OA16464A - New compositions of fungal inocula, their method of preparation and their application to the improvement of the growth of cultures. - Google Patents

Abstract

The present invention relates to novel fungal dinocula compositions, their method of preparation and their application to the improvement of the growth of cultures.

Description

The present invention relates to novel fungal inocula compositions, their preparation method and their application to the improvement of crop growth.

Mycorrhization is a mutually beneficial association between a plant and a mycorrhizal fungus

The importance of mycorrhizal fungi in forest plantations was first demonstrated in 1910 in attempts to introduce exotic pines to Kenya. While these plantations generally ended in spectacular failures (chlorotic plants, very high mortality), on the advice of the Royal Botanic Gardens, soil from old pine plantations was imported from South Africa for use as nursery growing medium.

In fact, this soil contained mycorrhoids which are mixed organs located on the roots of plants, formed by the juxtaposition of two types of organisms: the upper plant and the underground mycelial filaments of the fungus. The fungi will draw mineral elements from the soil, and more particularly the less mobile ones such as zinc, copper and phosphorus, which are essential for the development of the latter and which they will provide to the plant. In return, the plant gives the fungus sugars and growth substances, thus ensuring its survival. Mycorrhizae play an important role in the uptake of certain forms of nitrogen as well as in the uptake of water from the soil.

This technique employing a soil rich in mycorrhizal fungal propagules was quickly disclosed with success in other African countries such as Tanzania, Uganda, Malawi, etc.

Since then, several mycorrhization techniques have been developed and that using mycorrhizal fungi alone is now based on the following steps: (i) isolation and purification of fungal strains, (ii) selection, under controlled conditions, of fungal strains performing for a given parameter ( for example: effect of the strain on the growth of the host plant) and (iii) multiplication of the strain under axenic conditions and production of fungal inocula AA

There are two types of mycorrhizal fungi, ectomycorrhizal fungi which mainly concerns forest species and arbuscular mycorrhizal fungi which concern herbaceous plant species.

Regarding ectomycorrhizal fungi, several inoculum formulations have been tested and have shown spectacular effects in the nursery.

Thus, an inoculum of the calcium alginate bead type is described in patent JP 3,091,855 B2 which relates to a process for preparing mycorrhizae of mycorrhizal fungus by development and then removal of Japanese red pine roots, added to a mixture of mycelium, such as Trichola matsutake Sing. with sodium alginate, then adding a solution of calcium chloride to polymerize the mixture. The covered roots are then cultured to form the mycorrhizae of the mycorrhizal fungus.

However, this inoculum does not include phosphate.

US 5,021,350, US 5,096,481 and CN 2010 1212881 describe compositions comprising a mycorrhizal inoculum coated in a matrix and contain no phosphate or peat.

Consequently, this composition involves the use of external phosphate to provide the plant with phosphorus (P) which is an essential nutrient for it, but which has two major drawbacks which are, on the one hand, the very low solubility 20 aqueous phosphate in non-acidic sol, and on the other hand a loss of efficiency of the phosphate due to the dispersion of the powder in the culture substrate.

To reduce phosphorus deficiencies and ensure good plant productivity, large amounts of phosphorus-based fertilizers in soluble form are used. However, up to 80% of the phosphorus fertilizer is lost because amendment 25 is easily precipitated insoluble forms (CaHPO ^, Ca ₃ (PO 4) 2 and AlPO4 FePCL) and becomes unavailable to the plant.

International application WO 2007/066136 and application JP 1993-163583 describe compositions comprising a mycorrhizal inoculum, inorganic phosphate and respectively peat or compost.

Documents EP 0 092 990, EP 0 485 229, WO 2009/009805 and US 2008/0064598 describe compositions comprising a mycorrhizal inoculum formulated in the form of granules or particles but devoid of coating in a matrix.

Another formulation comprises an inoculum associated with inorganic phosphate. This type of inoculum is for example described in Ouahmane et al. (Responses of Pinus halepensis growth, or microbial catabolic functions and phosphate-solubilizing bacteria after rock phosphate amendment and ectomycorrhizal inoculation; Plant soil, DOl 10.1007 / sl 1104-008-9882-z) which describe the effect of an ectomycorrhizal fungus ( Pisolithus sp.) On the growth of pine plants (Pinus halepensis) in the presence or absence of rock phosphate. The results show that when said ectomycorrhizal fungus is used in the presence of phosphate, a synergy is then observed on the growth of the aerial and / or root biomass compared to the aerial and / or root growth observed with the phosphate alone or the fungus alone. .

However, the ectomycorrhizal fungus combination has one main drawback, which is the low impact of the ectomycorrhizal fungus on its ability to alter inorganic phosphate.

Consequently, there is a need to provide non-polluting formulations making it possible to supply plants with the phosphorus necessary for their growth.

One of the objects of the invention is therefore to provide a formulation of mycorrhizal fungus in the form of granules coated in a matrix allowing the mycorrhizal fungi to alter the complex inorganic phosphates to release P ions bioavailable for the plant via cultural processes. respectful of the environment while avoiding the dispersion of phosphate in the soil.

