Patent Application
20170020132
The present invention relates to the use of fructooligosaccharides to elicit mechanisms for absorption of inorganic nutrients and as agents for stimulating plant defenses against pathogens.
The invention also relates to fertilizing and biostimulating products containing such fructooligosaccharides, and also to a method for spreading these fructooligosaccharides on crops.
The necessity to increase the effectiveness of agricultural practices is subject to both economic and environmental constraints.
One of the strategies for increasing yields consists in producing more effective fertilizers, for example by reducing the loss of nutritive elements into the soil. Thus, urease and nitrification inhibitors have been used with the purpose of reducing the volatilization of ammonia and the leaching of nitrates.
Another supplementary strategy consists in developing molecules which increase the plant's capacity to absorb nutrients from the soil, or in stimulating the plant's natural defenses against pathogens. Recently, a fructooligosaccharide comprising thirteen fructose units has been described as a growth activator for plants at different stages of development (CN 2007/101006777 et J. Phytopathol., 157, pp 201-207, 2009). This effect is attributed to the activation of the production of salicylic acid and the glycosylated homolog thereof in the leaves of treated plants. The fructooligosaccharide is obtained by aqueous-phase extraction from burdock (Arctium lappa) roots followed by enzymatic deprotonation, precipitation in ethanol and purification by chromatography.
The invention relates to fructooligosaccharides of lower molecular weight than that of the fructooligosaccharide described in the prior art. Indeed, the inventors have discovered that these fructooligosaccharides cause a better rate of nutrient absorption by the roots of the plants, and further stimulate the plant's natural defenses. This effect relates to all inorganic nutrients, in particular nitrogen, phosphorus and calcium. Indeed, the fructooligosaccharides used within the context of the invention have better water-solubility than the fructooligosaccharides of the prior art. The inventors have found that inulin cannot be applied in the form of a fertilizer in aqueous solution, since this compound cannot remain stable in the stock solution before application thereof to the crops.
The product of the invention enables increased production of organic nitrogen in the plant, responding to the growth requirements of the crop, which is manifested in an increased yield. The product of the invention also enables an increase in the protein content and the nutritional value of protein-producers and fodder. The product of the invention also makes it possible to reduce the risks of toxicity caused by an excessive accumulation of ammonium ions in the leaves or to reduce the accumulation of nitrates in the leaves.
Thus, a subject of the present invention is a fertilizing and/or biostimulating composition containing fructooligosaccharides and also a process for treating plants or soils using them.
Within the context of the present description, the expression “fertilizing composition” is intended to mean any product, the use of which is intended to ensure, or improve, plant nutrition.
“Fructooligosaccharide” is intended to mean a compound comprising several fructose units and optionally another monosaccharide unit.
Within the context of the present description, the expression “biostimulating composition” is intended to mean any product, the use of which is intended to ensure the ability of the plants to stimulate their leaf or root metabolism.
According to a first aspect, a subject of the invention is a fertilizing and/or biostimulating composition containing at least one fructooligosaccharide (FOS) comprising from 2 to 10 monosaccharide units, which fructooligosaccharide is optionally sulfated. The composition may contain the fructooligosaccharide in combination with one or more fertilizing materials. The fructooligosaccharide is preferably in an effective amount in the composition in order to cause the desired effect.
The amount of FOS supplied to the plants must be sufficient to stimulate the eliciting of mechanisms for absorbing inorganic nutrients. This amount is variable depending on the nature of the plant to be treated and the mode of treatment (via the leaves or via the roots). This amount may especially be determined on a case by case basis by employing macroarray tests as defined below.
The fructooligosaccharides used within the context of the present invention are polymers comprising at least one fructose unit and at least one C3-C6 monosaccharide unit, preferably a C6 monosaccharide unit other than fructose, for example chosen from glucose, mannose and xylose.
The fructooligosaccharide (FOS) used within the context of the present invention comprises from 2 to 10 monosaccharide units (2 and 10 being included), said monosaccharide units comprising at least one fructose. It is preferred that the fructooligosaccharide (FOS) used within the context of the present invention comprise from 2 to 9 monosaccharide units (2 and 9 being included), or from 2 to 8 monosaccharide units (2 and 8 being included), or else from 2 to 7 monosaccharide units (2 and 7 being included), or else from 2 to 6 monosaccharide units (2 and 6 being included), for example from 2 to 6 monosaccharide units (2 and 6 being included), from 2 to 5 monosaccharide units (2 and 5 being included), from 2 to 4 monosaccharide units (2 and 4 being included). The fructooligosaccharide (FOS) used within the context of the present invention may be a mixture of the above fructooligosaccharides.
