EP2654435A1 - Novel fungal inocula compositions, method for preparing same, and use thereof for enhancing culture growth - Google Patents

Abstract

The present invention relates to novel fungal inocula compositions, to the method for preparing same, and to the use thereof for enhancing culture growth. (No drawings)

Description

 NOVEL FUNGAL INOCULA COMPOSITIONS, PROCESS FOR THE PREPARATION THEREOF AND THEIR APPLICATION TO ENHANCEMENT OF THE

 GROWTH OF CULTURES

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

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

 The importance of mycorrhizal fungi in forest plantations was first identified in 1910 in introductions of exotic pines to Kenya. While these plantations usually resulted 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 growing medium in nursery.

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

 This technique, using soil rich in mycorrhizal fungal propagules, was soon successfully disclosed in other African countries such as Tanzania, Uganda, Malawi, etc.

Since then, several mycorrhizal 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 that perform for a given parameter ( for example: effect of the strain on the growth of the host plant) and (iii) multiplication of the strain in axenic conditions and production of fungal inocula. There are two types of mycorrhizal fungi, the ectomycorrhizal fungi, which mainly concern forest species and mycorrhizal fungi with arbuscules, which concern herbaceous plant species.

 For ectomycorrhizal fungi, several inoculum formulations were tested and showed dramatic nursery effects.

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

 However, this inoculum does not include phosphate.

 US 5,021,350, US 5,096,481 and CN 2010 1212881 disclose compositions comprising a mycorrhizal inoculum embedded in a matrix and containing neither phosphate nor peat.

 Therefore, 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 aqueous phosphate in nonacidic soil, and secondly a loss of phosphate efficiency due to the dispersion of the powder in the culture substrate.

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

International Application WO 2007/066136 and JP1993-163583 describe compositions comprising a mycorrhizal inoculum, inorganic phosphate and respectively peat or compost. The 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 includes an inoculum associated with inorganic phosphate. This type of inoculum is for example described in Ouahmane et al. (Responses of Pinus halepensis growth, soil microbial catabolic functions and soil phosphate-solubilizing bacteria after rock phosphate amendment and ectomycorrhizal inoculation Plant Soil, DOI 10.1007 / sl 1 104-008-9882-z) which describe the effect of an ectomycorrhizal fungus (Pisolithus sp.) On the growth of pine (Pinus halepensis) plants with or without phosphate rock. The results show that when the said ectomycorrhizal fungus is used in the presence of phosphate, a synergy is then observed on the growth of the aboveground and / or root biomass compared to the aerial and / or root growth observed with the phosphate alone or the fungus alone. .

 However, the mushroom ectomycorrhizal fungi association has a major disadvantage, which is the weak impact of the ectomycorrhizal fungus on its ability to alter inorganic phosphate.

 Therefore, there is a need to provide non-polluting formulations to provide plants with the phosphorus necessary for their growth.

 One of the objects of the invention is therefore to provide a mycorrhizal fungus formulation in the form of granules embedded in a matrix allowing mycorrhizal fungi to alter the complex inorganic phosphates to release bioavailable P ions 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 grain that may range in size from about 2 to about 10 mm. By the term "inorganic phosphate" is meant any inorganic phosphate, such as rock phosphate (or natural phosphate), which can be found in different countries such as, for example, New Zealand, Peru, Egypt or Morocco without being limited to them, or lapouzolane, which will allow the release of phosphorus to the plant

 By the expression "embedded in a matrix", it is to be understood that the granules and their 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 brought into contact with the plant, and thus make the phosphor ions accessible to the plant, by releasing them outside the granules so that the plant can use them.

 By the term "nutrients" is meant plant nutrients, including chemical, organic or biological fertilizers such as nitrogen nutrients, especially nutrients containing ammonium compounds, amines or animal proteins.

 The phosphate or peat present in the granules are excluded from the definition of the nutrient term above. The expression "with the exception of nutrients" means "with the exception of added nutrients".

 Said inorganic phosphate granules embedded 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 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 kept for 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, especially a fixing agent of natural origin, plant or animal.

When the granules are brought into contact with a plant, the role of the 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 effect, mineral, etc.) but also indirectly by altering the inorganic phosphate thus making it possible to release phosphorus ions 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 above-ground and root biomass obtained with granules without alginate or without phosphate when the granules are put in 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 constituent.