Another object of the invention is to provide a process for preparing said granules.

The present invention relates to granules comprising inorganic phosphate, peat, at least one mycorrhizal inoculum, with the exception of nutrients or malt germ, said granules being coated in a matrix based on at least one polymerized hydrocolloid.

By the term "granules" is meant a small particle or a grain the size of which may be from about 2 to about 10 mm.

By the expression "inorganic phosphate" should be understood any inorganic phosphate, such as rock phosphate (or rock phosphate) which can be found in different countries such as for example New Zealand, Peru, Egypt or Morocco without being limited to these, or of pozzolana, and which will allow the release of phosphorus to the plant

By the expression “coated in a matrix”, it should be understood that the granules and its constituents are covered by the polymerized hydrocolloid and therefore remain in the matrix, thus making it possible to promote the capacity of the inoculum or fungus to solubilize the phosphate. inorganic when the granules are contacted with the plant, and thereby make the phosphorus ions more accessible to the plant, releasing them outside the granules for the plant to use.

By the term “nutrients”, it is necessary to understand fertilizers for plants, in particular chemical, organic or biological fertilizers such as for example nitrogenous nutrients, in particular nutrients containing ammonium compounds, amines or animal proteins.

Phosphate or peat present in the granules is excluded from the definition of the term nutrient above. The expression "except nutrient" therefore means "except for added nutrients".

Said inorganic phosphate granules coated in a matrix based on at least one polymerized hydrocolloid have the advantage of being stable in the matrix, which means that the granules and its constituents (phosphate, peat, mycorrhizal fungus, etc.) when they are kept cold, that is to say between 2 and 6 ° C, and are not in contact with the plant or an aqueous medium, do not degrade and can be stored thus from a few days to several months, up to 6 months.

The matrix represents the medium in which the constituents are immobilized and consists of hydrocolloids, in particular a binding agent of natural, plant or animal origin.

When the granules are brought into contact with a plant, the role of mycorrhizal symbiosis on the development of the host plant will be optimized by intimately combining the impact of the fungus on the plant directly (hormonal, mineral effect, etc.) but also indirectly by alteration of inorganic phosphate thus allowing phosphorus ions to be released to the plant.

The inventors have, surprisingly, found that the coating of phosphate with peat, a mycorrhizal fungus and a hydrocolloid in the form of polymerized granules not only allowed the solubilization of the phosphate and the hormonal and mineral effect of the fungus but also exerted a synergistic action on the aerial and root biomass of plants compared to the aerial and root biomass obtained with granules without alginate or without phosphate when the granules are brought into contact with a plant.

In an advantageous embodiment, the present invention relates to granules as defined above, consisting of inorganic phosphate, peat, at least one mycorrhizal inoculum, said granules being coated in a matrix based on at least one. polymerized hydrocolloid.

In this embodiment, the granules contain only peat, inorganic phosphate, inoculum and matrix with the exception of any other component.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the mycorrhizal inoculum comprises at least one ectomycorrhizal fungus, of the genus Hebeloma, and / or of the genus Scleroderma, and / or of the genus Laccaria. ,

By ectomycorrhizal fungus is meant a fungus whose mycelium develops between the cells of the root cortex, but does not penetrate into living cells, thus forming “the Hartig intercellular network”. It forms a fungal mantle around the root. (Smith, SE & Read, DJ (1997). Mycorrhizal symbiosis. ^2nd edition, UK, Academic Press.)

In an advantageous embodiment, the fungus of the genus Hebeloma is chosen from the group consisting of: Hebeloma anthracophilum, Hebeloma cistophilum, Hebeloma crustuliniforme, Hebeloma fastibile, Hebeloma mesophaeum, Hebeloma pallidum, Hebeloma radicosum, Hebeloma sarcophyllum and Hebeloma Hebeloma sinapizans, in particular crustuliniform.

In an advantageous embodiment, the fungus of the Scleroderma genus is chosen from the group consisting of: Scleroderma dyctiosporum and Scleroderma verrucosum and preferably Scleroderma verrucosum

In an advantageous embodiment, the fungus of the genus Laccaria is chosen from the group consisting of: Laccaria amethystea, Laccaria bicolor, Laccaria laccata, Laccaria proxima, Laccaria purpureo-badia and Laccaria tortilis, in particular Laccaria bicolor. m

In a preferred embodiment of the method described above, the ectomycorrhizal fungus is used in spaces intended for forest crops.

Forest crops include, in particular, Myrtaceae, Pinaceae, Abietaceae, Fagaceae, Tiliaceae, Ulmaceae and Salicaceae; this list is not exhaustive.

The term “spaces intended for cultures” designates here as well as throughout the present description, any type of container comprising elements suitable for culture such as pots, trays, jars, honeycomb plates without being limited thereto as well. that any type of land, piece of land, field, greenhouse ...

In an advantageous embodiment, the present invention relates to granules as defined above, in which the mycorrhizal inoculum comprises at least one arbuscular mycorrhizal fungus of the genus Glomus.

