EP4322980A1

EP4322980A1 - Extracts of belgian endive forced roots

Info

Publication number: EP4322980A1
Application number: EP22722707.1A
Authority: EP
Inventors: Danny GEELEN; Bruno Cammue; Geert HAESAERT; Stefaan Werbrouck; Aldana RAMIREZ; Bart VAN DROOGENBROECK
Original assignee: Het Instituut Voor Landbouw En Visserijonderzoek Ilvo; Universiteit Gent
Current assignee: Het Instituut Voor Landbouw En Visserijonderzoek Ilvo; Universiteit Gent
Priority date: 2021-04-12
Filing date: 2022-04-12
Publication date: 2024-02-21
Also published as: WO2022218984A1

Abstract

The present invention relates to the field of Cichorium extracts, more specifically, the present invention pertains to a method for the preparation of a Belgian endive forced root extract, the extract obtained thereof and the use of said extract as a biopesticide and/or biostimulant in agriculture.

Description

EXTRACTS OF BELGIAN ENDIVE FORCED ROOTS

FIELD OF THE INVENTION

The present invention relates to the field of plant extracts, more specifically, the present invention pertains to a method for the preparation of a Belgian endive forced root (Cichorium intybus var. foliosum) extracts, the extracts obtained thereof and the use of said extracts as a biopesticide and/or biostimulant in agriculture.

BACKGROUND TO THE INVENTION

Cichorium intybus L. is believed to be one of the oldest cultivated vegetables in human history. Particularly its roots have historically been used for 3 purposes, (i) as a food product and for the production of food ingredients, (ii) as a medicinal plant, and iii) for miscellaneous use, such as cosmetic applications and spiritual intentions. In Europe, more specifically in Belgium, the Netherlands, France, and Italy, crops from the Cichorium genus (Asteraceae) are very popular and economically important. The most valuable ones in the European market are Belgian endive chicons (Cichorium intybus var. foliosum), Radicchio rosso (Cichorium intybus var. foliosum) and endive (Cichorium endivia). An industrial application of chicory (Cichorium intybus var. sativum) is the extraction of the polyfructosaccharide inulin (Barcaccia, G. et al. 2016).

During cultivation and processing of Cichorium crops, waste and by-products are generated. Belgian endive chicons, for example, are produced by forcing roots to form shoots by heating (16-20 °C) for 21 days, in the dark. After the edible shoots (chicons) are harvested, the remaining roots are composted or cleaned to convert into animal feed. Every year, Europe produces 300,000^100,000 tons of forced roots, which, in spite of their attractive chemical composition (sugars, dietary fibers (DF), phenolic compounds (PC) and sesquiterpenes lactones (SLs) (Kips, L 2017) are currently underutilized, hence, creating a need to explore for new strategies to exploit and valorize this by-product, other than that of animal feed and compost.

In order to be in line with the current trends to promote circular economy and its goal of zero waste, which aims at putting waste, or less profitable by-products, back into the value chain, this Belgian endive forced roots by-product, imperatively needs alternative routes to recycle, reuse and upgrade. The use of crops as vegetables, food replacers or food ingredient mainly depends on their yield, largely threatened by the numerous biotic and abiotic stresses that affect its production. In order to avoid an increase and excessive use of chemical fertilizers and pesticides in agriculture (and its negative consequences on the environment and human well-being), new eco-friendly products need to be developed. ln a world increasingly concerned with the environmental impact of the chemicals included in plant protection products and fertilizers, there is a growing need for natural alternatives, that could possibly one day replace synthetic agrochemicals. Therefore, in light of environmental concerns, biostimulants and biopesticides should be developed.

Several applications have been described for extracts from Cichorium intybus var. sativum. W02020049173 discloses the use of root extract from Cichorium intybus var. sativum against phytopathogenic fungi Stemphylium and Cercospora by applying extract on the plants.

LuanZi (2010) studies the herbicidal activity of three root solvent extracts of chicory (Cichorium intybus L.) on Trifolium repens L., Lolium perenne L, C. intybus , and Abutilon theophrasti Medic using a seed germination method. The results show that the ethanol extract had the highest herbicidal activity on three species.

Nishimura H. (2006) provides antimicrobial sesquiterpenoids, 8a-angeloyloxycichoralexin and guaianolides isolated and identified from the root extracts of chicory (Cichorium intybus var. sativum), recovered with hexane or ether. These sesquiterpenoids exhibited direct antifungal activities against Pyricularia oryzae, Pellicularia sasaki and Alternaria kikuchiana. Ether soluble phenolics from the chicory root were found to exhibit direct nematocidal activity. The dry root powder is suggested as a natural food preservative.

However, studies on applications of root extracts of Belgian endives, i.e. Cichorium intybus var. foliosum are very limited. Twarogowska A, et al., 2020, studied the chemical composition and functional properties of dietary fiber root powders of Belgian endive (Cichorium intybus var. foliosum) and suggests it as a possible functional food ingredient. While this publication provides aqueous extracts, these have been prepared from soaked julienne roots. In contrast, it was found in the present invention that a drying step prior to the extraction step allows for several advantages, such as increasing preservation, preventing spoiling and facilitating storage and further processing (e.g. grinding) of the dried roots.

Kips (2017; pages 109-129) suggests a putative application of extracts of Belgian endive forced roots towards food, pharma or biocidal medicinal applications.

It is an objective of the present invention to provide extracts of Belgian endive forced roots and uses thereof as a biopesticide and/or (plant) biostimulant, as well as methods for providing said extracts and compositions comprising it. SUMMARY OF THE INVENTION

The present invention provides extracts of Belgian endive forced roots, a by-product from the Belgian endive chicons’ (Cichorium intybus var. foliosum) production, currently in particular used for compost or as feed for cattle. The inventors have found that aqueous extracts and organic solvent extracts prepared from Belgian endive forced roots are effective biopesticides. Further, the extracts provided have the beneficial effect of being biostimulants.

Even further, the present invention provides a method of preparation of said extracts and uses thereof. It has been found that extracts according to the present invention provide for increased resistance to certain pathogens. It has furthermore been demonstrated that the extracts of the invention improve the growth rate of plants and/or the root growth, in particular primary, lateral, adventitious and/or junction root growth and shoot growth.

In particular, in a first aspect the present invention provides a method for the preparation of a Belgian endive forced root extract, comprising an aqueous extraction step comprising the steps of: a) providing endive chicon roots; b) mixing the roots of step a) with an aqueous solution, while heating said mixture; c) separating the heated mixture of step b) into a solid phase and a liquid phase; and d) obtaining the liquid phase of step c) as a Belgian endive forced root aqueous extract.

In a particular aspect, the present invention provides a method for the preparation of a Belgian endive forced root extract, comprising the steps of: a) providing Belgian endive forced roots; b) slicing the roots of step a); c) drying the sliced roots of step b); d) mixing the dried roots of step c) with an aqueous solution, while heating said mixture; e) separating the heated mixture of step d) into a solid phase and a liquid phase; and f) obtaining the liquid phase of step e) as a Belgian endive forced root aqueous extract

In a further embodiment of the present invention, the method further comprises milling the dried roots before mixing them with an aqueous solution.

