Method for isolating environmental strains of arbuscular mycorrhizal fungi

Abstract

Method for Isolating Environmental Strains of Arbuscular Mycorrhizal Fungi. The invention relates to a method for isolating environmental strains of arbuscular mycorrhizal fungi comprising the following steps: an isolation phase including i. the treatment of a soil sample of interest by successive sievings of decreasing size to concentrate propagules of arbuscular mycorrhizal fungi present in the soil sample, ii. the encapsulation of a propagule obtained at the end of the treatment step in an alginate bead, iii. the culturing of the propagule encapsulated in an alginate bead in a solid substrate in contact with a previously germinated seedling intended to be mycorrhized by the propagule. It finds application in the field of agronomy for the purpose of improving the growth and resistance to stresses (biotic and abiotic) of plant species.This invention is part of a broader context of agricultural transition involving a change in farming practices towards cropping models that are less dependent on inputs (water, fertilizers, pesticides). Figure for the abstract: Fig. 1.

Description

Title of the invention: Method for isolating environmental strains of arbuscular mycorrhizal fungi technical field

[0001] The invention relates to a technique for isolating and advantageously preserving different species of arbuscular mycorrhizal fungi (AMF) from a soil sample.

[0002] It finds application in the agronomic field with the aim of improving the growth and resistance to stresses (biotic and abiotic) of plant species. This invention fits into a global context of agricultural transition involving a change in agricultural practices towards cropping models that are less dependent on inputs (water, fertilizers, pesticides). STATE OF THE ART

[0003] Agriculture is an activity that is particularly intensive in pesticides and fertilizers. Changes in public opinion and regulations are forcing farmers to find alternative solutions, among which is the use of the plant and soil microbiome and in particular Arbuscular Mycorrhizal Fungi (AMF) capable of establishing symbiotic interactions with plants by forming mycorrhizae.

[0004] Arborio fungi (AMF) are soil microorganisms that live in symbiosis with plants. The association between the two partners is characterized by the establishment of a network of hyphae and spores in the rhizosphere (the soil zone surrounding the roots) and the formation of organs characteristic of the symbiosis within the root cells, including vesicles and arbuscules. These symbiotic organs allow for the exchange of nutrients between the plant and the fungus. In this way, AMF enables the plant to more easily absorb certain nutrients (phosphorus, nitrogen, etc.) and helps it to overcome certain stresses more effectively (drought, presence of pathogens, etc.).

[0005] Arboriomorphic plants (AMPs) represent a promising biological tool for the agricultural sector. Given their beneficial impact on most plants, the application of propagules (organs responsible for multiplication: spores, vesicles, fragments of mycorrhizal roots) to various crops constitutes a credible alternative to fertilizers. Their efficient use as a biostimulant has been demonstrated in several sectors, including numerous aromatic and medicinal plants (MAPs; lavender, rose, jasmine, etc.), vineyards, market gardening (solanaceous plants), fruit trees (olive, citrus, plum, etc.), and major field crops (cereals, potatoes), or even certain ornamental shrubs used to reforest delicate areas. Besides acting as a biostimulant for the plant, depending on the genetic characteristics of the species used, arbuscular mycorrhizal fungi (AMF) can also improve the organoleptic and chemical qualities of a finished product by modulating the plant's secondary metabolism. Thus, a tomato grown from a mycorrhizal plant will have different tastes compared to a fruit from a non-mycorrhizal plant.

[0006] The use of arbuscular mycorrhizal fungi (AMF) in agriculture has been under trial for many years. However, a closer examination of these products reveals a problem with inoculum efficacy and reproducibility of results. Recently, an international study highlighted that of 25 commercial AMF-based products tested, 84% failed to establish mycorrhizal symbiosis. This failure can be partly explained by the nature of current inocula, which have several drawbacks. Indeed, some of these products contain only a single AMF strain. The species to which these strains belong is often the same, as it grows easily in vitro or in vivo and produces numerous propagules. However, it is rarely adapted to the context (combination of climate, soil physicochemical characteristics, and the nature of the host plant) in which it is placed.When the inoculum contains a consortium of AMF, the strains belong in the majority of cases to 3 different species, whereas it is estimated that in nature there are at least 350 species of AMF.

[0007] Considering that the genetic divergence of the different species of AMF allows them to occupy a different ecological niche, increasing the diversity of species within a consortium appears essential for at least two reasons: (i) increasing the probability of having a species beneficial to the host plant (ii) possibility of having certain complementary species that act in synergy.

