EP1322172A1

EP1322172A1 - Biological control of fungal contamination by applying geotrichum candidum

Info

Publication number: EP1322172A1
Application number: EP01965335A
Authority: EP
Inventors: Patrick Boivin
Original assignee: INSTITUT FRANCAIS DES BOISSONS de la BRASSERIE MALTERIE
Current assignee: INSTITUT FRANCAIS DES BOISSONS de la BRASSERIE MALTERIE
Priority date: 2000-08-22
Filing date: 2001-08-21
Publication date: 2003-07-02
Also published as: WO2002019827A1; US20040081639A1; FR2813166A1; CA2419421A1; AU2001285997A1; FR2813166B1

Abstract

The invention concerns a method for preventing or treating cereal contamination or fungi producing mold toxins by applying a micro-organism of the Geotrichum candidum family, by field spraying. The invention also concerns the use of a micro-organism of the Geotrichum candidum family.

Description

BIOLOGICAL CONTROL OF FUNGAL CONTAMINATION BY APPLYING GEOTRICHUM CANDIDUM </ 1>

The invention relates to the application of a microorganism, Geotrichum candidum. in the form of a field spray, for the treatment of cereals contaminated by mold, in particular by Fusarium strains.

The present invention will find its application in the field of agriculture.

Cereal diseases, in particular fusarium head blight, are the cause of major damage: loss of yield, depreciation of the quality of the harvest with deterioration in the technological values of cereals. These plant diseases are also responsible for the biosynthesis of mycotoxins that are found on grains at harvest.

It is in fact known that all cereals are contaminated with molds, in particular strains of Fusarium. The main Fusarium species found on cereals are the graminearum, culmorum, sambucinum, poae, proliferatum, or even nivale species. The strains contaminating cereals produce mycotoxins.

For example, the main mycotoxins produced by Fusarium are trichothecenes type A and B (Deoxynivalenol, Nivalenol or T2-toxin for example), supplemented by zearalenone and fumonisins for corn. The nature of the mycotoxins depends on the Fusarium species present. In particular, certain mycotoxins resist processing conditions and can thus be found in finished products. They therefore present a risk for food security. It is therefore important to prevent Fusarium head blight, mold contamination of the grain and the production of mycotoxins during the growing of grain.

The main known means of combating contamination by molds is the treatment of crops by the use of fungicides. The fact remains that the use of fungicides in agriculture poses different problems: Fungicides are biologically active chemicals and their application in fields can therefore present toxicological and ecological risks. On the other hand, the low stability of fungicide products requires regular renewal of treatments during the vegetation. The treatment must also have a sufficiently broad spectrum of action while being limited to fungi. Finally, the effectiveness of these treatments is not total on ears (20 to 80% efficiency) and weak on grains (0 to 30% efficiency). In addition, recent observatories carried out on different lots of cereals show that a large part of the lots have after treatment a still significant mycotoxin content of several hundred micrograms per kg.

There is therefore a real need to develop methods for treating mold contamination which constitute alternatives to methods of treatment with fungicides.

By seeding a non-mycotoxin-producing microorganism, the present invention aims to inhibit the development of fungal flora and in particular Fusarium species contaminating the ears and grains, as well as the production of secondary metabolites fungi, such as mycotoxins.

Similar treatment methods are described in the state of the art (WO 91/01641, US 4,842,871, US 5,668,008) under the name of "biological control" and include the use of a non-pathogenic and non-producing microorganism of mycotoxins on products of agriculture to limit the development of undesirable microbial flora.

In particular, the use of selected non-pathogenic microorganisms has been proposed to reduce the development of the fungal flora in malting. For example, lactic acid bacteria are used to reduce the development of the fungal flora and more specifically of Fusarium (WO 94/16053). The use of the strain Geotrichum candidum naturally present on barley during germination and on finished malt has also been proposed, on the one hand, to inhibit the development of the toxicogenic flora during malting and on the other hand , to improve the biochemical and physicochemical quality of malt (WO 96/34085). An advantage of the selected Geotrichum candidum strain is that it does not produce toxic and teratogenic substances for a vertebrate, nor substances exhibiting genotoxic activities.

