WO2001030160A1

WO2001030160A1 - Method for stimulating plant natural control

Info

Publication number: WO2001030160A1
Application number: PCT/FR2000/002970
Authority: WO
Inventors: Olivier Besnard
Original assignee: Mycos Sarl
Priority date: 1999-10-25
Filing date: 2000-10-25
Publication date: 2001-05-03
Also published as: FR2799935A1; AU1034701A

Abstract

The invention concerns a method for stimulating the natural control of plants by causing the latter to produce phytoalexins and peroxidases by the action of elicitors. The invention also concerns corresponding uses and compositions. The method is characterised in that it consists in applying on the leaves, on the roots or by injection, at least a protein of the enzymatic type belonging to cellulases, B-1,3-glucanases, xylanases, galactanases, mannanases, chitinases or of the non-enzymatic peptide type, one or the other being produced from any fungicidal or bacterial micro-organism.

Description

METHOD OF STIMULATING THE NATURAL DEFENSES OF PLANTS

DESCRIPTION

The invention relates to a method of stimulating the natural defenses of plants by the production by them of phytoalexins and peroxidases under the action of elicitors.

The invention also relates to the use of said specific elicitors for different cultures as well as the corresponding compositions.

The search for means of protecting plants against diseases has until now been rather directed towards a direct fight against pathogens by the use of different phytosanitary products, the plant being considered as a support more than a living being. able to defend themselves.

Unfortunately, the use of these products is not without consequences on the environment and on food because of the residues remaining in the plant.

Today, we are more interested in ways to trigger within the plant itself effective defense reactions or those already existing.

The stimulation of natural defenses results in the development of a set of biological modifications which give this plant immediate resistance and a pre-sensitization thanks to which it becomes able to react more effectively to a new attack. This pre-sensitization is designated by the expression "systematic resistance acquired".

We know of products called elicitors or sensitizers which, put in contact with the plant, are capable of provoking preventively in it, immediate resistance and presensitization or systematic resistance acquired which has just been mentioned. We observe a very rapid stimulation of secondary metabolisms which lead for example:

- the production of phytoalexins (Chappel et al., Plant Physiology (1991) 97, 693-698):

- a strengthening of the cell wall (Glazener, Physiological Plant Pathology (1982) 20, 11-25);

- the production of defense proteins (Kim et al., Physiological Plant Pathology (1994) 45,195-209).

Another possibility of inducing this resistance is to modify the genetic heritage of the plant so that it itself produces the compounds responsible for the establishment of defense mechanisms (Schϋlein et al., Patent FR-9611262).

This technology is quite common these days. It gives birth to new varieties commonly called "genetically modified organisms" (GMOs). For now, the impact on the environment and food security of these GMOs are little known.

With the aim of lowering the costs of agricultural production by reducing the use of phytosanitary products, the applicant has set itself up as a research program to discover and exploit new elicitors.

It appears from numerous bibliographic searches carried out that different compounds are described as elicitors. They are of chemical, biochemical, fungal or enzymatic origins. Chemical elicitors Many products are known as elicitors. In fact, in addition to mercuric ions, arachidonic acid and phosphorous acid are also known as stimulators of the natural defense mechanisms of plants sensitive to phycomycetes.

Copper in the form of hydroxide or sulphate from Bordeaux porridge triggers the vine's defense reactions. Likewise aluminum chloride (A1C1 ₃ ) triggers the synthesis of resveratrol in the vine. Many derivatives of salicylic acid have been tested for their potential as elicitors, oxi-fulvic acids are marketed as elicitors knowing that fulvic and humic acids are macromolecules consisting of carboxy-phenolic monomer units recalling salicylic acid. Laboratory tests have been carried out on young melon plants and the results are satisfactory. Biochemical elicitors

Natural compounds such as oligosaccharides are known for their activity as elicitors. Indeed, the chitosan (derivative of chitin) polymer of 1,4-BD-glycosamine is used to increase the resistance of plants to environmental stress and pathogens.

