WO2023166049A1

WO2023166049A1 - Combination of malt rootlets and microorganism

Info

Publication number: WO2023166049A1
Application number: PCT/EP2023/055170
Authority: WO
Inventors: Mathieu Allard; Erika SAMAIN
Original assignee: Etablissements J. Soufflet
Priority date: 2022-03-04
Filing date: 2023-03-01
Publication date: 2023-09-07
Also published as: EP4486714A1

Abstract

The present invention concerns a combination comprising malt rootlets with one or more microorganism, the one or more microorganism having biostimulation and/or biofertilization activities. The present invention also relates to uses of such combination, methods for treating seeds of crops or crops and methods of implantation of one or more microorganism at the vicinity of crops.

Description

COMBINATION OF MALT ROOTLETS AND MICROORGANISM

Malt rootlets are produced during the germination stage of malting. After the subsequent kilning, i.e. a drying process, these rootlets are removed. There are therefore no rootlets in the brewing malt. Rather, the malt rootlets are a by-product.

According to the prior art, malt germs and malt rootlets are used in their pure form or as a component of feed. More recently, the malt germ was also described as a fertilizer (see for example applications DE4424574A1 or W02018104531 (A1) or EP1 124776 (A 1)).

Summary of the invention

The inventors have surprisingly found that malt rootlets can also be used as a good substrate to microorganisms, promoting their survival and growth. The inventors also found that biostimulation and biofertilization activities of microorganisms are enhanced in presence of these malt rootlets, even when malt rootlets are used in concentrations where malt rootlets have no fertilizing activity.

In consequence, the use of microorganisms having biostimulation and/or biofertilization activities with malt rootlets, constitutes a new combination for promoting plant growth.

This invention also constitutes a new use of co-products of the malting industry and permit better valorization of malt rootlets.

Thus, in a first aspect, the present invention concerns a combination comprising malt rootlets with one or more microorganism, the one or more microorganism having biostimulation and/or biofertilization activities. Indeed, biostimulation and biofertilization activities of microorganisms are enhanced in presence of these malt rootlets.

In particular, the inventors demonstrated that in presence of malt rootlets as microorganism growing booster, bacteria and/or fungi with phosphorus solubilization activity demonstrate an acceleration and/or an increase of said phosphorus solubilization activity. Such increase of phosphorus solubility allows increasing the intake of phosphorus of plant.

Phosphorus (P) is vital to plant growth. It is involved in several key plant functions e.g. energy transfer, photosynthesis, transformation of sugars and starches, nutrient movement within the plant. Therefore, the combination according to the invention has the capacity to confer an improved nutrient absorption and thus to promote the growth of plants leading to improved crop quality, improved resistance to biotic and/or abiotic stresses, and plant vigor.

The invention also concerns the use of a combination according to the invention as a biostimulation agent and/or a biofertilizer.

In a further aspect, the invention provides the use of a combination according to the invention as an agent promoting the plant growth.

The present invention also relates to a method for treating seeds of crops or crops wherein the combination according to the invention is put on or in the substrate wherein seeds or crops are set, or wherein the combination according to the invention is put on the seeds or the crops, in a way permitting the release of the one or more microorganism at the vicinity of said seed or crops.

In a further aspect, the invention provides a method of implantation of one or more microorganism having biostimulation, or biofertilization activities at the vicinity of crops, comprising:

(a) bring together said one or more microorganism with malt rootlets, and administrating said microorganism with malt rootlets to said crops, or

(b) administrating malt rootlets to said crops and then administrating said one or more microorganism to said crops, or

(c) administrating said one or more microorganism to said crops and then administrating malt rootlets to said crops.

Detailed description of the invention

Malt rootlets

By "malt" is meant here a germinated cereal obtained through a malting process. The malt may be obtained by any malting technique well known to persons skilled in the art. Malting takes place typically in 4 steps, including soaking, which moistens the grain, the germination during which the grain begins to germinate and gives rise to "green malt", the roasting where the green malt is dried to obtain a dry malt, and the degermage where the malt is separated of its rootlets.

In the sense of the invention, the term "malt rootlets" includes herein the rootlets, the acrospires, the husks, the dust or fragments thereof, and mixtures thereof and corresponds to the co-products generated during the malting process. The coarse mixture of these coproducts is marketed as such or as granules (pellets) in animal feed under the term "malt sprouts".

In a particularly preferred manner, the malt rootlets used in the context of the invention are obtained after 3 to 11 days of germination.

The malt rootlets used in the context of the invention are preferably prepared from a cereal malt, and in particular from malt selected from barley, wheat, rye, spelt, corn, millet, sorghum, oat, triticale, rice and mixtures thereof. More preferably, the malt rootlets used in the context of the invention are prepared from barley malt and/or wheat malt.

In a particular embodiment, the malt rootlets used in the context of the invention are dried, and/or micronized and/or grinded, and optionally agglomerated. Indeed, the malt rootlets according to the invention can be in the form of flour, pellet or liquid extract.

By “dried”, it is meant that water of the malt rootlets is removed, typically by evaporation. Preferably, once dried the malt rootlet comprises less than 15% of water, and more preferably less than 10% of water in weight, and more preferably less than 5% of water in weight.

By “grinding”, it is meant reducing the average diameter of malt rootlets particle and in particular to fragments smaller than 2.5 mm.

By “micronized”, it is meant reducing the average diameter, preferably fragment smaller than 1 mm and in particular in fragments smaller than 0.5, 0.3 or smaller than 0.2 mm.