The fructooligosaccharide (FOS) used within the context of the present invention advantageously corresponds:
Compounds of formula GFn such that n=1, 2 or 3 are preferred. The fructooligosaccharide (FOS) used within the context of the present invention may be a mixture of Glucose-(Fructose)n, or Mannose-(Fructose)n, compounds such that n ranges from 1 to 3 (1 and 3 being included).
The fructooligosaccharides may be chosen from the group consisting of the compounds nystose (GF2), kestose (GF3), fructosylnystose (GF4), bifurcose (GF3), such that G=glucose, inulobiose (F2), inulotriose (F3) and inulotetraose (F4).
The FOSs may be obtained from the abovementioned species by a method comprising the steps of washing, grinding, extraction (solid-liquid separation) and optionally fractionation and concentration.
The FOSs may also be obtained by acid or enzymatic hydrolysis, from the abovementioned extracts.
According to one embodiment of the invention, the fructooligosaccharides are obtained by hydrolysis of inulin or by hydrolysis of oligosaccharides obtained from inulin. The hydrolysis may be carried out enzymatically, chemically or microbially (for example by Bifidobacterium).
According to one particular embodiment of the invention, the fructooligosaccharides are obtained by enzymatic hydrolysis of inulin.
Inulin is a polysaccharide consisting of D-fructose units bonded by β(2,1) linkages and of a D-glucose end group bonded by an α(1,2) linkage. The degree of polymerization of inulin is generally of the order of 40 to 60 monosaccharide units.
Inulin may be extracted from the roots of various plants, in particular Jerusalem artichoke and chicory.
The plant extract containing FOSs obtained according to one of the methods described above may be more or less concentrated depending on the intended use. Total dehydration of the extract enables it to be made available in water-soluble pulverulent form. The dehydration may be obtained by drum drying or spray drying.
Alternatively, the fructooligosaccharides may be obtained by chemical synthesis.
According to one variant, the fructooligosaccharides may be obtained by transfructosylation of sucrose, for example by the transfructosylation action of an Aspergillus niger β-fructosidase on sucrose.
The fructooligosaccharides may be sulfated, for example by reaction of an FOS as described above with sulfuric acid in the presence of pyridine.
The FOSs described above may be used alone or in a mixture with inorganic fertilizers, to prepare the fertilizing and/or biostimulating product of the invention.
It is known that fertilizers are defined as fertilizing materials, the main function of which is to supply plants with elements directly useful to their nutrition (major fertilizing elements, secondary fertilizing elements and trace elements).
To this end, root or leaf fertilizers generally use sources of nitrogen, phosphorus and potassium as well as trace elements and amino acids.
Plants mainly absorb nitrogen, which is an essential nutritive element for their growth. The nitrogen is generally supplied in the form of nitrate or ammonium, the use of which in large amounts poses problems from an ecological viewpoint.
The fertilizing and/or biostimulating product of the invention containing FOSs may therefore be used as a supplement in conventional fertilizing compositions such as fertilizers in order to activate inorganic nitrogen absorption and the assimilation thereof in the form of proteins.
The fertilizing and/or biostimulating product of the invention may therefore contain one or more fertilizing materials chosen from urea, ammonium sulfate, ammonium nitrate, natural phosphate, potassium chloride, ammonium sulfate, magnesium nitrate, manganese nitrate, zinc nitrate, copper nitrate, phosphoric acid and boric add.
The product of the invention may contain biostimulating agents chosen from humic acids, extracts of marine algae, amino acids, plant hormones or a mixture thereof.
The fructooligosaccharide (FOS) represents for example from 0.1 to 10% by weight, for example 1% by weight, of the weight of the composition.
When it is formulated in liquid form, the product of the invention contains for example from 0.1 to 5% by weight of FOS. When it is formulated in solid form, the product of the invention contains for example from 1 to 10% by weight of FOS.
The product of the invention may be in the form of a product chosen from calcareous enriching agents, organic enriching agents and crop supports, root fertilizers of NP, PK, NPK type, leaf fertilizers or else nutritive solutions for roots.
Another subject of the invention is a method for fertilizing plants to improve their absorption of inorganic nutrients, which comprises the application, to said plants or to the soils, of an effective amount of a fructooligosaccharide (FOS) comprising from 2 to 10 monosaccharide units or of a composition as has just been described.
Another subject of the invention is a method for the biostimulation of plants which comprises the application, to said plants or to the soils, of an effective amount of a fructooligosaccharide (FOS) comprising from 2 to 10 monosaccharide units or of a product as described above.
According to another aspect, the present application aims to protect a method for treating plants or soils intended to activate reactions for eliciting mechanisms for absorption of inorganic nutrients, characterized in that it comprises the application, to said plants or to said soils, of an effective amount of FOS.
In one of these three methods, the product of the invention or the FOS may be applied to the plants via the leaves or via the roots.