In accordance with an embodiment of the present invention, the solid phase is further processed by: a) mixing said solid phase with an organic extractant; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root organic solvent extract; wherein optionally steps a) to c) are repeated one or more times using a different extractant. ln accordance with a further embodiment of the present invention, said organic extractant is selected from the list comprising ethanol, ethyl acetate and hexane.

In accordance with yet another embodiment of the present invention, the steps a) to c) of the further processing on the solid phase are sequentially repeated using the following organic solvent extractants in the specified order: ethanol, ethyl acetate and hexane.

In accordance with a further embodiment of the present invention, the solid phase is further processed by: a) mixing said solid phase with ethanol; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root ethanol extract.

In accordance with a further embodiment of the present invention, the solid phase from the ethanol extraction is further processed by: a) mixing said solid phase with ethyl acetate; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root ethyl acetate extract.

In accordance with a further embodiment of the present invention, the solid phase from the ethyl acetate extraction is further processed by: a) mixing said solid phase with hexane; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root hexane extract.

In accordance with a further embodiment of the present invention, said Belgian endive forced root organic solvent extract is further subjected to an evaporation step.

In accordance with a further embodiment of the present invention, said heating performed onto the mixture obtained by mixing Belgian endive forced roots and an aqueous solution is performed at a temperature in a range from about 30 °C to about 120°C, preferably from 50°C to 100°C, preferably from 60°C to 90°C, more preferably at a temperature of about 80°C and/or for a sustained period of time, in a range from about 5 min to about 180 min, preferably from 10 min to 150 min, preferably from 50 min to 130 min, more preferably about 120 min.

In accordance with a further embodiment of the present invention, said Belgian endive forced roots are provided in dried and/or powder form. ln a second aspect, the present invention relates to Belgian endive forced root extracts obtainable by applying the method described by anyone of the embodiments of the present invention.

In a third aspect, the present invention relates to the use of Belgian endive forced root extracts obtainable by applying the method described by anyone of the embodiments of the present invention as a biopesticide and/or biostimulant, in particular in agriculture, horticulture, arboriculture and/or home gardening.

In a fourth aspect, the present invention relates to the use of a Belgian endive forced root extract as a biopesticide and/or biostimulant in agriculture, horticulture, arboriculture, public green, turf grass and/or home gardening.

In accordance with a further embodiment, the present invention relates to the use of a Belgian endive forced Belgian root extract wherein said extract is selected from the list comprising: a Belgian endive forced root aqueous extract or a fraction thereof, a Belgian endive forced root ethanol extract, a Belgian endive forced root ethyl acetate extract and a Belgian endive forced root hexane extract; in particular a Belgian endive forced root aqueous extract or a Belgian endive forced root ethanol extract.

BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Figure 1, also abbreviated as Fig. 1 : is a schematic representation of the sequential extraction procedure used in the preparation of the four Belgian endive forced root extracts: Liquid water (HO) extract, dried ethanol (EH) extract, dried ethyl acetate (EA) extract, and dried hexane (HE) extract.

Figure 2, also abbreviated as Fig. 2: Primary root length (cm) of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 4 Belgian Endive Forced Root extracts (HO, EH, EA, HE). Data represent the average of three biological and ten technical replicates (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 ;^****P <0.0001 ). HO: water extract, EH: ethanol extract, EA: ethyl acetate extract, HE: hexane extract.

Figure 3, also abbreviated as Fig. 3: Lateral roots numbers of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 4 Belgian Endive Forced Root extracts (HO, EH, EA, HE). Data represent the average of three biological and ten technical replicates per bar (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^**P<0,01 ;^****P <0.0001). HO: water extract, EH: ethanol extract, EA: ethyl acetate extract, HE: hexane extract.

Figure 4, also abbreviated as Fig. 4: Junction roots numbers of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 4 Belgian Endive Forced Root extracts (HO, EH, EA, HE). Data represent the average of three biological and ten technical replicates per bar (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 ; ^****P <0.0001 ). HO: water extract, EH: ethanol extract, EA: ethyl acetate extract, HE: hexane extract.

Figure 5A, also abbreviated as Fig. 5A: Adventitious roots numbers of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 4 Belgian Endive Forced Roots extracts (HO, EH, EA, HE). Data represent the average of three biological and ten technical replicates per bar (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^****P <0.0001 ). HO: water extract, EH: ethanol extract, EA: ethyl acetate extract, HE: hexane extract.

Figure 5B, also abbreviated as Fig. 5B: Shoot area of Arabidopsis seedlings treated with control (Water) or with 3 different doses of the Belgian Endive Forced Roots water (HO) extract. Data represent the average of three biological and ten technical replicates per bar (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^****p <0.0001 ). HO: water extract.

Figure 6, also abbreviated as Fig. 6: Primary root length (cm) of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 8 fractions obtained from the Belgian Endive Forced Root water (HO) extract. Data represent the average of three biological and ten technical replicates (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 ; ^****P <0.0001 ). F1 -8: Fractions 1 to 8.

Figure 7, also abbreviated as Fig. 7: Lateral roots numbers of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 8 fractions obtained from the Belgian endive forced root water (HO) extract. Data represent the average of three biological and ten technical replicates (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^**P<0,01 ; ^***P<0,001 ; ^****P <0.0001). F1-8: Fractions 1 to 8.

Figure 8, also abbreviated as Fig. 8: Adventitious roots primordia numbers of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 8 fractions obtained from the Belgian Endive Forced Root water (HO) extract. Data represent the average of three biological and ten technical replicates (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 ; *^**P<0,001 ; ^****P <0.0001 ). F1 -8: Fractions 1 to 8.

Figure 9A, also abbreviated as Fig. 9A: Adventitious roots numbers of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 8 fractions obtained from the Belgian Endive Forced Root water (HO) extract. Data represent the average of three biological and ten technical replicates (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 ). F1-8: Fractions 1 to 8.

Figure 9B, also abbreviated as Fig. 9B: Shoot area of Arabidopsis seedlings treated with control (Water or DMF 0,01%) or with 3 different doses of the 8 fractions obtained from the Belgian Endive Forced Root water (HO) extract. Data represent the average of three biological and ten technical replicates (30 seedlings in total, 10 per replicate). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 , ^***P<0,001 , ^****P <0.0001 ). F1 - 8: Fractions 1 to 8.

Figure 10, Figure 10, also abbreviated as Fig. 10: Root numbers (A), and root length (B) on Plectranthus escualentus shoot explants treated with water (Control), with 2 different doses (0.01 and 0.001 dilutions) of Belgian Endive Forced Root water (HO) extract, and with 0.00001 dilution of solid fractions F1 -F4 or 0.01 dilution of the liquid fractions F5-F8 obtained from the water (HO) extract. Data represent the average of two biological and 10 technical replicates (20 explants in total). Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^**P<0,01 ; ^****p <0.0001).

