FERTILIZING AND BIO STIMULATING PRODUCT CONTAINING FRUCTO OLIGOSACCHARIDES, APPLICATION METHOD AND USES

Abstract

The present invention relates to the use of a fructooligosaccharide (FOS) comprising from 2 to 10 monosaccharide units, as an elicitor of the mechanisms of absorption of mineral elements from the soil, and of the defense mechanisms against pathogens. It also relates to a fertilizing and biostimulating composition containing these fructooligosaccharides, and to the use thereof in a method for treating crop plants.

Claims

1-11. (canceled)

12. A method for eliciting mechanisms for absorption of inorganic nutrients in a plant, said method comprising applying to said plant or to a soil in which said plant is growing, an effective amount of at least one fructooligosaccharide comprising from 2 to 10 monosaccharide units.

13. The method according to claim 12, wherein the at least one fructooligosaccharide is applied to the plant via a leaf or via a root.

14. The method according to claim 12, wherein the at least one fructooligosaccharide is used in an amount from 0.1 to 100 g per litre when supplied in a liquid form via a leaf or in a nutritive solution via a root.

15. The method according to claim 12, wherein the at least one fructooligosaccharide is used in an amount of from 10 to 1000 g, when supplied in a solid form.

16. The method according to claim 15, wherein the at least one fructooligosaccharide is supplied in a pulverulent or a granulated fertilizer.

17. The method according to claim 12, wherein the at least one fructooligosaccharide is sulphated.

18. The method according to claim 12, wherein the at least one fructooligosaccharide corresponds to a formula Glucose-(Fructose).sub.n or Mannose-(Fructose).sub.n, also denoted as GF.sub.n, in which n ranges from 1 to 9, or to a formula (Fructose).sub.m, also denoted as F.sub.m, in which m ranges from 2 to 10.

19. The method according to claim 12, wherein the at least one fructooligosaccharide is derived from an enzymatic hydrolysis of inulin.

20. The method according to claim 12, wherein the at least one fructooligosaccharide is chosen from the group consisting of nystose (GF.sub.3), kestose (GF.sub.2), fructosylnystose (GF.sub.4), bifurcose (GF.sub.3), inulobiose (F.sub.2), inulotriose (F.sub.3), and inulotetraose (F.sub.4).

21. The method according to claim 12, wherein the at least one fructooligosaccharide has a formula GF.sub.n, wherein n=2, 3 or 4.

22. The method according to claim 12, wherein the at least one fructooligosaccharide is a mixture of nystose (GF.sub.3), kestose (GF.sub.2), and fructosylnystose (GF.sub.4).

23. A method for stimulating natural defenses of a plant, said method comprising applying to said plant or to a soil in which said plant is growing, an effective amount of at least one fructooligosaccharide comprising from 2 to 10 monosaccharide units.

24. The method according to claim 23, wherein the at least one fructooligosaccharide is applied to the plant via a leaf or via a root.

25. The method according to claim 23, wherein the at least one fructooligosaccharide is used in an amount from 0.1 to 100 g per litre when supplied in a liquid form via a leaf or in a nutritive solution via a root.

26. The method according to claim 23, wherein the at least one fructooligosaccharide is used in an amount of from 10 to 1000 g, when supplied in a solid form.

27. The method according to claim 26, wherein the at least one fructooligosaccharide is supplied in a pulverulent or a granulated fertilizer.

28. The method according to claim 23, wherein the at least one fructooligosaccharide is sulphated.

29. The method according to claim 23, wherein the at least one fructooligosaccharide corresponds to a formula Glucose-(Fructose).sub.n or Mannose-(Fructose).sub.n, also denoted as GF.sub.n, in which n ranges from 1 to 9, or to a formula (Fructose).sub.m, also denoted as F.sub.m, in which m ranges from 2 to 10.

30. The method according to claim 23, wherein the at least one fructooligosaccharide is derived from an enzymatic hydrolysis of inulin.

31. The method according to claim 23, wherein the at least one fructooligosaccharide is chosen from the group consisting of nystose (GF.sub.3), kestose (GF.sub.2), fructosylnystose (GF.sub.4), bifurcose (GF.sub.3), inulobiose (F.sub.2), inulotriose (F.sub.3), and inulotetraose (F.sub.4).

32. The method according to claim 23, wherein the at least one fructooligosaccharide has a formula GF.sub.n, wherein n=2, 3 or 4.

33. The method according to claim 23, wherein the at least one fructooligosaccharide is a mixture of nystose (GF.sub.3), kestose (GF.sub.2), and fructosylnystose (GF.sub.4).

