Use of phycobiliproteins or an extract containing same as fertilizer

Abstract

The invention relates to the use of phycobiliproteins or of an extract containing same as fertilizer, a method for stimulating tillering and/or root development and/or the yield of a plant, as well as fertilizer compositions comprising phycobiliproteins or an extract containing same and (i) an amendment and/or (ii) a fertilizer other than phycobiliproteins or an extract containing same.

Claims

1. A method for fertilizing a plant, comprising application to a soil containing the plant an effective amount of an extract containing phycobiliproteins, in which the extract containing phycobiliproteins is a phycobiliprotein-enriched extract, the dry mass of phycobiliproteins in the extract being at least 10% of the total weight of dry matter of the extract, thereby stimulating tillering and root development of the plant.

2. The method as claimed in claim 1, for stimulating the yield of the plant.

3. The method as claimed in claim 1, in which the plant belongs to the order of the monocotyledons.

4. The method as claimed in claim 1, in which the extract containing phycobiliproteins is an extract from cyanobacteria, an extract from Rhodophyceae, an extract from Glaucocystophyceae or an extract from Cryptophyceae.

5. The method as claimed in claim 1, in which the extract is an extract from cyanobacteria.

6. The method as claimed in claim 1, in which the extract containing phycobiliproteins is supplied to the soil in an amount sufficient to increase tillering by at least 5%.

7. The method as claimed in claim 1, in which the extract containing phycobiliproteins is supplied to the soil in an amount of phycobiliproteins from 0.1 to 5 kg/ha.

8. The method as claimed in claim 1, in which the plant belongs to the family Poaceae.

9. The method as claimed in claim 1, in which the plant is selected from the group consisting of wheat, rice, barley, oat, rye, sugar cane, pasture plants and maize.

10. The method as claimed in claim 1, in which the extract is an extract from cyanobacteria of the genus Arthrospira.

11. The method as claimed in claim 1, in which the extract is an extract from cyanobacteria of the genus Arthrospira platensis.

12. The method as claimed in claim 1, in which the phycobiliproteins or the extract containing same are supplied to the soil in an amount sufficient to increase tillering by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 40%.

13. The method as claimed in claim 1, in which the extract containing phycobiliproteins is supplied to the soil in an amount of phycobiliproteins from 0.2 to 3 kg/ha.

14. The method as claimed in claim 1, in which the extract containing phycobiliproteins is supplied to the soil in an amount of phycobiliproteins about 1 kg/ha.

15. A method for stimulating tillering and root development of a plant, comprising supplying an effective amount of an extract containing phycobiliproteins to a soil comprising the plant, in which the extract containing phycobiliproteins is a phycobiliprotein-enriched extract, the dry mass of phycobiliproteins in the extract being at least 10% of the total weight of dry matter of the extract, thereby stimulating tillering and root development of the plant.

16. The method as claimed in claim 15, in which the plant belongs to the order of the monocotyledons.

17. The method as claimed in claim 15, in which the extract containing phycobiliproteins is an extract from cyanobacteria, an extract from Rhodophyceae or an extract from Glaucocystophyceae.

18. The method as claimed in claim 15, in which the extract containing phycobiliproteins is supplied to the soil in an amount sufficient to increase tillering by at least 5%.

19. The method as claimed in claim 15, in which the extract containing phycobiliproteins is supplied to the soil in an amount of phycobiliproteins from 0.1 to 5 kg/ha.

20. The method as claimed in claim 15, in which the plant belongs to the family Poaceae.

21. The method as claimed in claim 15, in which the plant is selected from the group consisting of wheat, rice, barley, oat, rye, sugar cane, pasture plants and maize.

22. The method as claimed in claim 15, in which the extract is an extract from cyanobacteria of the genus Arthrospira.

23. The method as claimed in claim 15, in which the extract is an extract from cyanobacteria of the genus Arthrospira platensis.

24. The method as claimed in claim 15, in which the extract containing phycobiliproteins is supplied to the soil in an amount sufficient to increase tillering by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 40%.

25. The method as claimed in claim 15, in which the extract containing phycobiliproteins is supplied to the soil in an amount of phycobiliproteins from 0.2 to 3 kg/ha.

26. The method as claimed in claim 15, in which the extract containing phycobiliproteins is supplied to the soil in an amount of phycobiliproteins about 1 kg/ha.

27. A fertilizer composition comprising an extract containing phycobiliproteins and (i) an amendment and/or (ii) a fertilizer other than phycobiliproteins or an extract containing same, in which the extract containing phycobiliproteins is a phycobiliprotein-enriched extract, the dry mass of phycobiliproteins in the extract being at least 10% of the total weight of dry matter of the extract.