Figure 11, also abbreviated as Fig. 11 : Growth rate during week 3 (W3) of maize plants, treated with water (control) or with the Belgian Endive Forced Root water (HO) extract at a concentration of 10%. Data represent the average of three (control) or 4 (HO) biological replicates. Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05)

Figure 12, also abbreviated as Fig. 12: Root application of Belgian Endive Forced Root extracts (HO, EH, EA, HE) and Trichoderma hamatum T382 control (Ref 2; Krause et al. 2003) in the pathosystem Arabidopsis-Botrytis cinerea. Disease severity was evaluated by measuring the lesion diameter in 12 plants.

Figure 13, also abbreviated as Fig. 13: Leaf application of Belgian Endive Forced Root extracts (HO, EH, EA, HE) and Trichoderma hamatum T382 control (Ref 2; Krause et al. 2003) in the pathosystem Arabidopsis-Hyaloperonospora arabidopsidis. Disease severity was evaluated by quantifying the amount of newly produced pathogen spores on batches of 15 plants. Bars represent average spore formation of 7 batches.

Figure 14, also abbreviated as Fig. 14: Biocontrol activity of Belgian Endive Forced Root extracts (HO, EH, EA, HE), a control (no extract added) and reference fungicidal compound (Fun) on three different plant-pathogen systems: VMheaV Blumeria graminis ; Tomato/ Botrytis cinerea; and potato /Phytophthora infestans. On Wheat /Blumeria graminis Fun=Palazzo (BASF) plus Bravo (Syngenta); On Tomato/ Botrytis cinerea Fun=Mancozeb (Belchim Crop Protection); and on potato I Phytophthora infestans Fun=Ranman-top (Belchim Crop Protection). Data represent the average of three biological replicates. Error bars represent standard error. Asterisks indicate significant differences between control and treatment according to a Student’s t-test (^*P<0,05; ^**P<0,01 ; ^***P<0,001 ; ^****P <0.0001 ; ns P>0,05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10 % or less, preferably +/- 5 % or less, more preferably +/- 1 % or less, and still more preferably +/- 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "an extract" means one extract or more than one extract. ln particular, in a first aspect the present invention provides a method for the preparation of a Belgian endive forced root extract.

The present invention provides extracts of Belgian endive forced roots, a by-product from the Belgian endive chicons’ production chain, and used for compost or commercialized as feed for cattle. The inventors have found that aqueous extracts, including fractions thereof, and organic solvent extracts prepared from said roots are effective biopesticides. Further, the extracts provided have the beneficial effect of being biostimulants. Even further, the present invention provides a method of preparation of said extracts and uses thereof. It has been found that extracts according to the present invention provide for increased resistance to certain plant pathogens, in particular fungi and oomycetes. It has furthermore been demonstrated that the extracts of the invention improve plant growth and/or root growth, in particular primary, lateral adventitious and/or junction root growth and shoot growth.

Generally, the species Cichorium intybus can be split into three main cultivar groups: (i) the root chicory known as Cichorium intybus var. sativum, also called industrial chicory as these roots are used for industrial inulin extraction and coffee substitutes, (ii) the witloof chicory known as Cichorium intybus var. foliosum, and (iii) the leafy chicory, sub-classified into Sugarloaf (var. porphyreum), Radicchio (var. latifolium) and Catalogne (var. syivestre) (Barcaccia et al., 2016). Witloof chicory is also known as Belgian endive.

The white and bitter tasting Belgian endive chicon (Cichorium intybus var. foliosum) is an important Belgian vegetable that belongs to the Asteraceae family. To produce chicons, Belgian endive roots are forced in the absence of light. Non-forced roots are harvested on the field and stored cold (-2 °C) for up to several months, depending on cultivar. Subsequently, they are forced to produce edible chicons, which are compact heads of white to pale yellow leaves sitting on suppressed floral stems. The forcing process can take place in two different ways: (i) hydroculture or (ii) soil-based production (about 5 %), and is mainly by heating and in the absence of light.

In accordance with the present invention, by means of the term “Belgian endive forced root” is meant the root derived from the forced product of witloof chicory.

As used herein, the term “Belgian endive forced root extract”, refers to an extract obtained from Belgian endive forced roots, more in particular from Cichorium intybus var. foliosum. Such extract is obtained by means of an extraction process which is a separation process comprising the separation of substances (e.g. active agents) from a matrix. In one embodiment, Belgian endive forced roots are obtained from a chicon harvest. As soon as the chicons are full-grown they are harvested by cutting or breaking them off of the roots.

Fig 1. exemplifies an embodiment of the present invention, wherein at the left of the figure, an aqueous extraction process is illustrated, whilst at the right, extraction from Belgian endive forced roots with an organic extractant is illustrated. More specifically, Fig. 1 is a schematic representation of the sequential extraction procedure according to an embodiment of the present invention, used in the preparation of Belgian endive forced root extracts, more specifically a (sequential) aqueous extract (HO) at the left of the figure, and a hexane extract (HE), an ethyl acetate extract (EA), and an ethanol extract (EH), at the right of the figure.

The method of the present invention for the preparation of a Belgian endive forced root extract includes an aqueous extraction step comprising the steps of: a) providing Belgian endive forced roots; b) mixing the roots of step a) with an aqueous solution, while heating said mixture; c) separating the heated mixture of step b) into a solid phase and a liquid phase; and d) obtaining the liquid phase of step c) as an aqueous extract (HO).

At step a), the Belgian endive forced roots can be provided in various forms, such as after grinding, milling or crunching, and/or in the form of pellets, granules, powder or larger conglomerates, powder form is preferred. In the context of the present invention, the term “powder” is meant to be fine, dry particles produced by the grinding, crushing or disintegration of a solid substance. In a particular embodiment, the roots are dried. Particularly interesting results are obtained when the roots are dried prior to the mixing with an aqueous solution. Specifically, it was found that a drying step prior to the extraction step allows for increased preservation, prevents spoiling and facilitates storage and further processing (e.g. grinding) of the dried roots. Accordingly, in a particular embodiment, the present invention provides a method for the preparation of a Belgian endive forced root extract, comprising the steps of: a) providing Belgian endive forced roots; b) slicing the roots of step a) c) drying the sliced roots of step b) d) mixing the dried roots of step c) with an aqueous solution, while heating said mixture; e) separating the heated mixture of step d) into a solid phase and a liquid phase; and f) obtaining the liquid phase of step e) as a Belgian endive forced root aqueous extract. In one embodiment, Belgian endive forced roots are washed, cut into slices or pieces, dried and milled to obtain a powder. The drying temperature is between 30°C and 90°C, particular between 40°C and 80°C. The drying period is for at least 2-3 hours, in particular for at least 4 hours, more in particular for at least 5 hours and up to about 8 to 10 hours.

More specific, the drying temperature and duration is chosen so as to obtain dried roots (in any form) having a moisture content below 40%, in particular below 30%, more in particular below 20%, even more in particular below 10%.

