ZA200305963B - Entomopathogenic microorganism spores carrier and method for controlling harmful insects - Google Patents

Description

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TITLE

ENTOMOPATHOGENIC MICROORGANISM SPORES CARRIER AND METHOD

FOR COTROLLING HARMFUL INSECTS

TECHNICAL FIELD OF THE INVENTION

The present invention is set within the agricultural sector, particularly in the field of ©prccess for combating insects. More specificaliy, it refers to a carrier vehicle cf spores of at least one entomopathogenic microorganism adsorbed on a granular porous support or in powder form; to a device which includes said composition or said vehicle; to an incorporated attractant semiochemical component and to a method for combating insects by means of the use of said device.

BACKGROUND OF THE INVENTION

The use of insecticides in controlling plagues of insects presents problems such as toxicity; the lack of selectivity which leads to the destruction of beneficial insects or of natural predators of the plague it is sought to combat; and the resistance developed by the insects which causes an increase in the dose needed to maintain its efficacy.

The use of semiochemical substances has develcped substantially and, 1n particular, techniques such as surveillance in order to anticipate the appearance of plagues and extinguish them, sexual confusion in order to prevent reproduction of the insects, and mass capture in order to decrease the population of them.

Semiochemicals display low toxicity and very high selectivity, since they act on a single species of insect. Also, they do not provoke any resistance among insects cr contaminant effects fcr the environment.

As weil as the use oI semiochemicals in a process for combating insects, the entomcpathcgenic properties of certain tyres cf microcrganisms, such as fungi, can also be exploited. These properties are known, tut thelr effectiveness in biological conirel of 1nsect plagues depends largely on their method of application.

For the specific and directed application of entomovathogenic microorganisms, supports are needed for the spores that will keep them viable and without germinating for long pericds of time in the field and, moreover, combining them with specific semiochemicals that will attract the particular species to be combated, towards the contaminant support.

For the use of semiochemicals to be effective, it is necessary to have physical supports complying with requisites such as: - providing an adequate emission rate of the semiochemical, - permitting prolonged duration of the emission, - avoiding degradation of the semiochemical, - not producing contaminant waste, and - to be economical and to permit easy application of the semiochemical.

Of the emitter supports existing on the market, such as, for example, rubber septa, polyethylene pipes, porous plastic sheets, etc., none of them complies with the stated requisites.

Moreover, the methcds 50 far used for the application of entomopathogenic microorganisms present problems cf survival which diminish their efficacy.

Application WO0-9101736 claims a gel of natural polymers to which attractant entomopathogens, feeding stimulants and protectors against UV are incorporated. It also claims several forms of gelling the polymers by means of metal cations and the insects ingest the entomoprathogen with the gel.

Pe 2005/5563

The differences with the present proccess are that wnat is claimed is the adsorption cf entcrmopathogenic fungi spores in adsorbent materials selected among silicates, siliccalumirates, phosvhca’urinates and ion exchange resins, which further incorpcrate the insect- specific volatile attractant compounds (pheromones and other natural cr synthetic attractants) 1n such a way that a slcw, controlled and lasting emission of the same takes place (Patent UPV No. P 9701077) and they maintain a suitable degree of humidity for the survival of the spores.

WO-A-9208355 claims a process for drying microbial cultures, mixing them with a carrier like those used for formulating insecticides for dusting and drying, them with air. Its application is by means of coating seeds with microbial dust and 1s of nc use for protecting aerial parts.

Application W0-9211356 claims some particular strains of entomopathogenic fungi. It also claims a formulation wherein the spores are incorporated into an emulsified bait containing cotton seed flour (proteins and carbohydrates), extracts of parts of cotton plants, oil and an emulgent, all these as a “feeding stimulant”.

This bait is sprayed in liquid form or it is added to a solid carrier in powder form, in the usual form of insecticide formulations and 1s applied as a wettable powder or in dusting.

W0-9324013 claims a storage chamber for entomopathogenic fungi for their conservation until they are used for controlling insects such as cockroaches, flies, ants and plagues of larvae, plagues on lawns and caterpillars. In these chambers, the temperature, humidity and oxygen are regulated. An emulgent can also be added for applying the fungus in aguecus suspension or in powder form. It cites two patents for cockroach traps.

WO-951C597 claims a formulation of entormopathogenic fungi wherein the conidia are suspended in a mineral oil.

