WO2006067318A1

WO2006067318A1 - Novel use of bacillus sphaericus and bacillus thuringiensis spores, proteins or endotoxins in the form of plant natural defence stimulators

Info

Publication number: WO2006067318A1
Application number: PCT/FR2005/003177
Authority: WO
Inventors: Olivier Besnard
Original assignee: B2B Sarl
Priority date: 2004-12-17
Filing date: 2005-12-16
Publication date: 2006-06-29
Also published as: FR2879402B1; FR2879402A1

Abstract

The invention relates to the novel use of spores, proteins or delta-endotoxins produced from clones of Bacillus thuringiensis var kurstaki de Barjac et Lemille (serotypes 3a and 3b) deposited in the ATCC under the N° 33679, clones of Bacillus thuringiensis var israelensis de Barjac (serotype H 14) deposited in the ATCC under the N° 35646, clones of Bacillus sphaericus deposited in the ATCC under the N° 10208 in the form of a natural defence stimulator for entire plants by a foliar or root application or by injection against pathogens of fungal and/or bacterial and/or viral cultures.

Description

NOVEL USE OF SPORES, PROTEINS OR ENDOTOXINS OF BACILLUS SPHAERICUS AND BACILLUS THURINGIENSIS AS STIMULATORS OF NATURAL DEFENSE OF PLANTS

DESCRIPTION

The present invention relates to a novel use of spores, proteins or delta-endotoxins from Bacillus sp clones, as a stimulator of the natural defenses of whole plants by foliar, root or injection application against pathogens of fungal cultures.

And / or bacterial and / or viral.

It also relates to obtaining said components and the composition that incorporates them.

During their evolution, plants have developed various defense mechanisms that are effective against many pathogens such as fungi, bacteria, viruses and insects. If certain defense mechanisms are constitutive, others are induced when the plant has recognized either one of the components of the microorganism or an external elicitor (non-specific recognition). Cross-referencing of many studies related to metabolic changes

Associated with induced resistance has revealed important similarities in the responses of different plant species. As an example, we will mention three categories of metabolic variations:

an intense stimulation of the key enzymes Peroxidase and Phenylalanine ammonia lyase leading, by activation of the phenylpropanoid pathway, to reinforcement 0 of the natural mechanical barrier of plant cells (lignin, suberin, etc.), to the synthesis of phytoalexins, phenolic compounds and salicylic acid;

- activation of lipoxygenase (octanoid pathway) involved in the production of jasmonic acid

the production of a wide range of peptides and defense proteins (PR proteins or pathogenesis-related proteins) among which, chitinase, β-

1,3-glucanase, protease inhibitors and polygalacturonase inhibitors. It has been shown that many substances of different natures and chemical structures possess the property of stimulating the natural defenses of plants. In addition to oligosaccharides, certain proteins and oligopeptides have been shown to be effective and the effects of peptide and glycoprotein elicitors on higher plants are well documented (Ricci et al., 1989, Nϋrnberger et al., 1994, Pugin et al., 1997, Ebel and Mithôfer , 1998, Martinez et al, 1999, Picard et al, 2000, Martinez et al, 2001). Other molecules such as phosphonates (Bonomelli et al, 2002), β-aminobutyric acid (Daire et al, 2002, Zimmerli et al., 2001, Cohen et al., 1999, Reuveni et al, 1999) are have also turned out to be active as elicitors. The patents FR2821241 (Biophytech) and FR2832409 (CNRS and Biophytech) also report the elicitric properties of oligopeptides consisting of 3 to 20 amino acids and whose main characteristic is an alpha helical structure. In the first case, the elicitors described are peptaibols from the filamentous fungus Trichoderma harzianum. These peptaibols are small, hydrophobic, linear oligopeptides whose mass is between 1 and 2 kiloDaltons. They contain unconventional amino acids such as aminoisobutyric acid (Aib) or Isovaline (Ival). In the second case, it is a series of polymeric compounds of amino acids allowing the plants to fight against various pathogens. Bacillus thuringiensis (Bt) bacteria are now well known for the insecticidal toxins they produce and their uses. Until now the interest of the many commercialized formulations based on Bacillus thuringiensis resides on the one hand in their specificity of action vis-à-vis one or more orders of insects and on the other hand in their safety vis non-target organisms and the environment. The main components of this activity are attributed to crystalline inclusions produced during sporulation of Bt and containing the following delta-endotoxins: crystal proteins (Cry), according to which different types of Bt are classified and cytolytic toxins (Cyt) which increase the action of Cry proteins by enhancing their effectiveness against insects (hemolytic activity). Δ-endotoxins are produced extensively during sporulation and accumulate as parasporal cytoplasmic inclusions that can represent up to 25% of the dry weight of sporulated cells. It is thanks to these essential components that the properties of Bacillus thuringiensis are used in crop protection against insect pests, as an alternative solution to chemical insecticides.

