsifier commercially available from Quest International under the trade name HYMONO 8903 and 0,2 mg of gliotoxin as prepared in Example 1, were dissolved in acetone, and 250 mg of the mollusc attractant as prepared in Example 2, was added to the solution. The mixture was dried under vacuum, and the resultant substance formed into a tablet by pressing.

When the tablet was placed in water inhabited by snails, it was found that it was very attractive to the snails, and when ingested caused the snails to die. In separate experiments, it was found that different species of fish and tadpoles present in the water were unaffected.

AP Ο Ο ο 2 9 3

EXAMPLE S

PREPARATION OF A MOLLUSCICIDAL FORMULATION IN TABLET FORM; A tablet formulation was made up by admixing the following ingredients;

(a)	Gliotoxin	0,75 mg
(b)	Cutina CP’	122,00 mg
(c)	BaSO₄	50,00 mg
(d)	Glucose	50,00 mg
(^e)	Maize Starch	25,00 mg
(0	Attractant”	200/ri

Cutina CP (trade name) is a commercially available tablet binder.

The attractant was made up by dissolving 1 μί of the aforementioned Biobridge (trade name) fungal amylase concentrate in 150 ml of water.

Ingredients (a) - (d) were granulated by heating the Cutina to melting point (43°C - 51°C) and adding a pre-mixed mixture of gliotoxin, BaSO₄ and glucose. The mixture was cooled while constantly stirring to achieve granulation.

The correct amount of starch and the attractant were added to form a paste which was dried and compacted into tablets.

It was found that this procedure was not suitable for large scale tablet production. Furthermore, the attractive properties of the formulation were inadequate.

EXAMPLE 6

PRODUCTION OF A MOLLUSCICIDAL FORMULATION IN TABLET FORM (ON A SCALE SUFFICIENT TO SUPPORT EXTENSIVE FIELD TRIALS);

A tablet formulation was made up by admixing -the following ingredients: Gliotoxin 0,75 mg

Enzyme triturate’ 10,00 mg (ie 17 μξ of enzyme powder/tablet)

¹ *

	13
Starch	40,00 mg
Glucose	50,00 mg
Stearic acid	199.25 mg 350,00 mg

* The enzyme triturate is made up by mixing 10 mg of fungal amylase powder in

872,40 mg starch.

The mixture is pressed into a tablet, conveniently having a diameter of 8 mm, with a single break lip and a bevelled edge, and compacted to 9,SO newton.

The characteristics of the tablets eg stability in water, leaching out of attractant 10 and ease of compaction can be controlled by altering the amounts of stearic acid and starch included in the formulation.

The Applicants have found that a concentration of attractant of 17/zg of enzyme powder/350 mg tablet is optimum. Increasing the quantity of enzyme powder to 34wg/350 mg tablet begins to cause repulsion of the snails. Decreasing the quantity of enzyme to 8wg/350 mg tablet shows decreased attraction. The duration of attraction was found to be about 24 to 48 hours.

The Applicants have found that a solid molluscicidal formulation containing between about 0,07 to 0,21% of the toxic ingredient, and between about 0,0025 to 0,01% of the attractive ingredient gives favourable results in practice. A molluscicidal formulation in solid form may thus be provided, containing between about 0,71 and 2,14 mg of toxic ingredient per gram of solid formulation, and between about 22,9 and

Claims

1. A method for selectively combating or controlling molluscs, which includes the step of applying to an infested environment a mollusc attractant selected to attract target snails to a suitable toxic agent, the mollusc attractant comprising a fungal amylase as an

5 attractive ingredient.

2. A method as claimed in claim 1, in which the mollusc attractive ingredient is the product of a fermentation process with a suitable fungal species.

3. A method as claimed in claim 2, in which the fungal species is selected from the group comprising:

10 Aspergillus oryzae. and Aspgrgillus.nig.gr.

4. A method of selectively combating or controlling molluscs, which includes the step of applying to an infested environment a molluscicidal agent in which the toxic ingredient is selected from the group comprising gliotoxin and similarly effective epipolythiodioxo15 piperazine analogues thereof.

5. A method as claimed in claim 4, in which the mollusdcidal agent is applied together with a mollusc attractant comprising a fungal amylase as the attractive ingredient.