This formulation is used for being applied by spraying on crops. It can pe made emulsicnable for dispersion in water by the addition ©f an emulgent or adding an inert carrier to it for its application in the form of a suspendible powder as is usual in insecticide formulations.

EP-A-04C6103 claims the culture ct an entomcpathogenic fungus on an iner:i solid support such as montmorillonite or atapulgite and the use of the resulting mass for spraying against plagues or for fermenting in the soil under sporulation conditions.

The use cf adsorbent materials as controlied rate emitters c¢f inscct attractants - generally sexual pheromones - is described WO0-A-9944420-A and WO-A- 0000446. Nevertheless, the process described in these patent applications does not include the use of an entomopathogenic agent.

As is revealed from the above paragraphs, both the actual existing physical supports and, in general, the methods for combating insects based on the use of entomopathogenic microorganisms still display serious deficiencies in several basic aspects, such as the period of duration of the composition or vehicle used and the demonstrated efficacy, for which reason there exists a demand fcr the development of new insec% control systems.

CESCRIPTICN CF THE INVENTICN

The present inventicn aims to overcome the drawbacks of the state of the art by means of a carrier vehicle for spores of at least one entomopathogenic microorganism, consisting of an adsorbent suppcrt selected among a granular form, a powder form and mixtures of them, capable of retaining the spores and maintaining their viability and a attractant semiochemical component, wherein: the adsorbent support is a material selected among silicates, silicoaluminates, phcsphoalumirates, ion 5 exchange resins and combinations of them; the spores are suspended in an oll cemponent selected among mineral oils, vegetable oils, animal oils and mixtures of them, adsorbed onto the mineral support and located on the surface in a way that 1s accessible to the attracted insccts; and the semiochemical compcrenrnt is selected from among semicchemical substances specifically attractant of the species of insect it is wished to combat and is adscrbed on an adsorbent support.

The semiochemical component, selected among attractant semiochemical substances of insects susceptible of suffering the entomopathogenic effects of the spores, 1s also adsorbed on that support or on another analogous support.

The advantage of this invention 1s that 1t combines three different effects: a) The fixing of the biological material on a support which adsorbs it on its surface in an adequate way for contaminating insects by contact and for maintaining its viability during a long period of time. b) This support maintains by adsorption a degree of humidity that is adequate for preventing the piological material from drying out vet is insufficient for its germinaticn, giving a prolonged life. =) The same or anotler support, or a mixtuarz of them, adsorbs the attractant o»rodincing a controlled and

Lasting Emission tnerec: and ensuring the specificity. d) An adhesive fixes the adsorbent support, 1in powder or granular form, on a solid surface (plates,

SUR Sc ba spheres, etc.). This adrnesive can be an organic polymer

Or an aqueous gel which contributes to maintain a corstant and regulated supply of humidity to the adsorbent suppcrt located therecn. e) The local form of application is more specific

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and eco.cgical than general spraying. £) The carrier stpoecrt cf spores and/or of attractants Is adhered tc a solid surface (plates, srhieres, etc.) by means of a natural or synthetic pclymeric adhesive, and the device is located in the field ir the usual way for traps.

A first additional object of the present invention is a composition for combating insects wiiich lncorporates that vehicle comprising spores of at least one entomopathogenic microcrganism, at least cne adsorcent support selected Irom a granular form or in powder form and an attractant semiochemical component.

A second additional object of the present invention is a device for combating insects, characterised in that it cempriscs a receptacle comprising, in a way that is accessible for insects, a carrier vehicle of spores of at least one entomopathogeric microorganism, at least one adsorbent support in granular or powder form, and an attractant semiochemical component.

A third additional object of the present invention is a method for combating insects by means of infection of them with spores of at least one entomopathogenic microorganism, characterised in that an efficacious quantity of the vehicle or of the composition obtained, according tec the invention, is made available to the insects.

The entomopathogenic microorganism can be any fungus or bacterium capable of contaminating the insects. In a preferred manner, said microorganism is a fungus, for example Metarhizium anisopliae, Paecilomyces fumosoroseus, Beauveria bhassiana, e“c. The spores of the entomopathogenic microorganism are present in a quantity between 1x1C’ and 1x10? spores per gram of adscrbent support.

The support 1s a natural or synthetic adsorbent ee ————————————————————————— .