However, direct anti-fungal activity of certain strains of Bacillus thuringiensis has also been demonstrated, and this with regard to various pathogens and at the level of different cultures. As such, we can quote the studies carried out by Moar William J (patent US628072) on a producing strain chitinase (AU634) to control fungi such as Botrytis cinerea or Alternaria solani. Wigley, P. (patent US6077506) proposes a new strain (AQ52) that can be used as a control agent for a large number of pathogens such as Phytophthora infestans, Uncinula necator, Plasmopara viticola, Botrytis cinerea, Monilia fructigena ... on the other hand, nematicide (US5378460) and plant growth promotion (Patent CA2422343) have already been claimed.

Other Bacillus species such as pumilis and mycoides (Jacobsen, et al. - Montana State Univ), subtilis (Gu, Zhen-rong, Shanghai Acad of Agricultural Science), and licheniformis (Argento, US Patent No. 5665354), also have direct action against pathogens.

The peculiarity of the invention resides both in the mode and the mechanism of action of the Bacillus thuringiensis or sphaericus strain and / or proteins derived from the bacterium as well as in their activity. Indeed, according to the invention, it is no longer a question here of using the spores, proteins or endotoxins of Bacillus thuringiensis or sphaericus for their insecticidal or fungicidal properties but for their capacity to stimulate the natural defenses of whole plants. These new types of elicitors are therefore able to stimulate different enzymatic activities that are widely involved in the mechanisms of resistance induced against plant pests.

The specificity of the invention is therefore based on the use:

- on the one hand, spores, proteins or delta-endotoxins from a Barjac and Lemille Bacillus thuringiensis var kurstaki clone - serotype 3a and 3b deposited with the ATCC (American Type Culture Collection) - under number 33679 - on the other hand spores, proteins or delta-endotoxins derived from the Bacillus thuringiensis var israelensis clone of Barjac, deposited with ATCC under number 35646,

and finally spores, proteins or delta-endotoxins of a clone of Bacillus sphaericus Meyer and Neide thuringiensis var medellin) deposited with the ATCC under number 10208.

In the case of Bacillus thuringiensis Cyt proteins, pore formation occurs differently; these proteins are composed of a single domain consisting of 4 external alpha helices surrounding several β strands. The mechanism of pore formation by these Cyt δ-endotoxins would imply the oligomerization of several β-strands in the membrane to form a "β-barrel" leaving α-helices exposed on the membrane surface (Li et al, 2001). The subject of the present invention is therefore a method for controlling pathogenic agents (fungi and / or bacteria and / or viruses and / or insects) of agronomically useful plants, characterized in that it comprises the application to said plants of new elicitors.

According to the invention, the use of these compounds is mainly intended for viticulture, arboriculture and market gardening. The invention therefore relates to the production of bacteria and their δ-endotoxins Cry and Cyt as stimulators of the natural defenses of plants, the latter allowing a primarily preventive but also curative action. The helical and / or β-sheet structures are housed inside the cell membranes and form an ionic or pore channel that modifies the membrane permeability (depolarization), constituting an early signal for triggering the cascade of metabolic events leading to resistance of the plant to its aggressors. The method according to the invention is further characterized in that:

the spores, the proteins or the delta-endotoxins are incorporated into a vehicle authorized in agriculture of the wetting and penetrating type; the composition is in the liquid form, in particular in the form of an aqueous solution;

the composition is in the solid form, in particular in the form of powders, granules or seed coating.

It is also characterized in that the composition is used to reduce: - when it is applied to cereals, in particular wheat, maize and rice, the attack of oidiums, septoria, rust, fusariosis, blastosis and bacterial and viral diseases;

- when applied to fruit trees, especially the pear and apple, the attack of powdery mildew, scab, monilia, bacterial and viral diseases such as "Sharka";

- when applied to the vine, the attack of powdery mildew, mildew, Botrytis, wood diseases, telluric and viral diseases such as the "Court-Noue";

- when applied to lawns and horticulture, attacks of pythiids, sclerotia, fusarium, powdery mildew, bacterial and viral diseases; - when applied to oilseeds, including soya, sunflower, melons, carrots, cauliflower and potatoes, powdery mildew, Pythium mildew (Phytophtora, Pythium), mushrooms with sclerotia (Rhizoctonia, Sclerotinia Pyrenochaeta), vascular fungi (Fusarium, Verticillium), bacterial and viral diseases.