The term “arbuscular mycorrhizal fungus” is understood to mean a fungus, the mycelium of which penetrates the root cells and develops arbuscules or vesicles, when it is associated with the roots of plants. H does not form a fungal mantle around the root. (Smith, SE & Read, DJ. (1997). Mycorrhizal symbiosis. ^2nd edition, UK, Academic Press.)

According to an advantageous embodiment of the present invention, the arbuscular mycorrhizal fungus of the genus Glomus is chosen from the group consisting of: Glomus mosseae, Glomus fasciculatus, Glomus aggregatum and Glomus intraradices, in particular Glomus intraradices.

According to yet another embodiment of the above method, the arbuscular mycorrhizal fungus is used in areas intended for crops, in particular cereals, fodder, market gardens, fruit or horticulture and preferably market gardening, fruit or horticulture.

Cereal crops include barley, oats and wheat, but this list is not exhaustive.

By fodder culture we must understand legumes such as clover, alfalfa and trefoil trefoil, and grasses, such as timothy, orchard grass, this list not being exhaustive.

Vegetable crops include tomatoes, eggplants, potatoes, carrots, lettuce, cucumber, melon, cabbage, cauliflower ...

Fruit crops include apples, pears, peaches, grapes, cherries, plums, citrus fruits, this list not being exhaustive.

Horticultural crops include flowers, ornamental plants, and this list is not exhaustive.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the mycorrhizal inoculum comprises at least one ectomycorrhizal fungus of the genus Hebeloma, and / or of the genus Scleroderma, and / or of the genus Laccarta, and at least one arbuscular mycorrhizal fungus of the genus Glomus.

In an advantageous embodiment, the fungi of the genus Hebeloma, of the genus Scleroderma, of the genus Laccaria and of the genus Glomus are as defined above.

This association of ectomycorrhizal fungus and arbuscular mycorrhizal fungus is more particularly advantageous for fast-growing woody species of the genera Casuarina, Allocasuarina, Eucalyptus and Acacia of Australian origin.

These forest species are frequently used in reforestation programs in humid and dry tropical environments, and the combination of the two types of mycorrhizal fungi results in growth gains over those experienced when only one type of fungus is inoculated.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the weight of mycorrhizal inoculum is from about 0.05% (w / w) to 0.2% (w / p) in particular 0.11% (w / w) relative to the total weight of the granules.

The percentages are expressed in dry weight of inoculum relative to the weight of the granules.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the weight of peat is from about 2.2% (w / w) to about 3.3% (w / w ) in particular 2.8% (w / w) relative to the total weight of the granules.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the weight of inorganic phosphate is from about 36.7% (w / w) to about 37.8% (w / w). p) in particular 37% (w / w) relative to the total weight of the granules.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the inorganic phosphate is rock phosphate or pozzolan.

Pozzolana is a volcanic rock very rich in phosphorus, completely insoluble in water and which can only be solubilized by a mycorrhizal fungus.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the inorganic phosphate is sized to a diameter of about 2 mm to about 7 mm, in particular 5 mm.

The phosphate calibration makes it possible to homogenize the supply of granules in the soil. Too large granules (more than 7 mm) would not allow rapid contact between the symbiote and the plant (death of the mycelium).

Too small granules (less than 2 mm) would not offer a minimum capacity for the supply of fungal mycelium but also adequate protection of the fungus against environmental variations in the environment (abiotic stress).

In an advantageous embodiment, the present invention relates to granules as defined above, wherein the weight of polymerized hydrocolloid is from about 1.7% (w / w) to about 2.8% (w / w). / w), in particular 2.2% (w / w) relative to the total weight of the granules.

In an advantageous embodiment, the present invention relates to granules as defined above, in which said matrix is based on chitosan, carrageenans, carboxymethylcellulose, alginates and in particular sodium or calcium alginate. .

Calcium alginate can be obtained from sodium alginate by ion exchange, in particular by reaction of sodium alginate with a divalent cation such as calcium chloride, calcium acetate, sodium carbonate. calcium, calcium phosphate, preferably calcium chloride.

Chitosan is produced by deacetylation of chitin present in the exoskeleton of insects and other arthropods (crustaceans, arachnids, etc.). It is an amino polysaccharide made up of N-acetyl-D-glucose-2-amine groups linked together by a β- (1,4) type bond.

Carrageenans are linear polysaccharides made up of more or less substituted galactose molecules. The chain is made up of subunits called carrabioses comprising two galactoses linked by a β- (1,4) bond. These carrabioses are linked together in the chain by α- (l, 3) bonds.

Carboxymethylcellulose is a polymer derived from natural cellulose formed by the reaction of cellulose with a base and chloroacetic acid. It is based on a β- (1,4) -D-glucopyranose structure.

Alginates are polymers of alginic acid (consisting of mannuronic acid and guluronic acid) obtained from brown algae (Laminariales, Fucales). The sodium alginate is extracted from the algae with soda and then dried to obtain a white powder, with a molecular weight ranging from 32,000 to 200,000 Da.

In an advantageous embodiment, the present invention relates to granules as defined above, in which the polymerized hydrocolloid is calcium alginate.

According to another aspect, the invention relates to the use of granules comprising in particular at least one ectomycorrhizal fungus, as defined above, in spaces intended for forest crops.

