WO2021260034A1

WO2021260034A1 - Organic composition for bio-stimulation of a plant

Info

Publication number: WO2021260034A1
Application number: PCT/EP2021/067199
Authority: WO
Inventors: Adriano ALTISSIMO; Cristina SUDIRO; Leendert Cornelis SCHILPEROORD; Marinus Matheus Antonius VAN OERS; Laurent Aubertin
Original assignee: Thesis B.V.; Landlab Srl
Priority date: 2020-06-23
Filing date: 2021-06-23
Publication date: 2021-12-30
Also published as: NL2025884B1; EP4167739A1

Abstract

The present invention relates to an organic composition for bio-stimulation of a plant comprised of plant derived phytocomplexes. The present invention further relates to a method for the production of the organic composition of present invention, and a method for improving drought tolerance of a plant by applying to a plant the composition of present invention. Finally, the present invention relates to a plant nutrient solution comprising the composition of present invention.

Description

ORGANIC COMPOSITION FOR BIO-STIMULATION OF A PLANT Description

Conventional agriculture provides for 95% of food production in the world but is highly resource intensive and so far reluctant to adopt a full transformation towards more resource efficient practices, due to limited reliability of current solutions. Productivity growth is stagnating and in many areas declining, affected by pollution, declining soil quality and biodiversity loss. Increasing population and wealth standard worldwide provides todays agriculture with challenges to produce at improved yield per m² and cope with the rising food demand. Agriculture need to be more efficient and be able to counter resource-scarcity and in addition to preserve soils, water and human health.

Water demand is expected to face a 30% growth by 2030 and industrial agriculture is responsible for more than 2/3 of world’s water withdrawal. Abiotic stresses such as extreme temperatures, drought, and soil salinization are responsible for an average crop yield loss of over 50% for most of major crops. And among climate-related stresses, water scarcity is one of the largest; it is estimated that by 2030 half the world’s population will be living in areas with high water stress. While water shortage is a key factor triggering soil erosion, irrigation is highly energy consuming, hence improving plants efficiency in water uptake constitutes a big support to climate change adaptation, impacting on preservation of agricultural productivity, soil quality, water and energy consumption.

Since the beginning of the 20^th century, the use of herbicides and pesticides has been one of the main factors in the rising productivity of agriculture. However, increasing intensified use of chemical products in agriculture is becoming a problem in view of public health and environmental issues related to the use of these chemical products, especially in relation to long-term negative effects. Substitution of traditional fertilizers, pesticides, fungicides by plant efficiency enhancement products reduces pollution of soil and ground- and surface waters. Herewith, plant efficiency enhancement products reduce the heavy burden the traditional substances put on the ecosystem that harm the microbial and enzymatic activity of the soil, reducing populations of soil organisms that collaborate in the degradation of organic matter and of aquatic fungi that are involved in leaf litter decomposition. Plant efficiency enhancement products or biostimulants enable organic agriculture to become more profitable due to better and higher yields and conventional agriculture to better preserve their soil quality.

Furthermore, a more sustainable and efficient use of soils avoids the need to occupy more and more soil for crop production to feed the increasing global population. This helps to find a way out between the conflicting values of providing global food security and preserving our planet by assuring sufficient natural areas to host the largest possible biodiversity. In view of present climate change problem, agricultural lands drying out and being deprived of resources, in combination with water becoming an increasingly scares resource and precious commodity, there is a need of agricultural products that increase the efficiency of water usage of crops and to reduce irrigation and use of chemical commodities to boost crops production.

Considering the above, there is a need in the art for organic agricultural products that increase the efficiency of water usage of crops and to reduce irrigation and use of chemical commodities to boost crops production. In addition there is a need in the art for method for improving drought tolerance by plants using organic agricultural products to cope with the present climate change problem, agricultural lands drying out and being deprived of resources, in combination with water becoming an increasingly scares resource and precious commodity.