Teer ’ IEA material selected from among silicates, silicoaluminates, phosphocaluminates, icn exchange resins, or any combinaticn of them. In a preferred ranner, said support is a zeolite and mere preferably still it 1s a sepiclite.

The size of the structural channe.s of the adsorbent support must be adequate for housing the spores yet preventing them from penetrating to the interior of the structure cf the support when exposed and accessible to insects. This size of particie lies between 230-45C nm, preferably between 240 nm and 420 nm {figure I). The quantity of adsorbent support used is between 50 and 60 mg per cm’.

The device of the present invention can furthermore include an intermediate base for fixing the adsorbent suppcrt on a flat or curved selid surface. This intermediate base can be any organic adherent polymer, such as for example an elastomeric adhesive, or an emulsion of them, or an agueous adherent gel obtained with one or more natural or synthetic gelling agents, such as for example agar, alginates or other polymers of algae and fungi, carboxymethylcellulose, crystalline cellulose, quitosanes and derivatives, methylcellulcse and methylbutylcellulose.

When this adherent intermediate base 1s an agueous gel, a wetting agent can also be added to it, such as sorbitol, glycerol, manitol, xylitol and combinations therecft. Desiccation is Thereby avoided ana this intermediate base alsc helps to maintain the humidity of the adscrient suppgcrt in pewder cr granular form for the spores.

In a preferred embodiment, methyvlbutylcellulose is used. In ancther preferred embodiment, a mixture is used of carboxymethylcellulose (CMC, and methylcellulose (MC) in a proportion of CMC and MC of between 5 and 40 3% by welght. The mcistening agent is selected from among one

HIE . : 2003/5963 or more polyalcohols, in a preferred way it 1s selected from amecng sorbitcl, glycerol, manitol, xylitol and combinations therecf. In a still more preferred way, sorbitcl or glycerci is used.

The quantity cf said moistening agent in the device is between 20 % and 96 % by dry weight. It is preferably in a proportion of from 25 % to 83 % by dry weight of the device.

The attractant semiochemical component 1s specific for the species being cealt with and can be a pheromone or other natural or synthetic attractant that produces an adequate response. Specific examples of semiochemicals are trimedlure and 1l-4-tetramethylenediamine, specific attractants of Ceratitis capitata, or methyl-eugenol, an attractant of Ractrocera dorsalis. The semicchemical component is present in the adsorbent support in a proporticn between 0.005 - 1.0 gram per gram cf adsorrent support, preferably 1n a proportion between 0.02-0.7 grams per gram cf said adsorbent support.

The adsorbent support of the present invention can furthermore comprise an o©0il component selected among mineral oils, vegetable o0ils, animal oils and mixtures thereof, which contributes to fixing the attractant semiochemical component and protecting the spores. The function of the oil component is to help in the retention of the semiochemical and 1ts slow and controlled emission and to maintain the spores in an oily medium in order to increase their protection and extend their life-time.

Said cil ceompenent 1s present in the vehicle ky an amcunt between 20 % and 73 %.

The device of the present invention can adopt various forms according to the arrangement of its components, thereby ensuring the maximum duration of the attraction and contamination of the insect. So, the surface coated with the support can be flat (plates of different dimensions, folded or unfclded) or curved (spheres imitating fruits, cones, cylinders and other : shapes) .

In a first form, an adscroent support containing the spores and the semicchemical component with or without 0il is adhered to a solid surface by means of organic adhesives or aqueous gels.

In a second form, the spores and the semicchemical component are adscrioed cnto different adsorbent supports.

The mixture cf the two supports 1s achered to a solid surface by means of organic adhesives or agueous gels.

In a third form, the adsorbent support containing the semiochemical component is incorporated into the adhesive base layer and the adsorbent suppcrt ccntaining the spores is spread on the surface,

In a fourth form, the adsorbent support containing the semiochemical component is in the form of a vill, located in the centre of the adhesive base layer and surrounded by the adsorbent support containing the spores.

The vehicle of the present invention can include a

UV ray photoprotector in its composition.

For its transportation and use, the vehicle of the present invention is lccated on an object, for example, plates or spheres, the surface of which contains an adherent component and the adsorbent. These plates are located in the field underneath the traps, which protect them from the sun and rain.