The Belgian endive forced roots, in particular the sliced and/or dried once are subsequently mixed with an aqueous solution, which can be water, such as distilled water. The aqueous solution can be a saline solution, or a solution with other salts dissolved therein. The aqueous solution is preferably water. The ratio solid :aqueous solution of the obtained mixture can be variable. For example, in accordance with an embodiment of the present invention, the ratio of roots (kg):aqueous solution (L) is in the range of from 0.1 :5 to 5:5, in particular from 0.5:5 to 3:5, more in particular about 0.5:5. Heating has the advantage that the extraction is accelerated. The aqueous solution penetrates faster into the plant tissue, allowing the solubility of most compounds to be higher, further, it has the advantage of killing microbes which helps to protect the obtained extract from degradation.

In accordance with an embodiment of the present invention, the mixing of the roots with the aqueous solution is performed while heating the mixture comprising the aqueous solution and the roots.

In accordance with a further embodiment of the present invention, the step of heating the mixture obtained by mixing the roots and an aqueous solution is performed at a temperature in a range from about 30 °C to about 120°C, preferably from 50°C to 100°C, preferably from 60°C to 90°C, more preferably at a temperature of about 80°C and/or for a sustained period of time, in a range from about 5 min to about 180 min, preferably from 10 min to 150 min, preferably from 50 min to 130 min, more preferably about 120 min.

In accordance with a further specific embodiment of the present invention, the step of heating the mixture obtained by mixing the roots and an aqueous solution is performed at a temperature of about 80°C and for a period of time of about 120 min.

A person skilled in the art is able to determine the ideal time and temperature in order to obtain the most suitable extract for the intended applications. Moreover, similar bioactivity can be obtained by extracting a bit longer at lower temperature, or a bit shorter at higher temperature. In accordance with a preferred embodiment of the present invention, said heating is performed for about 2h at about 80°C.

As also illustrated in Fig. 1 , in order to provide for a solid phase after aqueous extraction, the mixture is filtrated or sieved. Several different techniques can be used to separate the solid phase from the liquid phase after aqueous extraction. Moreover, whereas the obtained extracts of the present invention may be used as such, they may also be further processed, such as evaporated, concentrated, freeze-dried, centrifuged,... prior to use. In accordance with a further embodiment of the present invention, the obtained solid phase is further processed by: a) mixing said solid phase with an organic extractant; the organic extractant being an organic solvent or organic agent to solubilize components in the roots; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root organic solvent extract; wherein optionally steps a) to c) are repeated one or more times using a different extractant.

In accordance with a further embodiment of the present invention, said organic extractant is any suitable organic extractant or solvent such as selected from the list comprising ethanol, ethyl acetate and hexane.

Therefore, in accordance with an embodiment of the present invention, after the Belgian endive forced root aqueous extract (HO) is obtained, the solid phase remaining is then subjected to three extractions steps with organic extractants, meaning ethanol (EH), ethyl acetate (EA) and hexane (HE).

In accordance with yet another embodiment of the present invention, the steps a) to c) of processing said solid phase are sequentially repeated using the following organic extractants in the specified order: ethanol, ethyl acetate and hexane.

More specifically, in accordance with a further embodiment of the present invention, the obtained solid phase from the aqueous extraction is further processed by: a) mixing said solid phase with ethanol; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root ethanol extract.

In accordance with a further embodiment of the present invention, the obtained solid phase from the ethanol extraction, is further processed by: a) mixing said solid phase with ethyl acetate; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root ethyl acetate extract.

In accordance with a further embodiment of the present invention, the obtained solid phase from the ethyl acetate extraction is further processed by: a) mixing said solid phase with hexane; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root hexane extract. In accordance with a further embodiment of the present invention, said Belgian endive forced root organic solvent extract is further subjected to an evaporation step.

In accordance with a further embodiment of the present invention, the aqueous (HO) extract is further fractionated using a liquid-liquid (e.g. ethyl acetate-toluene) extraction, under acidic (pH <7) or alkaline (pH>7) conditions.

In particular, the aqueous extract, either or not further concentrated, may be divided in different aliquots, all of which may further be treated differently. In some of these aliquots, the pH may be varied by using appropriate acids or bases such as HCI or KOH. In particular aliquots, the pH may be lowered to less than 7, such as less than or about 6, less than or about 5, less than or about 4, less then or about 3. In other aliquots, the pH may be increased to more than 7, such as more than or about 8, more than or about 9, more than or about 10. Furthermore, these aliquots may be subjected to a liquid-liquid fractionation procedure using organic solvents such a ethyl acetate or toluene. Either of which may be applied to the low or high pH aliquots. Any suitable ratio of watensolvent may be used such as about 1 :1 , 1 :2, 1 :3, 1 :4 or 1 :5. After mixing, the aliquots are separated into an organic and aqueous phase (optionally by centrifugation). The organic and/or aqueous phase may further be subjected to an evaporation step and/or freeze-drying step before further use; or they may directly be used as a biostimulant and/or biopesticide.

In a second aspect, the present invention relates to a Belgian endive forced root extract obtainable by applying the method described by anyone of the embodiments of the present invention.

In a third aspect, the present invention relates to the use of a Belgian endive forced root extract obtainable by applying the method described by anyone of the embodiments of the present invention as a biopesticide and/or plant biostimulant, in particular in agriculture. In accordance with the present invention, by means of the term “biopesticide”, reference is made to a pest management agent derived from natural sources.

Other than having biopesticide properties, the Belgian endive forced root extracts according to the present invention have biostimulant properties.

Any plant/crop can be treated. The term "plant (or plants)" is a synonym of the term "crop" which is to be understood as a plant of economic importance and/or a men-grown plant. The methods, extracts and compositions of the present invention may be applied to any plant, such as monocots, dicots, non-vascular (liverworts, mosses), ferns, gymnosperms, etc. In one embodiment the plant belongs to the Solanaceae (nightshade) family, in particular to the genus Solanum. In a further embodiment, the treated plant is tomato (Solanum lycopersicum), potato (Solanum tuberosum) or eggplant (Solanum melongena). In another embodiment, the treated plant is a grass (family Poaceae), in particular species from the genus Triticum or Zea, more in particular wheat or maize.

The extract or a composition comprising the extract is applied to a plant, directly or indirectly. Any appropriate plant part can be treated or used including plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells and progeny of the same. In the alternative, the extract or composition can be applied to the soil surrounding the plant, however with direct contact with the roots. The applying of the extract is prior to planting, at planting, or after planting. In one embodiment, contacting includes direct application to a plant. All or part of a plant including, without limitation, leaves, stems, roots, propagules (e.g., cuttings), fruit, seeds etc., may be contacted with the extract described herein. Contacting may also be carried out indirectly, via application, e.g., to soil or other plant substrates but making uptake by the plant possible.