It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by an organic composition for bio-stimulation of a plant comprised of plant derived phytocomplexes, wherein the phytocomplexes are comprising flavonoids at a concentration of between 0.1 to 2 wt% preferably between 0.15 to 1.5 wt%, more preferably between 0.1 to 1 wt%, even more preferably between 0.25 to 0.75 wt%, more preferably between 0.3 to 0.6 wt%, most preferably between 0.45 to 0.55 wt%, and organic acids at a concentration of between 8 to 40 wt%, preferably between 10% to 35 wt%, more preferably between 15 to 30 wt%, most preferably between 20 to 25 wt%, based on the total weight of the organic composition, wherein the phytocomplexes are derived from a plant in the family of Grossulariaceae or Apiaceae. It was surprisingly found that phytocomplexes of plants related to the specific family of Grossulariaceae or Apiaceae, more specifically Ribes nigrum or Daucus carota will provide increases resilience to drought stress. The composition of present invention comprises phytocomplexes, which are able to enhance crops’ resource efficiency, i.e. reducing significantly the amount of nutrients, water and pesticides needed by the crops to grow, herewith improving their resilience to climate change and diseases. The phytocomplexes present in the composition of present invention are a synergistic combination of the natural active ingredients (i.e. flavonoids and/or organic acids) extracted from organically cultivated plants of the Grossulariaceae or Apiaceae family that provide the enhanced crops’ resource efficiency. Experiments with tomato plants for example have demonstrated that the plants being treated with the composition of present invention have a higher number of fruits and flowers compared to the untreated plants and moreover perform better in terms of physiological parameters during stress.

The composition of present invention provides a new bio-stimulant for the plant to overcome drought stress related issues in a wide variety of crops. Treatments of plants with the composition have shown to oppose the negative effect of drought stress (both as severe drought stress and as reduction of irrigation) in crops such as sugarbeet, corn and tomato. Bio-stimulation according to present invention relates to the biological stimulation of the plant to increase the efficiency of water uptake and usage, and more specifically improving the drought tolerance of the plant.

According to a preferred embodiment, the present invention relates to the organic composition, wherein the flavonoids are one or more selected from the group consisting of delphinidin-, cyanidin-, malvidin-, petunidin-, and peonidin-based anthocyanins and catechin, preferably delphinidin- and/or cyaniding-based anthocyanins. Anthocyanins have free radical scavenging and anti-oxidative properties. Anthocyanin are involved in many important biological functions in plants such as attraction of pollinators, antioxidant capacity, protection against reactive oxygen species caused by abiotic stresses, protective effects against UV irradiation/DNA damage, and pathogen attack and plant immunity. Catechins include antioxidants like epicatechine, gallocatechine, epigallocatechine. Preferably the antocyanin one or more selected from delphinidine 3-O-glucoside, delphinidine 3-O-rutinoside, cyanidin 3-O-glucoside, and cyanidin 3- O-rutinoside.

According to another preferred embodiment, the present invention relates to the organic composition, wherein the organic acids are one or more selected from the group consisting of malic acid, ascorbic acid, citric acid, phenolic acid, shikimic acid, quininic acid, gallic acid, hydroxibenzoic acid, hydroxycinnamic acid, linoleic acid, preferably malic acid and/or citric acid. The organic acids have several function in plants physiology: e.g. ascorbic and citric acid are potent antioxidants (ROS-scavengers) while malic acid is involved in the modulation of plant transpiration. The composition of present invention enhances the ability of a plant to use water, nutrients more efficiently and to get in a stronger physiological status. The composition is based on a synergistic combination of secondary metabolites, more specifically flavonoids and/or organic acids, derived from organically grown plants. Results show that the composition of present invention increases the plants resilience to abiotic stress, and in particular the use of water by the plant is more efficient leading to significant reduction of water deficiency symptoms and increased crop yield and quality. E.g. tomato plants being treated with the composition of present invention in a water deficit environment were able, in contrast to control plant not treated, to increase their root biomass. In corn (or maize) it was shown that the composition resulted in a decrease in wilting in young corn plants and increasing yield in rain-fed and poorly irrigated crops. Additionally, it showed improved growth characteristics for plants grown in optimal growth conditions in combination with the composition of present invention, indicating that plants are exploiting more of their genetic potential in contrast to plants grown without the biostimulant composition of present invention.

According to yet another preferred embodiment, the present invention relates to the organic composition, wherein the phytocomplexes are derived from a black currant plant ( Ribes nigrum) or black carrot (Daucus carota).

According to a preferred embodiment, the present invention relates to the organic composition, wherein the phytocomplexes are extracts from organically grown plants, more preferably from leaves, roots, seeds and/or flowers of organically grown plants. Organically grown or organic production refers to production that integrates cultural, biological (non GMO, reduced use of pesticides, etc), and mechanical practices that foster cycling of resources, promote ecological balance, and conserve biodiversity, as set out in the directive on organic production and labelling of organic products and repealing Regulation (EEC) No 2092/91. Plant extracts are harvested from agricultural organically grown crops under controlled conditions to safeguard consistent high level of phytocomplexes. Present invention provides biostimulants to improve the resource -usage efficiency of crops, especially water usage by the crops. Experimental data supporting the positive effects of the composition of present invention on plant growth and ability to overcome drought related stress has been obtained by determining the activity of enzymes involved in ROS scavenging and measuring ROS production in plants, and by high-throughput phenotyping system providing morphological and physiological information on the plants, and by analysing plants transpiration and photosynthetic efficiency and finally by the conventional approach of open-field trials. Overall, the results show an improved performance of the treated plants compared to the untreated plants in the same stressed conditions in the majority of the parameters analysed, proving the efficacy of the composition of present invention.