6. A method as claimed in claim 5 for combating or controlling freshwater snails, in 20 which the molluscicidal agent and the mollusc attractant are provided in the form of a solid formulation, which solid formulation is placed in the water habitat of the snails.

7. A method as claimed in claim 6, in which the freshwater snails are snails capable of carrying Schistosome parasites, and in which the toxic ingredient is gliotoxin.

8. A method as claimed in claim 6 or claim 7, in which the mollusc attractant is selected and the solid formulation is made up to allow the attractant to be gradually leached from the formulation by water.

9. A method of producing a toxic ingredient for a molluscicidal agent, which includes the steps of allowing a selected fungus species to grow and ferment in a selected culture medium, and recovering the product formed.

10. A method as claimed in claim 9, in which the fungal species is selected from the group comprising:

Aspergillus chevalieri [(Mangin) Thom & Church]

Aspergillus fumigatus [Fresenius]

Aspergillus niger [van Tieghem]

Gliocladium deliquescens [Sopp = G, viride Matr]

Gliocladium virens [Miller, Giddens & Foster]

Penicillium cinerascens [Biourge]

Penicillium restrictum [Gilman & Abbot]

Penicillium terlikowski [Zaleski]

Trichoderma hamatum [Bonard (Bain)]

Trichoderma viride [Pers. ex Gray]

11. A molluscicidal agent which includes a mollusc attractant comprising a fungal amylase as the attractive ingredient, and a suitable toxic ingredient.

12. A molluscicidal agent as claimed in claim 11, in which the fungal amylase is the product of a fermentation process with a suitable fungal species.

13. A molluscicidal agent as claimed in claim 12, in which the fungal species is selected from the group comprising:

Aspergillus oryzae. and

Aspergillus niger.

APOOO293 ν'

14. A molluscicidal agent which includes a toxic ingredient selected from the group comprising gliotoxin and similarly effective epipolythiodioxo-piperazine analogues thereof.

15. A molluscicidal agent as claimed in claim 14, in which the toxic ingredient is the 5 product of a fermentation process with a suitable fungal species.

16. A molluscicidal agent as claimed in claim 15, in which the fungal species is selected from the group comprising:

_ Aspergillus chevatieri [(Mangin) Thom & Church]

V

Aspergillus fumigatus [Fresenius]

10 Aspergillus niger [van Tieghem]

Gliocladium deliquescens [Sopp = Guviridg Natr]

Gliocladium virens [Miller, Giddens & Foster]

Penicillium cinerascens [Biourge]

Penicillium restrictum [Gilman & Abbot]

15 Penicillium terlikowski [Zaleski]

Trichoderma hamatum [Bonard (Bain)]

Trichoderma viride [Pers. ex Gray]

17. A molluscicidal agent as claimed in any one of claims 14 to 16 for combating or controlling snails capable of carrying Schistosome parasites, in which the toxic ingredient

20 is gliotoxin, and which includes a fungal amylase as a mollusc attractant.

18. A molluscicidal agent as claimed in claim 17, which is in solid form.

19. A molluscicidal formulation which includes a molluscicidal agent as claimed in any one of claims 11 to 18, and an environmentally acceptable matrix.

20. A molluscicidal formulation as claimed in claim 19, which is in solid form and 25 includes a solid environmentally acceptable matrix selected and compacted to allow the gradual release of the active ingredient when placed in water while delaying the dissolution of the toxic ingredient.

21. A molluscicidal formulation as claimed in claim 20, in which the matrix is selected and compacted to allow release of the attractive ingredient over a period of at least 24 hours.

22. A molluscicidal formulation as claimed in claim 20 or 21, in which the solid matrix includes starch, glucose, stearic acid and barium sulphate.

23. A molluscicidal formulation as claimed in any one of claims 19 to 22, which contains between about 0,07% and 0,21% of the toxic ingredient, and between about 0,0025% and 0,01% of the mollusc attractive ingredient by weight in the solid formulation.

24. A molluscicidal formulation as claimed in claim 23, which contains between about 0,71 mg to 2,14 mg of the toxic ingredient per gram of solid formulation, and between about 22,9 /zg to 68,6 μ% of the attractive ingredient per gram of formulation

25. A mollusc attractant comprising a fungal amylase as the attractive ingredient.