28. The fertilizer composition as claimed in claim 27, in which the amendment is selected from the group consisting of: limestone, limestone and magnesia, compost, and dung.

29. The fertilizer composition as claimed in claim 27, in which the fertilizer is selected from the group consisting of: urea, ammonium sulfate, ammonium nitrate, phosphate, potassium chloride, ammonium sulfate, magnesium nitrate, manganese nitrate, zinc nitrate, copper nitrate, phosphoric acid, potassium nitrate and boric acid.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Diagram showing the number of tillers per plant of soft wheat after 29 days of supplying the “FLM17” extract at different concentrations in the hydroponic solution, i.e. 350 μl/L and 700 μl/L (and without supply of the “FLM17” extract (untreated control). The “FLM17” extract, at the two concentrations tested, makes it possible to stimulate tillering.

(2) The letters “a”, “b” and “c” correspond to ANOVA analysis of variance: “a”, “b” and “c” refer to different statistical groups at 5% and the groups “ab” and “bc” signify that the set of conditions is not different from the control, nor from the other set of conditions.

(3) FIG. 2: Diagrams showing the dry aerial and root biomasses produced by plants of soft wheat after 29 days of supplying the “FLM17” extract at different concentrations in the hydroponic solution, i.e. 350 μl/L and 700 μl/L and without supply of the “FLM17” extract (untreated control). The “FLM17” extract, at the two concentrations tested, stimulates root and aerial development.

(4) FIG. 3: Diagrams showing the dynamics of tillering with and without the “FLM17” extract established with: variation of the number of leaves per plant between 21 DPS (days post-sowing) and 32 DPS (FIG. 3a), and variation of the number of tillers between 21 DPS and 32 DPS (FIG. 3b).

(5) FIG. 3a shows that for one and the same number of leaves, treatment with FLM17 increases the number of tillers.

(6) FIG. 3b shows notably that tillering is earlier and more significant with supply of the “FLM17” extract at different concentrations in the hydroponic solution, i.e. 350 μl/L (middle bars) and 700 μl/L (bars on the right) compared to tillering without supply of the “FLM17” extract (bars on the left).

(7) FIG. 4: Diagram illustrating the morphology of the root system of the plants of soft wheat harvested after 28 days post-sowing (DPS), i.e. supplying the “FLM17” extract for 20 days. The diagram notably shows an increase in the area of the seminal roots and nodal roots with supply of the “FLM17” extract at different concentrations in the hydroponic solution, i.e. 350 μl/L and 700 μl/L compared to the area obtained without supply of the “FLM17” extract (TNT).

(8) FIG. 5: Diagram showing the variation of the number of tillers per plant of soft wheat until 43 days of supplying the “FLM17” extract (50 DPS) at different concentrations in the hydroponic solution, i.e. 350 μl/L (middle bars) and 700 μl/L (bars on the right). The “FLM17” extract, at the two concentrations tested, stimulates tillering throughout the development of the plant. The stimulation is even more marked at 50 DPS.

(9) The letters “a”, “b” and “c” correspond to ANOVA analysis of variance: “a”, “b” and “c” denote different statistical groups at 5% and the groups “ab” and “bc” signify that the set of conditions is not different from the control, nor from the other set of conditions.

(10) FIG. 6: Diagram showing the dry biomass of the plants treated with the “FLM17” extract and untreated (TNT) at 51 DPS.

(11) The letters “a”, “b” and “c” correspond to ANOVA analysis of variance: “a”, “b” and “c” refer to different statistical groups at 5% and the groups “ab” and “bc” signify that the set of conditions is not different from the control, nor from the other set of conditions.

(12) FIG. 7: Diagram showing the number of tillers per plant of soft wheat after 29 days of supplying the “FLM17” extract at a concentration of 1400 μl/L in the hydroponic solution, with supply of the extract “FLM17 B” at a concentration of 1400 μl/L in the hydroponic solution and without supply of the “FLM17” extract (untreated control). The diagram demonstrates that phycobiliprotein enrichment of the FLM17 extract improves the tillering of the wheat considerably.

EXAMPLES

Example 1: Preparation of an Extract Containing Phycobiliproteins

(13) 100 g of microalgae of the type Arthrospira platensis were incorporated in 0.9 liters of demineralized water. The mixture was then stirred at room temperature for about 3 h. Then the mixture obtained was centrifuged at 7000 revolutions/min for 30 minutes. This step allowed the phycobiliproteins present in the cells of the microalgae to be extracted in the water. The phycobiliproteins were then present in the supernatant. The supernatant was then recovered and filtered at 50 μm. The filtrate thus obtained, corresponding to an extract containing phycobiliproteins, comprised between 6 and 8 wt % of dry extract.