In a particular embodiment, a selected fraction of malt rootlets is used in the context of the invention, such as a fraction comprising fragments of a particular average diameter, such as having an average diameter of 0.2 to 0.3 mm, such as 0.3 to 0.5 mm, such as 0.5 to 1 mm, such as 1 to 2.5 mm. Agglomeration of dried malt rootlets can be performed by adding moisture to the dried rootlets. In a particular embodiment, the malt rootlets according to the invention are agglomerated in form of pellet.

By “pellet” or by “granule” it is meant agglomerated material, herein malt rootlets, typically in form of rods or sphere. Preferably, pellets according to the invention have an average diameter comprised between 1 to 5 cm and/or weight between 1 to 5 g, and more preferably weight between 1 and 2 g.

In order to limit contamination and quick deterioration of the product, the malt rootlets according to the invention are preferably decontaminated, more preferably before their use or before their combination with microorganisms.

By “decontaminating”, it is meant reducing initial endogenous contamination by 2 log at least, and preferably totally decontaminating the product, i.e. elimination of all forms of life in the product.

Decontamination may be performed by several techniques well-known to the skilled in the art, and in particular by pasteurization, by sterilization methods such as using dry heat (with oven), ionizing radiation, gas or by use of an autoclave. In a preferred embodiment according to the invention, the malt rootlets are autoclaved at least 100 °C during at least 30 min.

Microorganism

The term “microorganism” used herein refers to a living organism of microscopic size, which may exist in its single-celled form or as a colony of cells. Such term includes bacteria, fungi, yeast, archaea, microalgae. More preferably, microorganisms of the combination of the invention are selected among bacteria and fungi.

By “microorganism having biostimulation and/or biofertilization activities”, it is meant a microorganism known for these activities. These microorganisms are generally promoting the plant growth, notably through an increase in nutrient uptake and in phytohormones synthesis.

In particular by "microorganism having biostimulation activity", it is meant a non- pathogenic living microorganism capable of stimulating natural processes to improve/advantage nutrient absorption, nutrient efficiency, abiotic stress tolerance, and/or crops quality. Typically, “microorganism having biostimulation activity” have no direct action against pests, and therefore do not fall within the regulatory framework of pesticides. By "microorganism having biofertilization activity", it is meant a non-pathogenic living microorganism capable of increasing the availability of nutrients for plants.

Preferably, the one or more microorganism of the combination of the invention is a plant growth promoting microorganism, preferably selected among bacteria and fungi.

Preferably, the plant growth promoting microorganism is selected among phosphorus solubilizing microorganisms, auxin producing microorganisms, and diazotrophic microorganisms, more preferably the plant growth promoting microorganism is selected among phosphorus solubilizing microorganisms.

“Phosphorus solubilizing microorganisms” are well-known to the skilled in the art and for example comprises bacteria of the following genus: Pseudomonas, Bacillus, Rhizobium, Enterobacter, Agrobacterium, Azotobacter, Burkholderia, Erwinia, Kushneria, Paenibacillus, or fungi of the following genus : Penicillium, Aspergillus, Achrothcium, Alternaria, Arthrobotrys, Cephalosporium, Cladosporium, Curvularia, Glomus, Micromonospora, Mortierella, Myrothecium, Pythium, Rhizoctonia, Saccharomyces, Sclerotium, Trichoderma (see for more details : KHAN et al. Phosphorus solubilizing bacteria: occurrence, mechanisms and their role in crop production. J agric biol sci, 2009, vol. 1 , no 1 , p. 48-58, or ALORI et al. Microbial phosphorus solubilization and its potential for use in sustainable agriculture. Frontiers in microbiology, 2017, vol. 8, p. 971).

In a particular embodiment of the invention, the phosphorus solubilizing microorganism is selected from Pseudomonas fluorescens, Pseudomonas veronii, Paenibacillus hordei and Penicillium brasilianum. More preferably, the phosphorus solubilizing microorganism is selected from Pseudomonas fluorescens, Pseudomonas veronii and Paenibacillus hordei.

In an embodiment of the invention, the phosphorus solubilizing microorganism is from Penicillium brasilianum species, and in particular, is the microorganism deposited under accession number MLICL 54519 at the BCCM/MUCL (Belgian Coordinated Collections of Microorganism (BCCM), Universite catholique de Louvain, Mycotheque de I’Universite catholique de Louvain (MUCL), Croix du Sud 2, box L7.05.06, 1348 Louvain-la Neuve, Belgium). This microorganism was deposited on 30 November 2012 and converted to a deposit under the Regulations of the Budapest treaty on 11 July 2013. In the example section, this microorganism is named PB1.

In an embodiment of the invention, the phosphorus solubilizing microorganism is from the Pseudomonas Fluorescens group, and in particular, is the microorganism deposited under accession number CBS 148846 at the Westerdijk Fungal Biodiversity Institute (CBS) (Uppsalalaan 8, P.O. Box 85167, 3508 AD Utrecht, The Netherlands). This microorganism was deposited on 18 February 2022 under the Regulations of the Budapest treaty. In the example section, this microorganism is named PF2.

In an embodiment of the invention, the phosphorus solubilizing microorganism is from the Paenibacillus hordei species, and in particular, is the microorganism deposited under accession number CBS 148847 at the Westerdijk Fungal Biodiversity Institute (CBS) (Uppsalalaan 8, P.O. Box 85167, 3508 AD Utrecht, The Netherlands). This microorganism was deposited on 18 February 2022 under the Regulations of the Budapest treaty. In the example section, this microorganism is named PH1.

