CA2286637C - Method for potentiating and stimulating plant natural defence system - Google Patents

Abstract

The invention concerns a method for potentiating and stimulating the natural defence system of plants, in particular cereals and particularly wheat, as well as potatoes and grapes, using a phytosanitary composition containing one or several oligopectins compounds of less than 150 saccharide units, preferably 3 to 50 and more preferably, still, 3 to 20 saccharide units, said oligopectins being present in the composition in concentrations which do not directly induce defence reactions, i.e. in concentrations of the order of 1 to 20 mg/l, preferably 2 to 10 mg/l and more preferably, still, to 10 mg/l.

Description

METHOD FOR POTENTIATING AND STIMULATING
PLANT NATURAL DEFENCE SYSTEM

The invention consists in a method of potentiating and stimulating natural defences of plants, in particular cereals and especially wheat, potatoes and grapevines.
Stimulating the natural defences of plants is a pressing problem and the subject of much research.
For example, the person skilled in the art knows that incubating plants in the presence of substances known as elicitors stimulates defence reactions of the following types:
accumulation of natural antibiotics better known as phytoalexins, synthesis of defence proteins such as chitinases or glucanases, also referred to as PRP (pathogenesis-related proteins), - hardening of the cell walls by synthesis of lignin or cross-linking proteins, and synthesis of secondary messengers such as ethylene, hydrogen peroxide or salicylic aid.
The elicitors include oligopectins; they elicit the above defence reactions in various agronomic plants; the maximum responses are generally achieved for concentrations in the order of 100 mg/1 and remain at a comparable level up to concentrations of 4 g/l.
The invention also provides a use for potentiating and stimulating natural defences of plants of a phytosanitary composition containing one or more oligopectins composed of less than 150 saccharide units, the oligopectins being present in the composition in a concentration lower than that directly inducing defence reactions.

la The invention also provides a method of treating plants to potentiate and stimulate their natural defences, said method being characterised in that it comprises application of a composition containing one or more oligopectins composed of less than 150 saccharide units, the oligopectins being present in the composition in a concentration lower than that directly inducing defence reactions.
In this regard, and by way of example, the production and accumulation of phytoalexins, in this instance avenaluminum-1 in wheat, have been achieved, according to published Japanese Patent Application No. HEI 3-339080 of 20 December 1991 in the name of Fushimi Seiyakusho K.K., by applying to fragments of leaves of wheat aqueous solutions containing DP 6, DP 8, DP 10 and DP 12 pectin oligomers at concentrations in the

2 PCT/FR98/00795 range from 100 to 1 000 mg/1.
This approach, which produces defence reactions in the absence of any pathogenic agent, has a number of disadvantages which include non-negligible energy expenditure for the plant.
The most important object of the invention is to remedy the drawbacks of the prior art and to make available to users means for stimulating plant natural defences only when required, in other words when the plant is attacked by a pathogenic agent, such means consequently making the plant immune to the pathogenic agent.
The Applicant Company has found, following in-depth research, and in a manner that is totally surprising and unexpected, that not only is the above result achieved but also the natural defences are potentiated as soon as a composition is applied to the plant containing one or more oligopectins composed of less than 150 saccharide units, preferably of 3 to 50 saccharide units and more preferably of 3 to 20 saccharide units, said oligopectins being present in the composition at concentrations that do not induce defence reactions directly, i.e. concentrations in the order of 1 to 20 mg/l, preferably 2 to 10 mg/1 and more preferably 5 to 10 mg/l.
The Applicant Company has been able to show that when the above oligopectins are 'used in the form of compositions in which they are present in concentrations in a narrow range from 1 to 20 mg/1, their effect on the treated plants is to potentiate natural defences only from the time of attack by a pathogenic agent.
The advantage of this is that the metabolism of the plant is not side-tracked into setting up defence reactions at a time when this is of no utility; the metabolism is alerted and it is only when the plant is the subject of an attack that its defences are mobilised, and