The use of fragments of polysaccharides of all kinds as stimulators of the defense mechanism of plants has thus been the subject of numerous experiments. Fungal elicitors

The Fiedler et al. Patent, W0-8906687 is an essential reference for these types of elicitors. In it, the authors claim the process of increasing the enzymatic activities responsible for the synthesis of dyes, alkaloids or phytoalexins when we put micro-organisms and inactivated bacteria, their fragments or their excretions containing elicitors in contact with cell cultures of plants. An exhaustive list of microorganisms and bacteria is given there; many are not included, including trichoderma.

In addition, they claim that fragments of microorganisms or bacteria are brought into contact with cell cultures while the applicant's research program has focused on a composition for foliar or root application on an entire plant. The enzyme elicitors

Compared to chemical compounds, very few enzymes are used as elicitors. The first studies describe the synthesis of phytoalexins, in particular capsidiol, by the cells of tobacco plants when cellulase produced by Trichoderma viride is added to the culture medium.

Other work has confirmed this discovery on potato or pepper cells. Furthermore, on vine cells, the synthesis of other types of phytoalexins and in particular resveratrol is observed.

Similar discoveries have been made when using xylanase produced by Trichoderma viride.

All the work in question was carried out in vitro, on suspensions of plant cells. There has never been any question of direct application or spraying of a cellulase-based composition. or any other enzyme from the hydrolase class on plants to trigger plant defense reactions.

Only applications based on fungicidal compositions containing known phytosanitary products in combination with chitinolytic and glucanolytic enzymes

Indeed, the presence of these enzymes in the composition increases the activity of these fungicides because chitanases are known to degrade the wall of fungi consisting of chitin (polymer of N-acetyl-1,4-BD-glycosamine) and glucanases as for them, are capable of hydrolyzing polymers of 1, 3 (4) -B-glucans, essential constituents of the wall of certain fungi. They act in synergy with chitinases to degrade cells of pathogens and allow fungicides to act more effectively.

In these applications, there is also no question of stimulating the natural defense mechanisms of plants but rather of increasing the effectiveness of already existing phytosanitary products.

For example, the production of cellulases and other enzymes by Trichoderma harzianum deposited by the depositor (PRE1 Institut Pasteur) can be done on culture on solid support or in liquid medium.

Example No. 1: Production of enzymes in solid medium

The strain of trichoderma harzianum Al, obtained by selection, is cultivated industrially, according to the technique of fermentation in solid medium on beet pulp (carbon source). Indeed, the trichoderma harzianum Al cultivated in a Petri dish on potato-dextrose-agar medium (PDA) is inoculated at a concentration of 2.10 spores / g (dry substrate) in the following nutritive medium: (NH4) 2SO4: 4, 85 g; urea: 1.20 g; KH2PO4: 2.50g; beet pulp: 80g and water 100 ml. The starting culture medium is at pH 4.5. After 3 days of incubation at a temperature of 28 ° C, the medium containing the mycelium of the fungus and the solid substrates is treated with a hydraulic press (1 min at 230 bars). We thus obtain two fractions: - the soluble fraction (the juice) containing the soluble proteins and the mineral salts; - the insoluble fraction composed essentially of unused substrate and mycelium.

The soluble fraction is centrifuged at 15000 RPM for 30 min, at room temperature to remove the solid particles. The collected liquid phase is then subjected to tangential microfiltration on a 30 kDa cut-off membrane. The mixture of enzymes retained on the membrane is lyophilized.

Assays of enzymatic activities are made on the liquid fraction resulting from the centrifugation, the following results are obtained:

- A cellulase activity of 0.95 U per ml of solution was found. The unit of cellulase activity is defined as the amount that can release a micromole of glucose from cellulose in one hour at pH 5.0 and at a temperature of 37 ° C.

- A chitinasic activity of 0.017 U per ml of solution was found. The unit of chitinic activity is defined as the quantity which can release one mg of N-acetyl-D-glucosamine from cm-chitin-rbv for 48 hours at pH 6.0 and at a temperature of 25 ° C.

- A protease activity of 1.79 U per ml of solution was found. The unit of protease activity is defined as being the quantity which can hydrolyze casein to generate a micromole of tyrosine per mm at H 7.5 and at the temperature of 37 ° C.