In one embodiment, the extract of the present invention is used in a method of inducing (systemic) resistance to biotic stress in a plant. The method comprises applying the extract to the plant, after which systemic plant immunity will be activated. The applying step could be performed according to various embodiments. For instance, the plant extract or a composition comprising it could be sprayed on the plant, watered on the plant, added to the substrate, such as hydroponics, soil, peat, compost, vermiculite, perlite, sand or clay, in which the plant is growing, etc. In a particular embodiment there is no direct contact of the extract or composition with the pathogen or target organism. Hence, the current invention provides a method of treating or preventing, or at least inhibiting or alleviating, pathogen or pest damage in a plant, in particular through the activation of the plant defence mechanism. The plant extract is able to achieve this protecting effect in the whole plant even when sprayed only on a part of the plant, or when sprayed at relatively low concentrations, mostly without being directly toxic to said plant pathogen. Of particular advantage is that the present plant extract can be used pre-emptively (e.g. to seedlings or non-infected plants or plants having no visible signs of infection) and require only a simple formulation. The use as a priming agent will delay, or even prevent the damage to the plant when infected. The present invention relates to extracts, methods and compositions which can be used to stimulate or induce plant defence and/or immune responses against plant pathogens such as oomycetes and/or fungi. In one embodiment, the invention provides a method for controlling plant pathogens, said method comprising applying on or to said plant the extract provided herein. Examples of phytopathogenic oomycetes (formerly classified as fungi) are species of the genera Pythium, Phytophthora, Peronospora, and Hyaloperonospora. In one embodiment, the invention provides a method to reduce and/or prevent infection of a plant with phytopathogens from the genera Phytophthora and Hyaloperonospora. Examples of phytopathogenic fungi (including biotrophic, hemi-biotrophic, necrotrophic fungi) include the genera Magnaporthe, Botrytis, Puccinia, Fusarium, Blumeria, Mycosphaerella, Colletotrichum, Ustilago, Phakopsora, Alternaria, Sclerotinia, Cladosporium and Rhizoctonia. In one embodiment, the invention provides a method to reduce and/or prevent infection of a plant with the phytopathogen Botrytis cinerea /pathogen of tomato/, and/or Blumeria graminis (pathogen of wheat).

In one embodiment, the invention provides a method to reduce and/or prevent infection of a plant with oomycetes, said method comprising applying the water extract or the EH extract of the present invention to or on the plant by any one of the methods as described herein. In one embodiment, the oomycetes are from the genera Phytophthora and Hyaloperonospora. In one embodiment, the oomycete is Phytophthora infestans. In a further embodiment, the applying of the HO extract is on the leaves of the plant.

Furthermore, the invention provides a method to reduce and/or prevent infection of a plant with a fungus from the genus Blumeria or Botrytis, said method comprising applying the organic solvent extract of the present invention to or on the plant by any one of the methods as described herein. In one embodiment, the fungus is Blumeria graminis or Botrytis cinerea. In particular, the organic solvent extract is an EH, EA or HE extract a provided herein, more specific an EH or EA extract. In a further embodiment, the applying of the organic solvent extract is on the leaves of the plant.

In the context of the present invention, the term biostimulant is meant to be any substance, composition or product whose function is to stimulate plant nutrition processes independently of its nutrient content with the sole aim of improving one or more of the following characteristics of the plant or the plant rhizosphere: (a) nutrient use efficiency, (b) tolerance to abiotic stress, (c) quality traits, or (d) availability of confined nutrients in the soil or rhizosphere. Hence, a biostimulant is a product whose function is to stimulate plant nutrition processes independently of the nutrient content of said product. This in contrast to a fertilizer which is a product of natural or synthetic origin to be applied to soil or to plant tissues to supply one or more plant nutrients essential to the growth of plants. Different tests may be used to determine the biostimulant effect on plants, such as height, growth rate, chlorophyll content, root growth, root length, root branch numbers, adventitious rooting and shoot area, and/or if applicable silique length, seed amount per silique, seed weight, surface area or percentage of normal size pollen, pollen viability.

In one embodiment, the extract of the invention is used for modulating plant development and in particular for promoting the growth rate of the plant and/or growth of primary, lateral, adventitious and/or junction roots, this when compared to untreated plants. Hence, the extract can be used as a biostimulant, more specific in a method to control plant development such as e.g. increasing the tolerance of plants to stress (e.g. drought stress, heat stress, cold stress, salt stress), or to control physiological phenomena such as pre-harvest sprouting and premature senescence. In certain embodiments, the plant with altered root morphology exhibits improved tolerance to stress conditions selected from the group consisting of drought, flooding, high salt growth conditions, extreme cold, and (extreme) heat, compared to the average tolerance of a statistically significant control population that has not been treated with the extract. The term ‘adventitious root growth’ refers to the expansion of the root biomass mediated by cell division and cell expansion in the adventitious root meristems.

It was observed that primary root growth (PR) was promoted by the HO-extract, including fractions thereof, and by the EA and the HE-extracts. In addition, the number of adventitious roots (AR) and junction roots (JR) were strongly increased by the EH-extract and to a lesser extent by the HO, EA and H E extracts. Moreover, the HO extract and fractions thereof (especially F5-F8) induced an increase in the shoot area. Bioassays showed that the growth rate of maize, as well as the root length, and the root and shoot numbers of Plectranthus escualentus were stimulated by application of the HO-extract. As such, a biostimulant effect is present across all extracts.

In a fourth aspect, the present invention relates to the use of an endive forced root extract as a biopesticide and/or plant biostimulant, in particular in agriculture, horticulture, arboriculture, public green, turf grass and/or home gardening.

In accordance with a further embodiment, the present invention relates to the use of a Belgian endive forced root extract wherein said extract is selected from the list comprising: an endive forced root aqueous extract, an endive forced root ethanol extract, an endive forced root ethyl acetate extract and an endive forced root hexane extract; preferably an endive forced root aqueous extract or an endive forced root ethanol extract. Based on the results of biopesticide and/or biostimulant activity, the most interesting extracts in terms of activity are in order of relevance: endive forced root aqueous extract, endive forced root ethanol extract, endive forced root ethyl acetate extract and endive forced root hexane extract.

The present invention also encompasses (the use of) a composition or formulation comprising the extract of the invention. An “agrochemical composition” as used herein means a composition for agrochemical use, such as use in the agrochemical industry, including agriculture, horticulture, floriculture, arboriculture and home and garden uses for stimulating plant/root growth and/or for protecting plants or parts of plants, crops, bulbs, tubers, fruits (e.g. from harmful organisms, diseases or pests) as herein defined, comprising at least the extract as defined herein, and at least one agriculturally and/or horticulturally acceptable excipient. Typically, the extract of the invention may be administered to a plant in a suitable agriculturally acceptable formulation, including but not limited to, a growing medium such as soil or hydroponic liquid medium, dusts, granules, solution concentrates, emulsifiable concentrates and wettable powders. The term “agriculturally acceptable” indicates that the formulation is non-toxic for the plant and otherwise acceptable for application to a plant, whether applied indoors (e.g. in a contained environment) or outdoors (e.g. in a non-contained environment that is exposed to other plant, animal and human life).