The process according to the invention is also characterized in that the application of said compounds is carried out with a frequency of 10 to 15 days, the most common case in agriculture. Defense mechanisms are stimulated with each new treatment. The choice of the biochemical marker focused here on the measurement of peroxidase activity. These enzymes are involved in early responses to stimulation of the defenses. They are therefore markers of the triggering of a defense reaction such as:

- The strengthening of cell walls and the formation of parietal barriers that block the pathogen's progression inside the host cells.

- The production of toxic compounds involved in defense mechanisms.

Peroxidases catalyze the oxidation of many substrates by hydrogen peroxide: The assay of this enzyme is done by spectrophotometry using guaiacol as a substrate. Colorless at first, it quickly becomes orange-brown when oxidized. This property makes possible the study of enzymatic kinetics by spectrophotometry. The reaction can be monitored by measuring the absorbance variation over time at 470 nm The preparation of the plant extract for the extraction of these soluble proteins is carried out by grinding the fresh leaves in a 50 mM sodium acetate buffer , pH 6 containing PolyVinyIPyrolidone and in the presence of fountain sand. The extract is then centrifuged at 10,000 g for 5 min at 4 ° C. The kinetics are monitored after addition of guaiacol and hydrogen peroxide to the supernatant. A more detailed description of the invention is presented with the aid of the following examples. These examples are in no way intended to limit the scope of this invention. Example 1: Obtaining Spores of Bacillus thuringiensis and Proteins

The process for obtaining spores and proteins of Bacillus thuringiensis according to the invention is characterized in that it comprises the following steps:

a bacterial culture phase The culture medium is the usual glucose medium (UG) whose composition is as follows: KH ₂ PO ₄ (6.8 g / l) - MgSO ₄ , 7H ₂ O (1.13 g / l) 1) - MnSO ₄ , 4H ₂ O (2 mg / l) - ZnSO ₄ , 7H ₂ O (0.014 g / l) - Fe (SO ₄ ) ₃ (0.020 g / l) - CaCl ₂ , 4 H ₂ O ( 0.183 g / l). To this standard saline solution are added 0.75% (w / v) pancreatic peptone and 1% (w / v) glucose. The pH is then adjusted to 7.4. This medium is sterilized by autoclaving at 120 ^° C. for 20 minutes. The cultivation is carried out by seeding a preculture of spores in medium UG for 15 hours at 30 ⁰ C with stirring. The flasks containing the bacteria in culture are then placed at 30 ^° C. with gentle rotary stirring (200 rpm) until complete lysis of the bacteria (from 48 to 72 hours).

- a phase of separation of spores and crystals - protein analysis. This phase is carried out at 4 ° C. The contents of each vial are centrifuged at 4500 rpm for 30 minutes. The pellet is then taken up in 1M NaCl, washed twice with distilled water containing 1 mM phenylmethylsulfonyl fluoride and 10 mM EDTA and then resuspended in a volume of demineralized water. The protein portion is then purified on a discontinuous sucrose gradient according to the method of Thomas and Ellar (1983) [ultracentrifugation at 25,000 rpm and 48 ° C for 16 hours]. The protein concentration is determined by the Bradford method after alkaline solubilization of the extract and electrophoresis under denaturing conditions (SDS-PAGE) is performed for the detection of proteins according to the method already described by Thomas and Ellar (1983). Example 2: Physiological and agronomic effect of Bacillus thuringiensis var kurstaki (serotype 3a and 3b).

- Tomato case: potted tomato seedlings were treated with foliar spraying and a leaf sample for the peroxidase assay was carried out 24 hours and 7 days after treatment. The method of treatment with Bacillus thuringiensis according to the invention was compared with the peroxidase activity generated by a protein extract, an extract of algae and a water control. Each experiment was repeated 4 times for each modality. The results of the assays are expressed ΔDO / minute / gram of fresh material and are summarized in the table below:

In the case of the two samples (24 hours and 7 days), the peroxidase activity detected in the plants treated with Bacillus thuringiensis according to the invention is much greater than the control (more than 30%) as well as the other extracts tested for their activity revealed as an elicitor in several works. EXAMPLE 3 Physiological and Agronomic Effect of the Cyt 2 Ba (Bacillus thuringiensis var israelensis) and Cyt 2 Bc (Bacillus thuringiensis var medellin) Proteins - Case of the vine: the trials are carried out on vines in place (Syrah grape variety). Treatments are performed by spraying the elicitor. The plot is divided into 3 modalities including the proteins Cyt 2 Ba and Cyt 2 Bc as well as a water control. The concentration of the two Cyt proteins used in this test is 250 μg / ml. Adult leaves are collected during elicitation kinetics at time t = 0, 9 hours, 5 days and 15 days. Once the plant material crushed in the appropriate buffer, a peroxidase assay is performed. The results are expressed as ΔDO / minute / gram of fresh material and are summarized in the table below:

These tests show a double interest of this CYT 2 Ba protein: It has a rapid stimulation of the peroxidase activity after spraying. This increase in activity is preserved and even increased after 5 days EXAMPLE 4 Effect of anti-pathogenic protection of Bacillus sphaehcus by stimulating the natural defenses of the plant

The spore composition of Bacillus thuringiensis var kurstaki prepared according to Example 1 is sprayed on different plants to test their resistance to various pathogens. By way of example and in comparison with untreated controls, the protective effects of the composition according to the invention have proved to be effective during preventive treatments renewed regularly. Examples include the following plant / pathogen pairs: vine / mildew, vine / botrytis, melon / powdery mildew, wheat / powdery mildew.

Claims

1- Use of Spores, Proteins or Delta-Endotoxins Resulting from:

- clones from Bacillus thuringiensis var kurstaki Barjac and Lemille (serotype 3a and 3b) deposited with the ATCC under No. ⁰ 33 679;

Bacillus thuringiensis var israelensis clones of Barjac (serotype H14) deposited with ATCC under No. 35646;

Bacillus sphaericus clones deposited with ATCC under No. 10208; as a stimulator of the natural defenses of whole plants by foliar, root or injection application, against the pathogens of fungal and / or bacterial and / or viral types of cultures. 2- Process for obtaining spores, proteins or delta-endotoxins as defined in claim 1, characterized in that it comprises the following steps: a) a bacterial culture phase consisting of:

to use a glucose medium (UG) whose composition is: KH ₂ PO ₄ (6.8 g / l) - MgSO ₄ , 7H ₂ O (1.13 g / l) - MnSO ₄ , 4H ₂ O (2 mg / l) - ZnSO ₄ , 7H ₂ O (0.014 g / l) -

Fe (SO ₄ ) ₃ (0.020 g / l) - CaCl ₂ , 4 H ₂ O (0.183 g / l), in which 0.75% (w / v) of pancreatic peptone and 1% (w / v) are added. ) of glucose and whose pH is adjusted to 7.4; sterilizing said medium by autoclaving at 120 ^° C. for 20 minutes;

- Seeding a preculture of spores in medium UG for 15 hours at 30 ⁰ C with stirring;

placing the flasks containing the bacteria at 30 ^° C. with gentle rotary stirring (200 rpm) until complete lysis of the bacteria (from 48 to 72 hours); b) a phase of separation of spores and crystals consisting of:

- to achieve it at a temperature of 4 ° C; centrifuging the contents of each flask at 4500 rpm for 30 minutes;

- Take up the pellet in 1M NaCl and wash twice with distilled water containing 1 mM phenylmethylsulfonyl fluoride and 10 mM EDTA and then resuspend in a volume of demineralized water;

- purifying the protein portion on a discontinuous sucrose gradient according to the method of Thomas and Ellar (1983) [ultracentrifugation at 25,000 rpm and 48 ° C for 16 hours];

determining the protein concentration by the Bradford method after alkaline solubilization of the extract and electrophoresis under denaturing conditions (SDS-PAGE) performed for the detection of proteins according to the method already described by Thomas and Ellar (1983).

3- composition comprising spores, proteins or delta-endotoxins obtained by the implementation of the method according to claim 2, characterized in that they are incorporated in a wetting and penetrating type of agricultural authorized vehicle.

4. Composition according to claim 3, characterized in that it is in the liquid form, especially in the form of aqueous solution.

5. Composition according to claim 3, characterized in that it is in the solid form, especially in the form of powders, granules or seed coating.

Use of a composition, as defined in claim 3, 4 or 5, to reduce:

- when applied to cereals, especially wheat, maize and rice, the attack of oidiums, septoria, rust, fusariosis, blast, bacterial and viral diseases;

- when applied to fruit trees, especially the pear and apple, the attack of powdery mildew, scab, monilia, bacterial and viral diseases such as "Sharka"; - when applied to the vine, the attack of powdery mildew, mildew, Botrytis, wood diseases, telluric and viral diseases such as the "Court-Noue";

- when applied to lawns and horticulture, attacks of pythiids, sclerotia, fusarium, powdery mildew, bacterial and viral diseases;

- when applied to soya, sunflower, melons, carrots, cauliflower and potatoes, the attack of powdery mildews, Pythium mildews

(Phytophthora, Pythium), sclerotia fungi (Rhizoctonia, Sclerotinia Pyrenochaeta), vascular fungi (Fusarium, Verticillium), bacterial and viral diseases.