Description

DESCRIPTION OF THE FIGURES

[0060] FIG. 1 represents the concentration of salicylic acid (expressed as pmol/gPF) in the stems of young tobacco plants supplied with a nutritive solution containing a fructooligosaccharide in accordance with the invention (FOS) or the same nutritive solution without FOS (C).

[0061] FIG. 2 represents the concentration of jasmonic acid (expressed as pmol/gPF) in the stems of young tobacco plants supplied with a nutritive solution containing a fructooligosaccharide in accordance with the invention (FOS) or the same nutritive solution without FOS (C).

[0062] FIG. 3 represents the concentration of salicylic acid (expressed as pmol/gPF) in the stems of plants supplied with a nutritive solution containing a fructooligosaccharide not in accordance with the invention (Comparative FOS) or the same nutritive solution without FOS (Control).

[0063] FIG. 4 represents the concentration of jasmonic acid (expressed as pmol/gPF) in the stems of plants supplied with a nutritive solution containing a fructooligosaccharide not in accordance with the invention (Comparative FOS) or the same nutritive solution without FOS (Control).

EXAMPLE 1: SOLID GRANULATED NPK FERTILIZER CONTAINING A FRUCTOOLIGOSACCHARIDE

[0064] 10 kg of a fructooligosaccharide sold under the trade name Profeed L95 were mixed with 250 kg of ammonium sulfate, 50 kg of urea, 300 kg of superphosphate and 300 kg of potassium chloride in a drum pelletizer in the presence of 125 l of water at 45 C. All the starting materials, including the fructooligosaccharides, were ground beforehand.

[0065] The granules were dried in a drum dryer at 90 C. for 15 min.

EXAMPLE 2: LIQUID WATER-SOLUBLE NPK FERTILIZER CONTAINING A FRUCTOOLIGOSACCHARIDE

[0066] 100 l of water were mixed with 10 kg of a fructooligosaccharide manufactured and sold under the trade name Profeed L95. The mixture was kept under continuous agitation for 2-6 hours. The temperature was preferably between 25-70 C.

[0067] The liquid containing the fructooligosaccharide was mixed with 10 kg of urea and 25 kg of monopotassium phosphate. The mixture was kept under continuous agitation at 25 C. for 24 h and the final product was filtered.

EXAMPLE 3: EFFECT OF A FRUCTOOLIGOSACCHARIDE ON THE RATE OF ABSORPTION OF NUTRITIVE ELEMENTS BY YOUNG LETTUCE PLANTS

[0068] Methodology:

[0069] The tests were carried out on young, 15-day-old lettuce (Lactuca sativa) plants cultivated hydroponically. The tests were carried out in a greenhouse at temperatures of 22 C./16 C. day/night, 60% humidity and a photoperiod of 10 hours.

[0070] The lettuce seedlings (originating from a nursery) were washed to remove the compost around the root, and grown hydroponically for two days in water in order to remove the nutritive elements originating from the precultivation.

[0071] The rate of absorption of the minerals depending on whether the nutritive solution contained FOS (treatment) or not (control) was compared.

[0072] 9 young lettuce plants (three batches each comprising three young plants) were transferred to a fresh nutritive solution containing 100 mg.Math.l.sup.1 of a fructooligosaccharide sold by Beghin Meiji under the reference Profeed L95.

[0073] 9 young lettuce plants (three batches each comprising three young plants) were transferred to the same fresh nutritive solution not containing FOS (control).

[0074] The nutritive solution contained: 2 mM of NO.sub.3.sup., 2 mM of PO.sub.4.sup.2, 4 mM of K.sup.+, 1 mM of Ca.sup.2+, 2 mM of Mg.sup.2+, 100 M of Fe, 14 M of B, 16 M of Mn, 4 M of Mo and 0.8 M of Cu. The pH of the solution was adjusted to 6 with KOH.

[0075] The amount of the inorganic elements absorbed was calculated by monitoring the depletion of these elements in the nutritive solution over a period of 30 hours. The nitrate measurements were carried out by ion chromatography (Dionex 120) and the measurements of the other elements (P, Ca, Mg, K, Fe) were carried out by optical ICP (IRIS Intrepid II xdl, Thermo-Electron).

[0076] The plants were then harvested. The root and the aerial part were separated, weighed then dried in a forced air oven at 45 C. for 48 h in order to determine their dry weight.