- An activity of 1.5 U of B-1,3-glucanase per ml of solution was found. The unit of enzymatic activity is defined as the quantity capable of releasing one micromole of glucose per mm from laminarin 0.1% (W /) at pH 6.7 and at the temperature of 30 ° C. Example No. 2: Production of enzymes in liquid medium

Trichoderma harzianum Al cultivated in a Petri dish on PDA medium is inoculated at a concentration of 10 spores per liter in the following synthetic medium: (NH4) 2SO4 1.00 g; 0.30 g urea; 0.12g Na2HPO4; MgSO4 0.30 g; CaCl2 0.30 g; sucrose 2.00g; solution of trace elements and vitamins: 2 ml; distilled water: 1000 ml. The starting medium, at pH 5.6, is stirred at 200 rpm for 66 h at a temperature of 28 ° C.

After centrifugation at 15,000 RPM for 30 min, at room temperature to remove solid particles (mycelium, etc.), the liquid phase collected is then subjected to tangential microfiltration over a cut-off membrane at 30 kDa. The mixture of enzymes retained on the menbran is lyophilized.

Assays of cellulase, chitinase, protease and B1, 3-glucanasic activities were carried out and the following results are obtained: cellulase (5.64 U / ml), chitinase (0.2 U: ml), protease (2 U / ml), B1, 3-glucanase (2.3 U / ml). Example 3

To demonstrate the elicitor properties of cellulases and proteases, whatever their origin, on plants, various experiments have been carried out on different plant species in order to measure the induction of resistance against pathogens (melon, grape, tomato, cucumber ...).

Given the importance of peroxidase and chinitases (enzymes endogenous to plants) in the resistance of plants (proteins linked to pathogenesis), we have used these activities as a marker of resistance.

The infiltration of a cellulase solution (at 5 U / ml) in the leaves of young melon plants aged 15 days, causes the increase in peroxidase activity 5 times greater than that detected in the control plans infiltrated with the water. In order to dose the peroxidase activities, the leaves are ground in a tris-maleate buffer medium pH 6. The extract is then placed in the presence of gaiacol and hydrogen peroxide and a rapid reaction takes place: the gaiacol is transformed into The appearance of tetragaiacol in the medium allows us to calculate the peroxidase activity.

The assays are carried out in this same buffer using gaiacol as a substrate. The results are expressed in ΔDO / min / gMF. Spraying on the cellulase leaves combined with wetting agents causes an increase in peroxidase and chitinic activity 4 times greater than plants treated without cellulase. Example 4

Similar results are obtained on a large scale on plants developing the fruit in a greenhouse.

Following an infiltration or spraying of cellulase, the melon plants are inoculated by fusarium oxysporum fsp melonis. In parallel, the leaves of a seedling lot were infiltrated or sprayed with water. Ten days after inoculation, symptoms appear only in inoculated plants treated with water. Three weeks after infection, infected seedlings dry up and die. Plants inoculated and treated with cellulase do not show symptoms for 6 weeks and then continue to develop normally.

These results clearly express the elicitor power of cellulase on the resistance of melon plants to fusarium sp. Example 5

By infiltrating or spraying cellulase on the melon plants, a similar protective effect is observed compared to oodium. Indeed, 5 days after inoculation with powdery mildew, the symptoms appear on the plants treated beforehand with water, and yellow little by little. Plants treated with cellulase show little or no symptoms and continue to develop normally 3 weeks after inoculation. Example No. 6 The infiltration and spraying of cellulase on the vine leaves also causes a strong increase in peroxidase activity. The method according to the invention is characterized in that it comprises the foliar, root or injection of at least one protein of the enzymatic type belonging to cellulases, to B1, 3-glucanases, to xylanases, to galactanases, to mannanases, chitinases or non-enzymatic peptide type and in that one or the other is produced from any fungal or bacterial microorganism.

The active substance of the non-enzymatic peptide type comprises peptides whose number of animated acids is at least equal to 2. The enzymatic proteins and non-enzymatic peptides are obtained from any type of fungus and more particularly from filamentous fungi chosen from Trichoderma, Penicillium and Aspergilus.