(14) The amount of phycobiliproteins in the extracts was measured by the reference spectrophotometric method described in Bennett et al. (The Journal of Cell Biology, Volume 58, p. 419-435, 1973). The extracts obtained by the method described above were titrated between 5 and 8 g of phycobiliproteins per liter of extract. This corresponds to a content of phycobiliproteins equivalent to 10% of the dry matter contained in the extract, i.e. the dry mass of phycobiliproteins in the extract is 10% of the total weight of dry matter of the extract.

(15) An extract containing 7 g of phycobiliproteins/liter was designated “FLM17”.

(16) An alternative method consists of incorporating 100 g of microalgae of the type Arthrospira platensis in 0.9 liters of demineralized water buffered with 5% phosphate buffer. The mixture is then stirred at a temperature of 4° C. for about 24 h. Then the mixture obtained is centrifuged at 4000 g for 30 minutes. This step allows the phycobiliproteins present in the cells of the microalgae to be extracted in water. The phycobiliproteins are contained in the supernatant. The supernatant is then recovered and filtered at 50 μm.

Example 2: Preparation of a Phycobiliprotein-Enriched Extract

(17) Starting with 500 mL of the FLM17 extract obtained in Example 1, the phycobiliproteins were precipitated by adding ammonium sulfate at a dose of 500 g/L of filtrate. Precipitation occurred after stirring the mixture for 2 hours at +4° C.

(18) The precipitate was recovered by centrifugation for 30 minutes at 7000 rpm at +4° C.

(19) The precipitation pellet was taken up in 500 mL of demineralized water, then membrane ultrafiltration to constant volume was carried out on a ceramic membrane with a porosity of 1000 daltons, in order to remove the salts, to obtain a phycobiliprotein-enriched extract. Ultrafiltration also made it possible to remove small molecules with a molecular weight below 1000 daltons, notably phytohormones, such as phytohormones known by the name Auxin (IAA=indoleacetic acid: MW=175 Da), Zeatin (trans and cis: MW=219.2 Da) and Zeatin riboside (MW=351.3 Da). This phycobiliprotein-enriched extract was enriched with phycobiliproteins 2.5 times relative to the starting FLM17 extract, i.e. a content of phycobiliproteins of 17.5 g/L of enriched extract. This corresponds to a content of phycobiliproteins equivalent to 25% of the dry matter contained in the enriched extract, i.e. the dry mass (or weight) of phycobiliproteins in the extract is 25% of the total weight of dry matter of the extract.

(20) This phycobiliprotein-enriched extract was designated “FLM17 B”.

Example 3: Agronomic Effect of the Phycobiliprotein Extract FLM17 Obtained in Example 1, on the Tillering Stage of a Winter Soft Wheat in Controlled Conditions

(21) Materials and Methods

(22) Seeds of winter soft wheat (Triticum aestivum), cv. Rubisko, were sown in opaque black boxes containing vermiculite. Immediately after sowing, the substrate was watered with water and the boxes were kept away from the light for 2 days. The seedlings were then moved and kept in a growing chamber for 5 days until the time for transplanting.

(23) On the seventh day after sowing (BBCH 11-12), the roots of the seedlings were cleaned and the seedlings were planted out in opaque pots with a capacity of 5 liters, at a rate of 3 plants per pot. A hose with a diameter of about 7 mm connected to a pump was introduced into each pot in order to ensure constant aeration of the nutrient solution in contact with the roots. These pots were kept in the same growing chamber throughout the test.

(24) The nutrient solution was prepared according to the classical Hoagland recipe, adapted to the needs of the crop. Renewal of this solution for all the pots, as well as supply of the “FLM17” extract to the roots by the liquid route in the treatment conditions (Table 1), took place every 2 days.

(25) TABLE-US-00001 TABLE 1 Test conditions in the hydroponics test on winter soft wheat. Conditions Characterization of the conditions 1 Hoagland solution (untreated control) 2 Hoagland solution + 350 μl of FLM17/liter of Hoagland solution (corresponding to 2.45 mg of phycobiliproteins/liter of Hoagland solution) 3 Hoagland solution + 700 μl of FLM17/liter of Hoagland solution (corresponding to 4.9 mg of phycobiliproteins/liter of Hoagland solution)

(26) Each set of conditions tested comprised 3 repetitions (buckets) in which 3 plants were put, totalling 12 plants per set of conditions.