3 PCT/FR98/00795 then much more intensely than in the absence of any treatment.
The invention therefore consists in the use for potentiating and stimulating natural defences of plants, in particular cereals and notably wheat, potatoes and grapevines, of a phytosanitary composition containing one or more oligopectins comprising less than 150, preferably 3 to 50 and more preferably 3 to 20 saccharide units, the oligopectins being present in the composition in concentrations that do not directly induce defence reactions, i.e. concentrations in the order of 1 to 20 mg/1, preferably 2 to 10 mg/l and more preferably 5 to 10 mg/l.
In one advantageous embodiment of the invention said phytosanitary composition comprises, in combination with the oligopectins, at least one other phytosanitary product chosen from the group of fungicides.
The invention also consists in a method of treating plants, in particular cereals and especially wheat, potatoes and grapevines, to potentiate and stimulate their natural defences, i.e. to assure preventive protection against pathogenic agents, which method is characterised in that it comprises application, in particular to the leaves or to the seeds, of a composition containing one or more oligopectins composed of less than 150 saccharide units, preferably 3 to 50 saccharide units and more preferably 3 to 20 saccharide units, said oligopectins being present in the composition in concentrations that do not directly induce defence reactions, i.e. concentrations in the order of 1 to 20 mg/1, preferably 2 to 10 mg/l and more preferably 5 to 10 mg/1.
In an advantageous embodiment of the method in accordance with the invention the composition is applied to the treated plant in quantities per hectare that do not

4 PCT/FR98/00795 directly induce defence reactions, i.e. quantities in the order of 10 to 1 000 g, preferably 20 to 500 g and more preferably 50 to 200 g.
The invention further consists in a liquid, powder or granule concentrate capable of providing the composition used in accordance with the invention on dilution with an appropriate quantity of solvent, in particular water.
It should be noted that the term " oligopectins"
refers to hydrolysis products of pectins.
Pectins are the main polysaccharide constituents of the primary wall of terrestrial vegetables; they are polysaccharides having a molecular weight in the range from 100 000 to 150 000; they essentially consist of partially methylated galacturonic acid units joined by glucosidic bonds; they are linear polysaccharides having up to approximately two thousand saccharide units per molecule.
There are three main types of pectins, namely homopolygalacturonanes, type I rhamnogalacturonanes and type II rhamnogalacturonanes.
Homopolygalacturonanes, or polygalacturonic acid, frequently referred to as PGA, is a linear polymer of galacturonic acid whose degree of polymerisation can be as high as 2 000 and whose structural 'units are connected by a 1->4 bonds.
In these polysaccharides, the a 1->4 galacturonic acid skeleton can include branches based on xylose or be interspersed with a 1->2 rhamnose residues, which introduce " elbows" into the three-dimensional structure of the molecule. If the molar ratio of the rhamnose to the galacturonic acid is close to 1:1, the substance is type I
rhamnogalacturonane, generally referred to as RGI.
It is type II rhamnogalacturonane, generally WO =98/47375 5 PCT/FR98/00795 referred to as RGII, if the polysaccharides further have arabinose and galactose lateral chains; in type II
rhamnogalacturonane, the arrangement of the galactose, arabinose, rhamnose and galacturonic acid residues is even more complex than previously.
The invention will be better understood from the following additional description and non-limiting examples which relate to advantageous embodiments of the invention.
The following approach, or an equivalent approach, is used to stimulate the natural defences of said plants, in other words to confer on them a preventive immunity to pathogenic agents.
The plants identified above, whose natural defences are to be stimulated in the event of attack by a pathogenic agent, are treated with a composition that is applied to the leaves or to the seeds and includes, in addition to the standard vehicles and constituents of this type of composition, one or more oligopectins having 150, preferably 3 to 50 saccharide units and more preferably 3 to 10 saccharide units, present in concentrations that do not induce defence reactions directly, i.e. in concentrations in the order of 1 to 20 mg/l, preferably 2 to 10 mg/l and more preferably 5 to 10 mg/l.
The treatment is generally applied from the first vegetative stages of the plant,' possibly by several successive applications, and advantageously by spraying.
The precise moment(s) of application are chosen in particular according to the nature of the plant treated.
The vehicle or vector is generally water.
However, instead of water, the vector can be one chosen from the group comprising mineral oils, vegetable oils, all liquid fats and alcohols, in particular propylene glycol or glycerol.
The essential standard constituents of said compositions can vary according to the nature of the plant treated; they are generally chosen from the group comprising solvents, surfactants, dispersing agents and/or solid bulking agents.
The oligopectins constituting the active substance of said compositions can be prepared by enzyme or acid hydrolysis of pectins generally extracted from pome marc or citrus fruit peel, in particular lemon peel.
The concentration of pectins in the above four materials, in weight percent relative to the dry material, is:
- 15 wt.% in the case of pome marc, - 25 wt.% in the case of orange or grapevinefruit peel, - 35 wt.% in the case of lemon peel and - 50 wt.% in the case of lime peel.
In practice, the pectins are extracted from byproducts of the fruit juice industry; treatment can be with a mineral acid, for example hydrochloric acid, at a pH in the range from 1 to 2.5, for 2 to 10 hours at a temperature of 60 to 80 C.
The extract is purified, for example by filtering and centrifuging it; the extraction medium is neutralised and concentrated, after which the pectins are precipitated, using an alcohol such as ethanol or isopropanol, then dried and obtained in powder form.
The pectins can be degraded enzymatically to oligopectins using pectin lyases (in particular EC 4.2.2.1.0) or pectinases, also referred to as polygalacturonases (in particular EC 3.2.1.15), both extracted from Aspergillus niger.
The following examples describe:
the preparation by enzymatic degradation of a pectin constituting the raw material of three hydrolysates each containing at least one of the oligopectins that constitute the active substance of the compositions in accordance with the invention, and designated H18, H19 and H21, - two compositions in accordance with the invention, and - experiments illustrating the invention.

