AU631221B2 - Microoganism-based pesticide compositions, their production process and their application in agronomy - Google Patents

Abstract

Pesticidal composition composed of particles made of a solid support which is colonised by a microorganism, in particular by spores of an entomopathogenic fungus which can be used in the control of crop-destroying insects or for the fertilisation of soils. The support used can be, for example, montmorillonite or attapulgite, and the fungus can be Beauveria bassiana, which has a pathogenic action on larvae of the European corn borer Ostrinia nubilalis. The composition is prepared by a fermentation process in solid medium and can be used by spreading directly as it leaves the fermenter, without further treatment.

Description

WATERMARK PATENT TRADEMARK ATTORNEYS

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t- L

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s s Form COMMONWEALTH OF AUSTRALIA PA TENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: ,Complete Specification Lodged: 'JEr P o i Accepted: Published: !1 Priority

A.,

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Relaced Art I ,Name of Applicant Address of Applicant SActual Inventor Address for Service INSTITUJ NATIONAL DE LA RECHERCHE AGRONCMIQUE 147 rue de 1'Universite, 75341 Paris Cedex 07, France GUY RIBA, JACQUELILNE GOUSSARD, ALAIN DURAND and CATHERINE

DESGRANGES

WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: MICROORGANISM-BASED PESTIC DE CCMPOSITIONS, THEIR PRODUCTION PROCESS AND THEIR APPLICAITON IN AGRONCMY The following statement is a full description of this invention, including the best method 'f performing it known to us r~--rr MICROORGANISM-BASED PESTICIDE COMPOSITIONS, THEIR PRODUCTION PROCESS AND THEIR APPLICATION IN AGRONOMY.

The present invention relates to new compositions with a base of entomopathogenic mushrooms, their produ :ion process and their application in agronomy.

0 ti Numerous synthesized chemical compounds with rela- 0 tively se.ective insecticide properties are already known, and although they are highly efficacious, their use is preferably limited, whether for economic or environmental reasons.

S With previous methods it has been noted that app- Sropriately selected microorganisms, notably certain mushrooms, could make an enormous contribution to an infected agrosystem and play the same role as chemical pesticides by L *limiting the development of crop insect pests and retarding -i 20 multiplication of weeds or other mushroom plant pests.

Biological insecticides are more specific than the majority of chemical pesticides and therefore do not lead to secondary pnenomena furthermore, they are non-toxic Cg rPI__JP~mnYIUsm_ IO~~ it i e to either invertebrates and vertebrates or humans. On this basis their use would be totally advantageous. Nevertheless, the following problems are encountered first, the proliferation of entomopathogenic mushrooms is limited to warm and humid regions, whereas in temperate regions the fight against insects is not considered economically viable, given the high doses of mushrooms used secondly, in order to compensate for these drawbacks, a technology has been proposed, namely the use of solid supports, which are already known to professionals, as they are used in the production of compositions containing chemical insecticides or pesticides. GE 1002977 describes several compositions containing vermiculite as a solid support, each also containing a given pesticide, as well as various agents suchas wetting agents.

US 4530834 relates to a production process for dried mycelium, comprising a cerLain niumber of steps, including a step for cultivation in a medium defined by the inventors as 'any commercially available medium', a step for treatment by a protective chemical agent for increasing viability of the mycelium, and a step for incubation and drying. It is specified that the products to be used for cor.trol of in.

sects are ground and formed into wettable powder, then sprayed onto plants.

S 20 US 3013 946 also relates to a production p.ocess 3 for a musliroom composition having an insecticide effect, by cultivation of microorganisms of the Moniliales family on a solid vermiculite support. It is specified 'hat fermentation is carried out over a period of 14 to 21 days, but without control of the state of the mushroom, and especially of the presence of spores. The culture medium is dried after fermentation before being used for spreading.

SOo Mites are very well known for causing insect damage 1..0 to crops, such as corn mites of the pyralide family Ostrinia o o' .nubilalis and Ostrinia furnacalis. Several species of entomoo pathogenic mushrooms are vulnerbale to attack by mites thus 0 0e the eggs of this insect are very sensitive to stocks of Metarhizium anisopliae, while the pupae die easily from pink pebrine with Paecilomyces fumosoroseus. Another mushroom, Beauveria bass.ana, is responsible for white pebrine which 4 o attacks the nite larvae and exists in an endemic statt in populations of Ostrinia nubilalis. As with the majority of hyphomycetes, Beauveria bassiana generates two types of 20 infectious propagules blastospores by budding of mycelium, ensuirng vegetative multiplication of the mushroom exclusively present in sugmerged culture, and conidia present in submerged culture and in solid state culture.