The present invention relates to the organic composition, wherein the composition is preferably a liquid composition, but may very well be a powder or any other suitable form that is applicable for plant growth applications.

According to a preferred embodiment, the present invention relates to the organic composition, wherein the flavonoids are present in a concentration at least 1 mg of flavonoids per gram of composition, preferably at least 3 mg/g, most preferably at least 5 mg/g. The main flavonoids present in the organic composition of present invention are the anthocyanins, preferably one or more anthocyanins selected from delphinine 3-O-glucoside, delphinidine 3-O-rutinoside, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside. According to another preferred embodiment, the present invention relates to the organic composition, wherein the organic acids are present in a concentration of at least 80 mg of organic acid per gram of composition, preferably at least 150 mg/g, most preferably at least 250 mg/g. The main organic acids present in the organic composition of present invention are malic acid, citric acid and ascorbic acid.

The present invention, according to a second aspect, relates to a method for improving drought tolerance of a plant by applying to said plant a composition according to present invention, wherein the application on the plant is achieved by foliar spray, watering the soil, or adding the composition to the growth substrate of the plant, preferably by foliar spray. Applying the composition of present inventions according to the method of present invention a bio stimulant applied on the plant, preferably to the foliage of the plant, which has a positive effect on a plant's growth, enabling improved assimilation of nutrients thereby boosting the effectiveness of the nutrition, and improves resistance to drought stresses resulting in improved quality of the crop. The present invention applies to agriculture in general, gardening, horticulture, arboriculture, etc. More specifically, the composition used in the method of present invention improves plant resilience to abiotic stress, it boosts water use efficiency leading to water savings of up to 30% in irrigated crops and to significant reduction of water deficiency symptoms and increased crop yield and quality in rainfed crops in climatic zones with water deficiency.

According to yet another preferred embodiment, the present invention relates to the method wherein the composition is applied to the foliage of a plant. Preferably the application on the plant is achieved by foliar spray.

According to another preferred embodiment, the present invention relates to the method, wherein the composition is applied to said plant at least one time per year, preferably at least two times per year, more preferably at least three times per year.

According to a preferred embodiment, the present invention relates to the method wherein the plant is one ore more agricultural crop plants selected from the group consisting of tomato, corn, sugar beet, lettuce, pepper and cucumber.

The present invention, according to a further aspect, relates to a method for the production of the organic composition of present invention for bio-stimulation of a plant, wherein the method comprises the steps of a) refining and crushing of an organically grown plant into a mixture comprising fine solid and liquid biomass, b) optionally; heating of the mixture to a temperature between 20 to 65 °C, preferably 35 to 60 °C, more preferably 45 to 55 °C and adding enzymes to the mixture, c) separating the liquid fraction from the solid biomass fraction, preferably the separation is done by decantation, d) fractionating and stabilizing phytocomplexes from the liquid biomass fraction to obtain the organic composition for bio-stimulation of a plant.

Separating and fractionating can be done by cross flow filtration techniques. It is important that the phytocomplexes comprising the organic acids and flavonoids remain intact, i.e. stabilizing the phytocomplexes in the liquid biomass. This is especially of importance for the flavonoids, more specifically the anthocyanins. Anthocyanins are highly susceptible to (physio) chemical degradation, such as high pH, temperature, light, oxygen, metal ions, affect the colour and stability of anthocyanins. To avoid breakdown of the flavonoids, stabilizing additives are added to the liquid fraction, such as compounds that have an anti-oxidant effect such that the added compound “sacrifices” itself for the anthocyanin by being bonded to oxygen and being oxidized, capturing the oxygen in the new molecular structure. Furthermore or alternatively, co-pigmentation compounds may be added, wherein the compound forms an intermolecular (non-physical binding) structure with anthocyanin. The anthocyanin forms a molecule pair with the added co-pigment which results in a more stable structure than anthocyanin on its own. Furthermore or alternatively, compounds may be added that “shield” the anthocyanin molecule from degradative effects (due to UV, oxygen, pH, etc), i.e. by adding encapsulation compounds to the liquid fraction that can encapsulate the anthocyanins.