Preparation of hydrolysates H18 and H19.
The raw material was an apple pectin in the form of partially methoxylated polygalacturonic acid from Sigma, sold under the reference P-2157.
The enzyme used was P-7052 pectin lyase from Sigma, which is obtained from Aspergillus niger.
A 20 mM phosphate-citrate buffer solution with a pH
equal to 5 was prepared.
g of said pectin was dissolved in 5 litres of said buffer solution, 0.2M dibasic phosphate being regularly added in sufficient quantity to maintain the pH
20 at 5, the pH tending to fall as the pectin was rendered soluble.
20 units of said pectin lyase were added to 2 litres of the above solution thermostatically maintained at 40 C.
The resulting solution was maintained at 40 C for 30 25 minutes.
The solution was then ultrafiltered continuously and at a flowrate of 500 to 1 000 ml/h in a Millipore PELLICONTM tangential ultrafiltration device fitted with a cassette having a porosity of 10 000 Daltons. The input pressure was 1 bar.
During ultrafiltration, the volume to be ultrafiltered was maintained constant at 2 litres by automatic addition of 3 litres of pectin solution.
The 4 litres of ultrafiltrate obtained after this WO.98/47375 8 PCT/FR98/00795 first ultrafiltration operation contained oligopectins with a molecular weight less than 10 000 Daltons ; given that the molecular weight of a saccharide unit is approximately 200, the oligopectins in the ultrafiltrate are composed of at most 50 saccharide units.
The ultrafiltrate underwent further tangential ultrafiltration in the same device, this time fitted with a cassette having a porosity of 500 Daltons.
This second ultrafiltration desalted the preceding ultrafiltrate and eliminated oligopectins having a molecular weight less than 500 Daltons, i.e. consisting of at most two saccharide units; the second ultrafiltration was carried out at an input pressure of less than 2 bars and at a flowrate of 2 to 3 1/h.
The 4 litres of ultrafiltrate obtained in this way were washed twice with 5 litres of distilled water, concentrated to a volume of 50 ml by evaporation at 55 C
using a ROTOVAPOR device, neutralised by means of dilute soda solution and then freeze-dried.
Analysis by ionic chromatography coupled to current measurement and using DIONEX"' ion exchange resin from Dionex Chemical Corp., a subsidiary of Dow, and the technique described by Spino et al. in "Carbohydrate Research", 247 (1993), 9-20, showed that the oligopectins of said powder in fact had from 3 to 10 saccharide units, the average degree of polymerisation being from 4 to 6.
15 g of a cream-coloured powder were obtained; this powder constituted the hydrolysate H19.
The residue from the first ultrafiltration operation underwent a third ultrafiltration operation in the same device, this time fitted with a cassette having a porosity of 30 000 Daltons.
Consequently, the ultrafiltrate obtained in this way contained the oligopectins having a molecular weight less WO.98/47375 9 PCT/FR98/00795 than 30 000 Daltons (i.e. having at most 150 saccharide units) and, because of the first ultrafiltration operation, those having a molecular weight greater than 000 Daltons (i.e. having more than 50 saccharide