In an embodiment of the invention, the phosphorus solubilizing microorganism is from the Pseudomonas veronii species, and in particular, is the microorganism deposited under accession number CBS 148853 at the Westerdijk Fungal Biodiversity Institute (CBS) (Uppsalalaan 8, P.O. Box 85167, 3508 AD Utrecht, The Netherlands). This microorganism was deposited on 22 February 2022 under the Regulations of the Budapest treaty. In the example section, this microorganism is named PV5.

“Auxin producing microorganisms” are well-known to the skilled in the art and for example comprises bacteria of the following genus: Agrobacterium, Pseudomonas, Plantoea, Rhizobium, Azospirillum, Bacillus, Alcaligenes, Klebsiella, Enterobacter, Burkholderia, Xanthomonas, Erwinia, Arthrobacter, Acetobacter, Streptomyces, or fungi of the following genus : Trichoderma, Aspergillus, Penicillium, Piriformospora, Colletotrichum, Fusarium (see for more details : SPAEPEN et al.,, lndole-3-acetic acid in microbial and microorganism-plant signaling. FEMS microbiology reviews, 2007, vol. 31 , no 4, p. 425-448, KUMAR, et al., Plant growth promotion efficacy of indole acetic acid (IAA) produced by a mangrove associated fungi-Trichoderma viride VKF3. International Journal of Current Microbiology and Applied Sciences, 2017, vol. 6, no 11 , p. 2692-2701 , FORNI et al., Indole- 3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla. Microbiology, 1992, vol. 138, no 2, p. 377-381 , PATIL et al., Optimization of indole 3-acetic acid (IAA) production by Acetobacter diazotrophicus LI isolated from sugarcane. International Journal of Environmental Sciences, 2011 , vol. 2, no 1 , p. 295-302).

“Diazotrophic microorganisms” are well-known to the skilled in the art and for example comprises bacteria of the following genus: Azotobacter, Rhizobium, Bacillus, Clostridium, Klebsiella, Azospirillum, Arthrobacter, Burkholderia, or fungi of the following genus: Pleurotus, Macrotermes, Odontotermes (see for more details: SHRIDHAR et al. Nitrogen fixing microorganisms. Int J Microbiol Res, 2012, vol. 3, no 1 , p. 46-52, Jayasinghearachchi et al., Can mushrooms fix atmospheric nitrogen? J Biosci. 2004 Sep;29(3):293-6. doi: 10.1007/BF02702611. PM ID: 15381850, SAPOUNTZIS et al., Potential for nitrogen fixation in the fungus-growing termite symbiosis. Frontiers in Microbiology, 2016, vol. 7, p. 1993). In a particular embodiment of the invention, the one or more microorganism is a living microorganism in a germinative form or a microorganism in a sporulated form.

Combination comprising malt rootlets with microorganism

The present invention concerns a combination comprising malt rootlets with one or more microorganism, the one or more microorganism having biostimulation and/or biofertilization activities.

The term “combination” according to the invention refers to combination and combination products wherein the two components (herein malt rootlets and microorganisms) are: in contact (such as a pellet with microorganism sprayed on it or a pellet or a capsule soaked in a liquid comprising microorganism, or pellets or capsules coated with microorganism), or mixed together (such as a flour of malt rootlets mixed with microorganism) or

- joined in single package or as a unit (such as in a capsule), or packaged separately in a product that according to its labelling is intended for use only in combination as both are required to obtain the intended effect.

Thus, this term also defines a kit of parts for the combined administration.

Preferably, in the combination according to the invention, the ratio between quantities of microorganism and malt rootlets is comprised between 10⁵CFU/g of malt rootlets and 10¹⁰CFU/g of malt rootlets (dry weight), more preferably between 10⁷ CFU/g of malt rootlets and 10¹⁰CFU/g of malt rootlets.

Preferably, in the combination according to the invention, the ratio between quantities of microorganism and malt rootlets is superior or equal to 1 g of microorganism for 10 kg of malt rootlets, more preferably superior or equal to 1 g of microorganism for 1 kg of malt rootlet, more preferably superior or equal to 1 g of microorganism for 0.1 kg of malt rootlets (dry weight). In a preferred embodiment, it is comprised between 1 g of microorganism for 0.1kg of malt rootlets and 10 g of microorganism for 0.1 kg of malt rootlets (dry weight). In a preferred embodiment of the combination according to the invention, the malt rootlets and the one or more microorganism are co-encapsulated.

By the term “co-encapsulated”, it is meant that the malt rootlets and the microorganism are both simultaneously comprised in a capsule. In some embodiment, the malt rootlets and microorganism can be encapsulated in separate capsules, which are and comprised in a unique bigger capsule. By “capsule”, it is meant a structure comprising a core and a shell, wherein microorganisms and malt rootlets are comprised in the core of the capsule. Preferably, the shell is gelatinized and is made of biodegradable constituents.

According to one embodiment, the combination according to the invention is in the form of co-encapsulated malt rootlets and microorganisms. Such co-encapsulation is preferably performed with malt rootlets in the form of liquid extract, or flour in suspension in a liquid.

According to another embodiment, the combination according to the invention is realized by spraying of microorganisms on pellets of malt rootlets, or soaking pellets of malt rootlets in a liquid comprising suspended microorganisms, or coating pellets of malt rootlets with a liquid comprising suspended microorganisms.

According to another embodiment, the combination according to the invention is realized by mixing germinative form or sporulated form of the microorganism with malt rootlets flour.

