EA045371B1 - DOMINIKIA SP. STRAIN, COMPOSITIONS CONTAINING IT AND APPLICATION OPTIONS - Google Patents

Description

Field to which the invention relates

The present invention relates to the agronomic field. In particular, it concerns the strain

Dominikia sp, compositions containing it, and the use of these compositions as a biostimulant and bionematicide. Compositions containing the Dominikia sp. strain have been found to be particularly beneficial for cereal crops.

State of the art

Currently, about 300 species from 30 genera are described in the class Glomeromycota. However, nucleic acid (DNA, RNA)-based biodiversity studies indicate much higher diversity in the Glomeromycota and several new species and genera have recently been described.

Most Glomeromycota are arbuscular mycorrhizal fungi (AMFs), which are mutualistic symbionts of approximately 80% of all vascular plants. Nutrient uptake by plants colonized by AMF occurs directly through the root epidermis and root hairs, as well as through fungal-root interactions, which have the characteristic form of arbuscules or intraradical hyphal tangles. In addition to their role in enhancing host nutrient uptake, AMF play an important role in soil aggregation and plant protection from drought stress and soil pathogens. Because of this extremely beneficial feature, several mycorrhizal compositions are known in the art that have been developed to provide beneficial effects on the crops with which they are treated.

WO 2015/000612 and WO 2015/000613 disclose compositions containing the strain Glomus iranicum var. tenuihypharum var. nov. and their application.

In particular, WO 2015/000612 discloses a composition containing the strain Glomus iranicum var. tenuihypharum var. nov. and smectite clays in a ratio of 2:1. It has been revealed that the composition WO 2015/000612 has a positive effect on crop yields, i.e. the composition can be used as a biostimulator for lettuce crops.

Similarly, WO 2015/000613 discloses a composition containing the strain Glomus iranicum var. tenuihypharum var. nov. and smectite clays in a ratio of 2:1, metal ions and chitin. WO 2015/000613 discloses that this composition acts as a bionematicide for tomato crops.

However, the previously disclosed compositions do not provide an acceptable positive effect on crops, both as biostimulants and in terms of bionematicidal effect.

Brief description of the invention

It is therefore an object of the present invention to provide a strain of fungus that is suitable for inclusion in compositions, namely compositions suitable for providing a beneficial effect on agricultural crops, in particular cereal crops.

Another object of the invention is to provide a composition that is effective as a biostimulant for cereal crops.

Another object of the present invention is to provide a composition that is effective as a bionematicide for cereal crops.

Another object of the present invention is to provide a method for preparing compositions.

Detailed description

Thus, the object of the present invention is a strain of Glomeromycota, namely a strain of Dominikia sp., deposited under registration number MUCL 57072. The strain according to the invention was isolated from a very hydromorphic, highly compacted sodium-saline type of soil (solonetz gley) with a large amount of salt deposits on surface, in the region of Fortuna, Murcia (Spain).

Strain Dominikia sp. according to the invention, deposited on 03/21/2018 in the International Depository Body Belgian Coordinated Collections of Microorganisms (BCCM), Catholic University of Louvain, Mycothequeque de l'Universite catholique de Louvain (MUCL), Croix du Sud 2, box L7.05,06, 1348 Louvain-la-Neuve , Belgium, by Symborg S.L., at Campus de Espinardo, 7, edificio CEEIM, 30100 Murcia, Spain.

The strain Dominikia sp. according to the invention, the depositor was assigned the identifying reference SMB01, and the international depository was assigned the deposit number MUCL 57072.

The sporocarps of the strain of the invention are unknown. In the strain of the invention, spores in the soil occur in loose clusters and can be terminal or intercalary; spores may also form in the roots. The spores are transparent to light ocher, round to spherical in structure (rarely irregular), relatively small, i.e. from 24.0 to 42 µm in diameter, with an average of 30.7 ± 3.7 µm in diameter. The spores have a complex three-layer spore wall (1-4 µm thick). In particular, an outer layer, a middle layer and an inner layer can be recognized.

The outer layer of the spore wall is quite slimy and short-lived; this gives young spores a rough appearance, while older spores appear a little shaggy. The outer layer of the spore wall exhibits a dextrinoid reaction when stained with Meltzer's reagent, giving young spores a brownish-red color. The middle layer of the spore wall is quite constant and has a thickness of 0.5-2.0 microns.

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The inner layer of the spore wall is essentially laminar, 0.5-1.5 µm thick. The contents of the spores have a pale, spotted appearance. The hyphae holding the spores are transparent to pale ocher in color, straight or wavy, with a diameter of 2.5-4.5 µm (average 3.0 µm). The hypha is cylindrical or slightly funnel-shaped, which is connected to the open-porous layers of the spore wall, at least in mature spores. The germinating structure contains a growth tube that grows and develops through the attachment of the hypha to the spore. It forms vesicular arbuscular mycorrhiza.

Extraradical mycelium forms an extensive network.

The applicant has performed phylogenetic analysis of 134 18S rDNA sequences, including sequences from environmental samples and reference sequences. For most Dominikia species, no or relatively short partial 18S sequences were available. Sequences of Dominikia indica and D. iranica can be included in this phylogenetic analysis. The sequences of these two species were shorter than the rest of the sequences in the data set. Trimming the dataset to the length of these sequences resulted in rather incompletely resolved trees. The use of full-length sequences made it possible to obtain trees with branches representing higher taxonomic levels. However, with this data set it was not possible to construct a robust monophyletic clade consisting of sequences from Dominikia spp. Dominicia sp. forms a single, well-supported clade together with Dominikia iranica and anonymous Glomerales sequences from the environment, from a variety of host plants from various locations.

Phylogenetic analysis of the SSU-ITS1 dataset, which includes sequences from most described Dominikia species, indicates that the clade Dominikia sp. close to D. aurea.

The genus Dominikia is characterized by small spores (up to 65-70 µm in diameter), usually aggregated into loose or compact clusters. The spore wall of representatives of the genus Dominikia consists of two or three layers. The outer layer forming the surface of the spores is slimy, short-lived, stains with Meltzer's reagent or is solid (not divided into sublayers), permanent, and does not react with Meltzer's reagent. The hyphae that hold the spores are cylindrical or funnel-shaped with a pore that may be open or blocked by a septum. Dominikia minuta (Basidionym: Glomus minutum) was selected as the type species. The strain according to the invention, i.e. Dominicia sp. has transparent spores about the same size as Dominikia minuta, but unlike Dominikia sp. the pore of G. minutum (i.e. Dominikia minuta) is blocked by a septum. D. sp. also differs from Dominikia minuta in having a three-layer spore wall, as opposed to the two-layer spore wall of D. minuta, as well as in the presence of a dextrinoid reaction, which is absent in Dominikia minuta.

Dominicia sp. also differs from other described Dominikia species and strains. The outer and inner layers of Dominikia achra spores stain deep red in Meltzer's reagent, while Dominikia sp. only the outer layer exhibits a dextrinoid reaction. Dominikia indica is different from Dominikia sp. the formation of small transparent spores in hypogeous aggregates. The spore wall of D. indica consists of two hyaline layers: a thin, short-lived mucoid outer layer, stained pinkish or pink in Melzer's reagent, and a laminar, smooth, permanent inner layer, thicker than the three layers in Dominikia sp.

Based on 18S-ITS1 phylogeny, Dominikia aurea is the closest relative of the Dominikia sp. According to the invention, the two species differ in many morphological characteristics (see Table 1), the most obvious difference being the predominantly ovoid spores of Dominikia aurea, which are aggregated into irregular sporocarps.

Table 1

Dominikia aurea (Oehl et al. 2003) Dominicia sp. aggregation dispute irregular sporocarps without peridium loose clusters intraradical disputes not described present intercalary spores not described present color light orange or orange ocher form ovoid, rarely rounded round or spherical

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size ovoid spores: (36-) 55-65 x (30) 45-52 µm rounded spores: (27-) 40-60 µm (24.0) 30.7±3.7 (42) µm wall spores two-layer three-layer outer layer short-lived, transparent up to 1.5 microns, degenerate in mature spores, dextrinoid slimy, rough, degenerate (0.4-) 1.0 (1.5) µm, degenerate in mature spores, dextrinoid middle layer constant slightly rough 0.52.0 microns thick inner layer laminar, light orange, 1.5-3 (-4) µm laminar, smooth (0.5-1.5 µm) binding hyphae light orange to orange in color, straight or curved, cylindrical or slightly funnel-shaped; 6-10 microns transparent or pale ocher, straight or wavy, 2.5-5 microns germination unknown through the connecting hypha

Many sequences with high similarity to the invention sequence of Dominikia sp. can be discovered using a BLAST search against the gene bank (e-value 0.00, 99% identity). These sequences originate from a wide range of different hosts (e.g., liverworts, monocots) from different countries and continents, indicating their worldwide distribution. These results indicate that Dominikia sp. widely spread.

As mentioned above, it has been found to be particularly useful to include Dominikia sp. in compositions, in particular in compositions used for cereals.

As mentioned above, another object of the invention is a composition containing Dominikia sp., deposited on 03/21/2018 under registration number MUCL 57072.

Without being tied to a specific scientific explanation, it was unexpectedly discovered that Dominikia sp. and a composition containing it are particularly effective in providing beneficial effects on agricultural crops, in particular cereal crops.

In particular, it has been found that application of a composition containing Dominikia sp. to a cereal crop (eg corn, wheat, barley or rice) results in increased nutrient uptake and yield of the cereal crop compared to crops that have not been treated with Dominikia sp.

It is preferable that the concentration of Dominikia sp. in the composition was from 4.0 to 1.0% by weight, preferably from 3.0 to 2.0% by weight, even more preferably from 2.5 to 2.3% by weight.