[0077] Result

[0078] The results underwent statistical analysis (ANOVA) using the STATISTICA 6.0 program, taking P<0.05 as significant.

[0079] The degrees of root absorption of the nitrate, phosphate and calcium by the lettuces have been given in the following table. The results are expressed as % relative to the control (100%).

TABLE-US-00001 Nitrate Phosphate Calcium Control without FOS 100 100 100 Treatment with FOS 112.95 159.8 119

EXAMPLE 4: COMPARISON OF THE EFFECT OF A FRUCTOOLIGOSACCHARIDE ACCORDING TO THE INVENTION WITH A FRUCTOOLIGOSACCHARIDE NOT IN ACCORDANCE WITH THE INVENTION

[0080] The impact of a fructooligosaccharide in accordance with the invention and the impact of a fructooligosaccharide not in accordance with the invention on biostimulation were compared by measuring the content of jasmonic acid (plant hormone) in the stems of young tobacco plants.

[0081] This is because the metabolic pathway of jasmonic acid (JA) plays a role in resistance to insects and diseases. An increase in the content of jasmonic acid in the tissues is therefore a good indicator of the increase in the potential of the young plants to resist pathogenic agents.

[0082] The effect of a fructooligosaccharide (FOS) comprising from 2 to 10 monosaccharide units (sold under the reference Profeed 195) was compared to the effect of a comparative FOS (sold under the reference Probiofeed, containing 95% inulin and 5% monosaccharides).

[0083] Growth of the Plants

[0084] Young tobacco plants germinated in a mixture of peat/sand (1/1 proportion) were transplanted at the 5-leaf stage into a 1 l pot in a greenhouse. The plants were treated by radicular application of a 0.3% aqueous FOS solution, a 0.3% aqueous chicory inulin (Sigma I2255) solution, or the same aqueous solution not containing FOS or inulin. The experiment was carried out on five plants for each treatment applied.

[0085] Other oligosaccharides such as betamune (B), bio-mannanoligosaccharides (MS), mannanoligosaccharide (M), were tested either in aqueous solution (10 min at 100 C.) (B20, MS20, M20) or in acid solution (0.1N HCl, 10 min at 100 C., the pH of which was neutralized before application to the plants) (BCl, MSCl, MCl).

[0086] Analyses

[0087] The plants harvested following application of the treatment were frozen and ground with liquid nitrogen in order to carry out the quantitative analysis of the jasmonic acid and salicylic acid that they contain, according to the following protocol.

[0088] Reagents and Materials

[0089] The jasmonic acid (JAS) and salicylic acid (SAL) standards were provided by Sigma-Aldrich (St Louis, USA). The dihydrojasmonic acid (DHJAS) used as internal standard for the jasmonic acid was provided by OlChemin Ltd (Olomouc, Czech Republic), and the .sup.2H.sub.4-salicylic acid (D-SAL) used as internal standard for the salicylic add was provided by Sigma-Aldrich (St Louis, USA).

[0090] Agitations were carried out in a Multi Reax system from Heidolph Instruments (Schwabach, Germany) and centrifugations in a Centrikon T-124 centrifuge from Kontron Instruments (Zurich, Switzerland). Evaporations were carried out in a model 432-2100 vortex evaporator from Labconco Corporation (Kansas City, Mo., USA).

[0091] Extraction Method

[0092] 0.25 gram of plant tissue (fresh weight) are weighed out, to which are added 100 l of a solution of D-SAL and DHJAS (1000 ng/ml in methanol) and 2 ml of MeOH/H.sub.2O/HCOOH (90/9/1 by volume) containing 2.5 mM of sodium diethyldithiocarbamate at 20 C. Everything is agitated for 60 minutes at maximum speed, 2000 min.sup.1, and centrifugation is carried out at 11 000 min.sup.1 for 10 minutes at 20 C. 0.5 ml of supernatant is introduced into a test tube already containing 0.5 ml of 0.4% acetic acid, then this is agitated and centrifuged at 12 000 min.sup.1 for 10 minutes. A portion of the supernatant is poured into a chromatography vial, from which it is injected into the LC/MS/MS system.

[0093] Chromatographic Method

[0094] A Waters AllianceHT (Milford, Mass., USA) chromatograph is used, fitted with a 2795 XE separation module, an ALLCOLHTR column module with temperature regulation, and a 4 m Hydro-RP Synergi (150.2 cm) chromatographic column (Phenomenex, Torrance, Calif., USA). The gradient and the conditions used are as follows:

TABLE-US-00002 % 0.4% acetic Minutes ml/min % MeOH acid 0 0.2 35 65 1 0.2 35 65 6 0.2 60 40 14 0.2 64 36 15 0.2 90 10 16 0.2 90 10 17 0.2 35 65 20 0.2 35 65
Injection volume: 40 l.
Sample temperature: 20 C.
Column temperature: 30 C.