According to a preferred embodiment, the present invention relates to the method wherein stabilization of the phytocomplexes is provided by addition of one or more co pigmentation, anti-oxidation, and/or encapsulation compounds to the liquid fraction

According to another preferred embodiment, the present invention relates to the method wherein the one or more co-pigmentation, anti-oxidation, and encapsulation compounds are selected from the group consisting of ascorbic acid, citric acid, ferulic acid, hesperidin, chitosan, rutin, epigallocatechin, L-tryptophan, tannic acid, preferably hesperidin and/or chitosan.

According to yet another preferred embodiment, the present invention relates to the method wherein stabilization of the phytocomplexes is provided by exposing the liquid fraction to ultrasonic waves. Oxygen is one of the main effectors that harm the stability of anthocyanins. By using ultra sound, i.e. ultrasonic waves, the oxygen molecules can be removed from the liquid fraction thereby reducing the degradation of the anthocyanins.

According to a preferred embodiment, the present invention relates to the method wherein the enzymes are pectinase and/or polygalacturonase. Using the enzymes in the method is optional in case the plant based material has a very resistant cell wall which is hard to destroy by the refining and crushing in step a only. Therefore enzymes are added to the mixture to break down the cell walls and free the phytocomplexes from the biomass into the liquid fraction. The step of heating of the mixture and adding enzymes to the mixture will take approximately about 1 to 2 hours. A too short enzymatic digestion will result in low phytocomplex yield and a too long digestion will negatively affect the end product in view of its bio stimulatory effect.

According to another preferred embodiment, the present invention relates to the method wherein the organically grown plant is a black currant plant ( Ribes nigrum ) or black carrot ( Daucus car otd).

According to yet another preferred embodiment, the present invention relates to the method wherein the refining and crushing of the organically grown plant comprises one or more plant parts selected from the group consisting of leaves, roots, seeds and/or flowers.

According to a preferred embodiment, the present invention relates to the method, wherein the liquid biomass fraction obtained after separation in step c is subsequently inoculated with yeast for fermentation of said liquid biomass fraction. By performing a fermentation step of the liquid biomass fraction obtained in step c of the method of present invention, sugars present in this liquid biomass fraction are converted into alcohol through fermentation. These alcohols from the liquid biomass fraction is removed from by a concentration or (vacuum) evaporation step. Experiments show that the fermentation of the liquid biomass results in that the water activity of the composition is lowered, providing increased shelf life. But more important and surprisingly, through the fermentation a more optimized, potent and stable composition is obtained in terms of phytocomplexes and the flavonoids and organic acids present in the composition.

According to the method of present invention, fermentation is preferably performed at a temperature of between 30 to 41 °C, preferably between 34 to 40 °C, more preferably between 35 °C to 39 °C, most preferably between 36°C to 38 °C. The fermentation is preferably performed for approximately for 3 to 24 hours, preferably 6 to 18 hours, more preferably between 10 to 16 hours, most preferably between 12 to 15 hours, depending on the inoculation and temperature. The yeast for fermentation may be any suitable yeast, preferably S. Cerevisiae.

According to yet another preferred embodiment, the present invention relates to the method wherein fermentation occurs until the sugars that are initially present in the liquid biomass fraction are reduced by at least 70%, more preferably at least 80%, most preferably at least 90%. Experiments show that when approximately 90% of the sugars are being fermented from the starting liquid biomass fraction, the phytocomplexes in the composition of present invention are significantly increased as was indicated by an at least 50% increase of both the flavonoids and organic acids present in the compositions obtained after fermentation, resulting in a more potent composition for the bio stimulation of plants of present invention. The present invention, according to a further aspect, relates to a use of the composition of present invention for improving the drought tolerance of plants and/or promoting plant growth or root growth. (Organic) Farmers benefit greatly from the composition of present invention comprising the bio-stimulant phytocomplex derived from organically grown Grossulariaceae plant. Farmers are offered a food grade product to decrease the amount of fertilizers applied whose use is highly restricted excluding e.g. the use of municipal organic wastes and hampering e.g. the use of sewage sludge due to concerns about heavy metals and other pollutants. They can fortify their crops resistance and better withstand the a-biotic stress factors that impact on their production, especially drought. The composition of present invention improves yield, in terms of quality or quantity, with around 4- 6%, especially in areas that suffer the impacts of climate change, in particular drought. The composition of present invention is able to promote plant growth, i.e. increasing the biomass of plants in general, and/or increasing the size of plants, and/or increasing the size of the fruit, and/or increasing the weight of the fruit and may especially be suitable under conditions of drought related stress.

The present invention, according to a further aspect, relates to a plant nutrient solution comprising between 5 to 50 wt%, preferably 10 to 25 wt%, more preferably 15 to 20 wt% of the composition of present invention.