Kit comprising malt rootlets with microorganism

The present invention concerns a kit comprising malt rootlets and one or more microorganism, the one or more microorganism having biostimulation and/or biofertilization activities.

Preferably, said kit comprises an instruction leaflet. Such instructions leaflet can provide instructions on the use of the combination or the way to administrate malt rootlets and microorganisms in combination.

In a particular embodiment, said kit also comprises a hydration solution or a powder allowing the formation of a hydration solution, facilitating the spraying or the soaking or the coating of some component of the kit, and in particular of malt rootlets and/or microorganisms.

Uses of the invention

The present invention concerns the use of a combination according to the invention as a biostimulation agent and/or a biofertilizer. The present invention also concerns the use of a combination according to the invention as an agent promoting the plant growth.

The malt rootlets are used according to the invention in a quantity that do not permit to significantly fertilize the soil but is used herein as a substrate to microorganisms, promoting their survival and growth and enhancing their biostimulation and/or biofertilization activities and/or promoting plant growth.

Thus, in an embodiment of the uses of the invention, the amount of malt rootlets used per hectare is comprised from 100 grams to about 10 000 grams (dry matter d.m. or active ingredient, a.i.) and the amount of microorganisms used per hectare is comprised from 10⁹ to 10¹² CFU of microorganisms.

In an embodiment of the uses of the invention, the amount of malt rootlets used per hectare is comprised from 100 to about 10 000 grams (dry matter d.m. or active ingredient, a.i.) and the amount of microorganisms used per hectare is comprised from 1 g to 100 grams of microorganisms.

In particular, such combination can be used by spraying or spreading, or laying on or in the soil capsules comprising co-encapsulated malt rootlets and microorganisms, or by spreading or laying on or in the soil pellet of malt rootlets which have been sprayed with microorganisms.

An “agent promoting the plant growth” is an agent that affect at least one aspect of plant growth, including germination, root growth, leaves growth, flowering, maturity and fruit set, increasing the nutrient absorption of the crops, preferably the agent allows to improve yield, grain weight, grain quality and/or biomass of the plant

The term "plant" according to the invention covers plant crops, ornamentals, trees, grasses, annuals, perennials or any other commonly cultivated member of the kingdom Plantae. Herein the term "plants" is meant to include germinant seeds, cuttings, emerging seedlings, and established vegetation, including roots and aboveground portions, for example, leaves, stalks, flowers, fruits, branches, limbs, root, and the like.

In a preferred embodiment, the plant is a crop plant.

The term "crop plant(s)" or “crops” according to the invention includes any edible or non-edible plant, including decorative, plant species with commercial value, which is planted and cultivated for commercial use. Thus, crop plants include floral and non-floral plants, perennials and annuals, trees, shrubs, vegetable plants, fruit trees, turf, and ground cover. Non-limiting specific examples of crop plants include wheat, corn, soybean, dry bean, cotton, cereals, rice, corn, sorghum, canola, rape, soya, barley, potato, sweet potato, sunflower, rye, oats, sugar beet, safflower, alfalfa, cassava, cucurbits, pineapple, pastures, canola, flax, peas, lentils, beans, linola, mustard, chickpeas, seedling alfalfa, onions, soybeans, turf grass, stone fruits (cherries, plums, apricots, peaches, nectarines), blueberries, mangos, avocados, pastures, turfgrass, ornamentals, tree crops, eucalyptus, pine, tea, coffee, nut trees, citrus, tropical fruits, pome fruits, grapes and vines, perennial grasses, caneberries, bananas, or sugarcane.

Preferably, crops is chosen from the group consisted of commonly cultivated member of the Phylum of Angiosperms, and preferably selected among cereals, grapevines, industrial crops, vegetables, market gardening, and arboriculture, and more preferably selected among cereals, grapevines, and industrial crops.

Industrial crops consisted of commonly cultivated crops which are used to produce goods for manufacturing, rather than food. These crops can be used for production of biofuels and bioenergy, for construction, for producing fiber (e.g., for clothing, paper, rope) for pharmaceuticals or chemicals, or for the production of renewable biopolymers (rubbers and plastics). Industrial crops thus encompass among others potatoes, sugar beet, all seed crops and protein crops.

Combination according to the invention can be used by a foliar administration, roots administration or at the vicinity of root.

By the term “at the vicinity of root”, it is meant less than 10 cm from roots of the plant and preferably less than 5 cm from roots of the plant.

Preferably, uses of the invention are realized at any growth stage of said crops. For example, uses of the invention can be performed on seeds, on seedlings or on plant, preferably the uses of the invention are performed before flowering.

Methods of the invention

The invention also relates to a method for treating seeds of crops or crops wherein the combination of the invention is put on or in the substrate wherein seeds or crops are set, or wherein the combination of the invention is put on the seeds or the crops, in a way permitting the release of the one or more microorganism at the vicinity of said growing crops.

The definition of the term “vicinity” and “crops” are the same as in the section uses of the invention.

Preferably, said method for treating seeds of crops or crops is a method to promote plant growth. The invention also concerns a method of implantation of one or more microorganism having biostimulation, or biofertilization activities at the vicinity of crops, comprising:

By “bring together” it is meant putting into direct contact said one or more microorganism with malt rootlets. It comprises for example a pellet of malt rootlets sprayed with microorganism, pellets or capsules of malt rootlets soaked in a liquid comprising microorganism, pellets or capsules coated with microorganism, flour of malt rootlets mixed with microorganism, and capsules comprising malt rootlets and microorganism.