An additional oblect of che present invention is ga composition that includes a carrier vehicle for spores of at least one erntomopathogenic microcrganism adsorked on a support, as specified earlier, the application of which can be done in a suspension of the vehicle in a fluid, such as water for example, or by dusting, in an manner analogous to insecticide formulations. )

Ge . p 2005/5953

A second additional object of the present invention is a device for —ccmbating insects, ccrprising a receptacle which, in a manner accessible for the insects, cecmprises e& vehlcle cr a composition which incorporates spores of at least cne entomopathogenic microorganism adsorbed on an adsorbent support, as has been specified earlier.

By means of the present invention, a long period of activity of the spores 1s achieved since the device maintains =the necessary nurmidity in the adsorpent material for their survival and permits the degree of humidity to be maintained for prolcnged periods, of the order of 2 to 5 months of exposure in the field.

Another advantage over the prior art is the selective effect that is achieved, thanks to the use of an attractant semiochemical, specific to the species to be dealt with.

Another advantage of the present invention consists of the use of particles of adsorbent material as support for the spores, which grants efficacy to the method of combating insects since it ensures its contact with the insect anc contamination of it, as well as acting as an attractant vehicle and so that the humidity can reach the spores.

EXAMPLES

The following examples serve to 1ilustrate the different aspects of the invention.

EXAMPLE 1

Ar. adherent layer of 2 mm of polyiscbutylene is deposited on a plastic plate, and on this another layer of MCM-41 zeolite carrier of adsorbed spores of Metarhizium anisopliae suspended in mineral oil is deposited. The zeolitic support 1s also impregnated with specific pl attractants of Ceratitis capitata, for example, with trimedlure or 2-4-te*tramethylerediamine in a proportion of 0.5 and €.005 g per gram of zeolite.

Table © sncws the result crtalired with the spore carrier vehicle, expcsed in the field fcr three months.

In particular, the effect of ageing is shown on the physical and biological properties of the attractant- contaminant plates of Ceratitis caplitata, alcng with the results cf its action on the insects.

TABLE 1

Time (days) Power of Mortality Loss of

Attraction (3) © humidity

EN (mean * SD) (%) {mean + SD) 1s ls.2 0.5 [45.8 + 1.6 35.4 + 1.1 60 175 40.9 + 0.9 [70.8 % 3.2 al Mean count of males alighting on the plate, every 5 minutes, for 3 hours.

P! Number of dead flies as a result of mycosis following a 24 hour exposure period to the plate.

EXAMPLE 2

A plate Is rvrerared with a gelled adherent base of methybutylcellulose in a proportion that can vary from 10 to 45 %. Glycerol is added as a moistening agent in a proportion cf 20 to 75 % by dry weight. Deposited on the

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Te Loe 12 gel is a layer of sepiclite of particle size between 240~- 420 nm, which is impregnated with spores of Paecilomycées fumosoroseus, suspended in mineral oil. The attractant ser‘ccherical used ‘ss methyl-cugencl, an attractant of

Bactrocera dorsalis, which is adsorbed on another portion of sepiolite in a proportion between of 0.8 gram of sepiolite and is compacted to form a pill, which 1s partially submerged in the centre cf the plate carrying the gel component. 10 . The plates are located in the field underneath the traps, which protect them from the sun and rain.

Table 2 shows the effect of ageing in the physical and biological properties of the attractant-contaminant plates of Bactrocera dorsalis, along with the results of its action on the insects,

TABLE 2

Time (days) Power of Mortality Loss of

Attraction (2) ° humidity (3) (mean + SD) (3) (mean + SD) o leoxs [sooo Jo 000 50 38.3 & Z.Z 170.4 + 0.5 52.3 £ 1.3 * Mean count of males alighting on the plate, every 5 minutes, for 3 hours.

Pl Number of dead flies as a result of mycosis following a 24 hour exposure period to the plate.

Claims (21)

1. A carrier vehicle of spores of at least one entomopathogenic microorganism OL an adsorbent support selected among a granular form, a powder form and mixtures +tnereof, and of an attractant semicchemical component, characterised in that: the natural or synthetic adsorbent support is a material selected from among silicates, silicoaluminates, phosphoaluminates, ion exchange resins and combinations of them; tne spores suspended in an c1. compcnent selected among mineral oils, vegetable oils, animal oils and mixtures of them, and are adsorbed onto the mineral support and located on the surface in a way that is accessible to the attracted lnsects; and the semiochemical component 1s selected trom among semiochemical substances specifically attractant of the species ~f ipsect lu as wished 10 mowrat ana 1s adsorbed on an adsorbent support.