According to another aspect, the invention relates to the use of granules comprising in particular at least one arbuscular mycorrhizal fungus, as defined above, in spaces intended for crops, in particular cereals, fodder, market gardens, fruit or horticultural crops and preferably market gardening, fruit or horticulture

In an advantageous embodiment, the invention relates to the use of granules comprising in particular at least one ectomycorrhizal fungus, or an arbuscular mycorrhizal fungus, in which the proportion of granule per 100 ml of substrate containing a unitary culture element is included. from about 0.05% (w / v) to about 0.5% (w / v) in particular 0.1% (w / v).

Another advantage of the invention consists in the optimization of the conditions for mycorrhization and phosphate solubilization allowing the use of very small quantities of granules.

The containers used for the production of forest seedlings have a volume of 0.5 l to l. For a 0.5 l container, the weight of granules will be approximately l g per jar, i.e. 0.2 g per 100 ml of substrate

Below 0.05%, the proportion of granules is too low to observe a significant improvement in root biomass.

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Above 0.5%, increasing the amount of granules does not produce any substantial improvement over 0.5%.

The expression “unitary cultivation element” here designates a seed, seed, grain, fruit, pip, stone or a plant without being limited to these, making it possible to obtain a forest, cereal, fodder, market garden, fruit or horticultural crop. .

In an advantageous embodiment, the invention relates to the use of granules comprising in particular at least one ectomycorrhizal fungus, and at least one arbuscular mycorrhizal fungus, in which the proportion of granule per 100 ml of substrate containing a unitary culture element is from about 0.05% (w / w) to about 0.5% (w / w) in particular 0.1% (w / w).

The containers used are ^m me as above defined.

Below 0.05%, the proportion of granules is too low to observe a significant improvement in root biomass.

Above 0.5%, increasing the amount of granules does not produce any substantial improvement over 0.5%.

According to another aspect, the invention relates to a process for preparing granules as defined above, comprising a step of polymerization of inorganic phosphate granules impregnated with peat, at least one mycorrhizal inoculum and at least one hydrocolloid. with an aqueous solution of a divalent cation chosen from among calcium chloride, zinc acetate, calcium acetate, calcium carbonate, calcium phosphate, aluminum chloride or dialdehydes.

Contact of the hydrocolloid with the divalent cation causes polymerization of said hydrocolloid which then immobilizes the various constituents present (mycorrhizal inoculum, peat) coated on the phosphorus granules.

In an advantageous embodiment, the invention relates to a process for preparing granules, as defined above, comprising the following steps:

a) multiplication of at least one mycorrhizal mycelium in a fermenter on a nutrient medium suitable according to the fungal strain cultivated to obtain a multiplied mycorrhizal mycelium,

b) harvesting said mycelium by filtration, washing the mycelium with water and then grinding to obtain a harvested multiplied mycorrhizal mycelium, AJ

c) sizing of inorganic phosphate granules, in particular rock phosphate or pozzolan and washing the granules with water to obtain washed calibrated granules,

d) preparation of the immersion solution for the granules composed of:

i. at least one hydrocolloid, ii. crushed peat previously sterilized, and iiî. mycorrhizal mycelium,

e) immersion of the washed calibrated granules obtained in step c) in the immersion solution to obtain granules impregnated with hydrocolloid, peat and mycelium,

f) introduction of the impregnated granules into an aqueous solution of a divalent cation to obtain polymerized granules,

g) removal of traces of divalent cation by washing with water to obtain washed polymerized granules,

The multiplication and harvesting of the mycelium are carried out according to Duponnois & Garbaye (1991); DUPONNOIS, R. & GARBAYE, J. (I99l). Techniques for controlled synthesis of the Douglas fir-Laccaria laccata ectomycorrhizal symbiosis. Annales des Sciences Forestières, 48: 239-251.

The immersion solution is an aqueous solution.

The different constituents are mixed at room temperature, ie from 20 to ° C.

The hydrocolloid is chosen from chitosan, carrageenans, carboxymethylcellulose, alginates and in particular sodium or calcium alginate.

Preferably, the hydrocolloid is sodium alginate.

The hydrocolloid is present from 15g to 25g per liter of immersion solution, in particular 20g. Below 15g, the amount of hydrocolloid is insufficient to obtain correct polymerization.

Above 25g, the proportion of hydrocolloid is too high compared to the other constituents, thus leading to granules which can no longer release the various constituents.

The peat must first be crushed and sterilized. Grinding allows a

homogenization of the granule immersion solution. Sterilization eliminates microbial agents that may compete with the fungus. The grinding can be carried out by any device well known to those skilled in the art.

The proportion of peat is between 20 and 30 g / l of immersion solution, in particular 25 g / l.

Peat is used to support the mycelium, to acidify the environment for optimal development of the fungus (pH = 5.5)

The immersion of the granules in the immersion solution is carried out at room temperature for 60 min to 120 min, in particular 90 min.

The immersion step has the effect of impregnating, that is, adsorbing the hydrocolloid, inoculum and peat on the phosphate granules.