By “administrating to crops”, it is meant any way to provide said component, for example by placing said component in or on the substrate where the crops are, or by spraying, or by spreading.

Preferably, methods of the invention are realized at any growth stage of said crops. For example, methods of the invention can be performed on seeds, on seedlings or on plant, preferably the methods of the invention are performed before flowering.

In some embodiments, methods of the invention are performed several times on the same crops.

In a preferred embodiment, the malt rootlets and microorganism after performing (b) or (c) of the method of the invention are in direct contact or at the vicinity of one another. By the term “at the vicinity of one another”, it is meant less than 10 cm and preferably less than 5 cm from one another, and more preferably less than 1 cm from one another.

In a preferred embodiment, the malt rootlets and microorganism used in the methods of the invention are provided in an amount of sufficient to promote plant growth and/or to have biostimulation activity and/or biofertilization activity.

The term "an amount of sufficient to promote plant growth and/or to have biostimulation activity and/or biofertilization activity" means any amount of said two components (malt rootlets and microorganism) capable of increasing plant growth, seedling germination, biomass, yield, grain weight, grain quality, enhancing the chlorophyll content, and the like, relative to an untreated control. The quantity of malt rootlets used in the methods of the invention preferably do not permit to significantly fertilize the soil but is used herein as a substrate to microorganisms, promoting their survival and growth and thereby enhancing their biostimulation and/or biofertilization activities and/or promoting plant growth.

In general, the sufficient amount to stimulate plant growth may be within the application rate range of from about 100 grams to about 10 000 grams of malt rootlets extract (dry matter, d.m. or active ingredient, a.i.) per hectare (ha) and about 10⁹ to 10¹² CFU of microorganisms per hectare (ha). The amount sufficient to stimulate plant growth may be within the application rate range of from about 100 to about 10 000 grams of malt rootlets extract (dry matter d.m. or active ingredient, a.i.) per hectare (ha) and about 1 g to 0.1 kg of microorganisms per hectare (ha). Optimal application rates for enhancing/stimulating plant growth may depend on a given plant, the method of application, the microorganism used and other well-known factors.

Preferably, the ratio between quantities of microorganism and malt rootlets used in methods of the invention is superior or equal to 1 g of microorganism for 10 kg of malt rootlets, more preferably superior or equal to 1 g of microorganism for 1 kg of malt rootlet, superior or equal to 1 g of microorganism for 0.1 kg of malt rootlets (dry weight). In a preferred embodiment, it is comprised between 1 g of microorganism for 0.1 kg of malt rootlets and 10 g of microorganism for 0.1kg of malt rootlets (dry weight).

Throughout the instant application, the term “comprising” is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term “comprising” also discloses the embodiment wherein no features other than the specifically mentioned features are present (/.e., “consisting of”). Furthermore, the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention will be further illustrated by the following figures and examples. FIGURES

Figure 1 : Yield (g/pot) comparison of each represented modality. RO represents modalities wherein pellets of barley rootlets are added in the seedbed. Microorganisms were sprayed on the pellets in RO-modalities, or on the substrate without RO. Modalities are compared to the non-inoculated and non-amended control modality named “TNT”. The non-inoculated modalities were sprayed with water.

Figure 2: Grain specific weight comparison of each represented modality. RO represents modalities wherein pellets of barley rootlets are added in the seedbed. Microorganisms were sprayed on the pellets in RO-modalities, or on the substrate without RO. Modalities are compared to the non-inoculated and non-amended control modality TNT. The noninoculated modalities were sprayed with water.

Figure 3: 1000 grains weight (g) comparison of each represented modality. RO represents modalities wherein pellets of barley rootlets are added in the seedbed. Microorganisms were sprayed on the pellets in RO-modalities, or on the substrate without RO. Modalities are compared to the non-inoculated and non-amended control modality TNT. The noninoculated modalities were sprayed with water.

Figure 4: Biomasses (mg/pot) comparison at end of tillering growth stage, of each represented modality. RO represents pellets of barley rootlets, added in the seedbed. Microorganisms were sprayed on the pellets in RO-modalities, or on the substrate without RO. Modalities are compared to the non-inoculated and non-amended control modality TNT. The non-inoculated modalities were sprayed with water.

EXAMPLES

Example 1 : In vitro proof of concept, boost of microbial phosphorus solubilization

1. General materials and methods

1.1. Microorganisms isolation, identification and conservation

Three bacterial strains, a Pseudomonas fluorescens strain (PF2), a Pseudomonas veronii strain (PV5), a Paenibacillus hordei strain (PH1), and one fungal strain, a Penicillium brasilianum strain (noted PB1) were used in these examples.

The Penicillium brasilianum strain PB1 was deposited under accession number MUCL 54519 at the BCCM/MUCL (Belgian Coordinated Collections of Microorganism (BCCM), Universite catholique de Louvain, Mycotheque de I’Universite catholique de Louvain (MUCL), Croix du Sud 2, box L7.05.06, 1348 Louvain-la Neuve, Belgium). This microorganism was deposited on 30 November 2012 and converted to a deposit under the Regulations of the Budapest treaty on 11 July 2013.

The three bacterial strains were isolated. PH1 was isolated from wheat leaf, at flowering growth stage; PF2 was isolated from wheat roots, at ripening growth stage; and PV5 was isolated from wheat rhizospheric soil, collected 15 cm below the soil surface. These microorganisms were identified based on analysis on 16s ribosomal gene for the bacteria and ITS ribosomal gene region for the fungi. Finally, the isolated and identified strains PF2, PV5 and PH1 were transferred in liquid solution containing 20% of glycerol 99.5% while spores from PB1 were transferred in liquid solution containing 15% of glycerol. The four strains were conserved at -80°C.