In accordance with embodiments, the composition of the invention is a liquid, solid or gel composition.

Preferably, the composition according to the invention is a solid composition.

In accordance with embodiments, the composition of the invention may be in the form of a powder, an emulsifiable concentrate, granules or microgranules.

The composition according to the invention is solid, and the concentration of propagules of Dominikia sp. in the composition is measured according to the most probable number method (Porter, Aust. J. Soil Res., 1979, 17, 515-19) from 180 to 120 propagules per gram of composition, preferably from 150 to 120 propagules per gram of composition, even more preferably 125 to 120 propagules per gram of composition.

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Propagule concentration refers to the concentration of propagules in the final product.

According to a preferred embodiment, the composition of the invention is in the form of microgranules.

In accordance with embodiments, said microgranules have a size between

500 to 2000 µm, preferably 800 to 1500 µm, even more preferably 900 to 1200 µm.

Helpfully, the concentration of Dominikia sp. in the composition of the invention, as well as the form in which the composition is presented, for example microgranules, can be selected in accordance with the predetermined end use.

In accordance with embodiments, the composition of the invention further comprises at least one fungicide, and/or at least one biofungicide, and/or at least one insecticide, and/or at least one bioinsecticide, and/or at least one nematicide, and/or at least one biostimulant.

According to a preferred embodiment, the composition of the invention is formulated as a seed coating.

For example, said fungicide is selected from the group consisting of Maneb, Mancozeb, Metalaxil-Ridomil, Myclobutanil, Olpizan, Propamocarb, Quintozen, Streptomycin, Sulfur, Thiophanate-methyl, Thiram, Triforin, Vinclozolin, Zinc white, Zineb, Cyram, Benorad, associated copper, Chlorothalonil, Captan, Chloroneba, Cyproconazole, Zinc ethylene bisdithiocarbamate, etridiazole, fenaminosulf, fenarimol, flutolanil, folpet, fosetyl-AL and Iprodione.

Examples of biofungicides are: Trichodermas sp, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Streptomyces sp, Coniothyrium minitans and Pythium oligandrum.

In accordance with embodiments, the insecticide is selected from the group consisting of organophosphates, carbamates, and neonicotinoids.

In accordance with embodiments, said bioinsecticide is selected from the group consisting of Bacillus sp., Chromobacterium sp., Beauveria sp. and Metarhizium sp.

In accordance with embodiments, said nematicide is an organophosphate or carbamate.

In accordance with embodiments, said bionematicide is Pasteuria sp.

Another object of the present invention is a method for producing a composition containing a strain of the invention, Dominikia sp.

The method according to the composition invention includes the following steps:

a) providing a substrate;

b) introducing into the specified substrate the seeds of the host plant and the Dominikia sp. strain deposited under registration number MUCL 57072;

c) cultivating the specified host plant and watering to maintain the moisture level of the specified substrate at least 75% of the natural moisture capacity;

d) cessation of watering for a period of at least 7 days;

e) removal of the aboveground part of the host plant and substrate;

f) drying the removed substrate;

g) grinding the dried substrate to obtain granules with a particle size of less than 100 microns.

Preferably, the substrate contains clay.

In accordance with embodiments of Dominikia sp. introduced in step b) is an inoculum containing propagules of Dominikia sp. In a preferred embodiment, said inoculum is obtained by providing substantially pure Dominikia sp to the root system of a host plant, cultivating said host plant on a substrate preferably containing clay (preferably sterile smectite clay), preferably throughout its life cycle, and then extracting the root system to obtain the inoculum . In this case, the inoculum contains a certain amount of substrate, roots and propagules of Dominikia sp.

According to a preferred embodiment, the method according to the invention further comprises a microgranulation step h1).

Preferably, as a result of granulation, a composition containing the Dominikia sp strain according to the invention is obtained in the form of microgranules.

As mentioned above, said microgranules have a size of from 500 to 2000 μm, preferably from 800 to 1500 μm, even more preferably from 900 to 1200 μm.

According to a preferred embodiment, the method according to the invention further comprises step h2) of preparing a concentrated biological inoculum for cereal seed coating, namely a concentrated biological inoculum for coating cereal seeds from the ground product obtained in accordance with step g). It should be noted that in this context, step h2) of concentrating the biological inoculum for coating cereal seeds means the step in which the concentration of Dominikia sp. is increased. (e.g. propagule Dominikia sp.) in the composition.

For example, said concentration step h2) can be carried out by sieving the ground product obtained in accordance with step g) to separate granules having a particle size above a predetermined value, for example above 35 μm.

It is useful that the concentration step h2) allows you to increase the concentration of Dominikia sp. (e.g. Dominikia sp. propagules) up to 10 times the initial concentration, preferably up to 50 times the initial concentration (i.e. the concentration of Dominikia sp. in the product before step h2.), even more preferably up to 100 times the initial concentration, i.e. .e. 100 times relative to the concentration of the composition before step h2).

In accordance with embodiments, it is possible to increase the concentration of Dominikia sp. (e.g. propagule Dominikia sp.) more than 100 times the initial concentration (i.e. the concentration of Dominikia sp. in the product before step h2).

According to a preferred embodiment, the method according to the invention further comprises the step of coating the seeds i), namely coating the seeds with the concentrated product obtained in accordance with step h2).

Said seed coating step i) includes the following steps:

i1) coating the seeds with an adhesive substance;

i2) adding said concentrated biological inoculum;

i3) optionally carrying out one or more compatible treatments:

i3.1) treatment with fungicides, insecticides and/or herbicides compatible with mycorrhizal fungi;

i3.2) treatment with beneficial microorganisms, i3.3) treatment with macro- and/or micronutrients, i3.4) treatment with stimulants, i3.5) treatment with coloring pigments compatible with mycorrhizal fungi;

i4) drying seeds.

Another object of the present invention is the use of a composition containing Dominikia sp., deposited under registration number MUCL 57072, as a biostimulant.

As used herein, the term biostimulation refers to the stimulation that the composition of the present invention provides to plants that are treated with said composition.

Without reference to a specific scientific explanation, it can be assumed that the invention strain Dominikia sp. performs the function of moving nutrients, taking them from the soil or substrate and using them in its metabolic system. It moves said nutrients from the network of its mycelium and subsequently exchanges them in the root cells.

According to a preferred embodiment, the composition of the present invention is used as a biostimulant for cereal crops.

Another object of the present invention is the use of a composition containing Dominikia sp., deposited under registration number MUCL 57072, as a bionematicide.

In other words, the composition of the present invention can be used to protect plants from nematodes.

According to a preferred embodiment, the composition of the present invention is used as a bionematicide for cereal crops.

It is useful that the composition can be treated with the composition of plants, i.e. crop, such as a grain crop, in several ways.

In embodiments, the composition of the present invention can be applied to a plant by seed treatment (i.e., seed coating), root treatment, root emulsion, addition to irrigation water, irrigation, powder application to the root system, or injection application. emulsions into the root system.

It is preferred, in particular, that when it is to be applied to cereal crops, the composition according to the invention is applied by coating the seeds, or by combining with the seeds at the time of sowing, or in the form of a microgranulate and combining with the seeds at the time of sowing.

Brief description of drawings

In fig. Figure 1 shows the dynamics of spore formation and fungal colonization in plants, expressed as high optical density (%), treated with Dominikia sp.

In fig. Figure 2 shows the behavior of arbuscular external mycelium and arbuscular endophyte when treated with Dominikia sp.

In fig. Figure 3 shows the relationship between external arbuscular mycelium and arbuscular endophyte when treated with Dominikia sp.

experimental part

Example 1. Molecular analysis.

DNA extraction.

Isolated hyphae and spores were transferred into 1.5 ml Eppendorf tubes with 0.2 g glass beads (2 mm diameter) and 100 μl CTAB buffer (2% CTAB = cetyltrimethylammonium bromide, 1.4 M NaCl, 0.1 M Tris-HCl , pH 7.5, 0.2 M Na-EDTA). This mixture was homogenized using a ball mill

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Retsch MM 301 at 50 Hz for 30 s. Another 400 μl of CTAB buffer was added, the mixture was incubated at 65°C for 1 hour. 400 μl of a mixture of cholroform-isoamylol (24:1) was added to the suspension and mixed by inverting the reaction tubes, and then centrifuged for 5 min at 15000xg and the top layer was collected in a clean Eppendorf tube. This step was repeated twice. To this suspension was added 200 μl of 5 M ammonium acetate; the mixture was incubated at 4°C for at least 30 minutes, followed by 20 minutes of centrifugation at 4°C and 15,000xg. DNA was precipitated with 700 μl of isopropanol at -20°C overnight. The DNA precipitate obtained after precipitation with isopropanol was washed with ice-cold 70% ethanol, air-dried and redissolved in 50 μl of TE buffer (10 mM Tris, 10 mM EDTA, pH 8) + 4.5 units. RNase/ml.

PCR conditions.

18S rDNA was amplified using primers GEOA2, GEO11 (Schwarzott D., Schussler A. (2001), A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores, Mycorrhiza, 10:203-207 ). The primers used for PCR amplification and for sequencing the internal transcribed spacer region of 18S rDNA were Glom1310 and ITS4i (Redecker D. (2000), Specific PCR primers to identify arbuscular mycorrhizal fungi within colonized roots, Mycorrhiza, 10:73-80). Amplification was performed in 0.2 mM dNTP mixture, 1 mM of each primer, 10% PCR reaction buffer, and sterile molecular grade water. GoTaq® DNA polymerase (Promega, Mannheim, Germany) was added in an amount of 4 units/100 μl of the reaction mixture; 2 μl of genomic DNA template was used for every 20 μl reaction. Amplification was carried out in a Primus 96 advanced thermal cycler (peqLab Biotechnology) in 200 μl reaction tubes using the following PCR conditions: 96°C, initial denaturation 180 s; followed by 35 cycles: 96°C - 30 s, 58°C - 30 s, 72°C - 90 s; and final extension at 72°C for 10 min.