The introduction of the granules impregnated with hydrocolloid, inoculum and peat in an aqueous solution of divalent cation is then carried out at room temperature for 120 min to 180 min, in particular 150 min.

The divalent cation is chosen from calcium chloride, zinc acetate, calcium acetate, calcium carbonate, calcium phosphate, aluminum chloride or dialdehydes and preferably is calcium chloride.

This step has the effect of polymerizing the hydrocolloid on the phosphate granules, thus immobilizing the peat and inoculum coated on the phosphate granules.

After washing, the granules are recovered and can thus be stored for a few days to several months, up to six months, when they are kept cold, that is to say between 2 and 6 ° C, and preferably 4 ° C.

In an advantageous embodiment, the invention relates to a process for preparing granules, as defined above, in which the phosphate granules are sized to a diameter of about 2 to about 7 mm, in particular about 5 mm. .

The phosphate calibration allows is performed for the same reasons as above.

In an advantageous embodiment, the invention relates to a process for preparing granules, as defined above, in which the hydrocolloid is sodium alginate at a concentration in the immersion solution of approximately 10 to 30 g / l, in particular 20 g / l, the peat in the immersion solution is at a concentration of about 20 to 30 g / l, in particular 25 g / l, and the aqueous solution of divalent cation in the immersion solution is

Calcium chloride in a concentration of from about 50 to about 150 g / l, in particular 100 g / l.

According to another aspect, the present invention relates to a method of fertilizing forest species such as Pinus spp., Quercus spp., Isoberlinia glaucia, comprising bringing forest plants into contact with the polymerized granules comprising at least one ectomycorrhizal inoculum, such as as defined above, by unit crop element.

The expression "unitary crop element" here denotes a seed, seed, grain, fruit, pip, kernel or a plant without being limited to these making it possible to obtain a forest crop.

In particular, the unitary element of culture corresponds to a seed or a plant.

By bringing forest plants into contact with the granules, it is necessary to understand the introduction of the granules into, for example, a jar, container, pot ... containing a planting substrate and a young forest plant or a forest plant seed or a seed ....

In an advantageous embodiment, the present invention relates to a method of fertilizing forest species as defined above, comprising the following steps:

at. preparation of granules comprising an ectomycorrhizal inoculum, as defined above,

b. placing forest plants in contact with granules at a dose of about 1 g of granules to about 5 g of granules, in particular about 1 g of granules, per unit crop, in particular seed or plant.

In an advantageous embodiment, the ectomycorrhizal inoculum consists of at least one ectomycorrhizal fungus of the genus Hebeloma, and / or of the genus Scleroderma, and / or of the genus Laccaria, in particular the fungus of the genus Hebeloma is chosen from Hebeloma anthracophilum , Hebeloma cistophilum, Hebeloma crustuliniforme, Hebeloma fastibile, Hebeloma mesophaeum, Hebeloma pallidum, Hebeloma radicosum, Hebeloma sarcophyllum and Hebeloma sinapizans, in particular Hebeloma crustuliniforme, the fungus of the genus Scleroderma is chosen from the group consisting of: Sclerosclerosclerosclerosclerosperma and Sclerosclerodermenti and Sclerosclerodermi verrucosum and the fungus of the genus Laccaria is selected from the group consisting of: Laccaria amethystea, Laccaria bicolor, Laccaria laccata, Laccaria proxima, Laccaria purpureobadia and Laccaria tortilis, in particular Laccaria bicolor. fxl

According to another aspect, the present invention relates to a method of fertilizing cereal, fodder, market garden, fruit or horticultural species, comprising bringing cereal, fodder, market gardeners, fruit trees or horticultural plants into contact with the polymerized granules comprising at least one arbuscular mycorrhizal inoculum, as defined above, per unit culture element.

By bringing cereal, fodder, market gardeners, fruit trees or horticultural plants into contact with the granules, it is necessary to understand the introduction of the granules into, for example, a jar, container, pot ... containing a planting substrate and a young plant cereal, forage, market gardener, fruit or horticultural or a seed of cereal, forage, market gardening, fruit or horticultural plants or a seed ....

The expression "unitary crop element" here designates a seed, seed, grain, fruit, pip, stone or plant without being limited to these, making it possible to obtain a cereal, fodder, market garden, fruit or horticultural crop.

In particular, the unitary element of culture corresponds to a seed or a plant.

In an advantageous embodiment, the present invention relates to a method of fertilizing cereal, fodder, market garden, fruit or horticultural species, as defined above, comprising the following steps:

at. preparation of granules comprising at least one endomycorrhizal inoculum, as defined above,

b. bringing forest plants into contact with granules at a dose of approximately 1 g of granules to approximately 5 g of granules, in particular approximately 1 g of granules, per unit crop, in particular seed or plant

In an advantageous embodiment, the arbuscular mycorrhizal inoculum consists of at least one arbuscular mycorrhizal fungus of the genus Glomus, in particular the arbuscular mycorrhizal fungus of the genus Glomus is chosen from the group consisting of: Glomus mosseae, Glomus fasciculatus, Glomus aggregalum and Glomus intraradices, in particular Glomus intraradices.

