OA10048A

OA10048A - Method and means for selective pest control

Info

Publication number: OA10048A
Application number: OA60258A
Authority: OA
Inventors: Sarel Jackobus Pretorius; Roelof Marthinus Horak; Ilana Mouton
Original assignee: Csir; South African Medical Research Council
Priority date: 1991-08-09
Filing date: 1992-08-07
Publication date: 1996-10-14
Also published as: AP293A; AP9200415A0

Abstract

The invention relates to a method for selectively combating or

Description

1 0048 G/i THIS INVENTION relates to a method and means for sélective pest control.More particularly the invention relates to a method for selectively combating snails,particularly freshwater snails, and to molluscicidal agents and formulations effective tobe used in accordance with tbe invention.

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

According to another aspect of the invention there is provided a method for10 selectively combating or controlling molluscs, which includes the step of applying to aninfested environment a molluscicidal agent comprising a toxic ingrédient selected fromthe group comprising gliotoxin and similarly effective epipolythiodioxo-piperazine analogues thereof.

Gliotoxin is an epipolythiodioxo-piperazine the Chemical name (according to IUPAC) of15 which is 2,3,5a, 6 - tetrahydro - 6 - hydroxy - 3 - (hydroxymethyl) - 2 - methyl - 10H - 3,10a - epidithiopyrazino [1,2 - a] - indole - 1,4 - dione (C13H14N2O4S2) and baving theformula

CHjOH I (hereinafter compound I); 1 0048 and the similarly active epipolythiodioxo-piperazine analogues may be compounds of theformula

vn (hereinafter compound II) in which η = 1 - 4; R, R1 = H, OH, alkyl, alkoxy, acvloxy; R2, R3 = H, alkyl;or R2R3 = a fused ring System; or compounds of the formula.

(hereinafter compound III) in which η = 1 - 4; and R1, R2 = H, alkyl or acyl.

The Applicants hâve found that gliotoxin is particularly effective for combatingor controlling freshwater snails, and specifically the snails capable of carrying 10 Schistosome parasites. The molluscicidal agent incorporaîing gliotoxin as the toxicingrédient, is preferably applied to the infested water environment in the form of a solidformulation.

The mollusc attractant may advantageously be applied to an infested environmenttogether with or in combination with the molluscicidal agent according to the invention. 15 The mollusc attractant and the molluscicidal agent may be incorporated in amolluscicidal formulation, eg in solid form to be placed in water infested with watersnails. 1 0048

The toxic ingrédient gliotoxin and its similarly effective analogues as definedherein may be produced by appropriate Chemical synthetic processes. Alternatively andpreferably gliotoxin may be produced by a microbiological process, conveniently by wayof a fermentation process with a suitable fungal species. The mollusc attractant maylikewise be produced by a microbiological process, conveniently also by way of afermentation process with a suitable fungal species. As mentioned above, the molluscattractant may be an enzyme, namely a fungal amylase.

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

The Applicants hâve found that, surprisingly, certain fungal species can producegliotoxin as well as an enzyme, eg a fungal amylase, which is attractive to molluscs. Forexample, when the fungus Aspergillus fumigatus is grown on a particular culture mediumit will produce a substance which is very attractive to snails, but grown on a differentculture medium it will produce gliotoxin. This aspect will be reverted to in more detailbelow. Because Aspergillus fumigatus has pathogenic properties, it is generally preferredto produce the fungal amylase by fermentation of Aspergillus oryzae or Aspergillus niger.and the gliotoxin by fermentation of Trichoderma virtde.

The following fungi hâve been found to produce gliotoxin when grown on suitableculture media:

Aspergillus chevalieri [(Mangin) Thom & Church]

Aspergillus fumigatus [Fresenius]

Aspergillus niger [van Tieghem]

Gliocladium deliquescens [Sopp = £. viride Matr]

Gliocladium vire ns [Miller, Giddens & Foster] Pénicillium cinerascens [Biourge] Pénicillium restrictum [Gilman & Abbot] Pénicillium terlikowski [Zaleski] 1 0048

Trichoderma hamatum [Bonard (Bain)]

Trichoderma viride [Pers. ex Gray]