2. A vehicle according to claim 1, characterised in that it furthermore comprises an adherent intermediate pase for retaining the adsorbent powder and preventing its dispersion, said base being selected Dpetween a viscous polymer or an aquecus gel with a moistening agent, and sald adherent intermediate base being app.ied on p.ates, spheres or other so.id shapes fcr being located in the field.

3. A venicle accoraing To Claim Z, crnaracrerisea in that the attractant semiochemical component is adsorbed or +he adscrgent csuppcrt and sald supccrt, coated with the spores, 1s spread on the intermediate adherent.

4. A vehicle according to claim 2, characterised in that: Sra Lleet Lo on 2006 A —

the semiocnemical comporent is adsorbed on a natural cr synthetic suppert sclected among a granu.ar form, a powder form ard mixtures therewi, and selectca Irom among silicates, silicoaiuminates, phosphoaluminates, ion exchange resins and combirations of them, and this support can be the same as or aifferent from the spore carrier.

5. A venicle acccrding to ciaim 4, characterisea in that: the semiochemical component 1s adsorbed on the adsorbent support, the same as or different from the spore carrier. the adsorbent support together wlth the semicchenical is compacted in pills, which are surrounded py Tne farrier suppers for spores, pelng the TWO adsorbent supports the same or different.

6. A vehicle according to claim 1, 4, or 5, characterised ir that: the spores are suspended in an oil component selected among mineral oils, vegetable oils, animal oils and mixtures of them.

7. A vehicle according to claim 2, characterised in that: the intermediate adhesive layer 1s selected among organic polymeric adhesives or 1s an aqueous gel of natural cor synthetic polymers.

8. A vehicle according to claim 7, characterisca in that: tne adherent layer 1s formed from crganic adhesives or from gels, the gelling substance of which is selected among natural or synthetic hydrophilic polymers such as carboxymethylcellulose, crystalline cellulose, I SE T= SRS SER PP PE £6 carboxymethylchitosane, methylcellulose, methylbutyicellulose, gquitosanes, po.ymers of algae and plants and cecmbinations thereof.

9. A vehicle according to claim 2, characterised in that the moistening agent consists of one or more polyalcohols.

10. A vehicle according tc claim 9, characterised in that the mcistening agent 1s selectea amcng sorbitol, glycerol, manitol, xylitol and combinations thereof.

11. A vehicle according to claim 2, characterised in that the moistening agent 1s present in a quantity between 20 % and 96 % of the ary welght ot the gelling agent.

12. A vehicle according to any of the above claims, characterised in that the semiochemical component comprises at least one pheromone or a synthetic or natural attractant, specific fcr the species is it wished to combat.

13. A vehicle according to any of claims 1 to 5, characterised in that the semiochemical component is present in the adsorbent support in a guantity between

0.C05 and I g per gran.

14. A venicle accoralng To claim 4 or 3, characterised in that the semiochemical component 1s present 1n the second adsorbent cuppert in a cuantity between 02.000° and 1 g per gram of said second adsorbent support.

15. A vehicle according to claim 1, characterised in Toad hoot Luo oa uld that the entomopathogenic microorganism is a fungus or bacterium.

16. A vehic.e according to claim 1, characterised in that the spores of the entomopathogenic microorganism are present in a quantity between 1x10’ - 1x10'* spores per gram of adsorbent support.

17. A vehicle according to claim 1, characterised in that the oil component is present in a quantity between 20-75 % per gram of adsorbent support.

18. A composition for combating insects which contains a vehicle as defined according to any of claims 1 to 17.

19. A device for combating insects, characterised in that it comprises a receptacle containing, in a form accessible for the insects, a vehicle defined according to any of claims 1 to 17.

20. A device for combating insects, characterised in that it comprises a receptacle containing, in a form accessible for the insects, a composition defined in claim 18.

21. A method for combating insects by means of infection of the insects with spores of microorganisms, characterised in that the insects are exposed to an efficacious quantity of a vehicle according to any of claims 1 to 17 or composition according tec claim 18. Slmiidel mio Zo 0136