5 units).
The 0.75 litre of ultrafiltrate obtained in this way was washed twice with 5 litres of distilled water, concentrated to a volume of 50 ml by evaporation at 55 C
using said ROTOVAPOR device, neutralised by means of 10 dilute soda solution and then freeze-dried.
5 g of a cream-coloured powder were obtained which constituted the hydrolysate H18.
Using the Blumenkrantz and Asboe-Hansen method described in "Analytical Biochemistry" 54, pages 484-489 (1973) and metahydroxyphenyl, the uronic acid content was found to be 100%.
Analysis by ionic chromatography coupled to current measurement and using DIONEXTM ion exchange resin from Dionex Chemical Corp., a subsidiary of Dow, showed that the oligopectins of said powder in fact had from 8 to 30 saccharide units, the average degree of polymerisation being from 10 to 20.

Preparation of hydrolysate H21.
25 g of the pectin used in ekample 1 were dissolved in 5 litres of water.
0.5N soda was added continuously to maintain the pH
at 10.
After complete dissolution, the temperature was raised to 70 C and maintained at that value for 30 minutes.
This demethoxylated the polymer.
The demethoxylated polymer solution was neutralised using iN HC1 and desalted by tangential ultrafiltration WO.98/47375 10 PCT/FR98/00795 using the device used in example 1 equipped with a cassette having a porosity of 10 000 Daltons.
4 litres of residue were obtained and made up to litres with distilled water. The pH was adjusted to 4.5 5 using a 20 mM sodium acetate buffer solution.
350 units of Sigma P-9179 pectin hydrolase obtained from Aspergillus niger were added at room temperature.
The resulting mixture was left for 30 minutes.
The hydrolysate obtained in this way was subjected to continuous ultrafiltration in the same device fitted with the same cassette ; hydrolysis continued during ultrafiltration and the volume of hydrolysate was maintained at 5 litres using the above buffer; after two hours of hydrolysis, a further 350 units of pectin hydrolase were added.
After 5 hours of hydrolysis and 4.5 hours of concomitant ultrafiltration, approximately 12 litres of ultrafiltrate were obtained.
The ultrafiltrate was processed (flowrate: 2 to 3 1/h, pressure less than 2 bars) using said ultrafiltration device equipped with a cassette having a porosity of 500 Daltons.
Oligopectins having two saccharide units were removed from it in this way and it was desalted.
The residue was washed twice with 5 litres of distilled water, concentrated to a volume of 50 ml by evaporation at 55 C using the ROTOVAPOR device used in example 1, neutralised and freeze-dried.
20 g of a white powder was obtained made up of oligopectins having a molecular weight from 500 to 10 000 Daltons, i.e. having 3 to 50 saccharide units.
The yield was 50 to 60% relative to the pectin constituting the raw material.
Using the Blumenkrantz and Asboe-Hansen method described in "Analytical Biochemistry" 54, pages 484-489 (1973) and metahydroxyphenyl, the uronic acid content was found to be 85%.
Analysis by ionic chromatography coupled to current measurement and using.DIONEXT" ion exchange resin from Dionex Chemical Corp., a subsidiary of Dow, showed that the oligopectins of said powder in fact had from 3 to 9 saccharide units, the average degree of polymerisation being from 4 to 6.
The pectins constituting the raw material could be chemically degraded using a mineral acid, in particular hydrochloric acid or sulphuric acid.
Following de-esterification by saponification, the raw material consisting of said pectins was incubated at a high temperature, in particular from 9o to 1100C, in the presence of a strong (0.5M to 1M) acid, for 3 to 6 hours.
The reaction medium was then neutralised and the fractions corresponding to oligopectins having a molecular weight from 500 to 10 000 Daltons and oligopectins having a molecular weight of 10 000 to 30 000 Daltons were isolated by tangential ultrafiltration as described in examples 1 and 2.