The invention accordingly extends also to a method of producing the active toxicingrédient 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 growon a suitably selected culture medium, and recovering the product formed. Morespecifically the method for producing the toxic ingrédient gliotoxin includes the steps ofpreparing a suitable culture medium, inoculating the culture medium with a strain of afungus selected from the following species:

Aspergillus chevalieri [(Mangin) Thom & Church]

Aspergillus fumigatus [Fresenius]

Aspergillus niger [van Tieghem]

Gliocladium deliquescens [Sopp = G. viride Matr]

Gliocladium virens [Miller, Giddens & Foster] Pénicillium cinerascens [Biourge] Pénicillium restrictum [Gilman & Abbot] Pénicillium 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 considération, a fungal species should preferably be selected which wouldproduce the desired toxic Chemical in commercially viable yields. Secondly, it ispreferred to select a species which has no or little human pathogenic characteristics. Theculture medium should be selected and adapted so as to enable the selected fungalspecies to produce optimally, and preferably also to facilitate the extraction of thedesired product.

The Applicants hâve found that the species Trichoderma viride gives particularlygood results in practice when used to produce gliotoxin. 10048

The following fungi hâve been found to produce fungal amylases when grown onsuitable culture media:

Aspergillus orvzaeAspergillus niger [Van Tieghem]

According to another aspect of the invention there is provided a molluscicidalagent in which the toxic ingrédient is selected from the group comprising gliotoxin andsimilarly effective epipolythiodioxo-piperazine analogues thereof as defîned herein.

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

The molluscicidal agent according to the invention is particularly effective tocombat or control freshwater snails, and in particular the snails capable of carryingSchistosome parasites. It will be appreciated that a molluscicidal agent which is effectiveto control or eradicate these Schistosome host snails would hâve important implicationsfor the control or éradication of schistosomiasis as the disease caused by these parasitesis known.

Schistosomiasis is a chronic disease which affects humans and various animais,particularly in tropical and sub-tropical countries, and which is caused by the presencein the body of parasitic flat-worms of the genus Schistosoma. Two species ofSchistosome parasites are mainly responsible for schistosomiasis, namely Schistosomahaematobium which is carried by the Bulinids, eg Bulinus africanus (also known asBulinus globosus) and which causes urinary bilharziasis; and Schistosoma mansoni whichis carried by the Biomphalaria host snails, eg Biomphalaria pfeifferi, and which causesalimentary bilharziasis. 1 0048

The Applicants hâve found that the toxic ingrédient gliotoxin is effective toeradicate or at least control the host snail Bulinus africanus (also known as Bulinusglobosus). Furthermore the Applicants hâve found that the fungal amylase produced bythe fermentation of for example the fungi Aspergillus orvzae or Aspergillus niger is anattractant substance attractive to the host snail Bulinus africanus (also known as Bulinusglobosus).

Fungal amylase products are commercially available, and such commercialproducts may conveniently be used in accordance with the invention. Thus theApplicants hâve found that the fungal amylase commercially available under the tradename Biozyme F from Amano Pharmaceutical Companv of Japan gives favourableresults as an attractant in respect of host snails of the species Bulinus africanus. Asimilarly effective fungal amylase is commercially available under the trade nameBiobridge supplied by Enzyme SA of South Africa.

By incorporating a mollusc attractant in a solid molluscicidal formulation togetherwith the molluscicidal agent including the toxic ingrédient in accordance with theinvention, the target snails are attracted to the formulation and will proceed to eat thesubstance. When the gliotoxin is ingested, the snails will die. An advantage is that afungal amylase is not generally attractive to other animais, including water animais suchas fish, so that such other animais will not be tempted to eat the toxic substance.

The Applicants hâve found that the concentration of the fungal amylase attractantin the molluscicidal formulation should be carefully controlled, since if the amylase isprésent 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 détermine a suitable effective concentration of the fungalamylase attractant in a solid formulation for a spécifie target snail. The Applicants hâvefound that an effective concentration of fungal amylase in a solid formulation accordingto the invention may vary between about 0,0025% and 0,01% for the Bulinus africanussnail. For example, in a 350 mg molluscicidal tablet produced in accordance with theinvention, between about 8/zg and 34/zg of enzyme powder may be included. 100 48

Presently commercially available molhiscicides are usually applied as aqueoussolutions to dams and rivers inhabited inter alia by the schistosomiasis host snails. Suchapplication has several disadvantages: In the first place, because most existingcommercial molluscicides are inorganic substances, they are regarded by some asundesirable in their application to a natural environment. Furthermore, at molluscicidalconcentration, these substances are generally not only toxic to the target snails but alsoto other freshwater snails and to other water organisms. At higher concentrations suchexisting commercial molluscicides may also be toxic to fish, an aspect which precludestheir use in dams where a build-up of toxins can occur.