In a further embodiment of the present invention, the extract or a composition comprising the extract is applied to a plant or tree, directly or indirectly. Any appropriate plant part can be treated or used including plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells and progeny of the same. In the alternative, the extract or composition can be applied to the soil surrounding the plant, however with direct contact with the roots. The applying of the extract is prior to planting, at planting, or after planting. In one embodiment, contacting includes direct application to a plant. All or part of a plant including, without limitation, leaves, stems, roots, propagules (e.g., cuttings), fruit, seeds etc., may be contacted with the extract described herein. Contacting may also be carried out indirectly, via application, e.g., to soil or other plant substrates but making uptake by the plant possible. Suitable application methods include high or low-pressure spraying, immersion, atomizing, foaming, fogging, coating, and encrusting. Other suitable application procedures can be envisioned by those skilled in the art. In a particular embodiment, the extract of the invention is applied to the parts of the plant above ground or to the foliage of the plant by spraying e.g. by the use of mechanical sprayers. Sprayers convert a formulation of the invention which is mixed with a liquid carrier, such as water or fertilizer, into droplets. The droplets can be any size. Boom sprayers and air blast sprayers can also be used to apply formulations of the invention to pre-emerging or post-emerging crops. Air blast sprayers inject formulations of the invention mixed with a liquid carrier into a fast-moving air stream. Boom sprayers, aerial sprayers, ultra-low volume sprayers, drip irrigation, sprinkler irrigation, and foggers can also be used to apply formulations of the invention. Where the formulations of the invention are in a solid, powder or granule form, they can be applied with granule or dust application equipment. Formulations of the invention can also be applied as a fumigant to soil, plant media, plants, or plant tissues. In another embodiment, seeds of a plant are coated with the extract of the invention (“coated seeds”). Any appropriate seed coating method known the skilled person can be used.

In a specific embodiment, the extract of the present invention can be applied to a plant as provided herein alone, in combination or in a mixture with other compounds. Suitable other compounds include effective amounts of other agricultural or horticultural biologicals and/or chemicals, such as herbicides, insecticides, nematicides, molluscicides, bactericides, acaricides, fungicides, and/or plant growth regulators or fertilizers. The following examples are set forth below to illustrate the methods, compositions, and results according to the disclosed subject matter. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art. EXAMPLES

1. PREPARATION OF THE EXTRACTS

Raw material pre-processing treatment

Belgian endive forced roots was collected from the supplier and further processed at the ILVO’s Food Pilot facilities. First of all, forced roots were washed in cold water to remove the remaining soil. The outer ends of the roots are removed at the top and bottom. Further, the roots were julienned into 5 cm long and 2.5 2.5 mm width slices, using a Robot Coupe (CL50 Ultra, Mont- Sainte-Genevieve, France). The cut roots were placed in a hot air oven (60 °C, 6-8 h) to be dried to a moisture content below 10%. Dried samples were milled by using a ring sieve size 0.5 mm (Ultra centrifugal mill ZM 200, RETSCFI, Flaan, Germany) to obtain a powder which is, in turn, used for the production of water (FIO), ethanol (EH), ethyl acetate (EA), and hexane (FIE) extracts.

Extraction procedure

Three and a half kilograms of dried forced roots powder were mixed with 31 , 8L of water. The resulting solid/water mixture (of approximately 35L) was incubated 2h at 80°C in a so called “Stephan’s apparatus” (Food Pilot, ILVO, Belgium). Solid and liquid phases were separated by passing the mixture through a vibrating sieve. The liquid phase obtained in this way constitutes the “liquid water (HO) extract”, which was aliquoted and frozen until further use. The resulting solid phase of the water extraction, was then subjected to three sequential organic solvents extractions: ethanol (EH), ethyl acetate (EA) and hexane (HE) (Figure 1 ).

First, the solid left-over material from the first extraction was mixed and incubated with ethanol. After incubation, the solid/ethanol mixture was partitioned using a Buchner funnel. This procedure yielded the “liquid ethanol (EH) extract” and a new solid left-over phase. The ethanol contained in the “liquid ethanol (EH) extract” was evaporated to remove the ethanol, and stored as “dried ethanol (EH) extract”. The solid phase generated from the ethanol extraction went through an ethyl acetate (EA) extraction, and a subsequent hexane (HE) extraction, as described before. At the end of the whole extraction procedure, a liquid water extract (HO), and three solid solvent extracts (EH, EA, and HE) were obtained. Fractionation procedure

Previous to the fractionation, the 11 L of water (HO) extract previously obtained, were concentrated to a final volume of 2L, using a SpeedVac vacuum concentrator. The concentrated water extract was divided in four 500mL aliquots and the pH was adjusted to either pH3 (aliquots 1 and 3) or pH10 (aliquots 2 and 4) by using HCI or KOH, respectively. To proceed with the liquid-liquid fractionation, the volume of each aqueous aliquot was brought up to 1 L by the addition of water and later mixed with 2,2L of either, ethyl acetate (aliquots 1 and 2) or toluene (aliquots 3 and 4). After mixing, and to speed up the separation process, organic and aqueous phases were partitioned by centrifugation. The whole procedure yielded 8 fractions, four organic fractions (F1 : ethyl acetate-pH3; F2: ethyl acetate-pH10; F3: toluene-pH3; F4: toluene-pH10) and for aqueous fractions (F5: aqueous residue of F1 ; F6: aqueous residue of F2; F7: aqueous residue of F3; F8: aqueous residue of F4). The organic solvent (ethyl acetate (EtAc) and toluene (To)) contained in F1 , F2, F3 and F4 was later removed by evaporation, resulting in 4 organic/dried fractions. Aqueous fractions F5, F6, F6, and F8 remained intact (Table 1).

Table 1: Technical details of the fractionation procedure of the Belgian Endive forced roots water (HO) extract.

2. BIOSTIMULANT BIOASSAYS

2.1. In-vitro Arabidopsis ROOT and SHOOT assays

Materials and methods The 4 extracts and the 8 fractions prepared as described above were incorporated in MS (Murashige and Skoog) basal medium at the concentrations indicated in Table 2. The liquid water (HO) extract and aqueous fractions F5 to F8, were diluted to 2%, 1% and 0.5% v/v concentrations. The solid extracts (EH, EA and HE) and organic fractions F1 to F4 were first dissolved in 0.01 % DMF and then diluted to 10 times (1 :10), 100 times (1 :100) and 1000 times (1 :1000) (Table 2). Water and DMF 0.01% were used as controls.

Arabidopsis thaliana Col-0 seeds were sterilized, sown on MS petri dishes, vernalized in the dark at 5 °C for 4 days, and etiolated following an in-house developed protocol (Trinh HK’s protocol, Verstraeten, & Geelen, 20181 ). Etiolated seedlings were transferred to freshly prepared, treatment (MS medium with extracts) and control (MS medium without extracts/fractions), petri dishes. Root and shoot morphology traits (adventitious root numbers, adventitious root primordia numbers, junction root numbers, lateral root numbers, primary root length, and shoot area) were examined and recorded by digital photography after 10 days of incubation under the same light and temperature conditions. Table 2. Extracts and fractions concentrations used in the root/shoot bioassays.