Agricultural liquid concentrate based on oligopectins:
oligooectin H19 D.200 kg Tween 80 0.005 kg Tm Sodium salt of methylparaben O.OOS kg water 0.790 ko Total 1.000 kg The liquid concentrate is used after dilution of a quantity preferably in the range from 10 to 50 g and more preferably in the range from 25 to 50 g in 1 000 litres of water.

WO .98/47375 12 PCT/FR98/00795 This dilution provides a composition having a concentration of oligopectin H19 in the range from 2 to g and preferably in the range from 5 to 10 g per 1 000 litres of water.

Soluble concentrate powder containing hydrolysate H19 as active material with additives.
The weight for weight composition per 1 kg of the powder was as follows:
10 Oligopectin H19 0.150 kg Kaolin 0.500 kg Mannose 0.050 kg Sodium salt of methylparaben 0.005 kg Purified starch 0.295 ka Total 1.000 kg The concentrated powder was used after dilution in a sufficient quantity of water to obtain a composition in which the concentration of oligopectin H19 was in the range from 2 to 10 g and preferably in the range from 5 to 10 g per 1 000 litres of water.
The efficiency of hydrolysates H18 and H19 was demonstrated on wheat infected with septoriosis; the pathogenic agent responsible for septoriosis is Septoria tritici.
The corresponding experiments are described in example 5.

Study of the efficiency of hydrolysates H18 and H19 in wheat infected with Septoria tritici.
This experiment used 300 seedlings of "Tendral"
tender winter wheat known to be sensitive to Septoria tritici.
The seedlings were grown in 30 containers each WO.98/47375 13 PCT/FR98/00795 containing 10 seedlings.
sets A, B, C, D and E each consisting of 6 containers were assembled, in other words 6 batches of 10 seedlings.
5 In each set:
- the first and second batches of seedlings, which constituted control batches, were treated by spraying the leaves at the 2-leaf stage (GS 12 on the Zadocks scale) with distilled water containing 0.1% TWEEN 20, - the third and fifth batches were treated by spraying the leaves at the 2-leaf stage (GS 12 on the Zadocks scale) with 5 ml of a first composition comprising hydrolysate HIS as the active substance, and - the fourth and sixth batches were treated by spraying the leaves at the 2-leaf stage (GS 12 on the Zadocks scale) with 5 ml of a second composition comprising hydrolysate H19 as the active substance.
The first and second compositions contained, in distilled water in the presence of TWEEN 20:
- 1 mg/l of hydrolysate H18 or H19 in the experiment on set A, - 2 mg/l of hydrolysate HIS or H19 in the experiment on set B, - 10 mg/l of hydrolysate, H18 or H19 in the experiment on set C, - 100 mg/l of hydrolysate H18 or H19 in the experiment on set D, and - 1 000 mg/l of hydrolysate H18 or H19 in the experiment on set E.
After the above spraying treatment, the seedlings of the 6 batches of each set were maintained at room temperature for 4 hours to allow the droplets of the products to dry on the surfaces of the leaves. They were then placed in a controlled climate chamber maintained at WO,98/47375 14 PCT/FR98/00795 19 C during 12 hours of illumination and at 17 C during 12 hours of darkness, and at a relative humidity of 65%.
48 hours after the above treatment, the first, third and fifth batches of each set were inoculated by spraying them with a suspension in water in the presence of 0.10 TWEEN 20 containing 105 pycnospores per ml of a strain of Septoria tritici.
The second, fourth and sixth batches of each set were subject to the same inoculation with the pathogenic agent 72 hours after the treatment with hydrolysates H18 and H19.
The seedlings of the control batches and those of the batches inoculated with Septoria tritici were placed in a controlled climate enclosure at 19 C during 12 hours of illumination and 17 C during 12 hours of darkness, and at a relative humidity of 100% for the first 96 hours and 85% thereafter.
21 days after the first and second series of inoculations with Septoria tritici, the seedlings of wheat of the 4 sets of 6 batches were inspected.
The efficiency of the products was determined by the percentage protection PP and by the infection intensity II, both expressed as a percentage.
The two magnitudes were defined relative to the control batches.
The "protection percentage" was calculated using the following formula:

Number of control plants having _ Number of treated plants having at least one leaf necrosis at least one leaf necrosis Number of control plants having at least one leaf necrosis The "infection intensity" was calculated using the following formula:

WO.98/47375 15 PCT/FR98/00795 Proportion of control plant leaf _ Proportion of treated plant leaf surface suffering from necrosis surface suffering from necrosis Proportion of control plant leaf surface suffering from necrosis Table I sets out the results of the above determinations.
TABLE I
Hydrolysate Set Results after 21 days (hydrolysate Inoculated with Inoculated with concentration of spores 48 hours spores 72 hours treatment after treatment after treatment composition) pp % II % PP % II %

A 1 mg/1 5.5 10.2 20.9 31.7 B 2 mg/l 18.9 41.7 33.3 64.7 H18 C 10 mg/1 29.2 58.8 49.4 76.3 D 100 mg/1 7.5 11.5 19.4 27 E 1000 mg/l 5.2 0 JA 1 mg/l 30 37.2 36.7 26.8 3 2 mg/l 49.5 53.7 69.6 79.5 H19 C 10 mg/1 41.7 35.1 48.5 53.4 D 100 mg/l 2.8 3.5 11.9 17 E 1000 mg/i 0 0 The results set out in Table I showed that:
- the average degree of polymerisation of the oligopectins examined in the experiment had little influence on the results, - the treatment must be effected as soon as possible, before infection by a pathogenic agent, the results obtained for inoculation 72 hours after treatment being better than those obtained for inoculation 48 hours after treatment, and WO,98/47375 16 PCT/FR98/00795 - the concentrations of the compositions applied were advantageously from approximately 2 mg/l to approximately 10 mg/l.
It is noted in particular that the protection obtained at 100 mg/l of oligopectins was significantly weaker than that obtained at 10 mg/l. Also, and most importantly, for a dose of 1 000 mg/l, i.e. a dose for which the defence reactions are deemed to be maximal, regardless of the marker or the plant studied, the number of plants infected and size of the lesions were not significantly less than those observed in the untreated control batch.

Experiments showing potentiation of plant natural defences by hydrolysates H18 and H19.
Three experiments were performed.
In a first experiment, to study the direct elicitor effect of hydrolysates H18 and H19, compositions with increasing concentrations of H18 and H19 were applied to BY tobacco cell cultures, namely:
- for H18, compositions with concentrations successively equal to 2 mg/1, 10 mg/l, 20 mg/1, 40 mg/l and 200 mg/1, and - for H19, compositions with concentrations successively equal to 10 mg/l and 200 mg/1.
Four defence reaction markers were tested for each composition, namely:
- phenylalanine ammonia-lyase (PAL) activity, this being a key enzyme for the synthesis of phytoalexin in plants, - O-methyltransferase (OMT) activity, this enzyme being involved in the synthesis of lignin, - lipoxygenase (LOX) activity, this enzyme being involved in the generation of methyl jasmonate, i.e. an WO.98/47375 17 PCT/FR98/00795 element of the signalling cascade leading to the activity of the defence genes, and - salicylic acid (SA) concentration, this acid being another secondary messenger involved in the defence S reactions.
The results are expressed as a factor indicative of the induction of the markers studied, representing the ratio between the values measured in elicited cells and in control cells that were not elicited.
The results of the above measurements, which were carried out from 4 to 48 hours after the start of incubation, are set out in Table II below.
TABLE II