Sequence data and taxonomy of Glomeromycota.

The reference alignment published by Kruger et al. was used as the basis for the 18S phylogeny. (Kruger M., Kruger S., Walker S., Stockinger H., Schеβler A. (2012), Phylogenetic reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level, New Phytol., 193:970-984 ; downloadable at www.amf-phylogeny.com). To identify sequence similarities among environmental samples, the sequence of Dominikia sp. were analyzed through a BLAST search in GenBank, and highly similar sequences were included in the alignment forming the basis of the 18S tree. Published partial 18S-ITS1 - partial 5.8S sequences were used for the Dominikia phylogeny.

Data analysis.

Initial alignments were performed by Clustal W. Maximum likelihood phylogenetic analyzes were conducted via the CIPRES web portal (Miller M.A., Pfeiffer W., Schwartz T. (2010), Creating the CIPRES Science Gateway for inference of large phylogenetic tres. In Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, pp. 1-8, http://www.phylo.org/) with RAxML version 8.0 (Stamatakis et al., 2014) using 100 replicas bootstrap and GTRGAMMA model. The Bayesian consensus tree was constructed using MrBayes version 2.0.5. Two separate MS3 runs with randomly generated starting trees were performed for 2 M generations, each with one cold chain and three hot chains, using the GTR+I model. All parameters were estimated from the data. Trees were selected every 1000 generations. 200,000 generations were discarded as burn-in and consensus trees were constructed from the returned sample.

Example 2. Method for obtaining a variant of the composition according to the invention - a concentrated composition for coating seeds.

The first phase is greenhouse conditions.

Substrate. Smectite clays with a pH of 7.8 to 8 were chosen as the substrate, and before use they were sterilized in alternating cycles of 3 days.

AMG strain. Dominikia sp. propagules are used as the initial inoculum. in clean conditions. This type of inoculum is always maintained in continuous cycles of 90-180 days in either a growth chamber or controlled host plant greenhouses. Different plants are used to reproduce the inoculum in successive cycles to avoid disease transmission in the same host.

Summer autumn. Sorghum vulgare and Ocimum sp.

Winter-spring: Lolium perenne.

Culture pots. 15 liter pots are used.

Growing conditions for host plants.

Cultivation in a growth chamber or greenhouse begins with direct inoculation of the host plant's root system with a pure inoculum of a selected strain of arbuscular mycorrhizal fungus (Dominikia sp.) in a sterile smectite clay substrate. Plants are grown for a full life cycle according to plant type, which takes from 90 to 180 days. Plants

- 6 045371 is always kept well hydrated with daily irrigation (sterile water) at a temperature of 25 to 28°C and a relative humidity of 65%. Once completed, the root system containing the smectite substrate, rootlets and pure AMG shoots is removed and subsequently used for scaling in the second stage.

The following minimum specifications are used to determine inoculum quality:

total number of spores: 50-225 spores/g;

extramatric mycelium: >70 mg/kg substrate;

percentage of root colonization: Sorghum >50%, Lolium >45%, Ocimum >40%;

MPN concentration: >1x10 ⁴ propagules per 100 ml of substrate.

The second phase is scaling up.

Stage 1: preparing the beds.

The beds are made with plastic cushioning material, so that the bed is isolated from the surrounding soil; The beds are preferably made with a plastic covering so that drainage occurs and the growth of unwanted vegetation is prevented.

The beds should be filled with selected smectite clay (Arcilla Roja Galve). The moisture content of the clay should be approximately 15% to make it easier to work with when filling the bed. After filling, watering should be provided until saturated to improve the structure.

The beds are located in well-drained areas.

Beds can be of any size, allowing for access to facilitate the movement of people and equipment needed for maintenance.

The beds have an irrigation system according to local needs. The preferred system may be drip irrigation or a sprinkler, which should be automated and provide independent watering to selected areas of the bed.

Stage 2: host species.

Determination of host plant species and mycorrhizal fungi that can be used in the system: The selection and identification of host plant and fungal species will be tailored to the specific site conditions and production goals. Ryegrass (Lolium perenne) and AMG previously produced in the first stage are used.

Step 3: Host plant seeding and inoculation.

Before planting the host plant, the germination of seeds is checked. Based on the results of this test, the appropriate seeding rate is determined. For perennial ryegrass, seed is broadcast at 80 kg/ha using pre-tested pelleted seed. Also, together with the seeds, 20 g of AMF inoculum per ^m2 of bed is applied directly to the smectite clay.

Immediately after sowing, fine spray irrigation is used to prevent redistribution of seed and inoculum.

Water used for irrigation should preferably have the following characteristics:

pH values from >6 to <7.5;

electrical conductivity: <1.6 mS/m;

total soluble salts: <1000 ppm;

exchangeable sodium content (SAR) <10;

without heavy elements and pathogens; It is preferable that the water used for irrigation be potable.

Stage 4: Cultivation and Irrigation.

The irrigation applied should be sufficient to achieve 100% of natural water capacity, but avoid watering with too much water and causing it to accumulate or stagnate. The beds should be watered again when the clay moisture content drops to 75-80% of its natural moisture holding capacity.

Stage 5: management of the establishment of mycorrhizal symbiosis and study of the dynamics of the development of mycorrhizal fungal colonization.

During the growth and development of the host plant, AMF colonizes the roots and establishes a symbiosis between the plant and the fungus. To assess the development of this relationship, periodic sampling of the root system is carried out to assess mycorrhizal development. Methods used to assess colonization include Gerdeman & Nicolson (1963) (Gerdemann J.W., Nicolson T.N., 1963, Spores of mycorrhizal endogenous species extracted from soil by wet sieving and decanting, Transactions of the British Mycological Society, 46(2) ):235-44); McGonigle (1990) (McGonigle, T.P., Miller M.H., Evans D.G., Fairchild G.L., Swan J.A., 1990, A new method which gives an objective measure of colonization of roots by vesicular arbuscular mycorrhizal fungi, New Phytologist, 115(3):495 -501); and Phillips & Hayman (1970) (Phillips, J.M., Hayman D.S., 1970, Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection, Transactions of the British Mycological Society, 55:158-161).

Sampling begins two months after planting and continues monthly until the end of the growing season. Based on the information obtained from these samples, it is possible to determine the dynamics of the development of mycorrhizal symbiosis within the inoculum.

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Evaluation of the production method is based on mycorrhizal colonization of roots, extramatric mycelium concentration and periodic sampling of clay containing spores.

Knowledge of the dynamics of development of mycorrhizal products allows us to determine the optimal harvest time and make the most of the process of symbiosis between the host plant and mycorrhizal fungus.

Stage 6: Harvest.

If perennial Lolium is used as a host plant, the optimal harvest time is usually between 6 and 7 months after sowing because during this period the plants mature, complete their life cycle, tend to lose vigor and turn yellow.

Fifteen days before the scheduled harvest date, watering is stopped and leaf litter is stored to allow the clay to slowly lose moisture, ensuring the inoculation process is completed. If this operation coincides with the rainy season, it will be necessary to protect the bed from rain so that it can dry in time by covering it with waterproof plastic.

First, above-ground foliage is removed from the host plants by hand. Harvesting is done by removing clay from the bed. The substrate is removed by dividing the clay mass into as thin parts as possible to facilitate mixing of their contents throughout the depth of the profile, and placed in bags for transport.

Step 7: Drying and grinding the inoculum

Drying. The collected substrate and mycorrhizal propagules are exposed to sunlight and thermal disinfection for 30 days at 50°C. The drying period can be extended to a moisture content below 5% to facilitate grinding.

Grinding. The product is ground in an industrial mill and cooled to 2°C to prevent overheating of the mycorrhizal propagules. Grinding is continued until the particle size is less than 100 microns.

The third phase is concentration.

After step 7, the crushed biomass is brought to product concentration using a 35 micron sieve. Particles below this size (60-70% of the original product) are discarded, and 25-30% of the ultra-concentrated product does not pass through this size and will end up as concentrated product. Using this technology we went from 1.2 x 104 propagules per 100 ml to 1.2 x 106 propagules per 100 ml.

The relative humidity of the product at the outlet is below 5%.

Quality control. Purity and concentration of the final product are determined using Porter, W.M., 1979, The most probable number method for enumerating infective propagules of vesicular arbuscular mycorrhizal fungi in soils, Aust. J. Soil Res., 17: 515-519).

Package. Finished products are packaged and labeled for transportation. The final concentration of the concentrate composition is: MPN concentration: >1.2x106 propagules per 100 ml of product.

The fourth phase is seed coating.

Coating the seeds with mycorrhizal inoculum can be done in a special seed coating machine, in a conventional concrete mixer, or by hand in a mixer. This coating requires various steps, which are described below in the order in which they are performed.

1. At the initial stage, the seeds are coated with an adhesive substance. Adhesives that can be used in addition to water include organic adhesives (gelatin, ethylcellulose, propylene glycol, etc.) and inorganic adhesives (mineral oils, polyvinyls, plastic polymers, etc.). Preferably, the adhesives used are polymers or copolymers of the polyvinyl group, such as polyvinylpyrrolidone and polyvinyl acetate. The adhesive is added to the aqueous or alcoholic solution until its optimal solubility, varying the amount of adhesive used from 0.1 to 15%, preferably from 0.5 to 10% and even more preferably from 1.0 to 5% of the total weight of the seeds to be coated. The amount of adhesive used will depend on its chemical properties as well as the type of seed being treated. The time for treating the seeds with the adhesive can vary from 1 to 60 seconds, preferably from 5 to 50 seconds and even more preferably from 10 to 40 seconds per 100 kg of seeds used.