According to another aspect, the present invention relates to a method of fertilizing forest species such as Pinus spp., Quercus spp., Isoberlinia glaucia, or cereal, fodder, market garden, fruit or horticultural species, comprising bringing into contact

I5 forest plants or cereal plants, forage, market gardeners, fruit trees or horticultural plants with the polymerized granules comprising at least one ectomycorrhizal inoculum and at least one arbuscular mycorrhizal inoculum, as defined above, per unit culture element.

The bringing into contact is as defined above for forest plants and cereal, fodder, market gardeners, fruit trees or horticultural plants.

The expression "unitary element of culture" designates here a seed, seed, grain, fruit, pip, pit or a plant without being limited to these making it possible to obtain a forest culture or a cereal, fodder, market garden, fruit crop. or horticultural.

In particular, the unitary element of culture corresponds to a seed or a plant.

In an advantageous embodiment, the present invention relates to a method of fertilizing forest, cereal, fodder, market garden, fruit or horticultural species, as defined above, comprising the following steps:

at. preparation of granules comprising at least one ectomycorrhizal inoculum and at least one arbuscular mycorrhizal inoculum, as defined above

b. bringing forest plants into contact with granules at a dose of approximately 1 g of granules to approximately 5 g of granules, in particular approximately 1 g of granules, per unit crop, in particular seed or plant

In an advantageous embodiment, the ectomycorrhizal inoculum consists of at least one ectomycorrhizal fungus of the genus Hebeloma, and / or of the genus Scleroderma, and / or of the genus Laccaria, and of at least one arbuscular mycorrhizal fungus of the genus Glomus, in particular the fungus of the genus Hebeloma, is chosen from Hebeloma anthracophilum, Hebeloma cistophilum, Hebeloma crustuliniforme, Hebeloma fastibile, Hebeloma mesophaeum, Hebeloma pallidum, Hebeloma radicosum, Hebeloma sarcophyllum and Hebeloma sinapizans, in particular Sbeloma crustermignon duulinclera selected from the group consisting of: Scleroderma dyctiosporum and Scleroderma verrucosum and preferentially Scleroderma verrucosum and the fungus of the genus Laccaria is chosen from the group consisting of: Laccaria amethystea, Laccaria bicolor, Laccaria laccata, Laccaria proxima, Laccaria purpureo tortilisia, and in particular Laccaria badilis Laccaria bicolor and in particular the fungus

Arbuscular mycorrhizal of the genus Glomus is selected from the group consisting of: Glomus mosseae, Glomus fasciculatus, Glomus aggregatum and Glomus intraradices, in particular Glomus intraradices.

EXAMPLES:

Example 1: General procedure for preparing a mycorrhizal inoculum (ectomycorrhizal or arbuscular mycorrhizal or ectomycorrhizal and arbuscular mycorrhizal)

The procedures which can be used are described in the following references: DUPONNOIS, R. & GARBAYE, J. (1991). Techniques for controlled synthesis of the Douglas fir-Laccaria laccata ectomycorrhizal symbiosis. Annales des Sciences Forestières, 48: 239-251. DUPONNOIS, R. & GARBAYE, J. (1991). Mycorrhization heiper bacteria associated with the Douglas fir-Laccaria laccata symbiosis: effects in vitro and in glasshouse conditions. Annales des Sciences Forestières, 48: 239-251.

DUPONNOIS, R., PLENCHETTE, C. & BA, A.M. (2001). Growth stimulation of seventeen 15 fallow leguminous plants inoculated with Glomus aggregatum in Senegal. European Journal of Soil Biology, 37: 181-186.

Example 2: Preparation of granules comprising a mycorrhizal inoculum (ectomycorrhizal or arbuscular mycorrhizal or ectomycorrhizal and 20 arbuscular mycorrhizal)

Step 1: Preparation of the mycorrhizal inoculum according to Example 1

Step 2: Harvesting the mycelium by filtration, washing the mycelium in running water and then crushing.

Step 3: Calibration of the natural phosphate granules (diameter 5 mm) and washing of the granules in running water

Step 4: Preparation of the immersion solution for the granules composed of: sodium alginate (20 g.l ' ¹ ), crushed peat previously sterilized (50 gf ¹ ) and fungal mycelium (1 g dry weight per liter of solution)

Step 5: Immersion of the calibrated granules (900 g) in the immersion solution at room temperature (20-25 ° C) for 90 min.

Step 6: Immersion of the granules from step 5 in a solution of calcium chloride (CaCl ₂ , 100 gl ' ^l ) for 150 min at room temperature (20-25 ° C)

Step 7: Removal of traces of CaCl ₂ by washing with running water

Step 8: Storage of the granules at 4 ° C.