The application of Geotrichum candidum in malting was not a priori transposable to the field mainly due to the differences in environment and methods of sowing. In the case of malting, sowing is done in a liquid medium and in the presence of organic nutrients provided mainly by the barley grains and at a temperature between 15 and 20 ° C. In the field, sowing is done by spraying and under variable atmospheric conditions.

The present invention results in particular from studies on the contamination of the ears by Fusarium as a function of spraying tests in a crop field of Geotrichum candidum. Interestingly, these studies have shown an inhibitory effect of Geotrichum candidum on contamination by fungi such as Fusarium sp. They also show a decrease in the presence of mycotoxins and an increase in crop yields in the open field. More specifically, the invention provides a method of preventing or treating contaminations of cereals by fungi or molds producing mycotoxins, comprising spraying in fields an aqueous suspension of spores of a microorganism of the family of Geotrichum candidum with the following characteristics:

- an absence of mutagenic activity measured by the Ames test,

- an absence of production of mycotoxins,

Mycotoxin-producing fungi or molds are in particular of the Fusarium species.

In a preferred form of the invention, an aqueous suspension of spores of a strain of Geotrichum candidum derived from yeasts used in malting is sprayed. More preferably, these spores come from one of the strains deposited on September 6, 1994 under Nos. 1.1474 and 1.1975.

According to the invention, the spores of Geotrichum candidum are produced by the SKW biosystems system (BP20 77260 LA FERTE-SOUS-JOUARRE). The spores are conditioned in solution at an approximate concentration of 5 × 10 ⁸ units forming a colony per ml, (FCU) called mother solution. Before spraying, the concentration of Geotrichum candidum is diluted in an aqueous solution in a ratio of 1 to

100.

In another embodiment of the invention, other microorganisms, such as lactic acid bacteria, having a known effect in the biological control of the development of pathogenic microorganisms, are associated with Geotrichum candidum in the aqueous suspension.

The spores of Geotrichum candidum are sown by spraying an aqueous spore suspension in the field using sprayers conventionally used for herbicide, fungicide or insecticide treatments of field crops.

From 100 to 150 liters per hectare of dilution of the mother solution of spores of the family of Geotrichum candidum are sprayed. These values correspond to a spraying of 10 ⁹ to 10 ¹³ spores per hectare. The sowing period is chosen from the different stages of the vegetation (tillering, start of run, 1 knot, 2 knots, 3 knots, swelling, exit from last leaf, last leaf spread, bursting of the seed, heading and flowering). The seeding is carried out at once or in several times, at the different stages of the vegetation. Preferably, the seeding is carried out all at once, at the earing-flowering stage of the development of the cereals.

The invention thus makes it possible to treat cereals contaminated with fungi. The cereals processed are in particular wheat, barley, rye, oats, corn or triticale.

In a particular implementation of the method of the invention, the spraying of a suspension of spores of the Geotrichum candidum family is associated with a fungicidal treatment. The results provided in the experimental part of the present text show an optimum effectiveness in terms of reduction in the production of mycotoxins and yield of crops in the fields when the spraying is carried out in combination with a fungicide treatment. The fungicidal treatment includes the use of any type of fungicidal product alone or in combination according to known methods allowing a reduction in contamination by the fungal flora or in the production of mycotoxins. Examples of fungicide products are azoxystobin, bromuconazole, tebuconazole, epoxyconazole or kresoxymethyl. Of course, the list of usable fungicides presented here is not exhaustive. The fungicide treatment is preferably applied at the last leaf stage of the development of the cereal or at the flowering stage. An example of a fungicide treatment carried out at the last leaf stage includes kresoximmethyl and epoxiconazole. An example of a fungicide treatment carried out at the flowering stage includes azoxystobin and bromuconazole. The fungicide can be applied separately to the aqueous suspension comprising the spores or directly added to the aqueous suspension of spores of Geotrichum candidum.