2. After applying the adhesive, mycorrhizal inoculum is added. The proportion of inoculum added to the seeds can be selected in the range of 0.1 to 15%, preferably 0.5 to 10% and even more preferably 1 to 5% by weight of the seeds, which will depend on the type and variety. The time of treatment of seeds with mycorrhizal inoculum can vary from 1 to 50 seconds, preferably from 5 to 40 seconds and even more preferably from 10 to 30 seconds per 100 kg of seeds used.

Mycorrhiza treatments can be combined with other treatments, which are described in sections 2a-2e. These treatments can also be performed independently of the mycorrhizal inoculum treatment to produce a multi-layer coating. The steps required to apply each coating layer are the same as for mycorrhizal inoculum. These layers can be separated by covering the seeds with harmless substances of calcareous origin (calcium carbonate and the like), clay or polymer. The granulating or crusting agent should not exceed 50%, preferably 40% and even more preferably 30% of the weight of the seeds and is added to the seeds along with the adhesive as specified in section 1.

2a. Treatment with fungicides, insecticides and/or herbicides compatible with mycorrhizal fungi. In general, all commercial herbicides and insecticides are compatible with mycorrhizal fungi. However, not all fungicides are compatible with the survival of mycorrhizal fungi. The main fungicides used include azoxystrobin, carboxin, cyproconazole, chlorothalonil, metalaxyl, myclobutanil and prothioconazole. The amount of pesticide used will depend on the manufacturer's recommendations, the variety of pesticides used depends on the needs.

2b. Treatment with beneficial microorganisms including Trichoderma spp., Rhizobium bacteria and/or a combination of microorganisms beneficial to the rhizosphere such as Aspergillus, Penicillium and nitrogen-fixing bacteria.

2s. Treatment with macro- and/or micronutrients, in which nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) are the main macronutrients, and iron (Fe), zinc (Zn), manganese (Mn), boron (B), copper (Cu), molybdenum (Mo) and chlorine (Cl) are the main micronutrients. The dose of nutrients for coating may vary from 0.01 to 15%, preferably from 0.05 to 10% and even more preferably from 0.1 to 5% of the total weight of the seeds to be coated.

2d. Treatment with stimulants. Coating seeds with stimulants that induce seed germination and growth, as well as plant protection, sporulation and growth of mycorrhizal fungi. Stimulants can be phytohormones (abscisic acid, strigolactones, brassinosteroids, etc.) and their inducers and derivatives, secondary metabolites (flavonoids, terpenoids, etc.), cofactors (metal ions, etc.).

2e. Treatment with coloring pigments: These pigments must be compatible with the survival of the mycorrhizal fungus and provide a clear distinction between treated and untreated seeds.

3. Steps 1 and 2 mean that complete coverage of the mycorrhizal inoculum by the adhesive is achieved in 1 to 40 seconds, preferably 5 to 30 seconds, and even more preferably 10 to 20 seconds, complete coverage of the mycorrhizal inoculum by the adhesive is achieved.

4. After the mycorrhizal treatment, the seeds are dried for a time that may vary from 1 to 50 seconds, preferably from 5 to 40 seconds, and even more preferably from 10 to 30 seconds. This step can be completed after step 5.

5. Finally, the coated seeds are immersed in the coating component containers. The loading duration may vary from 5 to 30 seconds, preferably from 10 to 25 seconds and even more preferably from 15 to 20 seconds.

Example 3. Method for obtaining a variant of the composition according to the invention of a microgranular composition.

The first phase is greenhouse conditions.

Substrate. Smectite clays with a pH of 7.8 to 8 are chosen as a substrate and sterilized before use in alternating cycles of 3 days.

AMG strain. Dominikia sp. propagules are used as the initial inoculum. in clean conditions. This type of inoculum is always maintained in continuous cycles of 90-180 days in either a growth chamber or controlled host plant greenhouses. Different plants are used to reproduce the inoculum in successive cycles to avoid disease transmission in the same host.

Summer autumn. Sorghum vulgare and Ocimum sp.

Winter spring. Lolium perenne.

Culture pots. Use 15 liter pots.

Growing conditions for host plants.

Cultivation in a growth chamber or greenhouse begins with direct inoculation of the host plant's root system with a pure inoculum of a selected strain of arbuscular mycorrhizal fungus (Dominikia sp.) in a sterile smectite clay substrate. Plants are grown for a full life cycle, according to the plant type, which takes from 90 to 180 days. Plants are always kept well hydrated, with daily irrigation (sterile water) at a temperature of 25 to 28°C and a relative humidity of 65%. Once completed, the root system containing the smectite substrate, rootlets and pure AMG shoots is removed and subsequently used for scaling in the second stage.

The following minimum specifications are used to determine inoculum quality:

total number of spores: 50-225 spores/g;

extramatric mycelium: >70 mg/kg substrate;

percentage of root colonization: Sorghum >50%, Lolium >45%, Ocimum >40%;

MPN concentration: >1x10 ⁴ propagules per 100 ml of substrate.

The second phase is scaling up.

Stage 1: preparing the beds.

The beds are made with plastic cushioning material, so that the bed is isolated from the surrounding soil; The beds are constructed, preferably with a plastic cover, so that drainage occurs and the growth of unwanted vegetation is prevented.

The beds should be filled with the selected smectite clay (Arcilla Roja Galve). The moisture content of the clay should be approximately 15% to make it easier to work with when filling the bed. After filling, water until saturated to improve the structure.

The beds are located in well-drained areas.

Beds can be of any size, allowing for access to facilitate the movement of people and equipment needed for maintenance.

The beds have an irrigation system according to local needs. The preferred system may be drip irrigation or a sprinkler, which should be automated and provide independent watering to selected areas of the bed.

Stage 2: host species.

Determination of host plant species and mycorrhizal fungi that can be used in the system: The selection and identification of host plant and fungal species will be tailored to the specific site conditions and production goals. Ryegrass (Lolium perenne) and AMG previously produced in the first stage were used.

Step 3: Host plant seeding and inoculation.

Before planting the host plant, the germination of seeds is checked. The results of this test determine the appropriate seeding rate. For perennial ryegrass, seed is broadcast at 80 kg/ha using pre-tested pelleted seed. Also, together with the seeds, 20 g of AMF inoculum per ^m2 of bed is applied directly to the smectite clay.

Immediately after sowing, fine spray irrigation is used to prevent redistribution of seed and inoculum.

Water used for irrigation should preferably have the following characteristics:

pH values from >6 to <7.5;

electrical conductivity: <1.6 mS/m;

total soluble salts: <1000 ppm;

exchangeable sodium content (SAR) <10;

without heavy elements and pathogens; It is preferable that the water used for irrigation be potable.

Stage 4: Cultivation and Irrigation.

The irrigation applied should be sufficient to achieve 100% of natural water capacity, but avoid watering with too much water and causing it to accumulate or stagnate. The beds should be watered again when the clay moisture content drops to 75-80% of its natural moisture holding capacity.

Stage 5: managing the establishment of mycorrhizal symbiosis and studying the dynamics of the development of mycorrhizal fungal colonization.

During the growth and development of the host plant, AMF colonizes the roots and establishes a symbiosis between the plant and the fungus. To assess the development of this relationship, periodic sampling of the root system is carried out to assess mycorrhizal development. Methods used to assess colonization include Gerdeman & Nicolson (1963) (Gerdemann J.W., Nicolson T.N., 1963, Spores of mycorrhizal endogenous species extracted from soil by wet sieving and decanting, Transactions of the British Mycological Society, 46(2) ):235-44.); McGonigle (1990) (McGonigle, T.P., Miller M.H., Evans D.G., Fairchild G.L., Swan J.A., 1990, A new method which gives an objective measure of colonization of roots by vesicular arbuscular mycorrhizal fungi, New Phytologist, 115(3):495 -501); and Phillips & Hayman (1970) (Phillips, J.M., Hayman D.S., 1970, Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection, Transactions of the British Mycological Society, 55:158-161).

Sampling begins two months after planting and continues monthly until the end of the growing season. Based on the information obtained from these samples, it is possible to determine the dynamics of the development of mycorrhizal symbiosis within the inoculum.

Evaluation of the production method is based on mycorrhizal colonization of roots, extramatric mycelial concentration and periodic sampling of clay containing spores.

Knowledge of the dynamics of development of mycorrhizal products allows us to determine the optimal harvest time and make the most of the process of symbiosis between the host plant and mycorrhizal fungus.

Stage 6: Harvest.

Harvesting is the most critical stage in the production process. If perennial Lolium is used as a host plant, the optimal harvest time is usually between 6 and 7 months after sowing because during this period the plants mature, complete their life cycle, tend to lose viability and turn yellow.

Fifteen days before the scheduled harvest date, watering is stopped and the fallen leaves are stored so that the clay slowly loses moisture, ensuring the completion of the inoculation method. If this activity coincides with the rainy season, it will be necessary to protect the bed from rain so that it can dry in time by covering it with waterproof plastic.

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First, above-ground foliage is removed from the host plants by hand. Harvesting is done by removing clay from the bed. The substrate is removed by dividing the mass of clay into the thinnest possible portions to facilitate mixing of their contents throughout the depth of the profile, and placed in bags for transport.

Step 7: Drying and grinding the inoculum.

Drying. The collected substrate and mycorrhizal propagules are exposed to sunlight and thermal disinfection for 30 days at 50°C. The drying period can be extended to a moisture content below 5% to facilitate grinding.