FR 2.394.606 relaes to F. production process for microorganisms, including neauveria bassiana which can be 4 9 4 used in the fight against plant diseases, comprising mixing of an aqueous microorganism preparation with a clay suspension, which can be montmorillonite or bentonite.

A 1979 article by Ignuffo et al. (Journal of Economic Entomology, 72, 562-565) eascribes the production of Beauveria bassiana by fermentation in liquid medium, followed oy drying and mixing with talc or other inert material, such as pearlite, kaolin, bentonite or starch.

EP-A-O 226 394 describes a direct fermentation process in a single step for various microorganisms on particles, more specifically vermiculite particles. The support thus treated can used to inoculate cultures. In a characteristic realization the process comprises first mixing vermiculite, flour and water, sterilizing this mixture, then inoculating it with a microorganism such as a mushroom, storing it to mature and finally drying it to produce a particular product which can be used for treatment of crops.

The process described in EP-A-O 226 394 makes no mention of ocntrolling the microorganism culture, whereas this parameter is essential for allowing efficacy of the product under the conditions for spreading and for optimizing manufacture. This patent does not describe a product in which 'he microorganism has an aptitude for sporulation

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2 i aw. I_~b C; in spreading conditions. Moreover, it provides sterilization of the support. In the illustrative examples given in EP A-O 226 394 it is noted that significant growth times are specified 45 days in Example 1, 2 to 4 weeks in Examples 2 and 3. For the technician such time periods imply a lack of quality control and quantity control of the mushrooms in the final product. This process is also carried out in containers, the use of which produces growth of minimal thickness without any monitoring. The preferred support, such as illustrated in the examples of the prevoius patent, is vermiculite which proves unsuited to mechanized agronomic use.

Therefore, the process described in EP-A-O 266 394 does not ccme up with a product able to sporulate in spreading conditions, under industrial growth conditions.

FR-A-2 298 951 relates to products in granulose form obtained by extrusion, which serve as inoculant, fertilizer or in other similar applications. Microorganisms are added to a solid support and the mixture is treated until it reaches the desired solid form. The microorganisms, essentially of the Trichoderma type, are incubated on crushed barley corns or other grains, in closed containers.

This process disallows control of the quantity of biomass and of its quality.

6 FR-A-2 298 951 makes no mention of the use of particles or preplanted granules.

All these solutions to the problem of the biological fight against a crop insect pest, using mushrooms as insecticide, have the same disadvantages, that is, none of them is particularly efficacious in inhibiting propagation or growth of said insects.

0 The aim of the present invention is to obtain a product which offers high efficiency, stability, low production costs, due in particular to greatly reduced growth times and considerable ease of use.

A more particular object of the invention is a granular product, precolonized by microorganisms, whose maturing is controlled and which finds application in agronomic treatment.

The object of the present invention therefore is a 0.O product for agronomic processing with a base of at least one microorganism useful in the fight against insect pests and crop diseases or for soil fertili7ition, wherein it comprises particles formed from at least one solid support carrying the microorganism, said particles being colonized by the microorganism, the latter capable of sporulating _lpIl-il--~---D--ilC-~i under distribution conditions.

Said microorganism may be a bacteria permitting soil fertilization, such as Rhizobium, or an octomycorrhizian mushroom such as Laccaria. It may also be an entomopathogenic mushroom, effective against one or more crop insect pests, such as ground insects, especially scarabaeoidea and curculionides, stinging insects, especially cercopides, or boring insects, especially mites.

Said microorganism may also be a pathogenic mushroom vis-a-vis other mushrooms, such as Fusarium, non-pathogenic vis-a-vis plants but pathogenic vis-a-vis noxious Fusarium, or a Trichoderma pathogenic vis-a-vis Sclerotinia, Rhizo- S 15 otonia, Armillaria or Botrytis.

Said microorganism may also have a pathogenic effect on nematodes it may thus be an Arthrobotrys.

A product containing one of these microorganisms especially has the advantage, compared to pesticides or customarily used fertilizers, of not polluting the environment and thus preserving flora and fauna.

According to a particular embodiment of the invention the product can be spread in fields directly oi 8 after it is obtained by direct culture and solid state fermentation on said particles. This is a noteworthy advantage of the invention in fact it is not necessary to grind the product as it leaves the fermenter, nor to include any additives.

According to another embodiment of thb invention the microorganism is a mus'ioom entirely in the form of mycelium at the time of spreading, in which case said product is deemed immature, or the mushroom is in hIe form of mycelium and spores, in which case said product is deemed mature. THis control of the mushroom populatic. state, and thus of the efficacy of the product, is an essential characteristic of the invention. To the knowledge of the applican-, the former method does not describe products whose spore and mycelium composition is controlled precisely. The product according to the invention may aloo comprise a mixture of the mature and immature forms.