Oligopectin Defence marker induction factor PAL OMT LOX SA
H18 2 mg/l NS* NS NS NS
10 mg/1 NS NS NS NS
mg/1 12 NS 4.2 40 mg/l 60 2.7 11.3 200 mg/l 90 2.0 12.7 17.2 H19 10 mg/1 NS NS NS NS
200 mg/1 85 1.3 15.2 15 IS = not significant The above results showed that the hydrolysates H18 and H19 elicited defence reactions in the treated cells, as reflected in the accumulation of markers when said hydrolysates were used at concentrations from 20 to 20 200 mg/l; on the other hand, the same markers were not induced in significant quantities when the concentration of elicitor, i.e. of hydrolysate H18 or H19, was 2 mg/i or 10 mg/1.
In a second experiment, cultures of the same BY

WO.98/47375 18 PCT/FR98/00795 tobacco cells were pre-treated the day after pricking out using compositions with concentrations of hydrolysate H18 equal to 2 mg/1 and 10 mg/1.
S-even days later, the above cultures and untreated control cultures were treated with a composition containing 40 mg/1 of hydrolysate HIS.
This situation imitated attack by a pathogenic agent.
The kinetics of accumulation of salicylic acid (SA) .10 were monitored for 4 to 48 hours after the treatment.
The measurements showed that the cells of the treated cultures accumulated SA up to concentrations that did not exceed 50 ng per gram of wet weight.
They also showed that pre-treatment with HIS very strongly stimulated the accumulation of salicylic acid when the plant was elicited with this hydrolysate seven days later, which was totally unexpected; in the tobacco cells treated in this way, an accumulation of 700 ng per gram of wet weight was noted when the pre-treatment concentration of H18 was 2 mg/1 and an accumulation of 1 200 ng per gram of wet weight was noted when the pre-trea:ment concentration of HIS was 10 mg/l.
This demonstrated the potentiating effect of the use in accordance with the invention.
In a third experiment, hydrolysate H19 and fragments of leaves of wheat were used, the first leaf of wheat seedlings being excised at 8 days of age, abraded with fine emery paper and cut into 1 cm fragments which were floated in an appropriate buffer solution for 1 hour.
21 batches were prepared, of which 7 constituted control batches.
10 mg/l of hydrolysate H19 was added to the buffer solution of 7 of the 14 non-control batches; 100 mg/l of hydrolysate H19 was added to the buffer solution of the W0.98/47375 19 PCT/FR98/00795 other 7 batches.
This was followed by incubation for 18 hours.
At the end of incubation, the leaf fragments of the 21 batches were elicited with 200 mg/l of yeast P-glucane, this product being known to strongly stimulate defence reactions in plants.
The marker studied on this occasion was the accumulation of hydrogen peroxide in the minutes following the addition of the elicitor, the concentration of which was monitored by luminometry of the incubation medium for the leaf fragments.
Compared to the fragments of the batches that were not pre-treated, pre-treatment with H19 at a concentration of 100 mg/l did not induce greater accumulation of hydrogen peroxide.
In contrast, pre-treatment with H19 at a concentration of 10 mg/i increased the accumulation of peroxide by a factor of 2 to 20.
The above experiments showed that, when hydrolysate HIS (experiment 2) or H19 (experiment 3) was used for pre-treatment in concentrations of 2 mg/1 or 10 mg/l, i.e.
with no direct elicitation of defence reactions, they very strongly over-induced the defence reactions triggered by elicitation respectively 18 hours and 7 days after pre-treatment, which was totally unpredictable and surprising.
It was also noted that hydrolysate H19 had no potentiating effect when used for pre-treatment at a concentration of 100 mg/i.