Existing commercial molluscicides are usually applied as liquid formulations bymeans of pumps or spraying apparatus, methods of application which are clumsy andexpensive and with which overdosage may occur. Repeated, regular applications arenecessary in running water because there is no sustained activity. Because conventionalmolluscicides are generally water miscible, ail users of the treated water, humans as wellas animais, are exposed to the Chemicals concerned.

Gliotoxin, and the manner in which it may be applied in accordance with theinvention, does not entail these disadvantages. Gliotoxin is active against theschistosomiasis host snails at low concentrations. For example, it has been found inlaboratory trials that concentrations of gliotoxin in water from as low as 0,1 mg/0 areléthal to snails, and in particular the schistosomiasis host snails. Laboratory trialsindicated that a molluscicidal agent according to the invention containing gliotoxin as thetoxic active toxic ingrédient does not affect fish, tadpoles and water insects, even whenapplied in concentrations significantly higher than molluscicidal concentrations.

In practice, a molluscicidal agent according to the invention may be applied as anaqueous solution to the water habitat of the snails. Preferably, however, the toxicingrédient of the invention may be incorporated, advantageously with a mollusc attractantas set out above, in a solid environmentally acceptable matrix selected to allow the slowrelease of the attractant, while preventing the dissolution of the toxin, to make up a solidmolluscicidal formulation. The invention accordingly extends to a solid formulation 10048 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 mollusc attractant incorporated in a solid environmentally acceptable matrix. As already indicated above, the mollusc attractant may be a fungal amylase.

Pièces or chunks of suitable size, eg in the form of tablets or briquettes, of sucha solid formulation may then be placed in suitable locations in infested waters. Watersnails, and specifically the schistosomiasis host snails, attracted to the préparation by theattractant, will be killed when the toxin is ingested. Because only spécifie snails will beattracted by the préparation, the toxin concentration will be optimised in its effect onsuch snails, while other water animais, such as fish, tadpoles, etc will not be affected.

The active ingrédients may be incorporated in the matrix by admixing or blending.Such a solid matrix incorporating the toxic ingrédient according to the invention and themollusc attractive ingrédient may then be formed into tablets or briquettes. Such solidforms facilitate distribution and application, and application can be limited to spécifieinfested areas, and can be controlled.

Where the toxic ingrédient, eg gliotoxin, is incorporated in a solid matrix,overdosage will not easily take place. In the event that trace amounts of gliotoxin arereleased from the solid matrix, the gliotoxin will undergo a 50 per cent dégradation after100 hours in solution when exposed to sunlight, thereby eliminating or at least reducingthe possibility of a toxin build-up. Human and animal users of the water are thereforenot exposed to undue health risks.

Because the fungal amylase attractant gradually leaches out of the matrix, and sobecomes available to attract the target snails, the formulation will gradually loose itsattraction. When this occurs, the formulation must be replaced. It has been found thatthe duration of the attraction of the amylase may vary between about 24 and 48 hours. 1 0048 9

The invention and the manner in which it may be put into practice will now befurther described and explained by way of the following examples, based on laboratorytrials. EXAMPLE 1 PRODUCTION OF GLIOTOXIN WITH ASPERGILLUS FUMIGATUS: A liquid culture medium was prepared by admixing with 1? water the followingingrédients: 1 g kh2 po4 1,8 g Asparagine0,5 g MgSO415 g CaCO320 g Brown sugar