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 enter living cells, thus forming "the Hartig intercellular network". It forms a fungal mantle around the root. (Smith, SE & Read, DJ (1997), Mycorrhizal symbiosis, 2 ^nd edition, UK, Academy 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 sinapizans, in particular Hebeloma crustuliniforme. In one advantageous embodiment, the fungus of the genus Scleroderma is chosen from the group consisting of: Scleroderma dyctiosporum and Scleroderma verrucosum and preferentially Scleroderma verrucosum

 In an advantageous embodiment, the fungus of the genus Laccaria is selected from the group consisting of: Laccaria amethystea, Laccaria bicolor, Laccaria laccata,

Laccaria proxima, Laccaria purpureo-badia and Laccaria tortilis, especially Laccaria bicolor.

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

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

 The term "spaces intended for cultivation" means here as well as throughout the present description, any type of container comprising elements suitable for culture such as pots, trays, jars, blister packs without being limited thereto as well. that any type of ground, piece of ground, field, greenhouse ....

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

The arbuscular mycorrhizal fungus is a fungus whose mycelium penetrates the root cells and develops arbuscules or vesicles when associated with plant roots. It does not form a fungal mantle around the root (Smith, SE & Read, DJ (1997), Mycorrhizal symbiosis, 2 ^nd edition, UK, Academy Press.)

 According to an advantageous embodiment of the present invention, the arbuscular mycorrhizal fungus of the genus Glomus is selected 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 spaces intended for cultivation, in particular cereals, fodder, vegetable, fruit or horticultural crops and preferentially market garden, fruit or horticultural crops.

 Cereal crops include, but are not limited to, barley, oats and wheat.

 Forage crops include legumes such as clover, alfalfa and bird's-foot trefoil, and grasses, such as the meadow filly, orchardgrass, this list is not exhaustive.

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

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

 The horticultural crops include flowers, ornamental plants, 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 Laccaria, and at least one mycorrhizal arbuscular fungus of the genus Glomus.

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

 This association of ectomycorrhizal and mycorrhizal fungi with arbuscules is more particularly advantageous for fast-growing woody species of the Casuarina, Allocasuarina, Eucalyptus and Acacia genera 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 provides growth gains over those recorded when only one type of fungus is inoculated.

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

The percentages are expressed by dry weight of inoculum relative to the weight of the granules. In an advantageous embodiment, the present invention relates to granules as defined above, 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.

 In an advantageous embodiment, the present invention relates to granules as defined above, wherein the weight of inorganic phosphate ranges 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, wherein the inorganic phosphate is rock phosphate or pozzolan.

 Pozzolan is a volcanic rock very rich in phosphorus, completely insoluble in water and 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 calibrated to a diameter of about 2 mm to about 7 mm, in particular 5 mm.

 Calibration of the phosphate makes it possible to homogenise the supply of granules in the soil. Granules too big (more than 7 mm) would not allow to obtain a fast contact between the symbiont and the plant (death of the mycelium).

 Too small granules (less than 2 mm) would not offer a minimum capacity for fungal mycelium delivery but also adequate protection of the fungus against environmental environmental variations (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). / p), 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, calcium carbonate, 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 consisting 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 consists of subunits called carrabioses comprising two galactoses linked by a β- (1,4) bond. These carrabioses are linked together in the chain by α- (1,3) bonds.

 Carboxymethylcellulose is a polymer derived from natural cellulose formed by 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). 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 crops, vegetable gardens, fruit or horticultural crops and preferentially market garden, fruit or horticultural

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 granules per 100 ml of substrate containing a unitary unit of culture is from about 0.05% (w / v) to about 0.5% (w / v), especially 0.1% (w / v).

 Another advantage of the invention consists in the optimization of the conditions of mycorhization and phosphate solubilization allowing the use of very small amount of granules.

 The containers used for the production of forest seedlings have a volume of 0.5 1 to 1 liter. For a container of 0.5 1, the weight of granules will be about 1 g per pot or 0.2 g per 100 ml of substrate

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

 Above 0.5%, the increase in the amount of granules produces no substantial improvement over 0.5%.

 The expression "unitary unit of culture" here designates a seed, seed, grain, fruit, seed, kernel or plant without being limited thereto making it possible to obtain a forest, grain, fodder, vegetable, fruit or horticultural crop. .

In one 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, wherein the proportion of granules per 100 ml of substrate containing a unitary unit of culture is from about 0.05% (w / w) to about 0.5% (w / w), particularly 0.1% (w / w).

The containers used are ^m me as above defined.

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

 Above 0.5%, the increase in the amount of granules produces no substantial improvement over 0.5%.