Generally, the amount of FOS supplied to the plants is from 0.1 g to 100 g per litre, and preferentially of the order of 1 g per litre, when supplied in liquid form via the leaves or in nutritive solutions for the roots (hydroponically or dropwise).
The amount of FOS supplied to the plants is for example from 10 to 1000 g/ha, preferably of the order of 200 g/ha, when supplied in solid form, for example in pulverulent or granulated fertilizers.
The product of the invention may be used in one of the methods forming the subjects of the invention, at the following recommended doses:
Application to leaves: between 0.5 and 5 kg/ha at a degree of dilution of between 0.1-0.5%,
Application to the roots—application on the ground: 10-50 kg/ha,
Application to the roots—fertigation: 10-20 kg/ha by dropwise irrigation at a degree of dilution of between 0.1-0.5%.
The present invention can be used in the treatment of a very wide variety of plants. Among the latter, mention will in particular be made of:
The expression “plant” is intended to denote, in the present application, the plant considered as a whole, including its root system, its vegetative system, the grains, seeds and fruits.
According to yet another aspect, the present application aims to cover the use of FOSs comprising from 2 to 10 monosaccharide units to elicit mechanisms for absorption of inorganic nutrients in plants and as agent for stimulating natural plant defenses. The inorganic nutrients will in particular be nitrogen, phosphorus and calcium.
The present invention will now be illustrated by the following figures and nonlimiting examples. In these examples, unless otherwise indicated, the percentages are expressed by weight and the temperature is room temperature.
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.
The granules were dried in a drum dryer at 90° C. for 15 min.
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.
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.
Methodology:
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.
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.
The rate of absorption of the minerals depending on whether the nutritive solution contained FOS (treatment) or not (control) was compared.
9 young lettuce plants (three batches each comprising three young plants) were transferred to a fresh nutritive solution containing 100 mg.l−1 of a fructooligosaccharide sold by Beghin Meiji under the reference Profeed®L95.
9 young lettuce plants (three batches each comprising three young plants) were transferred to the same fresh nutritive solution not containing FOS (control).
The nutritive solution contained: 2 mM of NO3−, 2 mM of PO42−, 4 mM of K+, 1 mM of Ca2+, 2 mM of Mg2+, 100 μM of Fe, 14 μM of B, 16 μM of Mn, 4 μM of Mo and 0.8 μM of Cu. The pH of the solution was adjusted to 6 with KOH.
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).
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.
Results:
The results underwent statistical analysis (ANOVA) using the STATISTICA 6.0 program, taking P<0.05 as significant.
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%).
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.
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.
The effect of a fructooligosaccharide (FOS) comprising from 2 to 10 monosaccharide units (sold under the reference Profeed® L95) was compared to the effect of a comparative FOS (sold under the reference Probiofeed®, containing 95% inulin and 5% monosaccharides).
Growth of the Plants
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.
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).
Analyses
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.
Reagents and Materials
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 add was provided by OlChemin Ltd (Olomouc, Czech Republic), and the 2H4-salicylic acid (D-SAL) used as internal standard for the salicylic acid was provided by Sigma-Aldrich (St Louis, USA).
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 evaporater from Labconco Corporation (Kansas City, Mo., USA).
Extraction Method
0.25 gram of plant tissue (fresh weight) are weighed out, to which are added 100 μl of a solution of D-SAL and DHJAS (1000 ng/ml in methanol) and 2 ml of MeOH/H2O/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−1, and centrifugation is carried out at 11000 min−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 add, then this is agitated and centrifuged at 12000 min−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.
Chromatographic Method
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 μm Hydro-RP Synergi (15×0.2 cm) chromatographic column (Phenomenex, Torrance, Calif., USA). The gradient and the conditions used are as follows:
Injection volume: 40 μl.
Sample temperature: 20° C.
Column temperature: 30° C.
Mass Spectrometry Method
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.
Source Parameters:
CUR (curtain gas flow rate): 22.00 psi.
IS (ion spray voltage): −4000 V.
TEM: 550° C.
GS1 (nebulizer gas flow rate): 45.00 psi.
GS2 (reheater gas flow rate): 55.00 psi.
Mass Analyzer Parameters:
Scan mode: MRM (multiple reaction monitoring).
Q1 resolution: low.
Q3 resolution: 1.
CAD (collision-activated dissociation) gas: medium.
CXP (collision cell exit potential): −4.00 V.
Detector Parameters:
CEM (multichannel electron multiplier): −2800.0 V.
The data obtained are processed by means of the Analyst 1.4.2 software from Applied Biosystems.
Results
The results are given in the table below.
2H4-Salicylic acid (D-SAL)
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.
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:
The results have been given in
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1453157 | Apr 2014 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2015/050917 | 4/8/2015 | WO | 00 |