The liquid medium was inoculated with Aspergillus fumigatus and shaken for 9days at 25-28°C. The culture filtrate was then extracted with chloroform, and thegliotoxin was crystallised from ethyl acetate. A yield of 50-90 mg/? was obtained withdifferent trials. EXAMPLE 2 PRODUCTION OF GLIOTOXIN WITH TRICHODERMA VIRIDE: A liquid culture medium was prepared by admixing with 1 C of water the following ingrédients: Ug (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 daysat 27°C. The culture filtrate was then extracted with choloroform and 63 mg/î wasobtained in a représentative trial. 1 0048 10 EXAMPLE 3 PRODUCTION OF AMOLLUSC ATTRACTANT WITH ASPERGILLUS FUMIGATUS: A culture medium was prepared by boiling a potato, drying the boiled potato inan oven, and grinding or milling the dried potato to form a granulated dried product. 5 2 g of this product and 10 g of glucose were added to IC water, and the medium inoculated with Aspergillus fumigatus. The culture was allowed to stand for 7 days at37°C, whereupon the mycelia were filtered out. 1 g of CaSO4 was added to 80 ml offiltrate, and the product was freeze dried and pulverised. The résultant powder wasfound to be extremely attractive to fresh water snails. 10 The attraction of the product for snails was clearly demonstrated in tests.

An ordinary commercial potato dextrose broth used as a culture medium wasfound to give unsatisfactory results. EXAMPLE 4

PREPARATION OF A MOLLUSCICIDAL FORMULATION (ON A LABORATORY 15 SCALE): 250 mg of an emulsifier commercially available from Quest International underthe trade name HYMONO 8903 and 0,2 mg of gliotoxin as prepared in Example 1, weredissolved in acetone, and 250 mg of the mollusc attractant as prepared in Example 2, wasadded to the solution. The mixture was dried under vacuum, and the résultant substance 20 formed into a tablet by pressing.

When the tablet was placed in water inhabited by snails, it was found that it wasvery attractive to the snails, and when ingested caused the snails to die. In separateexperiments, it was found that different species of fish and tadpoles présent in the waterwere unaffected. 1 0048 11 EXAMPLE 5 PREPARATION OF A MOLLUSCICIDAL FORMULATION IN TABLET FORM: A tablet formulation was made up by admixing the following ingrédients: (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 (f) Attractant** 200/d

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

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

Ingrédients (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 mixturewas cooled while constantly stirring to achieve granulation.

The correct amount of starch and the attractant were added to form a paste whichwas 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 inadéquate. EXAMPLE 6 PRODUCTION OF A MOLLUSCICIDAL FORMULATION IN TABLET FORM (ONA SCALE SUFFICIENT TO SUPPORT EXTENSIVE FIELD TRIALS): A tablet formulation was made up by admixing the following ingrédients:Gliotoxin 0,75 mg

Enzyme triturate* 10,00 mg (ie 17 /rg of enzyme powder/tablet)

BaSO4 50,00 mg 10048 12

Starch

Glucose

Stearic acid 40,00 mg50,00 mg199.25 mg350,00 mg 5 * The enzyme triturate is made up by mixing 10 mg of fungal amylase powder in 5 872,40 mg starch.

The mixture is pressed into a tablet, conveniently having a diameter of 8 mm, witha single break lip and a bevelled edge, and compactée! to 9,80 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 andstarch included in the formulation.

The Applicants hâve found thaï a concentration of attractant of 17/zg of enzymepowder/350 mg tablet is optimum. Increasing the quantity of enzyme powder to34gg/350 mg tablet begins to cause repulsion of the snails. Decreasing the quantity of 15 enzyme to 8/zg/350 mg tablet shows decreased attraction. The duration of attraction wasfound to be about 24 to 48 hours.

The Applicants hâve found that a solid molluscicidal formulation containingbetween about 0,07 to 0,21% of the toxic ingrédient, and between about 0,0025 to 0,01%of the attractive ingrédient gives favourable results in practice. A molluscicidal 20 formulation in solid form may thus be provided, containing between about 0,71 and2,14 mg of toxic ingrédient per gram of solid formulation, and between about 22,9 and68,6 /zg of attractive ingrédient per gram of solid formulation.

Claims

1. A method for selectively combaîing or controlling molluscs, which includes thestep of applying to an infested environment a mollusc attractant selected to attract targetsnails to a suitable toxic agent, the mollusc attractant comprising a fungal amylase as an 5 attractive ingrédient.