1.2. Microorganisms cultivation

PF2, PV5 and PH1 were spread on Plate count agar (PCA). Plates were incubated at 30°C for 24 hours. A pregrowth phase was prepared by transferring one colony in Tryptic Soy Broth (TSB) and incubated at 30°C for 24 hours.

PB1 was spread on Potato dextrose agar Malt Extract Agar (MEA). Plates were incubated at 25°C for 10 days. Water was added to each Petri dish and the surface of the dish was gently scratched to detach spores. The spore suspension was recovered and filtered through a 40 - 100 pm filter to remove mycelium debris. The number of spores was counted to know the spore’s concentration. The spores and cells were used to inoculate three Solid state fermentation (SSF) media in a flask at a final concentration of 1x10⁷ spores/ml.

The three SSF media were composed as followed:

Medium 1 : Colza cake (CC)/wheat bran (WB). In 250 ml flask, 20g of dry matter was added, containing 11g of CC and 9g of WB. 10.33 ml of sterile water was transferred.

Medium 2: Sugar beet pulp (SBP)/corn germ (CG)/ Rice (R). In 250 ml flask, 20g of dry matter was added, containing 9% of SBP, 2% of CG and 9% of WB. 10.33 ml of sterile water was transferred.

Medium 3: pellets of barley rootlets (BR). In 250 ml flask, 20g of dry matter was added, containing 100% of BR. 10.40 ml of sterile water was transferred.

Flasks were autoclaved at 105°C for 35 minutes.

After inoculation, bacterial flasks (with respectively PF2, PV5 and PH1) were incubated at 30°C for 3 days, and the fungal flasks (with PB1), at 25°C for 6 days. Water was added to each Petri dish and the surface of the dish was gently scratched to detach spores.

1.3. NBRIP-BPB media’s preparation

The in vitro phosphate-solubilizing property of each bacterial and fungal strains was determined on NBRIP-BPB (National Botanical Research Institute's phosphate + colored pH indicator Bromophenol Blue BPB) medium containing bromophenol blue as a pH indicator composed by glucose (10 g/l), Ca3(PO4)2 (5 g/l), MgCl2 (5 g/l), MgSO4 (0.25 g/), KCI (0.2 g/l), (NH₄) 2 SO 4 (0.1 g/l), BPB (0.025 g/l), adjusted to a pH 7.0.

50ml-NBRIP-BPB media were inoculated at 1 *10⁷ cells/ml respectively for PF2, PV5 and PH1 or 1 xio⁷ spores/ml for PB1 in 250ml-flask. The flasks were incubated at 25°C under an agitation of 180 rpm. pH was measured 1 , 2 and 3 days after inoculation for bacteria and 3 days after inoculation for the fungus strain.

2. In vitro results and conclusion

The phosphoric solubilization property of a microorganism consists in decreasing the local pH to convert insoluble forms of phosphate into acidic form of phosphate which could be efficiently assimilated by the plants. The biochemical mechanism involved this microbial phosphoric solubilization property is mainly the secretion of organic acids such as fumaric, succinic, acetic, gluconic or citric acid. Here, the in vitro experiment consists in measuring the specific pH decrease for each microorganism, depending on the medium composition. This pH decrease is directly correlated to phosphoric solubilization. The pH of each condition was followed for 1 , 2, 3, or 6 days. A Control without inoculation was performed in parallel. Results are showed in table 1.

Table 1 : Percentage of pH reduction on various media vs non-inoculated control, measured on NBRIP-BPB, after 6 days of SSF for the several microbial assays and control. days of cultivation

Strain Media 1 2 3 6

BR 27,2% 22,6% 19,1 %

PV5 CC/WB 0% 24,5% 23%

SBP/GC/R 0% 0% 1 ,6%

BR 27,5% 10,7% 7,1 %

PF2 CC/WB 0% 18,5% 6,9%

SBP/GC/R 0% 0% 5,6%

BR 0% 18,9% 15,5%

PH1 CC/WB 0% 15,7% 14,8%

SBP/GC/R 0% 0% 2,8%

BR - - - 30,1 %

PB1 CC/WB - - - -15%

SBP/GC/R - - - 11 ,7%

A negative data corresponds to an increase of pH value.

The control modality without microorganism did not show any pH variation (data not shown). Results were expressed in percentage of control modality value. CC/WB are classical SSF media used in fermentation and were here compared to BR medium. Results demonstrated no pH variation at 1 day between control and modalities, excepted for PV5 and PF2 modalities, with SSF on barley rootlets. At 1 day, PV5 and PF2 showed the higher pH decrease with 27.2% and 27.5% compared to non-inoculated modality, respectively, over all modalities and timings of measure after inoculation for both microorganisms. PH1 did not demonstrated any acceleration but a higher decrease of pH was observed on barley rootlets, with 18.9% compared to 15.70% with CC/WB and compared to 0% with SBP/GC/R media respectively.

PB1 results did not show pH decrease for CC/WB medium modality but demonstrated 11.7% of decrease with SBP/GC/R medium at 6 days. However, the most important decrease of pH is observed with barley rootlets as culture medium of PB1 with a pH decrease of 30.1% at 6 days.

This experimentation points the impact of rootlets on microorganisms’ agronomical activities and development. In presence of rootlets as microorganism growing booster, bacteria and/or fungi demonstrate an acceleration and/or an increase of phosphorus solubility activity by decreasing pH.