[0008] This observation thus highlights the need to promote a diversity of species in CMA-based inocula, but also to better control this diversity.

[0009] To counter the inefficient use of these generic products, Mycophyto has developed a greenhouse production system that takes into account the diversity of arbuscular mycorrhizal fungi (AMF) in a soil and amplifies several species from a single soil sample. This system, based on the use of two types of successive trap plants and capable of amplifying a wide diversity of species, is the subject of patent application W02020 / 104501. This technique has made it possible to obtain several mixes containing on average about twenty AMF species.

[0010] The drawback of this technique is that the composition of the inoculum species is dependent on the starting sample, and it is not currently possible to exclude or add a single species based on these genetic characteristics. Furthermore, if This system has already proven itself; cultivating several species in parallel in the same system is not always easy, and over generations, some species can dominate others, and there is a risk of a loss of diversity within the mix.

[0011] At present, the isolation of AMF strains from a mix or soil sample is a long and tedious process and does not really follow a well-defined protocol; there are different alternatives, but none allows for the easy isolation of different species from the same mix.

[0012] A technique commonly used in research laboratories involves collecting several spores from a mixture of arbuscular mycorrhizal fungi (AMF) under a stereomicroscope and depositing a single spore onto a seedling under a binocular microscope. The spore attaches to the root by capillary action, and the seedling, with its attached spore, is then placed in a hole made in the center of a pot containing neutral substrate. After 2-3 months, mycorrhization is checked under a stereomicroscope. The success rate of this technique is very low, generally not exceeding 1%. With this technique, there is no way to verify the presence of the spore in the pot. Furthermore, before the spore germinates and attaches to the root, there is also a risk that the root will be washed away, preventing mycorrhization from occurring.

[0013] It is also possible, using another relatively similar method, to apply between 8 and 10 spores, previously grouped for their similar morphological characteristics, to the root by capillary action. While this technique offers a better success rate (the probability that at least one spore will reach the root is higher), there is nevertheless a significant risk of a mixture of species, since morphological characterization does not allow for the certain discrimination of different strains.

[0014] There is therefore a need to propose a solution to promote species diversity in CMA-based inocula, but also to better control this diversity. SUMMARY

[0015] To achieve this objective, according to one embodiment, a process for isolating environmental strains of arbuscular mycorrhizal fungi is provided, comprising the following steps: an isolation phase comprising, advantageously, a step of taking a sample of a soil of interest, a step of processing a sample of soil of interest to separate and concentrate propagules of arbuscular mycorrhizal fungi present in the soil sample of interest, a step of encasing a propagule obtained at the end of the processing step in an alginate bead, a step of culturing the propagule encased in an alginate bead in a solid substrate in contact with a seedling intended to be mycorrhized by the propagule.

[0016] The present method comprises isolating propagules from one another so as to cultivate them individually by associating a single propagule with a plant to avoid mixing species. The method improves the isolation of arbuscular mycorrhizal fungi (AMF) species from a mix obtained from a soil sample of interest providing a source of diverse and numerous environmental AMF. The invention improves the success rate of the isolation phase by 10 to 50 times, depending on the mix, particularly through the use of alginate beads to protect the isolated propagules. According to the present method, it is thus possible to isolate a single propagule in an alginate bead. Maintaining the propagule in close proximity to the mycorrhizal plant is therefore possible while limiting its leaching, facilitating the establishment of the symbiosis.

[0017] Advantageously, the process includes an amplification phase comprising the transplantation of the mycorrhizal seedling, obtained at the end of the isolation phase, to the center of the surface of a pot of substrate covered with plant seeds, advantageously of the same plant as the mycorrhizal seedling, and advantageously pre-germinated, to ensure centrifugal mycorrhization of the AMF and then cultivation under controlled conditions.

[0018] This amplification step, once the monospecific symbiosis has developed on the young seedling, facilitates the amplification of propagules to secure the new strain within the collection by extending the symbiosis to neighboring plants. The centrifugal propagation technique during the amplification phase allows for the production of several hundred propagules at the end of this phase. BRIEF DESCRIPTION OF THE FIGURES

[0019] The aims, objects, features and advantages of the invention will become clearer from the detailed description of an embodiment thereof, which is illustrated by the following accompanying drawings in which:

[0020] [Fig. 1] The [Fig. 1] represents the steps of the isolation phase of the process according to the invention.