The results presented in the examples made it possible to show the effectiveness of the use of Geotrichum candidum in the prevention of contamination of cultures by fungi or molds. These fungi or molds are the cause of many diseases such as foot rot, powdery mildew, brown rust, septotriosis, helminthosporiosis, rynchosporosis and fusarium wilt.

The invention therefore relates to the use of a microorganism from the family of Geotrichum candidum in the manufacture of a preparation capable of treating or preventing diseases resulting from the contamination of cultures by fungi or molds, in particular foot rot, powdery mildew, rust, septotriosis, helminthosporiosis, rynchosporosis and fusarium wilt.

Contamination by the development of mold affects all agricultural products and more particularly cereals, fruits, vegetables and vines. These contaminations can be accompanied by the production of mycotoxins. Spraying an aqueous suspension of Geotrichum candidum makes it possible to significantly reduce the quantity of mycotoxins present on agricultural products. Consequently, the invention also relates to the use of a Geotrichum candidum microorganism in the manufacture of a preparation capable of decreasing by at least 30% and preferably at least 50% the production of mycotoxins when growing cereal crops, fruits, vegetables and vines.

More specifically, the microorganisms of the Geotrichum candidum family are used in the preparation of aqueous spore suspensions allowing the spraying of 10 ⁹ to 10 ¹³ spores per hectare. Preferably, the use of a microorganism of the Geotrichum candidum family as described above is associated with one or more fungicides. More preferably, the associated fungicides are chosen from azoxystobin, bromuconazole, epoxyconazole, krezoxymethyl or a combination of these.

Biological control can also be achieved by the use of a combination of different microorganisms. Indeed, it may be advantageous to combine the inhibitory effects of several microorganisms when they are complementary, in particular a fungus and a bacterium. In particular, the invention relates to the use of Geotrichum candidum in combination with other microorganisms, such as lactic acid bacteria, which can play a beneficial role in the biological control of contamination or the production of mycotoxins.

The examples of implementation of the process of the invention make it possible to show the feasibility and the effectiveness of the treatment in comparison with other fungicidal treatments, without limiting the scope of the process of the invention. They make it possible in particular to compare the effectiveness of the treatment according to the stage of its application and according to its association with a fungicidal treatment.

Example 1: Comparison of the Percentage of Fusarium and Geotrichum Contamination on the Cobs as a Function of the Treatment Method Chosen To quantify the effect of spraying a suspension of Geotrichum candidum. 1 liter of stock solution per hectare containing 5 × 10 ¹¹ spores per liter (malting yeast IFBM, product of SKW Biosystem) was sprayed after dilution at different stages of the vegetation in order to obtain an implantation of Geotrichum candidum on the cobs. The results in percentage of Fusarium and Geotrichum contamination present on the ears are presented in Table 1 according to the stage at which the treatments are carried out (last leaf stage or flowering stage) and the process used (spore spraying process only “Geotrichum ", Or in combination with fungicide treatment). The percentage of contaminated grains is evaluated by depositing the grains on an agar medium and incubating the dishes allowing the development of contaminating molds. The fungicide treatment used is a spray treatment of 0.5 liters of azoxystobin at 250 g / l ("Amistar" from Zeneca-Sopra) and 0.6 liters of a mixture of bromuconazole at 167 g / l and tebuconazole at 107 g / l (“Soleil” by Rhône-Poulenc Agro).

TABLE 1:

It is necessary to remember that the method of seeding Geotrichum candidum by spraying, is different from that used in malting.

The results clearly show the possibility of obtaining an implantation of Geotrichum candidum on the ears by a mode of seeding by spraying. In addition, they show that this implantation is accompanied by a reduction in contamination by Fusarium. They also show that optimum efficiency is obtained when the spraying is done at the flowering stage. Finally, we note that a Geotrichum candidum treatment coupled with a fungicide treatment (Amistar + sun) at the flowering stage allows the implantation of Geotrichum candidum with a more significant reduction in contamination by Fusarium.

These results thus demonstrate the effectiveness of the use of Geotrichum candidum strains in the treatment or prevention of diseases developed by the contamination of cultures by fungi or molds by spraying with an aqueous suspension of spores.