According to a further embodiment of the invention Ssaid support has a density preferably between 0.6 and 1.

According to ailother embodiment of the invention the support is of mineral or organic origin, notably montmoril.lo.ite, attapulgite or sepiolite. The support must be compatible with a terrestrial or aerial mechanized application.

According to yet another embodiment of the invention said support has a minimum water retention capacity of According to another embodiment of the invention said particles have a size of between 100 and 1000 microns and preferably of around 500 microns.

0 o Another object of the present invention is a product in which said mushroom is a Beauveria bassiana stock, especially the stock registered with the National Collection of Microorganism Cultures under NO I 867, or a mutant of this stock. T-R&,7 -tas5 Iv 0 stAtu 15M c-\o &v R\c-cr-r Cc\o- C ov. \e on 7 According to anothe particular embodiment of the invention said product is dried for storing and spreading.

Furthermore, an object of the invention is a process for obtaining an agronomic teratment product with a base of at least one microorganism, to combat the depredation s o of insce pests and plant diseases, or for fertilization of soils, wherein it comprises direct culture by solid state fermentation of said microorganism on al least one solid support in particles, insoluble in tih fermentation medium, and the recuperation of said particles, which are 0Sec. I t 77 Tc _ZL colonized by the microorganism, while maturity of the culture is controlled to allow the microorganism to sporulate under the spreading conditionc.

The association therefore of the microorganism and the solid support particles occurs without intervention of any means or physical and/or chemical agent, which proves especially advantageous for the process according to the invention.

Moreover, the microorganisms precolonize the particles during fermentation, ensuring a more effective association of microorganisms and particles.

According to an embodiment of the process of the invention the microorganism is a mushroom. Fermentation is stopped when the mycelium is obtained, resulting in a so-called immature product, 'r when the mycelium and the spores are obtained, resulting in a so-called mature product.

i Stopping fermentation with a view to producing an i:ffmature product allows curtailing of fermentation by several days.

According to another embodiment of the process "f .risru---- I19PB ~WIIP7BBB~~ the invention said particles have or have not been previously sterilized. This is also an advantage of the invention, since processes according to previous methods require sterilization of supports, to the knowledge of the applicant.

This improvement in the fermentation process is thus very significant from the production and economic point of view, because it means that an energy-costly step can be avoided.

According to another embodiment of the process of the invention the mushroom used is an entomopathogenic mushroom, effective against one or more crop insect pests.

This mushroom can be, in particular, Beauveria bassiana, Beauveria tenella, Beuaveria brognartii or Paecilomyces fumosoroseus.

According to another embodiment of the process of the invention said mushroom is Beauveria bassiana. when fermentation take splace over 20 to 60 hours, and preferably over 48 hours, it provides an 'mmabure product when the fermentation time is 90 hours i..inimum a mature product is then obtained.

It will be noted also that the process of the invention can be implemented with increased thickness of support during fermentation, reazhing tens of cms in thickness, which is favourable for industrial production as well as I~ .1Llrr"*"" ~prrtr~F-P~WI~P 12 for control of the maturity of the crop.

A further object of the inventin is a biological control process against diseases and insect pests in plants, wherein an effective quantity of the above describea product, notably with a base of entomopathogenic mushrooms, is spread on plants to be treated, the disease in question being, for example, maize mite which is due to Ostrinia nubilalis, but also other mites or other plant diseases.

Yet a further object of the invention is a soil fertilization process, wherein an effective quantity of the fertilizer product with a microorganism base as previously described, is used for spreading.

The following description further illustrates examples of the invention's applications in a non-limiting way, with reference to the attached diagrams in which Figure 1 schematically represents a reactor-fermenter which can be used to obtain the product of the invention.

Figure 2 is a graphic representation of the evolution of biochemical parameters as a function of the growth stage following the process described in Example 1.

Figure 3 graphically illustrates results of the assays related to Example 4.

EXAMPLE 1 Production of products according to the invention a. Preparation of the inoculum A semi-synthetic liquid medium A (Table 1) is sown by means of conidia of Beauveria bassiana, resulting from a culture on a semi-synthetic medium B (Table 2) aged 5 days, at a rate of 106 conidia/ml. This medium is incubated at in flasks on an agitation table or in a liquid -rmenter as a function of quantities of culture to be inocula .d.

After 72 hours of growth the medium A contains between 8 1 and 2.10 blastospores/ml.