[0095] Mass Spectrometry Method

[0096] A 3200 Q TRAP LC/MS/MS System mass spectrometry detector (Applied Biosystems/MDS Sciex, Ontario, Canada) is used, fitted with a Turbo Ion Spray interface operating in negative mode.

[0097] Source Parameters:

[0098] CUR (curtain gas flow rate): 22.00 psi.

[0099] IS (ion spray voltage): 4000 V.

[0100] TEM: 550 C.

[0101] GS1 (nebulizer gas flow rate): 45.00 psi.

[0102] GS2 (reheater gas flow rate): 55.00 psi.

[0103] Mass Analyzer Parameters:

[0104] Scan mode: MRM (multiple reaction monitoring).

[0105] Q1 resolution: low.

[0106] Q3 resolution: 1.

[0107] CAD (collision-activated dissociation) gas: medium.

[0108] CXP (collision cell exit potential): 4.00 V.

[0109] Detector Parameters:

[0110] CEM (multichannel electron multiplier): 2800.0 V.

[0111] The data obtained are processed by means of the Analyst 1.4.2 software from Applied Blosystems.

[0112] Results

[0113] The results are given in the table below.

TABLE-US-00003 Transition Ionization and collision parameters Compound Q1 (amu) Q3 (amu) Dwell (ms) DP (V) EP (V) CEP (V) CE (V) JAS 209 59 40 35 7 12 22 209 165 40 35 7 12 20 DHJAS 211 59 40 34 8 13 21 211 167 40 34 8 13 20 SAL 137 93 40 25 6 10 20 137 65 40 25 6 10 38 D-SAL 141 97 40 30 5.5 10 20 141 69 40 30 5.5 10 36 amu = atomic mass unit Jasmonic acid (JAS) Salicylic acid (SAL) Dihydrojasmonic acid (DHJAS) .sup.2H.sub.4-Salicylic acid (D-SAL)

[0114] The total yield, including the matrix effect, was calculated in two ways. Firstly, plants were doped with different concentrations of hormones and the subtraction with the non-doped plant was compared to standards inserted between the samples as controls. As indicated in the tables, the doping was carried out with different concentrations depending on the hormone in question, taking into account the differences in sensitivity between them and also the concentration differences observed in the plant used, cucumber. The yield was also calculated by means of the internal standard, as the quotient between the mean of the areas of each internal standard in the samples to be examined and in the standards Inserted between the samples as controls. The results are as follows.

Total Yield in the Leaves

[0115]

TABLE-US-00004 Hormone +2.5 +5 +10 +25 +50 +100 Int. std. JAS 73.1 102.1 99.7 98.5 99.3 98.8 92.7 SAL 73.2 103.8 98.2 97.5 99.0 101.2 94.3

Total Yield in the Roots

[0116]

TABLE-US-00005 Hormone +2.5 +5 +10 +25 +50 +100 Int. std. JAS 93.7 92.5 92.4 92.4 91.8 93.6 97.5 SAL 89.0 95.0 95.2 98.5 94.5 95.2 98.1

[0117] The values for limit of detection (LOD) and limit of quantification (LOQ) were calculated by taking 10 times the mean level of background noise in the blanks, and likewise in the plant samples. The background noise was measured in the zones of the chromatogram close to the retention time of the hormones. In the case of the standards, the results are expressed in ng/ml and in picograms injected and, in the case of the plant extracts, in ng/g of plant. The results are as follows:

Limit of Detection (LD) and Limit of Quantification (LOQ)

[0118]

TABLE-US-00006 LOD LOQ standards LOD plant standards LOQ plant Hormone ng/ml (pg) ng/g ng/ml (pg) ng/g JAS 0.15 (6) 1.20 0.45 (18) 3.50 SAL 0.10 (4) 1.00 0.30 (12) 3.00

CONCLUSION

[0119] The results have been given in FIGS. 1 to 4. It can be seen that the inulinwhich comprises more than 10 monosaccharide unitsdoes not induce an increase in the jasmonic add concentration.

[0120] On the contrary, a fructooligosaccharide comprising from 2 to 10 monosaccharide units in accordance with the invention induces a plant hormone concentration 4 to 8 times greater, depending on the tissues.