Example 2: In planta proof of concept, boost of agronomical properties

1. General materials and methods

1.1. Microorganisms isolation, identification and conservation

Barley rootlets were provided in a form of pellets by Soufflet’s malthouse, located in Nogent sur Seine (France). As this malthouse by-product present a huge microbial contamination rate, it was autoclaved at 105 °C during 35 min.

The three bacteria (PF2, PV5, and PH1) and the fungus (PB1) were formulated using an encapsulation process via alginate polymerization process. The alginate encapsulation of the several microorganisms studied in this experiment was performed by the French company Kapsera S.A.S., as technical services. Encapsulated microorganisms are named PF2-A, PV5-A, PH1-A and PB1-A respectively. The concentration of dry material of each encapsulated microorganism is detailed in the table 2.

Table 2: Concentration in CFU of each microorganism in dry material after encapsulation

_ Microorganism _ Concentration (CFU/g of dry material)

PB1 1 ,27x10⁸

PF2 8,99x10⁸

PH1 1 ,96x10⁷

PV5 1 ,15x10⁸

1.2. Plant material and growth conditions

The experiment was conducted under greenhouse without controlled conditions, on the barley’s cultivar, RGT Planet. The temperature was followed in the greenhouse during the whole experimentation.

4L-pots were prepared, containing a soil mixture of loam soil (Tray 20/80) and sand (0-2 mm) (1 :1 , v/v). Before sowing, microorganism capsules suspensions were diluted in a solution of 5 ml demineralized water and sprayed on the substrate or on pellets of barley rootlets at 10⁷ CFU per pot (corresponding to 10¹² CFU/ha). Non-inoculated modalities were sprayed with 5 ml of demineralized water. Pellets of barley rootlets were added at 5g per pot, in the seedbed, in the 2 first cm of the subtract. 5g per pot were the minimum weight to use and allowing a satisfactory repeatability of the quantity of malt rootlets added to each pot. 10 grains per pots were sowed.

The irrigation program was optimal and has been adapted to plant growth stage. The experimentation has been homogeneously fertilized with liquid nitrogen at BBCH stage 29 with 80 nitrogen units per pot. BBCH scale is a well-known scale of plant development’ stages (see Witzenberger et al., 1989; Lancashire et al., 1991).

1.3. Growth observations

Biomasses of eight plants per modality were evaluated at the BBCH 29. The harvest happened at BBCH 99. Ears were cut, counted and the grains were separated from ears by using a threshing machine. The weight of 1000 grains and the specific weight were then determined.

2. Statistical analysis, results and conclusion

Eight technical replicates were used for the experiment. Significant differences were evaluated using ANOVA followed by Tukey’s post hoc test (a = 0.05) and the XLSTAT® statistics program (version 2014, Addinsoft, Paris, France). In the figures, if two modalities share the same lowercase letter then they are not statistically significantly different, and on the contrary, if two modalities do not share the same letter then they are statistically significantly different.

The objective of this in planta experiment is to observe the beneficial impact on barley growing of the combination of inoculated barley malt rootlets with phosphoric solubilizing microorganism. The fertilizing properties of this association is evaluated based on agronomical criteria:

- yield (Figure 1), grain specific weight (Figure 2),

1000 grains weight (Figure 3), and biomass at end of tillering growth stage (Figure 4).

The results of figure 1 demonstrated significant difference of yield for all microorganism + RO, compared to RO and control modalities. PB1-A + RO modality is significantly higher than RO and control modalities, whereas PB1-A showed no significant difference with RO modality. The PF2-A modalities (with and without RO) showed significant increases of yield compared to RO and control modalities. PB1-A, PH1-A and PV5-A demonstrated no significant difference with RO and/or control modality but, in presence of RO (PB1-A + RO, PH1-A + RO and PV5-A + RO modalities) yield is significantly higher than RO and control modalities (Figure 1). Furthermore, in presence of RO, PV5-A demonstrated a significant yield increase compared to PV5-A without RO modality; likewise, all microorganisms + RO modalities revealed an increasing trend of yield compared to microorganisms without RO modalities.

These results demonstrated that barley rootlets at the quantity used without inoculation do not allow increasing barley yield but, with inoculation of phosphorus solubilizing microorganisms, all tested modalities are significantly higher than RO and control modalities, demonstrating a biostimulation effect of the combination. In addition, on the four tested microorganisms, one (PV5-A) show significant increase of yield in presence of barley rootlets compared to modalities without barley rootlets, demonstrating a booster effect of barley rootlets on said microorganism. These results suggest the synergistic effect of barley rootlets and microorganisms on microorganism agronomical properties.

Figure 2 represents the grain specific weight comparison between modalities, including non-inoculated RO modality and the control modality. The results demonstrated non-significant increase of grain specific weight for the 4 microorganisms without RO compared to RO and control modalities whereas the 4 microorganisms associated with RO modalities showed significant increased compared to control modality. Furthermore, PV5- A + RO modality showed a significant increase of grain specific weight compared to PV5-A and RO modalities, likewise PH1-A + RO demonstrated a significant increase compared to PH1-A. Finally, PF2-A + RO and PB1-A + RO modalities revealed an increasing trend compared to these microorganisms without RO modalities.

These results demonstrated that barley rootlets without inoculation do not allow to increase grain specific weight of barley. With inoculation of phosphorus solubilizing microorganisms, all tested modalities are significantly higher than control modalities whereas, alone, the four tested microorganisms did not show significant increase compared to control modality. These results suggest the synergistic effect of barley rootlets and microorganisms on microorganism agronomical properties.