According to another aspect, the invention relates to a process for the preparation of 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 selected from calcium chloride, zinc acetate, calcium acetate, calcium carbonate, calcium phosphate, aluminum chloride or dialdehydes.

 The contact of the hydrocolloid with the divalent cation causes the 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 suitable nutrient medium 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 multiplied mycorrhizal mycelium harvested,

 c) calibrating granules of inorganic phosphate, 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. ground peat previously sterilized, and

 iii. mycorrhizal mycelium,

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

 f) introducing 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 the harvest of the mycelium are carried out according to Duponnois & Garbaye (1991); DUPONNOIS, R. & GARBAYE, J. (1991). Douglas fir-Laccaria laccata ectomycorrhizal symbiosis. Annals of Forestry Science, 48: 239-251. The immersion solution is an aqueous solution.

 The mixture of the various constituents is carried out at ambient temperature, ie from 20 to

25 ° C.

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

 Preferably, the hydrocolloid is a sodium alginate.

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

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

 The peat must first be crushed and sterilized. Grinding allows homogenization of the immersion solution of the granules. Sterilization eliminates microbial agents that may compete with the fungus. The grinding can be carried out by any apparatus well known to those skilled in the art.

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

 The peat is used to support the mycelium, to acidify the medium for an optimal development of the mushroom (pH = 5.5)

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

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

 Subsequently, granules impregnated with hydrocolloid, inoculum and peat are introduced into an aqueous divalent cation solution at ambient temperature for 120 minutes to 180 minutes, in particular 150 minutes.

The divalent cation is chosen from calcium chloride, zinc acetate, calcium acetate, calcium carbonate, calcium phosphate, aluminum chloride or dialdehydes and preferentially calcium chloride. This step has the effect of polymerizing the hydro colloid on the phosphate granules thus immobilizing the peat and inoculum coated on the phosphate granules.

 After washing, the granules are recovered and can be preserved for a few days to several months, up to six months, when these 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 calibrated to a diameter of about 2 to about 7 mm, in particular about 5 mm. .

 The calibration of the phosphate 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 about 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 at a concentration of from about 50 to about 150 g / l, especially 100 g / l.

 In another aspect, the present invention relates to a method of fertilizing forest species such as Pinus spp., Quercus spp., Isoberlinia glaucia, comprising contacting forest plants with polymerized granules comprising at least one ectomycorrhizal inoculum, such as as defined above, per unit unit of culture.

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

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

 By contacting forest plants 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 seed of a forest plant or a seed ....

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

at. preparation of granules comprising an ectomycorrhizal inoculum, as defined above, b. contacting forest seedlings 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 culture element, 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 selected from the group consisting of: Scleroderma dyctiosporum and Scleroderma verrucosum and preferentially Scleroderma verrucosum and the fungus of the genus Laccaria is selected from the group consisting of: Laccaria amethystea, Laccaria bicolor, Laccaria laccata, Laccaria proxima, Laccaria purpureo- badia and Laccaria tortilis, in particular Laccaria bicolor.

According to another aspect, the present invention relates to a method for fertilizing cereal, forage, vegetable, fruit or horticultural species, comprising contacting cereal, forage, vegetable, fruit or horticultural plants with the polymerized granules comprising at least one Mycorrhizal inoculum with arbuscules, as defined above, per unit culture element.

 By bringing grain, fodder, vegetable, fruit 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. grain, forage, market gardener, fruit or horticultural or a seed of cereal plants, forage, market gardener, fruit or horticultural or a seed ....

 The expression "unitary element of culture" here designates a seed, seed, grain, fruit, seed, kernel or a plant without being limited thereto making it possible to obtain a cereal, forage, 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 for fertilizing cereal, forage, vegetable, 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. contacting forest seedlings 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 culture element, in particular seed or plant. Advantageously, the arbuscular mycorrhizal inoculum consists of at least one mycorrhizal arbuscular fungus of the genus Glomus, in particular the arbuscular mycorrhizal fungus of the genus Glomus is selected from the group consisting of: Glomus mosseae, Glomus fasciculatus, Glomus aggregatum and Glomus intraradices, especially Glomus intraradices.

 According to another aspect, the present invention relates to a method for fertilizing forest species such as Pinus spp., Quercus spp., Isoberlinia glaucia, or cereal, forage, market garden, fruit or horticultural species, comprising contacting forest seedlings or grain, fodder, vegetable, fruit 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 contacting is as defined above for forest plants and grain plants, fodder, market gardeners, fruit or horticultural plants.