Example 3: Comparison of the controlled mycorrhization of Pinus halepensis by the ectomycorrhizal fungus strain Scleroderma verrucosum in different consitions 3.1 'After 4 months of culture in sandy soil (Table I)

Culture is carried out as described in the article Ouahmane et al. ((2009); Responses of 10 Pinus halepensis growth, soil microbial catabolîc functions and phosphate-solubîlizîng bacteria after rock phosphate amendment and ectomycorrhizal inoculation, Plant Soil DOI 10.1007 / sl 1104-008-9882-z)

Table I

Treatments Witness 5. verrucosum (inoc T / V) ^(,) 5.verrucosum (inoc BA) S. verrucosum + KRP (inoc BA) ⁽³⁾ Above-ground biomass (mg dry weight) 159.3 a ^<4) 246.9 b 250.4 b 342.6 c Root biomass (mg dry weight) 68.6 a 86.5 b 85.4 b 100.1 c Nitrogen content of needles (%) 1.3 a 1.6 b 1.7 b 1.8 b Phosphorus content of 7.7 a 9.4 b 9.2 b 11.1 c

needles (g.kg * ¹ )

^(|) : Peat vermiculite inoculum applied at T = 0 at a rate of 10% (v / v) in each pot.

⁽²⁾ : Inoculum Ca alginate beads applied at a rate of 10 beads per pot.

⁽³⁾ Calcium alginate balls manufactured with a physical support consisting of natural phosphate granules according to Example 2 (I0 balls per pot)

Values on the same row followed by the same letter are not significantly different based on the one-way analysis of variance (P <0.05).

Table I shows that after 4 months of culture:

pine grown with ectomycorrhizal Scleroderma verrucosum and peat (2 ^EMC treatments column) or an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (3 ^CME treatments column) developed ground biomass and root significantly higher than that observed with the witness;

there is no significant difference between the culture with an ectomycorrhizal fungus Scleroderma verrucosum and peat (2 ^EMC treatments column) compared to an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (3 ^ime treatments column);

the pines cultivated with the granules of the invention (4 ^Èmc column treatments) developed an aerial and root biomass significantly higher than that observed with the cultivation of pines with an ectomycorrhizal fungus Scleroderma verrucosum and peat (2 to ^mc column treatments) compared with an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (3 ^rd treatment column), showing the synergy between the different constituents of the invention;

3.2 rAfter 12 months of cultivation in sandy soil (Table II) (after transfer of the mycorrhizal plants to 10-liter pots)

TABLE II

Treatments Witness KRP ^U) 5. S. S. S.

verrucosum (T / V inoc) (2) verrucosum + KR.P (inoc T / V) ⁽²⁾ verrucosum (inoc BA) verrucosum + KRP (inoc BA) ⁽⁴⁾ Above-ground biomass (mg dry weight) 321.2 a ⁽⁵⁾ 318.6 a 426.7 b 534.6 c 446.7 b 648.9 d Root biomass (mg dry weight) 158.9 a 149.7 a 248.9 b 298.2 c 252.4 b 320.6 d Nitrogen content of needles (%) 1.2 a 1.1 a 1.6 b 1.7 b 1.7 b 1.8 b Phosphorus content of needles (g kg *) 4.1 a 4.3 a 7.2 b 9.7 c 7.4 b 9.9 c

^(l) : Natural phosphate applied at 0.1% (m / v) in the surface layer of the pots (0 - 10 cm) W: Peat vermiculite inoculum applied at T = 0 at a rate of 10% (v / v) in each jar: Inoculum Ca alginate beads applied at a rate of 10 beads per jar at T = 0 ⁽⁴⁾ Calcium alginate beads manufactured with a physical support consisting of natural phosphate granules according to Example 2 (10 beads per jar ) ^<5) The values of the same row followed by the same letter are not significantly different according to the one-way analysis of variance (P <0.05)

Table II shows that after 12 months of culture:

pine cultured with only natural phosphate (2 ^emo column treatment) showed no significant difference of air obtained biomass compared to the control (the ^ERC treatments column);

pine grown with ectomycorrhizal Scleroderma verrucosum and peat (3 ^ome treatments column) or an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (5 ^ven treatments column) developed ground biomass and root significantly higher than that observed with the control or the natural phosphate;

there is no significant difference between the culture with an ectomycorrhizal fungus Scleroderma verrucosum and peat ^(3rd column treatments) compared to an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (5 ^th column treatments);

pines cultivated with an ectomycorrhizal fungus Scleroderma verrucosum, peat and ^rock phosphate (4 ^ômc column treatments) developed a significantly higher aerial and root biomass than that observed in pines cultivated with an ectomycorrhizal fungus Scleroderma verrucosum and peat ( 3 ^emc treatment column) or with an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (5 ^emc treatment column);

the pines cultivated with the granules of the invention (6 ^th column treatments) developed an aerial and root biomass significantly higher than that observed with the culture of pines cultivated with an ectomycorrhizal fungus Scleroderma verrucosum, peat and natural phosphate ( 4 ^th treatment column), showing the synergy showing the synergy between the various constituents of the invention thus demonstrating not only the intrinsic action of the fungus on the development of the plant but also its action on the dissolution of phosphate which is also stimulated by coating on the granules compared to uncoated granules (4 ^th treatment column) which avoids the dispersion of the phosphate.