Original Concentration/Dilution

Extract/Fraction Code Type amounts

High Middle Low

(ml or mg)

Water extract HO 110000

- 2% 1% 0.5%

F5 2625

- liquid

F6 2800

Aqueous Fractions __ -

2560

F8 2785

Ethanol extract EH 65000

Ethyl acetate extract EA 817

_ 1 :10 1 :100 1 :1000

Hexane extract HE 53

F1 Solid 1026

F2 506

Organic Fractions — — 397

F4 100

Results

A highly and significant increase in the shoot area (Figure 5B), in the length of the primary root (Figure 2, HO) and in the number of lateral roots (Figure 3, HO) was observed when plants were treated with the Belgian endive forced roots water (HO) extract, specially at the lowest concentration tested (1% and 0,5%). A smaller increase in primary root length, but not in the number of lateral roots, was observed when plants were treated with the ethyl acetate (Figure 2, EA) and the hexane (Figure 2, HE) extracts.

The number of junction roots (Figure 4) was promoted by all 4 extracts, being the ethanol (EH) extract the most highly performant of the four, specially at higher concentrations (1 :10). Figure 5 shows that the ethanol (EH) extract, at the higher concentration tested (1 :10) was also and particularly highly proficient at stimulating the development of adventitious roots. In a less remarkable way, development of adventitious roots was induced by the hexane (Figure 5A, HE) extract.

Figure 6 shows the effect that different fractions obtained from the water (HO) extract had on the development of Arabidopsis primary root. Except for fractions 1 (F1 ), all other fractions have a primary root promoting effect, at low and/or medium concentrations depending on the fraction. The most pronounced promoting effect is giving by organic fraction F4 (Toluene, pH10), followed by organic fraction F2 (EtAc, pH10). Lateral root numbers (Figure 7), adventitious root primordia numbers (Figure 8) and shoot area (Figure 9B) are highly and significantly promoted by all aqueous fractions (F5, F6, F7, F8) at all the concentrations tested.

Finally, most fractions tend to induce the development of adventitious roots at high concentration, and this effect is significant for fractions F1 , F3, F4, F7 (Figure 9).

2.2. In-vitro ROOT and SHOOT Plectranthus escualentus regeneration bioassays

Materials and methods

Glass jars of 350 ml glass jars were filled with 100 ml Murashige & Skoog medium with half concentration of NH4N03 and KN03, including microelements and vitamins. This basal medium was supplemented with 30 g/l sucrose, 7 g/l agar-agar, and either the Belgian Endive Forced Root water (HO) extract, or its derived fractions (F1 - F8). The liquid HO extract was tested at two doses (0.01 and 0.001 dilution), the organic fractions (F1 - F4), which were solid, were tested at a final dilution of 0.00001 , and the liquid fractions (F5 - F8) were tested at a dilution of 0.01 . Controls consisted of no additions.

Plants were cut into uniform 1cm length including leaves and two axillary bud opposite of each other, and cultured 10 explants per jar (two jars per treatment = 20 explants).

Cultures were maintained under cool fluorescent light, provided by PHILIPS master TLD 36 W 830 Reflex ECO (40 pmol m-2 s-1 PAR) 16 h light and 8 h dark photoperiod at 22±2 °C. After 3 weeks the presence of new shoots, roots, and the length of the root, was assessed.

Results

Figure 10 shows that compared to the water control, addition of the Belgian endive forced roots water (HO) extract to the medium, significantly stimulates the development of new roots of Plectranthus escualentus explants, at both of the concentrations tested (Figure 10/A). Additionally, the length of the root (Figure 10/B) and the number of shoots (results not shown) were significantly promoted by addition of a low concentration of HO extract (0.001 dilution), a higher concentration does not result in a promoting effect. In addition, Fraction F2 showed a significant root promoting activity. 2.3. In-planta GROWTH parameters

Materials and Methods

The leaves of five weeks-old maize plants (Zea mays L LG31233) were sprayed with a 10% solution of the Belgian Endive Forced Roots water (HO) extract. Growth rate was measured by measuring plant height each week after spraying, and for a total of 3 or 4 weeks.

During this period plants were grown on pots with universal potting soil (Pflanzerde Potgrond Universeel - Jardino BASIC) containing 35% organic substrates, 1 .5 g KCI/I and pH value of 5.8 (adjusted with CaCl2). Plants were kept in walk-in climatized greenhouses, at an average night/day temperature ranging from 21 to 32 °C and natural light conditions. All plants were regularly watered. A mixture of nutrient solution (including triple superphosphate (TSP) 45%, and urea-ammonium nitrate fertilizer (UAN) 39%, Patentkali 30%) was given one time (50mL per pot) after 3 weeks of growing.

Results

Figure 11 shows that the Belgian Endive Forced Roots water (HO) extract, significantly increases the growth rate of maize plants when applied at a concentration of 10%.

3. ACTIVITY AGAINST OOMYCETES AND FUNGI.

3.1 In-planta (Arabidopsis) induced systemic resistance (ISR) bioassay. 3.1.1 Pathosvstem: Arabidopsis-Sofryf/s cinerea (necrotrophic fungal pathogen)

Materials and methods

Arabidopsis thaliana Col 0 plants were grown in square petri dishes containing ½ MS medium with 8 g/L sucrose. After stratification (2 days on 4°C) and sterilization (5 min in 30% bleach), six seeds were sown on the top of the solid medium, of which the upper part (1/3) was removed. Plates were sealed with Micropore™ Medical Tape (3M™, St. Paul, Minnesota, USA) and placed vertically in a growth chamber. After 21 days, the plants were treated with the candidate ISR- inducer. For the disease assay with Botrytis cinerea, two leaves per plant were infected with 2 mI_ drops 5^*10⁵ spores/ml spore suspension of Botrytis cinerea B05.10 in ½ PDB buffer. The infection took place three days after inoculation. Disease symptoms were scored three days after infection by measuring the diameter of the necrotic lesions parallel to the midrib. Results

Application of three (HO, EH, EA) of Belgian Endive Forced Roots extracts resulted in a significant reduction of lesions caused by infection Botrytis cinerea when applied on roots of Arabidopsis plants (Figure 12). Since extract application and pathogen inoculation are done on different organs of the same plant, the observed reduction does not result from direct antagonistic (or direct) effect of the extracts on the pathogen, but from an induced resistance (IR) in the plant triggered by the extracts.

3.1.2 Pathosvstem: Arabidopsis -Hyaloperonospora arabidopsidis (biotrophic oomycete pathogen).

Materials and methods

Arabidopsis plants (ecotype Col 0) were grown in soil with a dark/light regime of 12h/12h, a light intensity of 100 mM, a temperature of 21 °C and relative humidity of 70%. Leaves of 9 days-old plants were sprayed with the Belgian Endive Forced Roots extracts (HO:100%, EH: 250mg/mg, EA: 20mg/ml, HE: 20mg/ml) or distilled water (control) until run-off. One day later, inoculation of the leaves was done by spraying until run-off with a spore suspension (6^*10⁴ spores/ml in H2O) of Hyaloperonospora arabidopsidis Noksl . Plants were further grown for another 7 days under the same conditions as mentioned before (but at 17°C), and in an incubation box allowing maximal relative humidity and favoring disease progression.