Claims (28)

1. Use for potentiating and stimulating natural defences of plants of a phytosanitary composition containing one or more oligopectins composed of less than 150 saccharide units, said oligopectins being present in the composition in a concentration lower than that directly inducing defence reactions, said concentration being in the order of 1 to 20 mg/l.

2. The use of claim 1 wherein said plants are cereals, potatoes or grapevines.

3. The use of claim 2 wherein said cereal is wheat.

4. The use of any one of claims 1 to 3 wherein said oligopectins are composed of 3 to 50 saccharide units.

5. The use of claim 4 wherein said oligopectins are composed of 3 to 20 saccharide units.

6. The use of any one of claims 1 to 5, wherein said oligopectins are present in the composition in concentrations in the order of 2 to 10 mg/l.

7. The use of claim 6, wherein said oligopectins are present in the composition in concentrations in the order of 5 to mg/l.

8. Method of treating plants to potentiate and stimulate their natural defences, said method being characterised in that it comprises application of a composition containing one or more oligopectins composed of less than 150 saccharide units, said oligopectins being present in the composition in a concentration lower than that directly inducing defence reactions, said concentration being in the order of 1 to 20 mg/l.

9. The method of claim 8, wherein said plants are cereals, potatoes or grapevines

10. The method of claim 9, wherein said cereal is wheat.

11. The method of any one of claim 8 to 10, wherein said application is to the leaves or to the seeds.

12. The method of any one of claims 8 to 11, wherein said oligopectins are composed of 3 to 50 saccharide units.

13. The method of claim 11, wherein said oligopectins are composed of 3 to 20 saccharide units.

14. The method of any one of claims 8 to 14, wherein said oligopectins are present in the composition in concentrations in the order of 2 to 10 mg/l.

15. The method of claim 14, wherein said oligopectins are present in the composition in concentrations in the order of 5 to 10 mg/l.

16. Method according to any one of claims 8 to 15, characterised in that the composition containing one or more oligopectins is applied to the treated plant in a quantity per hectare lower than that directly inducing defence reactions.

17. The method of claim 16, wherein said quantity is in the order of 10 to 1 000 g.

18. The method of claim 17, wherein said quantity is in the order of 20 to 500 g.

19. The method of claim 18, wherein said quantity is in the order of 50 to 200 g.

20. A liquid, powder or granule concentrate, said liquid, powder or granule concentrate comprising oligopectins composed of less than 150 saccharide units, wherein said liquid, powder or granule concentrate, upon dilution with an appropriate quantity of solvent, provides a composition, wherein said oligopectins are present in a concentration being in the order of 1 to 20 mg/l.

21. The liquid, powder or granule concentrate of claim 20, wherein said solvent is water.

22. A composition comprising, in addition to phytosanitarily acceptable vehicles and phytosanitarily acceptable constituents, one or more oligopectins composed of less than 150 saccharide units, said oligopectins being present in the composition in a concentration lower than that directly inducing defence reactions, said concentration being in the order of 1 to 20 mg/l.

23. The composition according to claim 22, wherein said oligopectins have 3 to 50 saccharide units.

24. The composition according to claim 23, wherein said oligopectins have 3 to 20 saccharide units.

25. The composition of any one of claims 22 to 24, wherein said oligopectins are present in the composition in concentrations in the order of 2 to 10 mg/l.

26. The composition of claim 25, wherein said oligopectins are present in the composition in concentrations in the order of 5 to 10 mg/l.

27. The composition according to any one of claims 22 to 26, wherein the vehicle is water, a mineral oil, a vegetable oil, a liquid fat, an alcohol or a mixture thereof and wherein the constituent is a solvent, a surfactant, a dispersing agent, a solid bulking agent, or a mixture thereof.

28. The composition according to claim 27, wherein said alcohol is propylene glycol or glycerol.