[0021] [Fig.2] Fig.2 represents the steps of the amplification phase of the process according to the invention.

[0022] [Fig.3] Fig.3 represents the steps of the in vitro preservation phase of the process according to the invention.

[0023] [Fig.4] Fig.4 represents a box plot of the success rates of propagule isolation according to example 4.

[0024] [Fig. 5A] [Fig. 5A] illustrates a photograph of a pot showing optimal alfalfa density after 3 months in the amplification phase

[0025] [Fig. 5B] [Fig. 5B] illustrates a violin diagram representation of the mycorrhization rate results, validating the effectiveness of the centrifugal mycorrhization propagation system.

[0026] [Fig.6A] Fig.6A illustrates an in vitro culture box used in the in vitro preservation phase in the form of a biobank allowing the development of the aerial part of the plant outside the box and the sterile root part inside the box.

[0027] [Fig. 6B] Figures 6B and 6C illustrate the development of isolates in the in vitro system on alfalfa. The CSM0 strain shown in Figure 6B produces numerous spores, while the MAL3 strain in Figure 6C produces far fewer spores, but a much denser hyphal network.

[0028] [Fig.6C]

[0029] The drawings are given by way of example and are not limiting of the invention. They constitute schematic representations of principle intended to facilitate understanding of the invention and are not necessarily to scale with practical applications. DETAILED DESCRIPTION

[0030] Before proceeding to a detailed review of embodiments of the invention, optional features that may be used in combination or alternatively are listed below:

[0031] According to one example, the processing step comprises a sieving operation using three successive sieves, chosen between 800 µm and 20 µm. The successive sieves are of decreasing size so as to achieve increasingly finer sieving. Preferably, between 500 µm and 30 µm, respectively between 500 µm, 250 µm and 40 µm, or 32 µm, the propagules retained on the 250 µm and 40 µm or 32 µm sieves are collected; According to a cumulative or alternative example, the processing step comprises a sucrose gradient separation.

[0032] According to one example, the imprisonment step includes adding a propagule obtained at the end of the treatment step in an alginate bead in an alginate solution, advantageously at 2%, then adding this alginate solution including the propagule in a calcium chloride (CaCl2) solution, advantageously at 0.1M, ensuring the instantaneous solidification of the alginate trapping the propagule in the form of a bead;

[0033] According to one example, the solid substrate is a neutral substrate comprising sand, perlite, and vermiculite, preferably in proportions 70-15-15;

[0034] According to one example, the seedling intended to be mycorrhized is chosen from among the herbaceous plants of the family Fabaceae, Poaceae, Alliaceae, Solanaceae, Cucurbitaceae, Lamiaceae, Asteraceae, Plantaginaceae or Apiaceae;

[0035] According to one example, the seedling intended to be mycorrhized is an alfalfa seedling;

[0036] According to one example, the isolation phase lasts between 10 and 16 weeks, preferably 12 weeks under controlled conditions;

[0037] According to one example, during the amplification phase, the substrate pot is filled to 2 / 3 with a neutral substrate comprising sand, perlite and vermiculite, on which is added a layer containing the substrate, advantageously called mycorrhizal, of the isolation phase, before completing the pot with neutral substrate;

[0038] According to one example, the process comprises an in vitro preservation phase in the form of a biobank (which may also be called the in vitro biobank creation phase) comprising an in vitro plant / AMC pair system including the collection of propagules from the amplification phase, followed by the sterilization of the collected propagules. Advantageously, in parallel, plantlets, preferably previously germinated, preferably from the same plant as the mycorrhizal plantlet, are sterilized and cultured on an agar-coated in vitro culture dish. Advantageously, the in vitro culture dish comprises an agar medium and the plantlet comprises an aerial part and a root including an apex. Advantageously, the plantlets cultured on the culture dish are arranged such that the apex of the root is in contact with an agar medium of the culture dish and the aerial part of the plantlet is outside the dish.The sterilized propagules are placed in the culture dish containing an agar medium and the seedling, in contact with the agar medium and near the roots of the seedling. The whole constitutes a monospecific in vitro biobank;

[0039] Isolating and developing certain strains on an in vitro culture system makes it possible to eliminate all the auxiliary microorganisms that may be present around the root and to focus solely on the host plant and the arbuscular mycorrhizal fungus. This increases the "controlled" aspect of the system. Furthermore, certain strains will produce more propagules in an in vitro system, and the system requires less storage space;

[0040] Advantageously, the use of specific taller and better sealed dishes than traditional Petri dishes makes the in vitro culture system of the preservation phase in the form of a biobank more sustainable.