The proteins in question are used incorporated in a support or vehicle authorized in agriculture of the wetting and penetrating types. The aforementioned composition may be in the liquid form, in particular in the form of an aqueous solution.

It can also be presented in the solid form, in particular of powders or of granules or in coating of seeds.

The proteins in question have the effect of considerably reducing, when applied:

- in the vineyard, the attack of powdery mildew, downy mildew, Botrytis, wood diseases, telluric diseases;

- fruit trees, in particular pear and apple, the attack of oidiums, scab, monilioses, bacterial diseases; - cereals, in particular wheat, maize and rice, the attack of oidiums, septoria, rust, fusarioses, pyricularioses and bacterial diseases;

- oilseeds, in particular soybeans, sunflowers and rapeseed, the attack of oidiums, Phoma, bacterial diseases; - with vegetable plants, in particular tomatoes, melons, carrots, cauliflower and potatoes, attack by oidiums, mildew, pythiaceae (Phytophtora, Pythium), sclerotia mushrooms (Rhizoctonia, Sclerotinia , Pyrenochaeta, vascular fungi (Fusarium, Verticillium), bacterial diseases; - in lawns and horticulture, attacks by pythiaceae, sclerotia fungi, fusarium wilt, powdery mildew, bacterial diseases.

The composition according to the invention is therefore characterized in that it comprises:

- at least one protein of the enzymatic type belonging to the class of hydrolases;

- And / or at least one protein of the non-enzymatic peptide type.

Claims

1- Process for stimulating the natural defenses of plants by the production by them of phytoalexins and peroxidases under the action of elicitors; characterized in that it comprises the foliar, root or injection of said plants of at least one protein of the enzymatic type belonging to cellulases, to Bl, 3-glucanases, to xylanases, to galactanases, to mannanases, to chitinases or of the non-enzymatic peptide type and in that one or the other is produced from any fungal or bacterial microorganism. 2- A method according to claim 1, characterized in that the active substance of the non-enzymatic peptide type comprises peptides whose number of animated acids is at least equal to 2.

3- A method according to claim 1 or claim 2, characterized in that the enzymatic proteins and non-enzymatic peptides are obtained from any type of fungus and more particularly filamentous fungi chosen from Trichoderma, Penicillium and Aspergilus .

4- Use of a protein as defined in any one of the preceding claims, characterized in that it is incorporated into a support or vehicle authorized in agriculture of the wetting and penetrating types.

5- Use of a protein according to claim 4, characterized in that the above composition is in the liquid form, in particular in the form of an aqueous solution. 6- Use of a protein according to claim 4, characterized in that the aforementioned composition is in the solid form, in particular of powders or of granules or in coating of seeds.

7- Use of a protein as defined in any one of the preceding claims, characterized in that it has the effect of considerably reducing, when applied:

- in the vineyard, the attack of powdery mildew, downy mildew, Botrytis, wood diseases, telluric diseases; - to fruit trees, in particular pear and apple, the attack of oidiums, scab, monilioses, bacterial diseases;

- cereals, in particular wheat, maize and rice, the attack of oidiums, septoria, rust, fusarioses, pyricularioses and bacterial diseases;

- oilseeds, in particular soybeans, sunflowers and rapeseed, the attack of oidiums, Phoma, bacterial diseases;

- with vegetable plants, in particular tomatoes, melons, carrots, cauliflower and potatoes, attack by oidiums, mildew, pythiaceae (Phytophtora, Pythium), sclerotia mushrooms (Rhizoctonia, Sclerotinia , Pyrenochaeta, vascular fungi (Fusarium, Verticillium), bacterial diseases;

- in lawns and in horticulture, attacks by pythiaceae, sclerotia fungi, fusarioses, oidiums, bacterial diseases. 8- Composition for the implementation of the method according to any one of claims 1 to 3 and for the uses according to any one of claims 4 to 7, characterized in that it comprises at least one protein of the enzymatic type belonging to the class of hydrolases. 9- Composition for the implementation of the process according to any one of Claims 1 to 3 and for the uses according to any one of Claims 4 to 7, characterized in that it comprises at least one protein of the non-peptide type enzyme.