The present invention will be further detailed in the following examples and figures wherein:

Figure 1: shows the Catalase (CAT), Guaiacol Peroxidase (GPX), and H₂0₂ levels in plants under drought stress with and without the composition added to the plant. Plant samples were measured after induction of drought stress at day 3 (1st sampling) and day 14 (2nd sampling) on Catalase, Guaiacol Peroxidase activity, and H₂0₂ concentration. In the plants treated with the composition in comparison to the untreated control group and the positive control being the plants not subjected to drought stress, a decrease in H₂0₂ levels was observed, indicating a strong decrease in oxidative stress. Furthermore, both Catalase, Guaiacol Peroxidase activity was strongly reduced in the plants treated with the composition in comparison to the untreated control group, indicating a low level of ROS detoxification and oxidative stress.

Figure 2: shows wilting of the leaves of corn plants upon induced drought stress treated with and without the composition of present invention. Wilting was evaluated by counting the number of leaves per plant that were in a wilting stage. Treatment of the plants with the composition of present invention resulted in decreased wilting over time compared to the negative control during drought stress. Examples

Example I: Drought stress in Sugar beet

To study drought stress in sugar beet, the changes in activity of some antioxidant enzymes and the changes in fresh biomass weight upon in drought stress was monitored in sugar beet plants treated with and without the composition of present invention.

Plants were treated with the composition of present invention (comprising 0.37 wt% flavonoids, and 18 wt% organic acids), i.e. by applying the composition of present invention on to the foliage of the plant. One week after application of the composition, drought stress was being induced by not watering the plants. Plant samples (shoots and their roots) were collected 3 days after induction of drought stress and at the peak of drought stress approximately 14 days after induction of drought stress and the samples of day 3 (1^st sampling) and day 14 (2^nd sampling) were tested on Catalase, Guaiacol Peroxidase activity, and H₂0₂ concentration was determined. Catalase and Guaiacol Peroxidase activity are indicative for ROS detoxification, and H₂0₂ levels are indicative for oxidative stress in plants.

GPX (guaiacol peroxidase): GPX activity was determined by measuring the oxidation of guaiacol in the presence of H₂0₂ (extinction coefficient, 26.6 mM cm-1) at 1=470 nm over a 3 min interval. The extraction mixture contained 62.5 mM KH₂P0₄, and the reaction mixture contained 0.05 mL of guaiacol (20 mM), 2.9 mL of K-phosphate buffer (10 mM, pH 7.0) and 50 pL of enzyme extract. The reaction was initiated by adding 2 mL of 0.036% mM H₂0₂ (w/w) to the mixture.

H₂0₂ determination: frozen plant material was homogenized in 0.1% (w/v) TCA. Extracts were centrifuged at 15,000xg for 15 min at 4°C. From each supernatant, an aliquot of 0.5 ml was added to 0.5 ml of 10 mM phosphate buffer (pH 7.0) and 1.0 ml of 1 M KI. Absorbance was measured for 1 min at 1 = 390 nm. H₂0₂ is quantified by a refence with a calibration curve made using solutions with known H₂0₂ concentrations.

CAT assay: CAT activity was measured using a well known method in the art (Chance and Maehly, 1955). The extraction mixture contained 50 mM phosphate buffer (pH 7.0), 20 mM Polivinilpirrolidone (PVP), 250 pL Triton X-100. The reaction mixture contained 64 mM KH₂P0₄, 10 mM H₂0₂ and 50 pL of enzyme extract. The reaction was initiated by adding the enzyme extract. CAT activity was determined by following the consumption of H₂0₂ (extinction coefficient 39.4 mM cm-1) at 1=240 nm over a 3 min interval.

Results of 1^st and 2^nd sampling (figure 1) show that treatment with the composition of present invention resulted in decreased H₂0₂ content, indicating a strong decrease in oxidative stress in the plants treated with the composition in comparison to the untreated control group and the positive control being the plants were not subjected to drought stress. Furthermore, both Catalase, Guaiacol Peroxidase activity was strongly reduced in the plants treated with the composition in comparison to the untreated control group, indicating a low level of ROS detoxification and oxidative stress. These enzymatic levels were comparable or even reduced in comparison to the plants that were not subjected to drought stress, indicating that the composition of present invention improves the plants drought tolerance by reducing the oxidative stress levels of the plant.