TABLE 1 Composition of medium A Cereal maceration liquid 20.0g EScrose 30.0g

KNO

3

KH

2

PO

4 G.8g CaCO 3 MgSO 4 7.

2 0 0.lg Permutated water 1 1 ~~lW l~ a~ a~ -up~n~ TABLE 2 Composition of medium B Glucose 10.0g Yeast extract

KH

2

PO

4 0.4g Na 2 HPO4 12H20 1.4g Mg SO 4 7H20 0.6g KC1

NH

4

NO

3 0.7g 4 3 Chloramphenicol Agar 15.0g I Permutated water 1 1 b. Preparation of solil state culture medium The culture medium used comprises two phases a solid phase constituting the granulose support a liquid phase constituting the nutritive substratum of the mi-roorganism imbuing the solid phase.

The suppoit may be previously sterilized (3 hours at 120 0 C) or not sterilied a sterile process is not essential, since very few contaminants develop fully in the growth conditiors utilized a d since satisfying results are obtained- _I i l(~-La~-xurrrr^-- This support is impregnated with one of the nutritive media C, D or E (Tables 3, 4 or 5) these sterilized nutritive media C, D or E (30 minutes F.t 115°C) are added from the inoculum at the rate of 3.10 blastospores/ ml of medium C, D or E.

Impregnation and mixing can be performed in a kneading trough type of apparatus or in the reactor itself, so as to oLtain a dry matter rate of around 60% (weight of humid S O support/weight). Under these conditions an inoculation 96 rate of around 2.10 blastospores/g of dry matter is obtained. Where necessary the stock is incubated between 18

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and 27°C.

TABLE 3 Composition of medium C SSucrose 132."g Residual non-sugar* 27.0g Permutated water 1 1 i i The residual non-sugar is a by-product from extraction I of the sugar composed mainly from nitrogenous substances and commercialized by General Sugar Refineries.

TABLE 4 Composition of medium D Solulys** 150g j 16 (-ociet6 Roquette) Permutated water 1 1 Solulys is a by-product from extraction and refining of corn starch which contains amino acids and lactic acid.

TABLE Composition of medium E Sucrose 124.0g Solulys 38.5g 10 Permutated water 1 1 The entire homogeneous mixture is Lransferred to the reactor (Figure if the mixing is not performed there directly.

Figure 1 represents the reactor with its monitoring and control device.

The reactor comprises a container filled with a semi-solid medium, in which or more return propellers (1) macerate, carried on a mobile trolley and driven by a motor The trolley is equipped with a feed motor (3) and also carries a pulverization ramp fed by solution inlet pipes (19, 20, 21).

-I ua~~Ea~-u~smnrun~-- 17 Near the bottom of the reactor and parallel to it is an upper perforated plaque (22) constituting a coarse screen which effectively holds the culture medium layer in place this layer may be from 0.50 to 1 metre in thickness.

There is also a lower perforated plaque (23) forming a finer screen and allowing even distribution of air through the interior of the container The two screens may be replaced to advantage by a single screen comprising a mesh of plaited stainless steel (DYNAPORE plate), serving simul- •ig taneously to diffuse air and support the culture medium.

o The air enters the fermentation device in the direction indicated by the arrow F2 and exits from it in the direction of the arrow indicated by Fl. The air is filtered initially by the filter then, if necessary, cooled with the assistance of the heater battery (17) and humidified in the humidifier (13) Circulation of air is ensured by the ventilator the yield is in the order of 7.51.mn-kg of dry matter The mechanism measuring growth conditions in the reactor comprises an oxygen probe a pH probe and a temperature probe Measuring of air temperature and humidity is en,-ured by probes (11) as air enters and leaves the reactor at Compression strain gauges (12) allow 18 measuring of growth mass in the reactor, while measuring of air output is performed with the assistance of an output a meter.

Fermentation control and monitoring of the reactor are ensured by the micro-calculator (24) which receives i information supplied by the oxygen probe pH probe temperature probe temperature and humidity *probes (10) and compression strain sensors (12), 1 i output meter (14) and which as a function of this inform- S 'ation operates the trolley teed motor humidifier (13), ventilator cooler battery heater battery (17) Sadd valves controlling output of the solution inlet pipes (19, 20, 21) i i During growth the temperature is maintained at around i 25'C ond growth humidity at around 40% by ventilation of warm, humid air.

it I 20 d. Control of growth state during production 4 During grcwth it is important to be able to follow the evolution of the production, that is, the stage of the mushroom's growth, in order to decide whether to stop growth or not.

Following the growth stage it is necessary to be 3 IYP IIP~- I able to estimate both the mycelium growth and conidiogenesis. The stage following conidiogenesi.s is carried out meticulously by means of a standardized method for numbering conidia.

Method for numbering conidia Around Ig of culture is weighed precisely and introduced into a flask containing 50 ml of Tween 80 at 1% i, and about 30g of glass balls of 4mm diameter. The flasks agitated in a rotary mixer for 20 minutes.

The solution thus obtained is diluted by ten in a methylene blue solution. After an hour of incubation at ambient temperature the counts are realized on a Malassez cell.