(27) At 29 days post-transplanting (and therefore of treatment), the plants were harvested. The average number of tillers per plant as well as the aerial and root biomass were quantified.

(28) Results

(29) Tillering

(30) Independently of the dose tested, the “FLM17” extract promoted an increase in the average number of tillers per plant, relative to the plants in the untreated control group. With a 42% increase in the number of tillers per plant, the plants treated at 700 μl of FLM17/liter of Hoagland solution showed average tillering significantly (ANOVA at the threshold=5%) greater than the untreated control (FIG. 1).

(31) Production of Dry Biomass

(32) An average gain in the production of aerial and root biomass of the order of 15% and 4% was observed for the plants that had been treated with the “FLM17” extract at 700 μL/liter of Hoagland solution, respectively (FIG. 2). This converges and can be explained by the increase in the number of tillers.

(33) The FLM17 extract obtained in Example 1 causes a stimulation of tillering of the winter wheat by an increase in the number of tillers and an increase in total biomass.

Example 4: Agronomic Effect of the Phycobiliprotein Extract FLM17 Obtained in Example 1, on the Tillering Stage of a Winter Soft Wheat in Controlled Conditions

(34) Materials and Methods

(35) Experimental Setup

(36) Seeds of winter soft wheat (Triticum aestivum), cv. Rubisko, were sown in opaque black boxes containing vermiculite. Immediately after sowing, the substrate was watered and the boxes were kept away from the light for 2 days. The seedlings were then moved and kept in a growing chamber for 5 days until the time for transplanting.

(37) On the seventh day after sowing (BBCH 11-12), the roots of the seedlings were cleaned and the seedlings were planted out in opaque pots with a capacity of 5 liters, at a rate of 6 plants per pot. A hose with a diameter of about 7 mm connected to a pump was introduced into each pot in order to ensure constant aeration of the nutrient solution in contact with the roots. These pots were kept in a greenhouse in the following conditions:

(38) TABLE-US-00002 TABLE 2 Greenhouse conditions during the test Temperature 28° C. in the daytime and 21° C. at night Relative humidity 70%-80% RH Photoperiod 16 hours of light and 8 hours of darkness

(39) The nutrient solution was prepared in situ according to the classical Hoagland recipe, adapted to the needs of the crop. Renewal of this solution for all the pots, as well as supply of the “FLM17” extract to the roots by the liquid route in the treatment conditions (Table 3) took place 3 times per week.

(40) TABLE-US-00003 TABLE 3 Test conditions in the hydroponics test on winter soft wheat Conditions Characterization of the conditions 1 Hoagland solution (untreated control or TNT) 2 Hoagland solution + 350 μl of FLM17/liter of Hoagland solution (corresponding to 2.45 mg of phycobiliproteins/liter of Hoagland solution) 3 Hoagland solution + 700 μl of FLM17/liter of Hoagland solution (corresponding to 4.9 mg of phycobiliproteins/liter of Hoagland solution)

(41) Each set of conditions tested comprised 8 repetitions (buckets) in which 6 plants were introduced, totalizing 48 plants per set of conditions.

(42) Starting from 14 days of treatment in hydroponics (21 days post-sowing, i.e. 21 DPS), counting of the number of leaves per plant, of the total number of plants with tillering in each set of conditions as well as of the number of tillers per plant was begun. It was thus possible to establish the dynamics of tillering. Two separate harvests were carried out in the context of this test: 1. Intermediate harvesting at 28 DPS, and 2. Final harvesting at 51 DPS.

(43) At intermediate harvesting, 12 plants from each set of conditions were harvested, packaged and immersed in liquid nitrogen before being stored frozen at −80° C. The second half of the plants was harvested, packaged and put in an oven to dry, at 70° C.

(44) The following parameters were studied during intermediate harvesting: Root morphology using an acquisition tool (EPSON Expression 10000 XL scanner, Japan) and image processing (Winrhizo™, Canada); Aerial and root dry mass.

(45) At final harvesting, 8 plants from each set of conditions were harvested, packaged and immersed in liquid nitrogen before being stored frozen at −80° C. The remaining 16 plants from each set of conditions were collected, packaged and put in an oven to dry, at 70° C.

(46) During final harvesting, the aerial and root dry mass was measured.

(47) Statistical analysis of the data collected for this test was carried out using the R Studio software. The alpha threshold employed in the analyses of variance (ANOVA) and the post-hoc tests (Student-Newman-Keuls, SNK) was 5%.