Example 2: Comparison of the quantity of mycotoxins produced and the yields of the cultures according to the chosen treatment method

The results presented in Tables 2 and 3 are intended to show the effect of the treatment of Geotrichum candidum on the reduction of the production of mycotoxins during vegetation. This demonstration is carried out through the assay of deoxynivalenoi, the mycotoxin produced by Fusarium. most encountered on cereals. The experiments were carried out on wheat crops in 100 m ² test plots (Table 2) in combination with a fungicide treatment (Amistar and sun) and in the open field (several hectares) (Table 3) in combination with a fungicide treatment by spraying with epoxiconazole at 125 g / l and kresoximmethyl at 125 g / l (OGAM treatment by BASF). TABLE 2:

TABLE 3

It is noted that the various fungicidal / Geotrichum candidum associations have an effect on reducing the production of mycotoxins with an amplified effect when Geotrichum candidum is sprayed at flowering. It should also be noted that this improvement is accompanied by a significant and significant increase in yields.

(table 2). These results indicate that the use of Geotrichum candidum possibly in combination with a fungicide treatment allows:

(a) reduce the percentage of ears contaminated by Fusarium; (b) reduce the production of mycotoxins;

(c) significantly increase the yields obtained.

Claims

1. A method of preventing or treating contaminations of cereals by fungi or molds producing mycotoxins, comprising spraying in the field of an aqueous suspension of spores of a microorganism of the family of

Geotrichum candidum with the following characteristics:

0 an absence of mutagenic activity measured by the Ames test, 0 an absence of production of mycotoxins.

2. Method according to claim 1 wherein Geotrichum candidum is a malting yeast.

3. Method according to claim 2 in which Geotrichum candidum is one of the strains deposited on September 6, 1994 under the numbers 1.1474 and 1.1975.

4. Method according to one of claims 1 to 3 wherein the mycotoxin-producing fungi are of the species

Fusarium.

5. Method according to one of claims 1 to 4 wherein the spraying is carried out at a rate of 10 ⁹ to 10 ¹³ spores per hectare.

6. Method according to one of the preceding claims wherein the spraying is carried out in one go at the peeling -forging stage of the development of the cereals or in several times during the vegetation between tillering and flowering.

7. The method of claim 1 wherein the cereals are selected from wheat, barley, rye, oats, corn or triticale.

8. Method according to one of the preceding claims in which Geotrichum candidum is associated with other microorganisms, in particular lactic acid bacteria.

9. The method of claim 1 wherein the spraying is combined with a fungicidal treatment.

10. The method of claim 9 wherein the fungicide is selected from azoxystobin, bromuconazole, tebuconazole, epoxyconazole, kresoxymethyle or a combination thereof.

11. The method of claim 10 wherein the fungicide is applied in the last leaf stage of the development of the cereal.

12. The method of claim 10 wherein the fungicide is applied at the flowering stage.

13. The method of claim 12 wherein the spraying is carried out at the flowering stage.

14. The method of claim 10 wherein the fungicide is added to the aqueous suspension of spores of Geotrichum candidum.

15. Use of a microorganism of the Geotrichum candidum family in the manufacture of a preparation capable of treating or preventing diseases resulting from the contamination of cultures by fungi or molds, in particular foot rot, powdery mildew, rust brown, septotriosis, helminthosporiosis, rhynchosporosis and fusarium wilt.

16. Use of a microorganism from the Geotrichum candidum family in the manufacture of a preparation capable of reducing by at least at least 30% and preferably at least 50% the production of mycotoxins when growing cereal crops, fruits, vegetables, vines.

17. Use according to one of claims 15 or 16 in which the preparation allows a spraying of 10 ⁹ to 10 ¹³ spores per hectare.

18. Use according to one of claims 15 or 16 in which Geotrichum candidum is associated with other microorganisms, in particular lactic acid bacteria.

19. Use according to one of claims 15 or 16 in which

Geotrichum candidum is associated with one or more fungicides.

20. Use according to claim 19 in which the fungicide is chosen from azoxystobin, bromuconazole, tebuconazole, epoxyconazole, kresoxymethyle, or a combination thereof.