Composition of the methylene blue solution Methylene blue 0.6g

KH

2

PO

4 81.3g Na2HPO 4 12H20 5.7g Water 1.01 Estimation of mycelium growth Direct estimation of mycelium growth cannot be carried out online. However, evolution of respiration can be followed automatically, giving a perfect picture um of the physiological state of the microorganism.

Measuring the fungal respiration is the only method Swhich at the present time allows the cultures to be followed in real time. Gases are pumped at the outlet of Lhe fermenter, pumped off by means of a 'hygrostop' (Hartman and Braun). Their composition is determined by a URAS 3G type jinfra-red gas analyser (Hartman and Braun) for the CO 2 and ;i by a Magnos 3 type paramagnetic oxygen analyser (Hartman and braun). The unit is managed by a microcomputer which .calculates different parameters online the CPR or CO 2 production rate, and the OUR or oxygen consumption rate.

These two parameters are calculated and expressed in mole of CO 2 or 02 per hour and per gram of dry matter.

i Furthermore, pre-performed analyses mean that evoi lutions of glucosamine and ergosterol rates can be traced.

ii An example of the result obtained at the time of Igrowth on medium C is shown in Figure 2 in which the growth time has been set out in abscissa of inoculation of the fermenter the CPR and rate of glucosamine and ergosterol are set out in ordinates. Curve A represents evolution of the glucosamine rate, curve B evolution of the ergosterol rate and curve C evolution of the CPR.

w ur lcclYlllllax; 21 e. Quantity control of the product obtained at the end of fermentation.

The micrograin simultaneously carries mycelium and conidia. It is difficult to separate the two forms from each other quantitatively, and also to separate the mycelium from the support. Only indirect measuring methods can be performed. These consist of titrating biochemical consitiuents of mycelium and spores, chitin Pnd ergosterol, after extraction.

It Chitin and ergosterol.

Although they allow no online control of the product's maturing, since they are too protracted, these methods are quite satisfactory in characterizing the product at the end of fermentation. Numbering of conidia is carried out in paralle according to the numbering method described above in This method is applicable only from 72 hours of growth, as before this time the spore count is too weak for numbering. Total biomass is estimated by titration of chitin and ergosterol.

Titration of chitin.

Chitin, which consiiutes the cell wall, is hydrolized in an acid medium and at heat in N acetyl glucosamine tSakurai et al., 1977, Agric,, Biol. Chem. 41, 4, 619-624). Clucosamine reacts after desamination with a coloured reagent, namely methyl 2-benzothiazone hydrazone 22 (MBTHI. Intensity of the blue colouring is proportional to the quantity of giucosamine (Tsuji et al., 1969, Chem.

Pharm. Bulletin, 17, 7, 1505-1510 and Ride and Drysdale, 1972, Physiological Plant Pathology, 2, 7, Hydrolosis is performed as follows The test sample for processing is first washed three times with a solution of KC1 to 1 mole.l1 to elimi- 0. nate any interference drv to residual nutritive medium C oo ,.10 then 19 of the washed test sample is weighed precisely and 10 ml of concentrated HC1 are added. This is left at ambient temperature for 16 hours, then 10 ml of permutated water are added and this solution is sterilized for 2 hours at 130 0

C.

Titration of chitin is performed in two steps Neutralization of the hydrolysate 1 ml of hydrolysate is neutralized by adding 2.5 ml NaOH to 1 mole.l and 1.5 ml permutated w?.ter.

Titration Si 1 ml KHSO 4 at 5% and 1 ml NANO 2 at 5% are added to 1 ml of the above solution. For 15 minutes the tubes are shaken, then 1 ml NH 4

SO

3

NH

2 at 12.5% is added, after which the tubes are shaken for another 5 minutes. 1 ml MBTH at 0.5% is added and the mixture is put in a bain-marie 13 36 Z 0 I PiHO N I 0 23 for 5 minutes, after which it is cooled 1 ml FeCd 3 at is added and after 30 minutes the result is calculated at 650 nm.

The quantity of glucosamine in the test samples is determined in relation to a range of N acetyl glucosamine (0 to 30 pg/ml) realized simultaneously.

The results are expressed in pg of glucosamine/g of test sample dry matter.

Titration of ergosterol Ergosterol, a consitiuent of the cellular membrane, is extracted from the raw product by saponification of the product, then by liquid/ I liquid separation, the sterols are extracted and sepa- ;rated from the fatty acids and all unsaponifiable substances.

i by elution in high performance liquid chromotography, the ergosterol is separated from other sterols.

ii by absorption spectrophotometry iL UV, the Squantity of ergosterol is determined precisely. Effectively, ergosterol has a specific absorption wavelength of 282 nm and the peak surface obtained on the chromatogram is proportional to the quantity of ergosterol present.

j I I I (Seitz et al., 1979, Phytppathology, 69, 11, 1202-1203 seitz et 1977, Cereal Chemistry, 54, 6, 1207-1217 Kaminski et al., 1983, Fat Science Proceedings, 16th, 1st Congress, Budapest).