Grinding. The product is ground in an industrial mill and cooled to 2°C to prevent overheating of the mycorrhizal propagules. Grinding continues to a particle size of less than 100 microns.

The third phase is microgranulation.

The microgranulation method is divided into three stages.

Adding granular media to the rotating biconical mixer. A mixture of mica, attapulgite and limestone in the range of 40 to 90 wt%, more preferably 50 to 80 wt%, even more preferably 60 to 70 wt%, was used to prepare the support.

Addition of mycorrhiza (arbuscular mycorrhizal fungus) inside the rotating biconical mixer: AMH is dosed in the range of 10 to 60 wt.%, more preferably 20 to 50 wt.%, even more preferably 30 to 40 wt.% of previously produced Dominikia sp. (step 7) together with a binder (wax, linseed oil, gum arabic, gum tragacanth, methylcellulose, polyvinyl alcohol, tapioca flour, lactose, sucrose, microcrystalline cellulose, polyvinylpyrrolidone, lactose powder, sucrose powder, tapioca starch (cassava flour) and microcrystalline cellulose, gum or protein such as egg white or casein) in the range of 1 to 25% by weight, more preferably 5 to 20% by weight, more preferably 10 to 15% by weight. During this operation, the mixer continues to move until the granules are completely homogenized.

Package. After complete homogenization, the prepared product is discharged onto a sieve to remove lumps and formed dust. Finally, the product is collected into a packaging hopper, from which it automatically proceeds to filling the appropriate containers.

Quality control and packaging.

Quality control. Purity and concentration of the final product are determined using Porter, W.M., 1979, The most probable number method for enumerating infective propagules of vesicular arbuscular mycorrhizal fungi in soils, Aust. J. Soil Res., 17: 515-519).

Package. Finished products are packaged and labeled for transportation.

The final concentration of the microgranular composition is MPN concentration: >1x10 ⁴ propagules per 100 ml of product.

Example 4. Effect of the composition according to the invention on the development of wheat (Triticum durum).

To demonstrate the effectiveness of the composition of the present invention, which includes the arbuscular mycorrhizal fungus (AMF) Dominikia sp., deposited under registration number MUCL 57072 on wheat, a test was carried out in the Tres caminos experimental field belonging to CEBAS-CSIC in Murcia.

The experimental design was a randomized block with four replicates. The experimental plots were 3 m long and 4.2 m wide.

The following processing methods were used.

Control: without the use of chemical fertilizers and mushrooms.

Composition according to the invention. Available in microgranular form at a rate of 10 kg/ha in combination with chemical fertilizer (Guerra, B.E., Micorriza arbuscular. Recurso microbiologico en la Agricultura sostenible, Tecnologia en Marcha, 2008, vol. 21, no. 1, pp. 191-201).

Standard product: chemical fertilizer (Guerra, B.E., Micorriza arbuscular. Recurso microbiologico en la Agricultura sostenible, Tecnologia en Marcha, 2008, vol. 21, no. 1, pp. 191-201): 150 kg/ha nitrogen, 54 kg phosphorus/ ha, potassium 100 kg/ha, calcium 15 kg/ha, magnesium 15 kg/ha, sulfur 23 kg/ha and other trace elements.

Planting took place on November 1, 2013, and harvesting took place on June 1, 2014, for a total of 240 days. The type of sowing and inoculation used was mechanical and was carried out by passing a grain seeder, and the microgranulate composition according to the invention was applied at a dose of 10 kg/ha along with the seeds.

The soil used was classified as Nitisols (IUSS Working Group. WRB. World reference base for soil resources, 2006, World Soil Resources Reports, Rome: FAO, 2006). For the chemical characteristics of the soil, the following analytical methods were used:

pH: soil to solution ratio 1: 2.5;

organic matter (OM): Walkley and Black.

R ₂ O ₅ : Oniani;

exchangeable cations: NH ⁴ AC extraction, 1 mol/l, pH 7 and complexometric titration (Ca and Mg) and flame photometry (Na and K).

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These methods are described in the manual on analytical methods for the analysis of soil, foliage, organic fertilizers and chemical fertilizers (Institute National de Ciencias Agricolas, Manual de tecnicas

Analiticas para analisis de suelo, foliar, abonos organicos y Ferizantes quimicos: La Habana, 1989).

The results of soil chemical characteristics (Table 2) show medium to high fertility, noting an average organic matter content that is consistent with that described for this soil type (Hernandez, A., Morell, F., Ascanio, M.O., Borges, Y ., Morales, M., y Yong, A., Cambios globales de los suelos Ferraliticos Rojos Lixiviados (Nitisoles R0dicos Eutricos) de la provincia La Habana, Cultivos Tropicales, 2006, vol. 27, no. 2, pp. 41-50).

table 2

Soil chemical characteristics

Organic matter (%) 3.21 pH 7.02 R ₂ o ₅ 397.25 K (cmol/kg soil) 0.96 Ca (cmol/kg soil) 14.26 Mg (cmol/kg soil) 3.70 Na (cmol/kg soil) 0.11

Fungus strain of invention Dominikia sp. was isolated from saline soil, the fungus is resistant to high salt concentrations.

The analyzed parameters were: the number of AMF spores per gram of rhizosphere (Gederman, J.W., in Trappe, J.M., The endogonaceae in the pacific northwest, Mycologia Memoir, No. 5, The New York Botanic Garden, 1974, No. 5), the percentage of mycorrhizal colonization using root staining techniques (Phillips, J.M., in Hayman, D.S., Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection, Tranfer. Britanic: Mycology Society, 1970, vol. 55, pp. 158-161 ) percentage of visual density using the method of segments (Giovannetti, M., in Mosse, V., An evaluation of techniques to measure vesicular-arbuscular infection in roots, New Phytology, 1980, vol. 84, pp. 489-500) and general glomalin (glycoprotein) content, which was obtained by pressure cooking (Wright, S.E., Nichols, K.A., in Schmidt, W.F., Comparison of efficacy of three extractants to solubilize glomalin on hyphae and in soil, Chemosphere, 2006, t 64, No. 7, pp. 1219-1224).

Leaf nutrient content (% N, P and K) and total protein content were determined by the methods described in the INCA laboratory manual of analytical methods (Instituto Nacional de Ciencias Agricolas, Manual de tecnicas Analiticas para analisis de suelo, foliar, abonos organicos y fertilizantes quimicos : La Habana, 1989).

Productivity was assessed as follows: the number of grains per ear was assessed; number of ears per ^m2 ; weight per 1000 grains and yield (t/ha).

Statistical processing of the experimental results was carried out by analyzing one-way analysis of variance, using the Duncan test (Duncan, D.B., Multiple range and multiple F tests. Biometrics, 1955, vol. 11, No. 1), and where there were differences between the processed values in all cases statistical processor SPSS 11.5 was used.

Results and discussion.

In table Figure 3 shows the results of various treatments of mycorrhizal parameters. These values clearly show an increase in the number of spores where the composition of the present invention was applied compared to the control (without chemical fertilizer) and the standard product.

Table 3

The influence of treatments on the studied parameters of mycorrhiza.

Processing method Spores/gram soil Colonization (%) VP (%) Glomalin (mg/g soil) Control 42 b 12.3 b 0.12b 1.8b Composition by invention 156 a 65.3 a 3.25 a 9.27 a Standard product 36b 11.2b 0.11 b 1.78b R 0.014** 0.04*** 0.021** 0.017** F 8.5 9.2 7.1 6.5

*Different letters in the same column are significantly different at p < 0.05.

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The above may be due to strong colonization activity, which makes it difficult for other species of arbuscular mycorrhizal fungi to colonize roots. On the other hand, this shows that mycorrhizal colonization was also more effective with the composition of the invention, as reflected by higher values compared to other treatments.

It should be noted that native strains in soil showed low colonization values. Observation of visual density values (a variable that is a measure of the intensity of mycorrhizal colonization) shows that the highest percentage was obtained with the composition of the invention.

Another variable that should be highlighted was the total glycoprotein content, which was higher with the composition of the invention. This phenomenon confirms the greater symbiosis of mycorrhizae when using the composition according to the invention and the possible influence of the inoculum on increasing the formation of aggregates in the soil. Moreover, treatments in which no mycorrhizal inoculum was used had low values for this parameter, which may be due to the low efficiency of native mycorrhiza.

In table Figure 4 shows the mineral content in the leaves for each treatment. For nitrogen and phosphorus, the content was higher when using the composition according to the invention, while the sodium content was higher when applying chemical fertilizers.

Table 4

Leaf nitrogen, phosphorus, potassium and total protein

Processing method %N %R %TO Total protein, (%) Control 1.25b 0.26b 1.12 8.04b Composition according to the invention 1.45 a 0.40 a 1.18 10.1 a Standard product 1.41 a 0.36 a 1.23 9.89 a R 0.001** 0.023** 1.23ns 0.013* F 7.2 5.6 8.8 6.5

*Different letters in the same column are significantly different at p < 0.05.

It should be noted that the stated values (L0pez-Bellido, L., Cultivos herbaceos. Cereales, ed. Mundi-Prensa, 1991. pp. 151-158) for this parameter (% foliage N) are above the critical wheat yield index between 4 and 5 t/ha.