Figure 3 represents the 1000 grains weight comparison between modalities, including non-inoculated RO modality and the control modality. The results demonstrated non-significant increase of 1000 grains weight for two microorganisms (PB1-A + RO and PF2-A + RO) compared to these microorganisms without RO, whereas the four microorganisms associated with RO modalities showed significant increase compared to control and RO modalities. Furthermore, all microorganisms + RO modalities revealed an increasing trend compared to microorganisms without RO modalities.

These results demonstrated that barley rootlets without inoculation do not allow increasing 1000 grains weight of barley. With inoculation of phosphorus solubilizing microorganisms, all tested modalities are significantly higher than control modalities whereas, alone, two tested microorganisms did not show significant increase compared to control modality. These results suggest the synergistic effect of barley rootlets and microorganisms on microorganism agronomical properties.

Figure 4 represents the biomasses comparison between modalities, including noninoculated RO modality, and the control modality. The results demonstrated non-significant increase of biomass for the four microorganisms without RO compared to RO and control modalities, whereas the four microorganisms associated with RO modalities showed significant increased compared to control modality. Furthermore, in presence of RO, PV5- A + RO demonstrated a significant biomass increase compared to PV5 without RO modality; likewise, all other microorganisms + RO modalities revealed an increasing trend compared to microorganisms without RO modalities. All the microorganisms with RO show a significative increase of the biomass compared to control modality.

These results demonstrated that barley rootlets without inoculation do not allow increasing biomass of barley at end of tillering growth stage. With phosphorus solubilizing microorganisms inoculation, all tested modalities are significantly higher than control modalities whereas, alone, the four tested microorganisms did not show significant increase compared to control modality. These results suggest the synergistic effect of barley rootlets and microorganisms on microorganism agronomical properties.

Finally, all statistically significant increase of each of the agronomical properties measured in this example is summarized in Table 3. There is no significant difference between control and RO for all agronomical properties measured. Table 3: Significant improvement of agronomical properties for each microorganism in the different modalities

Claims

1.- A combination comprising malt rootlets with one or more microorganism, the one or more microorganism having biostimulation and/or biofertilization activities, wherein the ratio between quantities of microorganism and malt rootlets is superior or equal to 1 g of microorganism for 10 kg of malt rootlets in dry weight, wherein the one or more microorganism is selected among bacteria, phosphorus solubilizing fungi of the following genus: Penicillium, Aspergillus, Achrothcium, Alternaria, Arthrobotrys, Cephalosporium, Cladosporium, Curvularia, Micromonospora, Mortierella, Myrothecium, Pythium, Saccharomyces, Sclerotium and Trichodermades, auxin producing fungi of the following genus: Trichoderma, Aspergillus, Penicillium, Piriformospora, Colletotrichum, Fusarium, and diazotrophic fungi of the following genus: Pleurotus, Macrotermes, Odontotermes.

2 - The combination according to claim 1 , wherein the malt rootlets are prepared from malt selected from barley, wheat, rye, spelt, corn, millet, sorghum, oat, triticale, rice and mixtures thereof.

3.- The combination according to claim 1 or 2, wherein the malt rootlets are dried, and/or micronized and/or grinded, and optionally agglomerated.

4.- The combination according to any one of claims 1 to 3, wherein the malt rootlets are decontaminated by pasteurization or sterilization methods.

5.- The combination according to any one of claims 1 to 4, wherein the malt rootlets are in the form of in the form of flour, pellet or liquid extract.

6.- The combination according to any one of claims 1 to 5, wherein the malt rootlets and the one or more microorganism are co-encapsulated.

7.- The combination according to any one of claims 1 to 6, wherein the one or more microorganism is a plant growth promoting microorganism.

8.- The combination according to any one of claims 1 to 7, wherein the one or more microorganism is a living microorganism in a germinative form or a microorganism in a sporulated form.

9.- Use of a combination according to any one of claims 1 to 8 as a biostimulation agent and/or a biofertilizer.

10.- Use of a combination according to any one of claims 1 to 8 as an agent promoting the plant growth.

11 .- A method for treating seeds of crops or crops wherein the combination of any one of claims 1 to 8 is put on or in the substrate wherein seeds or crops are set, or wherein the combination of any one of claims 1 to 8 is put on the seeds or the crops, in a way permitting the release of the one or more microorganism at the vicinity of said seed or crops.

12.- A method of implantation of one or more microorganism having biostimulation, or biofertilization activities at the vicinity of crops, comprising:

(c) administrating said one or more microorganism to said crops and then administrating malt rootlets to said crops, wherein the one or more microorganism is selected among bacteria, phosphorus solubilizing fungi of the following genus: Penicillium, Aspergillus, Achrothcium, Alternaria, Arthrobotrys, Cephalosporium, Cladosporium, Curvularia, Micromonospora, Mortierella, Myrothecium, Pythium, Saccharomyces, Sclerotium and Trichodermades, auxin producing fungi of the following genus: Trichoderma, Aspergillus, Penicillium, Piriformospora, Colletotrichum, Fusarium, and diazotrophic fungi of the following genus: Pleurotus, Macrotermes, Odontotermes.

13.- The method according to any one of claims 11 to 12, wherein said crops is chosen from the group consisted of commonly cultivated member of the Phylum of Angiosperms, and preferably selected among cereals, grapevines, industrial crops, vegetables, market gardening, and arboriculture.

14.- The method according to any one of claims 11 to 13, wherein said method is realized at any growth stage of said crops.