Extraction is performed preactically, as follows A quantity of test sample is weighed precisely in a globular flask (a sufficient quantity fo the extract to contain 0.1 to 30 pg/10 ml). 100 ml methanol and 50 ml o0 alcohol potash (KOH 20g ethyl alcohol 50 ml) are added.

The whole is saponified under reflux for 30 minutes, then -1 0 centrifu-'d for 10 minutes at 6000 t.min. The flotsam *t is mixed with 100 ml water and 100 ml petroleum ether.

The whole is left in a decanting phial, then the ether phase (upper phase) is recuperated and evaporated. The dry residue is recovered by 10 ml dichloromethane.

The ergosterol is titrated in the extract thus obtained by high performance liquid chromatography on a column lined with Lichrososorb ST 60 silica having a grain size of 5pm (Merck). The mobile phase is a mixture of methylene chloride isopropanol (99.5/0.5 The results are expressed in pg of ergosterol per gram of dry matter.

f. Control of product viability after fermentation.

N The titration methods described in allow quantification of the total biomass at the end of :he process, but not verification of the viability of the product, either after growth or following a given storage period. Usually, realization of a germinatin test in Petri dishes can determine viability of a sporulated product, the expressed result being the number of germinated .onidia giving birth to a colony in relation to the total number of conidia measured on the Malassez cell.

1 4 Given that the microgranulose product contains essentially mycelium, and that there is thus no possibility of any correlation between the mycelian biomass and the number of colonies which have appeared, a method of analysis allowing estimation of the product's viability has been clarified. It concerns the measuring of the aptitude of the mycelium to be sporulated.

The dried growth test sample is humidified and incubated. Duiring incubation the mycelium continues to grow, although this was stopped by drying, and sporulates. After 144 hours of incubation in total, which takes place during growth and the viability test, the conidia count has reached maximum and remains constant. For growth always carried out under the same coniditons, the maximum count of conidia is always the same. The conidia count need be done only after 96 hours of incubation the resulting value is an indication of the quality of the product.

Measuring method 50g of dried culture are placed in a 500 ml Erlenmeyer flask and humidified with 22 ml sterile water to reach a count of dry matter between 60 and 70%. The flasks Sare then incubated in a climatized room at 25 0 C for 96 hours.

After this incubation period the conidia count is determined as described in g. Storage The granulose compositions according to the invention are stored after drying in an atmosph3re whose humidity is controlled by inert gas, such as azote and carbon dioxide, or under a vacuum preferably at a temperature between 5 and 0

C.

EXAMPLE 2 Field comparison of efficacy of microgranules obtained at various stages of maturity.

Preparations of montmorillonite granules of varying maturity corresponding to incubation times of 48, 72, 96 and 120 hours have been prepared according to the general method of operation described with reference to Example 1 ^~l~e 27 and ha e been compared in Table 6.

Efficacy was estimaated in a complete block assay wi' four repetitions. Ea h plot is infested weekly for three weeks. Each time an ooplaque of mites is introduced per plant.

Efficacy is expressed in percentage of decrease in relation to the proof whose total number is 1.9 caterpillar per plant.

0 e 0 e s The granules obtained after feriLentation of 48, 72 and 96 hours are considered immature, while those obtained 0"'0 over 120 hours are considered mature.

0. ,The mixture is composed of 70% granules of 48 hours and 30% mature granules.

SIt eventuates that immature microgranules, dried before conidiogenesis, are stringly infectious on the ground without the slightest retarding effect.

It should be noted that these immature microgranules contain no infectious propagules (conidia), but are capable of generating then after application in the field.

I

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28 TABLE 6 Study of g.anules with controlled maturity M '.urity in Application EFFICACY hoars of dose of decrease of decrease incubation kg/ha namber of number of attacks larvae 48 25 87.3 72 25 75.9 90.8 96 25 91.7 94 1 0 120 25 68.2 79.7 mixture 25 88.1 93.3 EXAMPLE 3 5 Field comparison of efficacy of product according to the invention and products resulting from other technologies (liquid or solid formulations).

A preparation constituted by a mixture of granules as defined in Example 2 (70% cganules of 48 hours and 0 mature granules of 120 hours) and obtained in accordance with the method of operation in Example 1 has been tested comparatively with formulations prepared according to processes described in previous specifications.

The assays were carried out on maize fields.