Similar results (Cornejo, P., Borie, F., Rubio, R., in Azcon, R., Influence of nitrogen source on the viability, functionality and persistence of Glomus etunicatum fungal propagules in an Andisol, Applied Soil Ecology, 2007, vol. 35, no. 2, pp. 423-431; Echeverria, E., in Stiddert, G. A., El contenido de nitrogeno en la hoja bandera del trigo como predictivo del incremento de proteina en el grano por aplicaciones de nitrogeno en la espigazon ( The nitrogen content in the wheat flag leaf as predictive of the increase in protein in the grain by nitrogen applications to the ear), Revista de la Facultad de Agronomia, 1998, vol. 103, no. 1, p. 10) indicate the presence of this nutrients in leaf tissue due to the process of mycorrhizal symbiosis, which allows the absorption and transport of nutrients through the mycelium. There were no significant differences in phosphorus content for any of the treatments, which may be due to the content of this element in the soil (Table 4), which is absorbed by plants from the soil solution in this particular case through the root system through the mechanism of three-way interaction between the plant, arbuscular mycorrhiza (mycorrhizal symbiosis) and soil, a higher value of this nutrient is indicated than the critical index in durum wheat leaves (Lopez-Bellido, L. Cultivos herbaceos. Cereales., ed. MundiPrensa, 1991, pp. 151-158) and noted good cultural development. In addition, this value deviated more where the composition according to the invention was used.

Another important parameter is the protein content of the leaves, which is higher when treated with the composition according to the invention (even higher than when treated with a chemical fertilizer). Finally, the control treatment had a significantly lower percentage.

The results of yield and its components (Table 4) clearly reflect the response of durum wheat to the application of the composition according to the invention and the standard product, equally with a high N° content.

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Table 5

The influence of treatments on yield and its parameters

Treatment Grain/ear No. of ears/ ^m2 Weight of 1000 grains (g) Yield (t/ha) Increase (%) Control 25.00 b 576 29.0 b 3.25b — Composition according to the invention 33.00 a 641 33.4 a 5.22 a 8.8 Standard product 30.00 a 635 32.0 a 4.60 a — R 0.02** 11.25 ns 0.015* 0.009** F AND 2 10.2 6.1 7.8

*Different letters in the same column are significantly different at p < 0.05.

Treatment with the composition of the invention and the standard product gave the highest values for each parameter compared to the control. This can be explained by the greater efficiency of solid inoculum and the use of high doses of nitrogen.

It should be noted that growing wheat in combination with the composition according to the invention made it possible to achieve adequate productivity with satisfactory indicators of the impact of mycorrhiza not only on improving the biological activity of the soil, but also contributed to a significant increase in yield by 8.8% compared to the control and standard product.

Example 5. Effect of the composition according to the invention on the development of corn (Zea mays).

To demonstrate the effectiveness of the composition of the present invention, which includes the arbuscular mycorrhizal fungus (AMF) Dominikia sp., deposited under registration number MUCL 57072, on maize, a field trial was carried out near the city of Egea de los Caballeros. A microgranular form of the composition of the present invention was tested. A standard product was used to compare the effects of both products on corn production, var DKC 6717, Monsanto; on soils of the Cambisol group.

Planting took place on April 12 and harvesting took place on December 14, 2014, for a total of 230 days. The type of sowing and inoculation used was mechanical and was carried out by passing a grain seeder, and the microgranulate composition of the invention was applied at a dose of 10 kg/ha along with the seeds. The spray system used was 18 x 18 m in size.

The experimental design was a randomized block with three replicates. The experimental plots were 20 m long and 20 m wide.

The following processing methods were used.

Composition according to the invention: applied at the rate of 10 kg/ha and chemical fertilizer (Guerra, V.E., Micorriza arbuscular. Recurso microbiol0gico en la Agriculture sostenible, Tecnologia en Marcha, 2008, v. 21, no. 1, pp. 191-201 ).

Standard product: chemical fertilizer (Guerra, B.E., Micorriza arbuscular. Recurso microbiologico en la Agricultura sostenible, Tecnologia en Marcha, 2008, vol. 21, no. 1, pp. 191-201): 325 kg/ha nitrogen, 80 kg phosphorus/ ha and potassium 200 kg/ha, with the initial application of organic matter in the form of slurry 10 t/ha.

The soil used was classified as Cambisol (IUSS Working Group. WRB. World reference base for soil resources 2006. World Soil Resources Reports. Rome: FAO, 2006). The following analytical methods were used for the chemical characterization of the soil:

pH: soil to solution ratio 1:2.5;

organic matter (OM): Walkley and Black;

R ₂ O ₅ : Oniani.

exchange cations: NH4 extraction AC, 1 mol/l, pH 7 and complexometric titration (Ca and Mg) and flame photometry (Na and K).

These methods are described in the manual on analytical methods for the analysis of soil, foliage, organic and chemical fertilizers (Instituto Nacional de Ciencias Agricolas, Manual de tecnicas Analiticas para analisis de suelo, foliar, abonos organicos y Ferizantes quimicos: La Habana, 1989).

The results of the chemical characteristics of the soil (Table 6) show medium to high fertility, there is an average content of organic matter, which corresponds to that described for this type of soil (Hernandez, A., Morell, F., Ascanio, M.O., Borges, Y ., Morales, M., y Yong, A., Cambios globales de los suelos Ferraliticos Rojos Lixiviados (Nitisoles R0dicos Eutricos) de la provincia La Habana, Cultivos Tropicales, 2006, vol. 27, no. 2, pp. 41-50).

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Table 6

Soil chemical characteristics

Organic matter (%) 2.51 pH 8.02 R ₂ O ₅ 258.60 K (cmol/kg soil) 1.40 Ca (cmol/kg soil) 15.44 Mg (cmol/kg soil) 2.90 Na (cmol/kg soil) 0.30

Fungus strain Dominikia sp. according to the invention was isolated from saline soil; this mushroom is resistant to high salt concentrations.

The parameters analyzed were: percentage of mycorrhizal colonization using the root staining technique (Phillips, J.M., in Hayman, D.S., Improved procedures for clearing roots and staining parasitic and vesiculararbuscular mycorrhizal fungi for rapid assessment of infection, Tranfer. Britanic: Mycology Society, 1970, t 55, pp. 158-161) and percent visual density (VP) using the intercept method (Giovannetti, M., in Mosse, V., An evaluation of techniques to measure vesicular-arbuscular infection in roots, New Phytology, 1980, vol. 84, pp. 489-500). Leaf nutrient content (% N, P and K) was determined by the methods described in the laboratory manual on analytical methods (Institute National de Ciencias Agricolas, Manual de tecnicas Analiticas para analisis de suelo, foliar, abonos organicos y Ferizantes quimicos: La Habana, 1989 G.).

Productivity was assessed as follows: the number of grains per ear was assessed; number of ears per square meter; weight per 1000 grains and yield (t/ha).

Statistical processing of experimental results was carried out using one-way analysis of variance and Duncan's test (Duncan, D.B., Multiple range and multi range and test of Duncan), when there were differences between processing tools, in all cases the statistical processor SPSS 11.5 was used.

Results and discussion.

In table Figure 7 shows the results of the development of mycorrhizal activity 45 and 120 days after planting. These values clearly showed increased activity where the composition of the invention was applied, achieving not only a greater percentage of mycorrhizal colonization, but also a greater intensity of colonization, as reflected through a higher visual density.

Table 7

Effect of treatment on the studied parameters of mycorrhiza

Colonization (%) 45 days Colonization (%) 120 days VP (%) 45 days VP (%) 120 days Composition according to the invention 36.2 a 64.12 a 3.4 a 4.28 a Standard product 10.1 b 11.36b 0.3b 0.85b R 0.011** 0.02*** 0.01* 0.006* F 7.2 8.2 8.1 9.4

*Different letters in the same column are significantly different at p < 0.05.

This analysis showed that the most effective inoculum was the composition according to the invention, as indicated by higher values compared to other treatments.

Visual density values (a parameter that measures the intensity of mycorrhizal colonization) show that the highest percentage was also associated with the composition of the invention and is closely related to effectiveness.

The mineral content of the leaves was measured (Table 8). The results showed no significant differences between both treatments, the composition according to the invention and the standard product. However, there was a tendency for nutrient supply to increase, especially in the case of nitrogen in the presence of Dominikia sp. (composition according to the invention).

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Table 8

Leaf nitrogen, phosphorus, potassium after 120 days in corn

Treatment %N %R %TO Composition by invention 1.48 0.41 1.81 Standard product 1.40 0.35 1.9 R 0.12ns 0.1 ns 0.22ns F 8.5 8.2 7.9

*Different letters in the same column are significantly different at p < 0.05.

It should be noted that the nitrogen content values obtained when treated with the composition according to the invention are higher than the critical wheat index for yields between 4 and 5 t/ha (L0pez-Bellido, L., Cultivos herbaceos, Cereales, ed. Mundi-Prensa, 1991, p. . 151-158).

There were no significant differences in the phosphorus content in the leaves for any of the treatments, which may be due to the content of this element in the soil (Table 8), which is absorbed by plants from the soil solution in this particular case through the root system using a three-way interaction between plant, arbuscular mycorrhiza (mycorrhizal symbiosis) and soil, a higher value of this nutrient is indicated than the critical index in durum wheat leaves (Lopez-Bellido, L., Cultivos herbaceos, Cereales, ed. Mundi-Prensa, 1991. p. 151- 158) and good cultural development was noted. In addition, this value deviated more where the composition according to the invention was used.

Similar results for wheat and corn demonstrate the effect of mineral fertilizers on the percentage of colonized root length where low doses of phosphorus were applied. This confirms the importance and usefulness of the composition according to the invention for corn and wheat in terms of increasing the absorption of phosphorus from the soil, both in the presence and absence of nitrogen and phosphorus, thereby minimizing the applied doses of fertilizers.