(48) Results

(49) Intermediate Harvesting

(50) Dynamics of Tillering

(51) The average number of tillers per plant in the various treatment conditions was greater overall than that of the untreated control starting from the first count, where 41% of the treated plants had tillered (both doses combined) against only 17% for the untreated control (FIG. 3). This difference was maintained until the last count, which took place on the day before intermediate harvesting.

(52) This precocity may be beneficial as these structures have more time before the end of the tillering stage to become independent of the main stem, i.e. to establish roots for their subsistence.

(53) Root Development

(54) The density of the nodal roots (or “crown roots”) in the vegetative stage is strongly correlated with the number of tillers developed by certain cereals because they are important for physical and nutritional support of the plants. The seminal roots are also associated with absorption of nutrients and water, contributing directly to the overall nutrition of the plants (NAKHFOROOSH et al., Wheat root diversity and root functional characterization, 2014; ROGERS & BENFEY, Regulation of plant root system architecture: implications for crop advancement, 2015).

(55) Although the appearance and the branching of the seminal roots, called embryonic, are highly dependent on the genetics of the plant in question, those of the nodal roots, called post-embryonic, evolve as a function of the surrounding conditions (KUHHAM and BARRACLOUGH, Comparison between the seminal and nodal root systems of winter wheat in their activity for N and K uptake, 1986).

(56) FIG. 4 shows that the “FLM17” extract stimulates root development, of the nodal roots in particular.

(57) Final Harvesting

(58) Dynamics of Tillering

(59) Appearance of new tillers was monitored until the day before the final harvesting, i.e. 50 DPS. Stimulation of tillering with “FLM17” was maintained throughout the treatment (FIG. 5).

(60) Production of Dry Biomass

(61) A slight increase in dry mass produced by the plants at 51 DPS (final harvesting) was measured (FIG. 6).

(62) In conclusion, the FLM17 extract obtained in Example 1 causes stimulation of tillering of the winter wheat by an increase in the number of tillers, acceleration of the dynamics of tillering and an increase in total biomass.

Example 5: Comparison of the Agronomic Effect of the Phycobiliprotein Extract FLM17 Obtained in Example 1 and the Agronomic Effect of the Phycobiliprotein-Enriched Extract FLM17 B Obtained in Example 2, on the Tillering Stage of a Winter Soft Wheat in Controlled Conditions

(63) Materials and Methods

(64) Seeds of winter soft wheat (Triticum aestivum), cv. Rubisko, were sown in opaque black boxes containing vermiculite. Immediately after sowing, the substrate was watered with water and the boxes were kept away from the light for 2 days. The seedlings were then moved and kept in a growing chamber for 5 days until the time for transplanting.

(65) On the seventh day after sowing (BBCH 11-12), the roots of the seedlings were cleaned and the seedlings were planted out in opaque pots with a capacity of 5 liters, at a rate of 3 plants per pot. A hose with a diameter of about 7 mm connected to a pump was introduced into each pot in order to ensure constant aeration of the nutrient solution in contact with the roots. These pots were kept in the same growing chamber throughout the test.

(66) The nutrient solution was prepared according to the classical Hoagland recipe, adapted to the needs of the crop. Renewal of this solution for all the pots, as well as supply of the extracts “FLM17” and “FLM17 B” to the roots by the liquid route in the treatment conditions (Table 4) took place every 2 days.

(67) TABLE-US-00004 TABLE 4 Test conditions in the hydroponics test on winter soft wheat Dose in μl/ liter of Content of Characterization of the Hoagland phycobiliproteins/L Conditions conditions solution Hoagland solution 1 Hoagland solution 0 0 (untreated control) 2 Hoagland solution + 1400 μl   9 mg 1400 μl of FLM17/Liter of Hoagland solution 3 Hoagland solution + 1400 μl 22.5 mg 1400 μl of FLM17B/Liter of Hoagland solution

(68) Each set of conditions tested comprised 3 repetitions (buckets) in which 3 plants were put, totalling 12 plants per set of conditions.

(69) At 29 days post-transplanting (and therefore of treatment), the plants were harvested. The average number of tillers per plant as well as the aerial and root biomass were quantified.

(70) Results

(71) Tillering

(72) The “FLM17” extract promoted an increase in the average number of tillers per plant, relative to the plants in the untreated control group. With a 44% increase in the number of tillers per plant, the plants treated with the FLM17 B extract showed tillering significantly (ANOVA at the threshold⋅=5%) greater than the untreated control and tillering 2.8 times greater relative to the FLM17 extract (FIG. 7).

(73) These results show that phycobiliprotein enrichment improves the tillering of the wheat considerably.