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

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

In one advantageous embodiment, the present invention relates to a method for fertilizing forest, cereal, forage, vegetable, 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. contacting forest seedlings 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 culture element, 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 at least one mycorrhizal fungus with arbuscules 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 Hebeloma crustuliniforme, the fungus of the genus Scleroderma is chosen from group consisting of: Scleroderma dyctiosporum and Scleroderma verrucosum and preferentially Scleroderma verrucosum and the fungus of the genus Laccaria is selected from the group consisting of: Laccaria amethystea, Laccaria bicolor, Laccaria laccata, Laccaria proxima, Laccaria purpureo- badia and Laccaria tortilis, in particular Laccaria bicolor and in particular the arbuscular mycorrhizal fungus 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 mycorrhizal arbuscules or ectomycorrhizal and mycorrhizal arbuscules)

The procedures that can be used are described in the following references: DUPONNOIS, R. & GARBAYE, J. (1991). Douglas fir-Laccaria laccata ectomycorrhizal symbiosis. Annals of Forestry Science, 48: 239-251. DUPO OIS, R. & GARBAYE, J. (1991). Mycorrhization helper bacteria associated with the Douglas fir-Laccaria laccata symbiosis: in vitro effects and in glasshouse conditions. Annals of Forestry Science, 48: 239-251.

 DUPONNOIS, R., PLENCHETTE, C. & BA, A.M. (2001). Growth stimulation of seventeen 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 mycorrhizal with arbuscules or ectomycorrhizal and mycorrhizal with arbuscules)

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

 Step 2: Harvest mycelium by filtration, wash the mycelium with running water and then grind.

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

Step 4: Preparation of the immersion solution for the granules composed of: sodium alginate (20 μl ^-1 ), ground peat previously sterilized (50 μl ¹ ) and fungal mycelium (1 g dry weight per liter of solution)

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

Step 6: Immersion of the granules of step 5 in a solution of calcium chloride (CaCl ₂ , 100 gl ^-1 ) for 150 min at room temperature (20-25 ° C.)

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

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

Example 3 Comparison of the Controlled Mycorrhization of Pinus HaLepensis by the Sectomycorrhizal Fungus Strain Scleroderma verrucosum in Various Consitions

3.1: After 4 months of cultivation in sandy soil (Table I) The culture is carried out as described in the article Ouahmane et al. ((2009); Responses of Pinus halepensis growth, soil microbial catabolic functions and phosphate-solubilizing bacteria after rock phosphate amendment and ectomycorrhizal inoculation, Plant Soil DOI 10.1007 / sl l04-008-9882-z)

 Table I

 : Inoculum Vermiculite peat applied at T = 0 at the rate of 10% (v / v) in each pot.

 : Inoculum Ca alginate balls applied at a rate of 10 beads per pot.

 Calcium alginate balls made with a physical support consisting of natural phosphate granules according to Example 2 (10 beads per pot)

 ^ The values of the same line followed by the same letter are not significantly different from the one-way analysis of variance (P <0.05).

Table I shows that after 4 months of cultivation: pine cultured with an ectomycorrhizal fungus Scleroderma verrucosum and peat ^(2nd column treatment) or with an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate ^(3rd column treatment) developed aerial and root biomass 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 ^nd column treatments) compared to an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate ^(3rd column treatments); pine cultured with the granules of the invention (treatments 4 ^th column) developed aerial and root biomass significantly higher than that observed with culture pins with an ectomycorrhizal fungus Scleroderma verrucosum and peat ^(2nd column treatment) compared with an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate ^(3rd column treatment), showing the synergy between the various components of the invention;

3.2: After 12 months of cultivation in sandy soil (Table II) (after transfer of mycorrhizal plants into 10-liter pots)

 TABLE II

 treatments

S. S. S.

 S.

KRP ⁽¹⁾ verrucosum verrucosum verrucosum

Witness verrucosum

 (inoc T / V) + KRP + KRP

(inoc BA) ⁽³⁾

(2) (inoc T / V) ⁽²⁾ (inoc BA) ⁽⁴⁾

biomass

(mg 321, 2 a ⁽⁵⁾ 318.6 to 426.7 b 534.6 c 446.7 b 648.9 d dry weight) biomass

root (mg 158.9 to 149.7 to 248.9 b 298.2 c 252.4 b 320.6 d dry weight)

Nitrogen content of 1, 2 to 1, 1 to 1, 6 b 1, 7 b 1, 7 b 1, 8 b needles (%)

 Phosphorus content

4.1 to 4.3 to 7.2 b 9.7 c 7.4 b 9.9 c needles (g-kg ¹ )