Leaf potassium content was similar among the treatments tested, showing satisfactory levels for this crop that are comparable to cereal data for this macronutrient (Lopez-Bellido, L., Cultivos herbaceos, Cereales, ed. Mundi-Prensa, 1991, p. 151-158). Moreover, high concentrations of this element are found both in plants with and without mycorrhiza (Bolleta, A., in Krugger, H., Fertilizacion e inoculacion con hongos micorrizicos arbusculares en trigo, Buenos Aires: Institute Nacional de Tecnologia Agropecuaria, 2004 .; Saleque, M.A., Timsina, J., Panaullah, G.M., Ishaque, M., Pathan, D.J., Saha, P.K., Quayyum, M.A., Humphreys, E.yu Meisner, C.A., Nutrient uptake and apparent balances for rice -wheat sequences. II. Phosphorus. Journal of Plant Nutrition, 2006, vol. 29, no. 1, pp. 157-172), which may be due to the fact that this element moves more easily in the soil solution.

The table below summarizes the results obtained (Table 9), which clearly reflect the response of corn to the use of the composition according to the invention and the standard product.

Table 9

Treatment Productivity (kg/ha) Grain moisture (%) Increase (%) Composition by invention 14700.2 a 22 9.18 Standard product 13500.3 b 20 — R 0.042** F 29.40

*Different letters in the same column are significantly different at p<0.05.

Plants treated with the composition of the invention achieved the highest values compared to the standard product, which can be explained by the greater efficiency of the solid inoculum in establishing symbiosis with the plant and thus better uptake of high doses of nitrogen.

Finally, in conclusion, it should be noted that the cultivation of corn, in combination with the composition according to the invention, made it possible to achieve a positive effect on productivity in terms of efficiency, achieving an increase in yield by 9.18% compared to the application of mineral fertilizers. In addition, nutrient values showed higher dynamics, and the use of the arbuscular mycorrhizal fungus according to the invention not only improved the biological activity of the soil, but also contributed to a significant increase in productivity based on more sustainable management.

Example 6 Effectiveness of corn seed coating with Dominikia sp. and an adhesive agent for mycorrhizal activity.

Objective: To investigate the effectiveness of maize coating with Dominikia sp. and an adhesive substance for mycorrhizal activity.

To achieve this goal, three pots (each considered a replicate) were sown with maize seeds coated with Dominikia sp. deposited under registration number MUCL 57072 and adhesive.

Seeds were planted on August 3, 2016, and mycorrhizal root colonization tests were performed 21 and 35 days after planting using the Phillips and Hayman staining method (Phillips and Hayman, 1970).

Table 10

Percentage of mycorrhizal colonization (% CM) of corn seed roots from grain coated with a combination of Dominikia sp. and adhesive substance 21 and 35 days after planting (dpp), respectively

Date of assessment Pot 1 (% KM) Pot 2 (% KM) Pot 3 (% KM) Average (X) 28/3/2016 (21 dpp) 7 9 10 8.6 10/4/2016 (35 dpp) 12 eleven 15 12.6

Table Figure 10 shows that each of the samples examined had a positive percentage of mycorrhizal colonization, which increased as the plants grew. At this stage, only the initial points of maize colonization were discovered, formed by a network of extramatric mycelium that had just begun to form.

Conclusions.

Coating corn seeds with an inoculum concentrate containing Dominikia sp. and the adhesive was effective because it created mycorrhizal structures in the root as extramatric mycelium during the early stages of plant development. Germinated spores were found to form an internal network of germination and colonization. Colonization values as a function of corn growth time are also shown.

Example 7. Results obtained in the field using the composition according to the invention in microgranular form on wheat and barley.

Results obtained in the field using the composition according to the invention containing Dominikia sp. deposited under registration number MUCL 57072, in microgranular form on wheat and barley, are presented in the following table. eleven.

Table 11

Culture Variety Irrigation Treated (kg ha ¹ ) Untreated (kg ha ¹ ) Improvement (%) Barley Will Rain agriculture 2600 3000 15 Barley Signoro Irrigated agriculture 7100 7700 8.5 Wheat Bonifacio Irrigated agriculture 6350 7000 10.2 Wheat Badra Irrigated agriculture 4981 5463 9.7 Wheat Chambo Rain agriculture 3350 3700 10.4 Wheat solid Rain agriculture 4300 4900 14

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Wheat solid Rain agriculture 4570 5110 11.8 Wheat solid Irrigated agriculture 6345 7135 12

Example 8. Results obtained in the field when using the composition according to the invention in microgranular form on corn.

Results obtained in the field using the composition according to the invention containing Dominikia sp. deposited under registration number MUCL 57072, in microgranular form on corn, are presented in the following table. 12.

Table 12

VarietyCycle Irrigation Unprocessed (dry matter) Processed (dry matter) Improvement (kg/ha) Zone 1 Lg 30369 - 365 Irrigated 7.87 8.25 809.091 Zone 2 Lg 30369 - 365 Irrigated 4.24 5.27 2408.655 Zone 3 Lg 30369 -335 Irrigated 4.85 5.26 963,080 Zone 4 Lg 30369 - 365 Irrigated 6.84 7.89 2085.287 Zone 5 Maysadur -350 Irrigated 7.40 8.09 1379.649

Example 9. Mycorrhizal activity of Dominikia sp. when growing rice (Oriza sativa) on floodplain soil.

An experiment was conducted to determine the mycorrhizal activity of the composition of the present invention containing Dominikia sp., deposited under registration number MUCL 57072, in concentrated form, in saline floodplain soil conditions. The composition used had a concentration of between 1-4x106 propagules/g substrate. On the other hand, in accordance with embodiments, the concentration of the composition may be 1-2x104, preferably 1.2-1.8x104 propagules/g substrate.

Materials and methods.

The experiment was carried out at the Tres Caminos experimental farm, owned by CEBASCSIC and located in the Matanza area, municipality of Santomera (Murcia). Plants were grown in a single-layer experimental tunnel-type greenhouse with an approximate area of 60 m ² covered with polycarbonate with a top window protected by an anti-slip mesh. It was equipped with a cooling system and a system of aluminized shading screens. The test was carried out on rice (Oriza sativa) cultivar J 104 plants subjected to two mycorrhizal inoculum treatments and a corresponding control without mycorrhizal inoculum treatment. The mycorrhizal treatment was carried out with a composition according to the invention containing Dominikia sp., deposited under registration number MUCL 57072, in the form of a concentrate at a dose of 1 kg/ha, coated with rice seeds.

Once inoculation was ensured on individual shoots, 15 days after germination, the plants were transplanted at a density of 10 plants per 2 m ² concrete channel using Hidrom0rfico Gley Nodular Salinizado soil according to the UNESCO soil classification (Hernadez, A., Perez , JM, Bosch, D., Rivero, L., Nueva Version de Clasificaci0n Genetica de los Suelos de Cuba, Soil Institute, AGRINFOR, La Habana, 1999, p. 64) as substrate in both containers. The main characteristics are shown in table. 1. Growing work was carried out, a layer of water was added 18 days after sowing the seeds in all treatment options.

Table 13

Some chemical properties and number of spores in 50 g of soil ^-1

ov (%) pH P (cmol, kg ¹ ) Sa Mg TO Na S. E (pS. cm ¹ ) No spores, g soil ¹ resin, kg ¹ 2.3 7.5 13.2 10.2 5.6 0.9 2.2 2876 1.2

¹ The soil used in the experiment was Hidrom0rfico Gley Nodular Salinizado

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Characteristics of the composition according to the invention in concentrated form.

In this example, the composition according to the invention in concentrated form with a mycorrhizal inoculum contains the arbuscular mycorrhizal fungus Dominikia sp. deposited under registration number MUCL 57072. The composition used had a concentration of between 1-4x106 propagules/g substrate.

Completed definitions.

Growth dynamics were determined during 90 days after transplantation (dpp), where plant height and root system depth were measured, as well as yield and some of its components.

Mycorrhizal activity was determined during crop development by measuring percent colonization (%), visual density (%), arbuscular extramatric mycelium and arbuscular endophytes using a stereomicroscope (Zeiss, West Germany -5-) and an Axiostar biological microscope (Zeiss, West Germany - 5-). The ratio between arbuscular external and endophytic mycelium (MEA:EA) was calculated.

Mycorrhizal assessment of samples was performed using a root staining technique (Phillips, DM, Hayman, DS, Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection, Trans. Br. Mycol. Soc., 55, 158- 161, 1970), and the percentage of colonization was determined by the intercept method (Giovannetti, M., Mosse, V. (1980), An evaluation of techniques to measure vesicular-arbuscular infection in roots, New Phytology., 84:489-500). Mathematical calculation of visual density, arbuscular endophytes and mycorrhizal activity was carried out in accordance with the proposed protocols (Trouvelot, A., Kough, J., Gianinazzi Pearson, V. (1986), Mesure du Taux de Mycorhization VA d'un Systeme Radiculaire. Recherche de Methodes d'Estimation ayant une Signification Fonctionnelle, Proceedings of the 1st European Symposium on Mycorrhizae: Physiological and Genetical Aspects of Mycorrhizae, Dij0n, 15 July, 1985. (eds. V. Gianinazzi Pearson and S. Gianinazzi). INRA, Paris, p. 217-222; Herrera-Peraza, R., Eduardo Furrazola, Roberto L. Ferrer, Rigel Fernandez Valle and Yamir Torres Arias, 2004, Functional strategies of root hairs and arbuscular mycorrhizae in an evergreen tropical forest, Sierra del Rosario, Cuba. Revista CENIC Ciencias Biolgicas, vol. 35, no. 2, 2004). The total number of spores was also determined, g soil ^-1 .

Statistical analysis.

Statistical processing of the results was carried out by simple analysis of variance classification and Tukey's test in the presence of significant differences between the mean values using the Statgraphics® Plus, 4.1 program. SigmaPlot 4 software was used to generate the graphs (i.e., Figures 1, 2, and 3).