C- I~PIPL---- 29 The liquid formulations tested are the following wettable powders a suspension of conidia coated in clay in accordance with the process described in FR-A-2 394 606 a suspension of conidia coated in alginate granules, whether added or not, of nutritive elements a mycelian suspension obtained according to the process described in US-4.530.834.

10 These liquid suspensions are applied to the dose of i' "13 10 conidia or 100 g of mycelium, in 200 1, per hectare.

The results of the assays are set out in Table 7.

Efficacy was estimated in a complete block assay with i four week repetitions. Each plot is infested weekly for 3 I1 weeks. Each time an ooplaque of mites is intr)duced per I plant.

Efficacy is expressed in percentage of decrease in Srelation to the proof whose total number is 1.9 caterpillar per plant.

These results show that the granulose formulation obtained according to the invention is clearly more effective than the wettable powders tested, with respect to decrease in the number of attacks on plants and decrease in the larva population.

The solid formulations tested comprise two types fixation or adsorption of infectious propagules (conidia or blastospores) on microgranules, whether impregnated or not, of a nutritive medium. Such a process is specified in GB-1.002.977 for chemical priucts. Experimentation results S are set out in Table 8. The ccnidia were absorbed in sus- 4 pension in a complete medium with a glucose base and a yeast extract

'I

Efficacy was calculated as previously.

ii These results show a very clear increase in effect- S 15 ively reducing attacks on maize by Ostrinia nubilalis and j in reducing the larva population of Ostrinia nubilalis at the time of treatment by the precolonized prodnct according 3 to the invention.

_I TABLE 7 Comparative study of granulose formulation with various wettable powders Formulation Application Production Efficacy dose/ha decrease in decrease in no. of attacks larva population Granule 25 kg according to (mixture) invention (montmorillonite) 88.1 93.3 Wettable powder conidia blastospores Smycelium conidia 2% chitin conidia 1% glucose .conidia conidia 2% chitin 1% yeast extract L t 1013 10 13 100 g 10 13 1013 10 1 10 1 3 i13 io0 FR-A 2394.606 US 4.530.834 alginate coating alginate coating alginate coating 3 0 9.3 8.1 0 14 28.3 8.3 0 alginate coating 15.8

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a*IBI~~BB~iiQBIBI* ~6~11~1~ TABLE 8 Effect of precolonization Preparation Support Dose sp/ha Production Efficacy decrease in no. of attacks decreases in larva population ADSORPTION 10 13 US 4.263.036 48 63 blastospores 10 36.3 52 ADSORPTION Expanded 101 3 1 application 36.2 54 conidia pearlite 1012 2 applications 52.3 81 1 application 27 72 Fixing Mon.morill- 1013 collage with 34.1 60.8 onrce 12 polyvinyl alcohol PRECOLONIZED Montmorill- GRANULE onite 25 kg/ha according to 88.1 93.3 (mixture) the invention i

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33 EXAMPLE 4 Comparison of product according to the inventin and chemical reference formulations.

A preparation constituted by a mixture of granules, as defined in Example 2 (70% of granules at 48 hours and of granules at 120 hours), and obtained according to the method of operation in Example 1 was tested comparatively with reference insecticides (Table9).

The chemical reference insecticides are the granule DURSBAN G, which is a chlorpyriphos (made by quinoline), applied at the rate of 25 kg/ha, and DECIS G (Soci6t6 Procida), applied at the rate of 25 kg/ha.

TABLE 9 Comparative efficacy of the product of the invention and chemical reference insecticides.

Product Application EFFICACY rate of decrease of decrease in attacks in larva population GRANULE 25 88.1 93.3 (mixture on montmorillonite) according to the invention DECIS G 25 90 DURSBAN G 25 91.3 90.8 34 Effectiveness in decreasing attacks and larva population were calculated as in the preceding example. These results show no evidence of significant differences between the actions of the product as defined by the invention and the two tested insecticides this shows that hte product as defined by the invention has an effect at least comparable to those of these two products.

Tests were made on remanences in the field of the insecticide effect of the mature and immature products of the invention and on the chemical formulation DECIS G.

In Figure 3 the time after the start of the experi- S. ment has been set out in abscissa, expressed in days, and 15 decrease in the larva population has been set out in ordinates, expressed in percentage.

The curves A, B and C represent respectively evoi lution of the insecticide effect of the immature and mature granules as defined by the invention is comparable to that of the chemical insecticide DECIS G, which is a reference U i product.

EXAMPLE Comparison of efficacy of granulose supports.

Preparations obtained by the general method of

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operation described in Example 1 by fermentation as far as the mature stage of Beauveria bassiana on various supports were tested for their efficacy in decreasing attacks on plants and decreasing the larva population. The results are summarized in Table 10 their effectLveness in decrease was calculated as in the preceding examples.