⁽ ': Natural phosphate applied at 0.1% (m / v) in the top layer of the pots (0-10 cm) ⁽²⁾ : Inoculum Vermiculite peat applied at T = 0 at a rate of 10% (v / v) in each pot: Inoculum Ca alginate balls applied at a rate of 10 beads per pot at T = 0

( ⁴⁾ Calcium alginate beads made with a physical carrier consisting of natural phosphate granules according to Example 2 (10 beads per pot)

 ^ Single-line values followed by the same letter are not significantly different from one-way analysis of variance (P <0.05)

 Table II shows that after 12 months of cultivation:

the pines grown with phosphate rock only (2 ^nd column treatments) do not show a significant difference in the aerial biomass obtained compared to that of the control ( ^1st column treatments);

pines grown with an ectomycorrhizal fungus Scleroderma verrucosum and peat (3 ^rd column treatments) or with an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (5 ^th column treatments) developed a significantly higher aerial and root biomass than that observed with the control or the natural phosphate; there is no significant difference between culture with an ectomycorrhizal fungus Scleroderma verrucosum and peat (3 ^rd column treatments) compared to an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (5 ^th column treatments);

pine cultured with an ectomycorrhizal fungus Scleroderma verrucosum, peat and natural phosphate (4 ^th treatment column) developed aerial and root biomass significantly higher than that observed pines cultured with an ectomycorrhizal fungus Scleroderma verrucosum and peat ( ^3rd column treatment) or with an ectomycorrhizal fungus Scleroderma verrucosum and calcium alginate (5 ^th column treatment);

the pines grown with the granules of the invention (6 ^th column treatments) developed an above-ground biomass and root biomass significantly higher than that observed with the cultivation of pines grown with an ectomycorrhizal fungus Scleroderma verrucosum, peat and phosphate rock ( 4 ^th column treatments), 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 otherwise stimulated by coating on the pellets compared to the uncoated granules (treatments 4 ^th column) which avoids the dispersion of phosphate.

Claims

1 CLAIMS 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 embedded in a matrix based on at least one polymerized hydrocolloid. Granules according to claim 1 or 2, wherein the mycorrhizal inoculum comprises at least one ectomycorrhizal fungus, genus Hebeloma, and / or genus Scleroderma, and / or genus Laccaria, 4. Granules according to claim 1 or 2, wherein the mycorrhizal inoculum comprises at least one mycorrhizal fungus with arbuscules 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) based on the total weight of the granules. 6. Granules according to one of claims 1 to 5, wherein the peat weight is from about 2.2% (w / w) to about 3.3% (w / w) especially 2.8% (w / w) relative to the total weight of the granules. Granules according to one of Claims 1 to 6, in which the weight of inorganic phosphate ranges from about 36.7% (w / w) to about 37.8% (w / w), in particular 37% ( p / p) 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. 2 Granules according to claim 8 or 9, wherein the inorganic phosphate is calibrated to a diameter of about 2 mm to about 7 mm, in particular 5 mm. 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. 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 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 1 1, in spaces for crops, including cereals, fodder, market garden, fruit or horticultural crops. and preferentially market gardeners, fruit or horticultural The use according to claim 12 or 13, wherein the proportion of granules per 100 ml of substrate containing a unitary unit of culture is from about 0.05% (w / v) to about 0.5% (w / w). v) in particular 0.1% (w / v). 15. Process for the preparation of granules as defined in one of claims 1 to 11, comprising a polymerization step 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 selected from calcium chloride, zinc acetate, calcium acetate, calcium carbonate, calcium phosphate, aluminum chloride or dialdehydes. 3 The process for preparing granules according to claim 15, comprising the following steps: a) multiplying at least one mycorrhizal mycelium in a fermenter on a nutrient medium adapted 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 multiplied mycorrhizal mycelium harvested,  c) calibrating granules of inorganic phosphate, 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 a hy dro coeloid,  ii. ground peat previously sterilized, and  iii. mycorrhizal mycelium,  e) immersing the washed granules obtained in step c) in the immersion solution to obtain granules impregnated with hydrocolloid, peat and mycelium,  f) introducing 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 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. The 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, peat in the The immersion solution is at a concentration of about 20 to 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 about 50 to about 150 g / l, especially 100 g / l 19. A method of fertilizing forest species such as Pinus spp., Quercus spp., Isoberlinia glaucia, comprising contacting forest seedlings with the polymerized granules as defined in claims I to 3 and 5 to 11, per element unit of culture.