Percent mycorrhizal colonization values were converted using the expression 2arcsen^x.

Results and discussion.

In the table above. Figure 13 shows some of the chemical properties of the soil used in the experiment. The soil had a slightly alkaline pH, the average level of organic matter, P and Ca ²⁺ was about 10 cmol/kg. Regarding salinity, Na content was high and electrical conductivity was high, indicating severe salinity, although fertility was acceptable for rice development.

The number of spores detected in this substrate was very low, which is typical for intensively used agricultural soils where the diversity and intensity of arbuscular mycorrhizal fungi (AMF) are reduced due to intensive tillage, over-exploitation and typical processes of chemicalization and salinization etc. (Rao, D.L.N., 1998, Biological amelioration of salt-affected soils, in: Microbial Interactions in Agriculture and Forestry, vol. 1, Science Publishers, Enfield, USA, pp. 21-238).

Analysis of the dynamics of rice growth under these conditions showed excellent behavior of the studied parameters during processing. Plant height constantly increased, accelerating development after the 27th day (Table 14).

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Table 14

Height (cm), root system depth (cm) and mycorrhizal colonization (%MC) of plants treated with Dominikia sp. (D. t) and control (K) plants for 90 days DPO in saline soil conditions

RKK: depth of the root system;

DPO: days after treatment;

SD: standard deviation.

Identical letters in the same column are not significantly different at p<0.05.

Both treatment methods show a significant difference in the growth process. The highest values were obtained from the control treatment until 18 days, after which a change in behavior occurred, and the highest values for plant height were obtained from the arbuscular mycorrhizal fungus inoculum treatment.

Analysis of the growth dynamics of rice plants under these conditions revealed different behavior of the variables during treatment. The height of the plants increased steadily, accelerating their growth after the 27th day.

Both treatment methods show a significant difference in the growth process. The highest values were obtained with the control treatment up to 18 days, after which a change in behavior occurred, and the highest plant height values were obtained with inoculation with the effective arbuscular mycorrhizal fungus.

A similar behavior of the height parameter was observed deep in the root system, which was

- 20 045371 more in treated than in control plants after 32 DPO. In this case, the behavior was highlighted because it is the part of the plant on which the fungus develops.

The mycorrhizal colonization study showed different behavior than that found under previously analyzed parameters. In this case and in both treatments, colonization was progressive and always reached the highest values in the inoculated treatment compared to the control treatment, which, although not inoculated, showed natural levels of mycorrhizal colonization, a typical result of experiments conducted in natural conditions.

The development was well expressed in the case of inocular treatment with AMF, reaching high values considering that the experiment was carried out under flooding conditions or with a layer of water after 20 days. At the end of cultivation, root colonization reached a value of 44%, which is considered high compared to other studies of rice mycorrhiza, where colonization maximums do not exceed 25% when sown on solid ground (Fernandez, F., Ortiz, R., Martinez, M.A., Costales, A., Llonin, D., The effect of commercial arbuscular mycorrhizal fungi (AMF) inoculants on rice (Oryza sativa) in different types of soils, Cultivos Tropicales, 18(1):5-9, 1997).

In fig. 1 shows the development of two very important parameters of mycorrhizal activity of a fungus when treated with an inoculum of the composition according to the invention in concentrated form - spore population and fungal colonization, expressed through the percentage of visual density, which is nothing more than the intensity with which the mycelium colonizes the interior of the root.

Visual density showed typical microbial behavior with a well-defined latent phase where the fungus slowly colonized the interior of the root from 0 to 20 days and then showed exponential growth until 40 days, the time when it reached the stationary phase and the end of growth.

In the case of a spore population, they were detected in the soil in the first days after inoculation (up to 15 days in the stems), then gradually disappeared over time; the product of germination under favorable conditions of humidity and high temperature was detected until the spore population in the soil fell. After 30 days, the formation of new spores began as a result of the development of external biomass of fungi and the development of symbiosis with the plant. In this case, the population continued to grow to values close to 12 spores g ^-1 soil.

The analysis of the arbuscular external mycelium and the parameters of the arbuscular endophytes (Fig. 2) is very interesting because it shows how the internal and external behavior of the mycorrhizal symbiont occurs as the symbiosis develops in annual culture cycles.

In this case, high values of external mycelium were observed in the early stages of the development of symbiosis caused by fungal growth due to plant growth, expressed after a few years (Bethlenfavay, G.J., Brown, M.S., Franson, R.L., Mihara, K.L., 1989, The glycine-glomus- bradyrhizobium symbiosis. IX. Nutritional, morphological and physiological response of nodulated soybean to geographic isolates of the mycorrhizal fungus of Glomus mosseae, Physiol. Plant., 76, 226-232), into a clear parasitic process resulting from rapid growth of the mycelium in early stages of mycorrhizal colonization of plants (hours) in the stage of low photosynthesis and high metabolic costs. The opposite trend is observed in the development of arbuscular endophytes. During the first few days, rates were very low, with significant growth only observed after 25 days of growth, a stage considered transitional in the arbuscular mycorrhizal symbiosis.

After 30 days, a decrease and stabilization of the external mycelium and a gradual increase in the endophyte are observed, associated with plant growth and the development of symbiosis.

In fig. Figure 3 shows the relationship established between mycorrhizal components in rice crops (ratio between arbuscular external mycelium and endophytic mycelium (MEA:EA)). This correspondence between the two main components of the symbiosis, the external mycelium and the endophyte, expresses the activity of this association, which passes through different stages of development. (Hirrel, M.C., 1981, The effect of sodium and chloride salts on the germination of Gigaspora margaria, Mycology, 43, 610-617). The initial stage, when high values of external mycelium correspond to very low values of endophytes, which balances overt parasitism, expressed not only in these parameters, but also in a decrease in plant growth compared to an uninoculated or ineffectively mycorrhizal control that does not cause significant changes in plant development (Table 2).

An intermediate or transitional phase, when both parts begin to balance, and a mutualistic exchange phase, when the components balance at a value of 1 or even less, so that there is marked growth within the root, allowing for proper exchange of nutrients at the arbuscule level within the cells.

In particular, two phases can be clearly distinguished for this crop; the initial phase of the transition period, up to 20-25 days and the mutualistic phase after this time. In parallel, plant height analysis shows an increase in plant growth compared to the uninoculated control after 27 days of sowing, which coincides with the mutualistic phase of mycorrhizal symbiosis under these salinity conditions.

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Claims (13)

This effect can subsequently be shown in the analysis of yield and its components (Table 15). Table 15 Number of panicles per plant (n), panicle weight (g), 100 grain weight (g) and yield (g plant ^-1 ) on rice plants treated with Dominikia sp. (D. t) and control untreated plants (C) in saline soil Treatment NPP RR R 100 g R D.t 8.33 a 2.70 a 3.69 a 21.66 a WITH 5.40 b 1.87b 2.70b 15.40 b SD 0.12 *** 0.05*** 0.001*** 1 ₃₄ *** S. V (%) AND 2 9.2 6.5 14.6 Notation. NPP: number of panicles per plant (n); PP: panicle weight (g); P 100g: weight of 100 grains (g); R: yield (g plant ^-1 ); SD: standard deviation. Identical letters in the same column are not significantly different at p < 0.05. There was an increase in all components of yield measured in plants treated with Dominikia sp. compared to control uninoculated plants. This was especially interesting under salinity conditions. Thus, the use of this mycorrhizal fungus strain was effective in these soil conditions. This viable and sustainable alternative is very interesting in unfavorable conditions caused by salt stress. CLAIM 1. Dominikia tenuihyphara strain for beneficial effects on crops, deposited in the Belgian Coordinated Collection of Microorganisms (BCCM) under registration number MUCL 57072. 2. A composition for providing a positive effect on agricultural crops, containing the Dominikia tenuihyphara strain according to claim 1, wherein the concentration of Dominikia tenuihyphara in said composition is from 4.0 to 1.0% by weight. 3. The composition according to claim 2, wherein the concentration of Dominikia tenuihyphara in said composition is from 3.0 to 2.0% by weight, preferably from 2.5 to 2.3% by weight. 4. The composition according to claim 2, wherein said composition is a solid composition. 5. The composition according to claim 4, wherein said composition is presented in the form of a powder, an emulsifiable concentrate, granules or microgranules. 6. The composition according to claim 5, wherein said composition is presented in the form of microgranules. 7. The composition according to claim 6, characterized in that said microgranules have a size from 500 to 2000 µm, preferably from 800 to 1500 µm, more preferably from 900 to 1200 µm. 8. The composition according to any one of claims 2 to 7, further containing at least one fungicide, and/or at least one biofungicide, and/or at least one insecticide, and/or at least one bioinsecticide, and/or at least one nematicide, and/or at least one biostimulant. 9. A method for preparing a composition according to claim 2, containing the following steps: a) providing a substrate; b) introducing into the specified substrate the seeds of the host plant and the Dominikia tenuihyphara strain deposited under registration number MUCL 57072; c) cultivating the specified host plant and watering to maintain the moisture level of the specified substrate at least 75% of the natural moisture capacity; d) cessation of watering for a period of at least 7 days; e) removal of the aboveground part of the host plant and substrate; f) drying the removed substrate; g) grinding the dried substrate to obtain granules with a particle size of less than 100 microns. 10. The method according to claim 9, wherein said Dominikia tenuihyphara strain of step b) is an inoculum containing propagules of the Dominikia tenuihyphara strain. 11. The method according to claim 9 or 10, further comprising step h1) microgranulation. 12. The method according to claim 9 or 10, further comprising step h2) preparing a concentrated biological inoculum for coating cereal seeds. 13. The method according to claim 12, in which said step h2) of preparing concentrated biological -