JUNE 2013 tfïÎNET CAZENAVE sari

Claims (19)

1. Granules comprising inorganic phosphate, peat, at least one mycorrhizal inoculum, with the exception of nutrients or malt germ, said granules being coated in a matrix based on at least one polymerized hydrocolloid. 2. Granules according to claim 1, consisting of inorganic phosphate, peat, at least one mycorrhizal inoculum, said granules being coated in a matrix based on at least one polymerized hydrocolloid. 3. Granules according to claim 1 or 2, wherein the mycorrhizal inoculum comprises at least one ectomycorrhizal fungus, of the genus Hebeloma, and / or of the genus Scleroderma, and / or of the genus Laccaria, 4. Granules according to claim 1 or 2, wherein the mycorrhizal inoculum comprises at least one arbuscular mycorrhizal fungus of the genus Glomus. 5. Granules according to one of claims 1 to 4, wherein the weight of mycorrhizal inoculum is from about 0.05% (w / w) to 0.2% (w / w) in particular 0.11 % (w / w) relative to the total weight of the granules. 6. Granules according to one of claims 1 to 5, wherein the weight of peat is from about 2.2% (w / w) to about 3.3% (w / w), in particular 2.8%. (w / w) relative to the total weight of the granules. 7. Granules according to one of claims 1 to 6, wherein the weight of inorganic phosphate is from about 36.7% (w / w) to about 37.8% (w / w) in particular 37% ( w / w) relative to the total weight of the granules. 8. Granules according to one of claims 1 to 7, wherein the inorganic phosphate is rock phosphate or pozzolan. F 9. Granules according to claim 8 or 9, wherein the inorganic phosphate is sized to a diameter of about 2mm to about 7mm, in particular 5mm. 10. Granules according to one of claims 1 to 9, wherein the weight of polymerized hydrocolloid is from about 1.7% (w / w) to about 2.8% (w / w), in particular 2 , 2% (w / w) relative to the total weight of the granules. 11. Granules according to claim 10, wherein the polymerized hydrocolloid is calcium alginate. 12. Use of granules comprising in particular at least one ectomycorrhizal fungus, as defined in one of claims 1 to 3 and 5 to 11, in spaces intended for forest crops. 13. Use of granules comprising in particular at least one arbuscular mycorrhizal fungus, as defined in one of claims 1, 2 and 4 to 11, in spaces intended for crops, in particular cereals, fodder, market gardening, fruit or horticulture and preferably market gardening, fruit or horticulture 14. Use according to claim 12 or 13, wherein the proportion of granule per 100 ml of substrate containing a unitary culture element is from about 0.05% (w / v) to about 0.5% (w / v). v) in particular 0.1% (w / v). 15. Process for preparing granules as defined in one of claims 1 to 11, comprising a step of polymerization of inorganic phosphate granules impregnated with peat, at least one mycorrhizal inoculum and at least one hydrocolloid with a aqueous solution of a divalent cation chosen from calcium chloride, zinc acetate, calcium acetate, calcium carbonate, calcium phosphate, aluminum chloride or dialdehydes 16. Process for preparing granules according to claim 15, comprising the following steps: a) multiplication of at least one mycorrhizal mycelium in a fermenter on a nutrient medium suitable according to the fungal strain cultivated to obtain a multiplied mycorrhizal mycelium, b) harvesting said mycelium by filtration, washing the mycelium with water and then grinding to obtain a harvested multiplied mycorrhizal mycelium, c) sizing of inorganic phosphate granules, in particular rock phosphate or pozzolan and washing the granules with water to obtain washed calibrated granules, d) preparation of the immersion solution for the granules composed of: i. at least one hydrocolloid, ii. crushed peat previously sterilized, and iii. mycorrhizal mycelium, e) immersion of the washed calibrated granules obtained in step c) in the immersion solution to obtain granules impregnated with hydrocolloid, peat and mycelium, f) introduction of the impregnated granules into an aqueous solution of a divalent cation to obtain polymerized granules, g) removal of traces of divalent cation by washing with water to obtain washed polymerized granules, 17. The method of preparing granules according to claim 16, wherein the phosphate granules are sized to a diameter of about 2 to about 7 mm, in particular about 5 mm. 18. Process for preparing granules according to claim 16 or 17, wherein the hydrocolloid is sodium alginate at a concentration in the immersion solution of from about 10 to 30 g / l, in particular 20 g. / 1, the peat in the immersion solution is at a concentration of about 20-100 g / l, especially 50 g / l, and the aqueous solution of divalent cation in the immersion solution is calcium chloride at a concentration of from about 50 to about 150 g / l, in particular 100 g / l. 19. Process for fertilizing forest species such as Pînus spp., Quercus spp., Isoberlinia glaucia, comprising bringing forest plants into contact with the polymerized granules as defined in claims 1 to 3 and 5 to 11, by element unitary culture. 24 pages ORIGINAL 1- INSTITUTE OF RESEARCH FOR DEVELOPMENT (I.R.D.) 2- CADI AYYAD UNIVERSITY-FACULTY OF SCIENCES SEMLALIA, MARRAKECH-MOROCCO 3- CENTER FOR INTERNATIONAL COOPERATION IN AGRONOMIC RESEARCH FOR DEVELOPMENT (CIRAD)