Additionally, root and shoot fresh biomass weight was measured. It was determined that fresh biomass was unaffected by drought stress in the plants treated with the composition, whereas the control plants showed reduced increase of fresh biomass weight over time, especially approximately 14 days after induction of drought the root fresh biomass differ greatly among groups. Control plants that were not treated with the composition of present invention and were well watered showed a increase of root fresh biomass of about 39%, whereas plants subjected to drought stress and not being treated with the composition showed only a slight increase in root fresh biomass of about 14%, and plants subjected to drought stress and treated with the composition showed a increase in root fresh biomass of about 44%, as determined approximately 14 days post induction of drought. Results therefore indicate that treatment with the composition of present invention improves the plants drought tolerance.

Example II: Drought stress in tomato plants

To study drought stress in tomato plants, phenotypic changes of the plant, i.e. effect on flowers and fruit, plant senescence reflectance index (PSRI), chlorophyll index (NPCI) upon induced drought stress was monitored in young tomato plants, treated with and without the composition of present invention. Using the Landlab phenotyping platform (LLPhP), wherein the LLPhP is based on a high-resolution 3D Laser Scanner (PlantEye) designed for plant phenotyping linked to a high precision gantry, moving above the plant in x and y axis directions. It is a high- throughput technology that allows to scan plants and follow their modifications through time in different environments. The instrument automatically computes a various set of morphological and physiological plant parameters and provides raw information as 3D point clouds. The NPCI number provides a normalized pigment chlorophyll ratio index that provides insight in the chlorophyll content of a crop, i.e. the higher the number, the more valuable and healthy the crop is. The PSRI maximizes the sensitivity of the index to the ratio of bulk carotenoids (i.e. alpha- carotene and beta-carotene) to chlorophyll. An increase in PSRI values indicate increased physiological plant stress, and relate to decreased vegetation health, crop production and yield.

Plants were treated with the composition of present invention, i.e. by applying the composition of present invention on to the foliage of the plant. One week after application of the composition, drought stress was being induced by not watering the plants. Plants were phenotypically examined focussing on number of flowers and fruit (determined by eye), PSRI and NPCI, 14 days after induction of drought stress.

Table 1 provides an overview of the results. Results indicate that plants treated with the composition of present invention under drought stress conditions outperform watered and untreated drought stressed induced plants in view of the number of fruits and flowers. Furthermore, treated plants under drought stress had a higher concentration of chlorophyll (NPCI) and more resistant to physiological plant stresses (PSRI) when compared to untreated plants, indicating that treatment with the composition of present invention improves the plants drought tolerance.

Table 1.

Furthermore, in an additional experiment performed in microplots in open field with tomato plants, the fruit production, brix level, fruit firmness and fruit weight over time was monitored during drought stress of tomato plants. Brix levels were determined by refractometer, fruit firmness was determined by penetrometer. Plants were treated with the composition of present invention twice (week -1, 0, high or low dose) or three times (week -2, -1, and 0, high dose), i.e. by applying the composition of present invention on to the foliage of the plant. Drought stress was induced (at week 0) by reduced irrigation of the plants of approximately 30% over an 8 week time period (Table 2). At week 6.5 (i) and week 8 (ii), average numbers of fruit production, brix level, fruit firmness and fruit weight was determined of the well watered plants, plants under drought stress conditions, and plants treated with the composition of present invention under drought stress conditions.

Table 2.

Results show that tomato plants being treated with the composition of present invention have a decreased drought stress effect on fruits production and slightly increased Brix levels, especially at the second harvest (ii). The production of tomato fruits of the treated plants, (especially treated for three times) was comparable with the plants under well watered conditions. Results show that that treatment with the composition of present invention improves the plants drought tolerance resulting in improved crop yield (both in weight and numbers) and fruit brix levels in comparison to untreated plants under drought stress conditions.

Example III: Drought stress in Corn plants

To study drought stress in corn plants, transpiration and photosynthetic efficiency, and wilting of the plant was determined upon induced drought stress was monitored in corn plants, treated with and without the composition of present invention.

Photosynthetic efficiency is determined to assess photosynthetic performance in plants. Photosynthetic activity was recorded by Fluorpen FP 100 on light-adapted (LA) and dark adapted (DA) leaves during the central hours of the day, and it is called Fv/Fm LA or Fv/Fm DA. This parameter gives an indication of the photosynthetic efficiency, the process that determines the conversion rate of light energy to biomass. Fv/Fm LA gives the information related to photosystem efficiency in the moment of the measurement. If biotic or abiotic stresses are present, a decrease in the photosystem efficiency is usually observed (Baker et al., 2004).

Transpiration is the process of water movement through a plant and its evaporation from aerial parts, such as leaves, stems and flowers. Transpiration changes osmotic pressure of cells, and enables mass flow of mineral nutrients and water from roots to shoots and determining transpiration efficiency is therefore an indicator for efficient water management of the plant. In this trial, transpiration was measured using an Automatic Weighting System, that measured pots weight in continuous.