These results show that expanded pearlite and vermiculite permit good production of germs with respect to effectiveness in decreasing attacks on plants and the number of larvae.

However, these two supports are j:icompatible with the mechanized application. Chamotte and artal are less efficacious. Montmorillonite is the support which provided the best results for production and effectiveness in application.

The invention has been .'llustrated in the above in its particular application to the mushroom Beauveria bassiana. It is understood, however, that it is in no way limited to this particular type of mushroom.

-PFI II II eBLB~~I~~ 1- 4 TABLE Comparison of efficacy of granulose supports SUPPORT APPLICATION CHARACTERISTICS EFFICACY DOSE kg/ha size density decrease in decrease in in mn no. of attacks larva population Expanded pearlite 25 1-2 0.1 86.8 and vermiculite 2.5 79.6 86.7 Montmorillonite (mature 25 0.5-1 0.6 68.2 79.2 granules) Chamotte 25 0.5-1 1.35 64.2 70.0 (mature) Artal 25 0.5-1 1.2 38.4 70.0 imature) i o

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Claims (24)

1. A plant protection composition containing particles foLed from at least one solid support carrying at least one microcrganism effective against insect pests or crop diseases or for soil fertilization, said particles being colonized by the microorganism, the latter having an aptitude to sporulate under spreading conditions.

2. A composition as claimed in Cleim 1, wherein it can be spread in a field immediately after it is obtained by direct growth and solid state fermentation on said particles.

3. A composition as claimed in either of Claims 1 and 2, wherein the microorganism is a mushroom.

4. A composition as claimed in Claim 3, wherein the mushroom is ,,entirely in mycelium form at the time of distribution, in whici case S' said product is considered immature.

5. A composition as claimed in Claim 3, wherein the mushroom is in the form of mycelium and spores, in which case said product is considered mature.

6. A composition as claimed in Claim 3, wherein it comprises a mixture of immature and mature forms.

7. A composition as claimed in any of Claims 1 to 6, wherein said support has a density between 0.6 and 1.

8. A composition as claimed in any of Claims 1 t- 7, wherein said support is of mineral or organic origin.

9. A composition as claimed in Claim 8, wherein said support is selected from montmorillonite attapulgite.

Product as claimed in any of Claims 1 to 9, wherein said support has minimum water retention capacity of

11. Product as claimed in any of Claims 1 to 11, wherein said particles are of a size between 100 and 1000 microns, preferably around 500 microns.

12. Stock of Beauveria bassiana registered with the National Collection of Microorganism Cultures under No. I 867.

13. Stock of Beauveria basspina, wherein it results from one or more ,r as t=v' Scwia- 0-.i'l Q.£s mutations of the stock as claime in Claim 12.

14. Product as claimed in any of Claims 3 to 11, wherein the mushrocmused is an entomopathogenic mushroom, effective ugainst one or more crop insect pests. Product as claimed in any of Claims 3 to 11 and 14, whecrin the /K mushroom is Beauveria bassiana.

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16. Process for preparing a product to combat insect pests or plant diseases or for soil fertilization, which comprises direct growth by solid state fermentation of at least one microorganism on at least one solid support in pc ticles, insoluble in the fermentation medium and the recuperation of said particles which are colonized by the microorganism, maturing of the growth being controlled to allow the microorganism to sporulate under distribution conditions.

17. Process as claimed in Claim 16, wherein the microorganism is a mushroom.

18. Process as claimed in Claim 17, wherein fermentation is stopped when tne mycelium is obtained, resulting in a so-called immature product.

19. Process as claimed in Claim 17, wherein fermentation is continued until the mycelium and spores are obtained, resulting in a so-called mature product.

Process as claimed in any of Claims 17 to 19, wherein the mushroom used is an entcmopathogenic mushroom, effective against crop insect pests.

21. Process as claimed in any cZ Claims 17 to 20, wherein the mushroom is Beauveria bassiana and fermentation takes place over 20 to hours, preferably 48 hours, in which case an imnature product is obtained.

22. Process as claimed in any of Claims 17, 19 and 20, wherein the mushroom is Beauveria bassiana and fermentation takes place over a minium of 90 hours, in which case a mature product is obtained.

23. A method of protecting plants against diseases or insect pests, wherein an effective quantity of the product as claimed in any of Claims 1 to 11 and 14 is spread on plants requiring treatment.

24. Process as claimed in Claim 23, wherein plants are treated against Ostrinia nubilalis, known as maize mite. Soil fertilization process, wherein an effective quantity of the product as claimed in any of Claims 1 to 11 is spread on soils requiring treatment. DATED this 27th day of June 1990. INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE WATERMARK PATENT TRADEMARK ATTORNEYS "THE ATRIUM" 290 BURWOOD ROAD HAWTHORN. VIC. 3122.