Disease severity was evaluated by quantifying the amount of newly produced pathogen spores on batches of 15 plants. Bars represent average spore formation of 7 batches (each representing 15 plants)

Results

Only the application of the water (HO) extract, resulted in a significant reduction of spore formation caused by infection of the oomycete Hyaloperonospora arabidopsidis when applied on leaves of Arabidopsis plants (Figure 13). The ethanol (EH) and ethyl acetate (EA) extracts had no significant effects. The hexane (HE) extract was not included in the experiment.

3.2. In-detached leaf (Tomato and potato) and in-plant (wheat) bioassays.

Materials and methods The (indirect) biocidal activity of Belgian Endive Forced Roots extracts was tested against three important widespread plant pathogens: Phytophthora infestans (oomycete pathogen of potato), Blumeria graminis (fungal pathogen of wheat) and Botrytis cinerea (fungal pathogen of tomato). Detached leaf bioassav

A detached leaf bioassay of 6-7 weeks-old tomato and potato plants was used for pathogens: Botrytis cinerea and Phytophthora infestans, respectively. Foliar spraying of tomato and potato plants with the extracts was done 24h before inoculation, in order to take protective mode of action into account. Control plants were treated with water (negative control), with 5330 mg/I Mancozeb (tomato positive control, Belchim Crop Protection, Londerzeel, Belgium), or with 160 g/l Ranman-Top® (potato positive control, Belchim Crop Protection, Londerzeel, Belgium). Note: the EH, the EA, and the HE extracts were not tested.

After spraying, leaves were removed (3 compound leaves per replicate) and inoculated with a single (15 pL) droplet of spore suspension of pathogens (10^*5 spore/ml). Leaflet were kept under appropriate incubation conditions, in a plant growth chamber. Disease incidence was assessed 5-7 days after inoculation, on treated and control leaflets according to an arbitrary grading scale and by converting to disease severity index (DSI), on a percentage basis.

To quantify the disease severity over time, the area under the disease progress curve (AUDPC) was calculated for potato plants during 32 days of infection according to the equation: AUDCP = å[(Xi+ Xi + 1 )/2]ti. Where Xi and Xi + 1 are severity on date i and date i + 1 , respectively, and ti is the number of days between date i and date i + 1 .

Whole plant bioassav

Whole plant bioassay of wheat was used for the pathogen: Blumeria graminis. Foliar spraying of 2 weeks-old wheat plants with the extracts was done 24h before inoculation in order to take protective mode of action into account. Control plants were treated with water (negative control), or with a mixture of 1 ,7l/ha Palazzo (BASF, Antwerpen, Belgium) plus 1 l/ha Bravo (Syngenta, Seneffe, Belgium) as positive control. Sprayed whole plants were inoculated by spraying spore suspension of Blumeria graminis (10^*5 spore/ml) and kept under appropriate conditions in the greenhouse. Disease intensity was assessed on the bases of disease symptoms (pathogen white flecks) and the severity is calculated 20 days after inoculation as described above. Results

Application of all the Belgian Endive Forced Roots extracts to whole wheat plants resulted in a reduction (from 30% for the HO extract, and up to 70% for the HE extracts) of disease symptoms caused by Blumeria graminis, with a significant reduction of the disease symptoms when applying the EH, the EA and the HE extracts (Figure 14, bottom panel). On the tomato detached leaves, the EH and the EA extracts were effective at reducing the disease symptoms caused by Botrytis cinerea, and this reduction was significant when applying the EH extract (Figure 14, top panel). Finally, on the potato detached leaves, the water (HO) extract was highly effective at reducing the disease symptoms caused by Phytophthora infestans (Figure 14, top panel).

REFERENCES

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Claims

1. A method for the preparation of a Belgian endive forced root extract (Cichorium intybus var. foliosum), comprising the steps of: a) providing Belgian endive forced roots; b) slicing the roots of step a) c) drying the sliced roots of step b) d) mixing the dried roots of step c) with an aqueous solution, while heating said mixture at a temperature of at least 30°C; e) separating the heated mixture of step d) into a solid phase and a liquid phase; and f) obtaining the liquid phase of step e) as a Belgian endive forced root aqueous extract.

2. The method of claim 1 , wherein step d) is performed at a temperature of at least 80°C.

3. The method of claims 1 or 2, further comprising milling the dried roots of step c) prior to the mixing of step d).

4. The method as defined in any one of claims 1 - 3 , wherein the solid phase obtained in step e) is further processed by: a) mixing said solid phase with an organic extractant; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root organic solvent extract; wherein optionally steps a) to c) are repeated one or more times using a different extractant.

5. The method as defined in claim 4, wherein said organic extractant is selected from the list comprising ethanol, ethyl acetate and hexane.

6. The method as defined in claims 4 or 5, wherein steps a) to c) are sequentially repeated using the following organic extractants in the specified order: ethanol, ethyl acetate and hexane.

7. The method as defined in claims 1 or 2, wherein the solid phase obtained in step e) is further processed by: a) mixing said solid phase with ethanol; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root ethanol extract.

8. The method as defined in claim 7, wherein the solid phase obtained in step b) is further processed by: a) mixing said solid phase with ethyl acetate; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root ethyl acetate extract.

9. The method as defined in claim 8, wherein the solid phase obtained in step b) is further processed by: a) mixing said solid phase with hexane; b) separating the mixture of step a) into a solid phase and a liquid phase; and c) obtaining the liquid phase as a Belgian endive forced root hexane extract.

10. The method as defined in anyone of claims 4 to 9 wherein said Belgian endive forced root organic solvent extract is further subjected to an evaporation step.

11. The method as defined in claim 1 , wherein said heating is performed at a temperature in a range from about 30 °C to about 120°C, preferably from 50°C to 100°C, preferably from 60°C to 90°C, more preferably at a temperature of about 80°C and/or for a sustained period of time, in a range from about 5 min to about 180 min, preferably from 10 min to 150 min, preferably from 50 min to 130 min, more preferably about 120 min.

12. A Belgian endive forced root extract obtainable by applying the method of anyone of claims 1 to 11 .

13. Use of a Belgian endive forced root extract as obtained by the method of anyone of claims 1 to 11 or as defined in claim 12 as a plant biostimulant and/or for reducing or preventing infection of a plant with phytopathogenic fungi and/or oomycetes.

14. Use of a Belgian endive forced root extract as a plant biostimulant and/or for reducing or preventing infection of a plant with phytopathogenic fungi and/or oomycetes.

15. The use as defined in anyone of claims 13 to 14, wherein said phytopathogenic fungi and/or oomycetes are selected from the list comprising: Phytophthora infestans, Blumeria graminis, Botrytis cinereal, and Hyaloperonospora arabidopsides.

16. The use as defined in anyone of claims 13 to 15, wherein the extract is applied on a plant, seeds or part(s) thereof, or in the growth medium or soil of a plant.