Wilting is the loss of rigidity of non-woody parts of plants and often occurs during drought conditions as the result of reduction of the turgor pressure in non-lignified plant cells, as a result of diminished water in the cells, and was evaluated by counting the number of leaves per plant that were in a wilting stage. Wilting also results in the leaves expose less surface area. The process of wilting modifies the leaf angle distribution of the plant (or canopy) towards more erectophile conditions. Therefore by observing differences in the wiling of plants, the drought tolerance can be assessed.

Plants (sixteen) were treated twice (day 1 and day 10) with the composition of present invention, i.e. by applying the composition of present invention on to the foliage of the plant. Approximately one week after the second application of the composition, drought stress was being induced by not watering the plants. Wilting was evaluated every 3/4 days after the start of first symptoms of drought stress, while the photosynthetic efficiency was analysed one day before the recovery (end of drought stress) and twice the day after.

Treatment of the plants with the composition of present invention resulted in decreased wilting over time compared to the negative control during drought stress (Figure 2). Furthermore, photosynthetic activity measurements (PAM) show (Table 3) that plant treated with the composition of present invention, was less impacted and even showed slight improved photosynthetic activity during drought stress than the negative control plants (not being treated with the composition).

Table 3.

Overall, these results show an improved performance of the plants treated with the composition of present invention compared to the untreated plants in drought stressed conditions in the majority of the parameters analysed.

Example IV: Measurement acid and flavonoid content

Samples of the organic composition of present invention derived from a black currant (Ribes nigrum) were analysed for their main acid and anthocyanin content and compared with two different control samples obtained from the The James Hutton Institute: JHL9889-6, which is a high-anthocyanin sample and “Ben Finlay”, which is a black currant standard cultivar to obtain reliable measurements. Organic acids (malic acid and citric acid) and ascorbic acid were quantified by HPLC and monitoring of absorbance at 210 nm and 245 nm, respectively. The main anthocyanins (i.e. delphinine 3-O-glucoside, delphinine 3-O-rutinoside, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside) were measured in LC-MS by integrating selected ion chromatograms of selected masses and quantitated against an external calibration curve. Table 4 provides an overview of the main acid and anthocyanin content of the composition of present invention.

Table 4.

Example V: Fermentation of the liquid biomass fraction

A production method was tested to optimize the organic composition of present invention, by addition of a fermentation step. Briefly, the organic composition for bio-stimulation of a plant of present invention is obtained by refining and crushing of an organically grown black currant (Ribes nigrum) providing a mixture comprising fine solid and liquid biomass.

Subsequently, the mixture is heated up to about 50 °C and enzymes pectinase and galacturonase are added to the mixture to degrade the cell wall of the plant cells and to provide the phytocomplexes inside the cell; i.e. to free the phytocomplexes from the biomass into the liquid fraction. After approximately 2 hours, the mixture is cooled down to room temperature and the liquid fraction is separated from the solid biomass fraction by decantation, to obtain the liquid biomass fraction.

This liquid biomass fraction is then fermented to obtain a more optimized organic composition, this test the beneficial effect of fermentation. Therefore, the liquid biomass fraction was divided in two groups, group A has been used in the fermentation process, whereas the other group B was not fermented. The fermentation process is carried out on the liquid biomass fraction of group A, which is heated to about 37°C. Then the liquid fraction is inoculated with yeast (S. Cerevisiae), about 1.5xl0⁷CFU/g. The fermentation process is performed for approximately 14 hours, until approximately 90% of the sugars are being consumed from the liquid biomass fraction. The sugar content is determined based upon the °Brix value measured in the liquid fraction and is typically used in the art to determine sugar content. The more sugars are being reduced the higher the increase in flavonoids and organic acids will be. Then the fermented product is filtered to remove the yeast and obtain a liquid biomass fraction, free of yeast.

Next, for both group A and B, the phytocomplexes were fractioned by cross flow filtration and stabilized from the liquid biomass fraction to obtain the final composition suitable for biostimulation of a plant. Hesperidin is added to the liquid fraction to protect the anthocyanin molecule from degradative effects. It is important that the phytocomplexes comprising the organic acids and flavonoids remain intact.

Both composition of group A and B were analyzed on their phytocomplexes and its content of organic acids and flavonoids (Table 5), as described in Example IV. The addition of a fermentation step results in a significant increase in both the flavonoids and organic acids present in the composition in present invention, thereby providing a more potent composition.

Table 5.