1 0048 13 CLAIMS

2. A method as claimed in claim 1, in which the mollusc attractive ingrédient is theproduct 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 thegroup comprising: 10 Aspergillus oryzae. andAspergillus niger.

4. A method of selectively combating or controlling molluscs, which includes the stepof applying to an infested environment a molluscicidal agent in which the toxic ingrédientis selected from thé group comprising gliotoxin and similarly effective epipolythiodioxo- 15 piperazine analogues thereof as herein defined.

5. A method as claimed in claim 4, in which the molluscicidal agent is appliedtogether with a mollusc attractant comprising a fungal amylase as the attractiveingrédient.

6. A method as claimed in claim 5 for combating or controlling freshwater snails, in20 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 capableof carrying Schistosome parasites, and in which the toxic ingrédient is gliotoxin. 10048 14

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

9. A method of producing a toxic ingrédient for a molluscicidal agent, which includesthe steps of allowing a selected fungus species to grow and ferment in a selected culturemedium, and recovering the product formed.

10. A method as claimed in claim 9, in which the fungal species is selected from thegroup comprising: Aspergillus chevalieri [(Mangin) Thom & Church] Aspergillus fumigatus [Fresenius] Aspergillus niger [van Tieghem] Gliocladium deliquescens [Sopp = G. viride Matr] Gliocladiuro virens [Miller, Giddens & Foster] Pénicillium cinerascens [Biourge] Pénicillium restrictum [Gilman & Abbot] Pénicillium terlikowski [Zaleski] Trichoderma hamatum [Bonard (Bain)] Trichoderma viride [Pers. ex Gray]

11. A molluscicidal agent which includes a mollusc attractant comprising a fungalamylase as the attractive ingrédient, and a suitable toxic ingrédient.

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

13. A molluscicidal agent as claimed in claim 12, in which the fungal species isselected from the group comprising: Aspergillus orvzae. and Aspergillus niger. 10048 15

14. A molluscicidal agent which includes a toxic ingrédient selected from the groupcomprising gliotoxin and similarly effective epipolythiodioxo-piperazine analogues thereofas herein defined.

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

16. A molluscicidal agent as claimed in claim 15, in which the fungal species isselected from the group comprising: Aspergillus chevalieri [(Mangin) Thom & Church] Aspergillus fumigatus [Fresenius] Aspergillus niger [van Tieghem] Gliocladium deliquescens [Sopp = G. viride Natr] Gliocladium virens [Miller, Giddens & Foster] Pénicillium cinerascens [Biourge] Pénicillium restrictum [Gilman & Abbot] Pénicillium terlikowski [Zaleski] Trichoderma hamatum [Bonard (Bain)] Trichoderma viride [Pers. ex Gray]

17. A molluscicidal agent as claimed in any one of daims 14 to 16 for combating orcontrolling snails capable of carrying Schistosome parasites, in which the toxic ingrédientis 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 anyone of daims 11 to 18, and an environmentally acceptable matrix.

20. A molluscicidal formulation as claimed in claim 19, which is in solid form andincludes a solid environmentally acceptable matrix selected and compacted to allow the 1 0048 16 graduai release of the active ingrédient when placed in water while delaying thedissolution of the toxic ingrédient.

21. A molluscicidal formulation as claimed in claim 20, in which the matrix is selectedand compacted to allow release of the attractive ingrédient over a period of at least 24 5 hours.

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

23. A molluscicidal formulation as claimed in any one of daims 19 to 22, whichcontains between about 0,07% and 0,21% of the toxic ingrédient, and between about 10 0,0025% and 0,01% of the mollusc attractive ingrédient by weight in the solid formulation.

24. A molluscicidal formulation as claimed in claim 23, which contains between about0,71 mg to 2,14 mg of the toxic ingrédient per gram of solid formulation, and betweenabout 22,9 //g to 68,6 /rg of the attractive ingrédient per gram of formulation 15

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

26. A method of selectively combating or controlling snails capable of carryingSchistosome parasites, substantially as described and exemplified herein.

27. A molluscicidal agent, substantially as described and exemplified herein.

28. A molluscicidal formulation, substantially as described and exemplified herein. 20

29. A mollusc attractant, substantially as described and exemplified herein.

30. A method of producing a toxic ingrédient for a molluscicidal agent, substantiallyas described and exemplified herein.