Claims

1. An organic composition for bio-stimulation of a plant comprised of plant derived phytocomplexes, wherein the phytocomplexes are comprising flavonoids at a concentration of between 0.1 to 2 wt%, preferably 0.1 to 1 wt%, and organic acids at a concentration of between 8 to 40 wt%, based on the total weight of the organic composition, wherein the phytocomplexes are derived from a plant in the family of Grossulariaceae or Apiaceae.

2. Organic composition according to claim 1 , wherein the flavonoids are one or more selected from the group consisting of delphinidin-, cyanidin-, malvidin-, petunidin-, and peonidin- based anthocyanins and catechin, preferably delphinidin- and/or cyanidin-based anthocyanins.

3. Organic composition according to claim 1 or 2, wherein the organic acids are one or more selected from the group consisting of malic acid, ascorbic acid, citric acid, phenolic acid, shikimic acid, quininic acid, gallic acid, hydroxibenzoic acid, hydroxycinnamic acid, linoleic acid, preferably malic acid and/or citric acid.

4. Organic composition according to any one of claim 1 to 3, wherein the phytocomplexes are derived from a black currant plant (Ribes nigrum) or black carrot (Daucus carotd).

5. Organic composition according to any of claim 1 to 4, wherein the phytocomplexes are extracts from organically grown plants, more preferably from leaves, roots, seeds and/or flowers of organically grown plants.

6. Organic composition according to any of claim 1 to 5, wherein the flavonoids are present in a concentration of at least 1 mg/g.

7. Organic composition according to any of claim 1 to 6, wherein the organic acids are present in a concentration of at least 80 mg/g.

8. A method for improving drought tolerance of a plant by applying to said plant a composition according to any one of the claims 1 to 7, wherein the application on the plant is achieved by foliar spray, watering the soil, or adding the composition to the growth substrate of the plant, preferably by foliar spray.

9. Method for improving drought tolerance of a plant according to claim 7, wherein the composition is applied to said plant at least one time per year, preferably at least two times per year, more preferably at least three times per year.

10. Method for improving drought tolerance of a plant according to claim 8 or 9, wherein the plant is one or more agricultural crop plants selected from the group consisting of tomato, corn, sugar beet, lettuce, pepper and cucumber.

11. A method for the production of an organic composition for bio-stimulation of a plant according to any one of the claims 1 to 7, wherein the method comprises the steps of a) refining and crushing of an organically grown plant into a mixture comprising fine solid and liquid biomass, b) optionally; heating of the mixture to a temperature between 20 to 65 °C, preferably 35 to 60 °C, more preferably 45 to 55 °C and adding enzymes to the mixture , c) separating the liquid fraction from the solid biomass fraction, preferably the separation is done by decantation, d) fractionating and stabilizing phytocomplexes from the liquid biomass fraction to obtain the organic composition for bio-stimulation of a plant.

12. Method according to claim 11, wherein stabilization of the phytocomplexes is provided by addition of one or more co-pigmentation, anti-oxidation, and/or encapsulation compounds to the liquid fraction.

13. Method according to claim 12, wherein the one or more co-pigmentation, anti-oxidation, and encapsulation compounds are selected from the group consisting of ascorbic acid, citric acid, ferulic acid, hesperidin, chitosan, rutin, epigallocatechin, L-tryptophan, tannic acid, preferably hesperidin and/or chitosan.

14. Method according to any of the claims 11 to 13, wherein stabilization of the phytocomplexes is provided by exposing the liquid fraction to ultrasonic waves.

15. Method according to any of the claims 11 to 14, wherein the enzymes are pectinase and/or polygalacturonase.

16. Method according to any of the claims 11 to 15, wherein the organically grown plant is a black currant plant (Ribes nigrum) or black carrot (Daucus carotd).

17. Method according to any of the claim 11 to 16, wherein the refining an crushing of the organically grown plant comprises one or more plant parts selected from the group consisting of leaves, roots, seeds and/or flowers.

18. Method according to any of the claim 11 to 17, wherein the liquid biomass fraction obtained after separation in step c is subsequently inoculated with yeast for fermentation of said liquid biomass fraction.

19. Method according to claim 18, wherein fermentation occurs until the sugars that are initially present in the liquid biomass fraction are reduced by at least 70%, more preferably at least 80%, most preferably at least 90%.

20. Use of a composition according to any of claims 1 to 7 for improving the drought tolerance of plants.

21. Use of a composition according to any of claims 1 to 7 for promoting plant growth or root growth.

22. Plant nutrient solution comprising between 5 to 50 wt%, preferably 10 to 25 wt%, more preferably 15 to 20 wt% of a composition according to any one of claim 1 to 7.