[0041] An arbuscular mycorrhizal fungus (AMF) is a eukaryotic soil microorganism, an obligate symbiont of many terrestrial plants.

[0042] Arbuscular mycorrhizal means a fungus that penetrates the roots of a plant by forming arbuscules to establish a symbiotic association.

[0043] A 'trap' plant is defined as a plant used to 'trap' the arbuscular mycorrhizal fungi (AMF) from the soil sample of interest.

[0044] A spore will be defined as a storage and propagation organ of AMF. It germinates and gives rise to mycelial filaments (hyphae) which explore the soil to connect with the roots of a compatible plant.

[0045] Symbiosis is understood to mean a relationship between two heterospecific organisms (different species) which results in reciprocal beneficial effects.

[0046] Environmental, which may have indigenous as a synonym, means that the element is originally present in the soil sample taken; conversely, collection or laboratory, possibly non-indigenous, means an exogenous element, that is to say, one that is artificially introduced, i.e., strains from collections or laboratories.

[0047] Environmental strains are understood to mean indigenous AMF strains.

[0048] An inoculum shall be defined as a sample which includes propagules of at least one strain of arbuscular mycorrhizal fungus.

[0049] A soil sample of interest is defined as a soil sample, for example from a given plot of land, in which environmental strains of arbuscular mycorrhizal fungi (AMF) of interest are estimated to be present. A sample is defined as a mixture of soil and environmental strains of AMF, either taken directly from the soil or having undergone pre-cultivation on trap plants in a neutral substrate to multiply the environmental strains.

[0050] A propagule is understood to mean any cell or group of cells of the AMF which ensures its vegetative multiplication; a propagule can be a spore, a cluster of spores or even microscopic mycorrhizal root fragments.

[0051] The term seedling refers to a young plant resulting from the germination of a seed.

[0052] The term isolate refers to a strain of AMC obtained from the isolation phase and the amplification phase and which is used in the conservation phase.

[0053] Will be defined as sand, preferably a fine sand of type B5 from quarries and previously washed.

[0054] Perlite will be defined as inert and rot-proof materials, with an almost neutral pH, very light, hydrophilic.

[0055] Will be defined by vermiculite as clay minerals with insulating properties and offering good aeration.

[0056] Mycorrhizal fungi contribute to plant root development, thereby increasing the surface area of ​​exchange between the plant and the soil by up to 1000 times. This allows plants to establish deeper roots and access essential nutrients much further away. For example, the applicant observed a 30% to 60% increase in fresh root weight in lavender plants after inoculation and cultivation for two months, compared to control plants. This enables them to draw nutrients more efficiently. Mycorrhizae exchange nutrients, including phosphorus, nitrogen, potassium, copper, calcium, magnesium, zinc, and water. They also form associations with soil bacteria to dissolve minerals and make phosphorus available to plants. This ability to regulate the flow of phosphorus from the soil to the plant is an important function of mycorrhizae in symbiotic exchanges.

[0057] The increased root surface area also acts as a barrier against pathogens, thus protecting the plant. The presence of mycorrhizae also reduces the plant's fertilizer requirements. The difficulty in producing these arbuscular mycorrhizal fungi (AMF) lies in the fact that they are obligate biotrophic organisms. In other words, these AMF are unable to complete their entire life cycle without establishing a symbiotic relationship with the plant. This mycorrhization also plays a role in stimulating the plant's defenses.

[0058] According to a first possibility, the process begins with a soil sample of interest. That is to say, soil in which the plant species to be mycorrhized grows or in which strains of AMF of interest are thought to be present.

[0059] According to this first possibility, a quantity of soil between 200 and 600 g is collected. For example, on an IHa plot, an average of 10 samples are taken to obtain an overall representation of the cultivated land. Physico-chemical analyses are carried out to determine the heterogeneity or homogeneity of the plot. Targeted metabarcoding or metagenomic analyses, in which a DNA fragment present in all arbuscular mycorrhizal fungi (AMF) but exhibiting sufficient polymorphism (variations in DNA bases) to discriminate between the different species, is sequenced and compared to a database to evaluate the strains present in the sample, can be performed.

[0060] According to a second possibility, the process begins with a soil sample that has undergone pre-culturing. Preferably, the soil sample, obtained for example according to the first possibility, is cultured with at least one trap plant, for example for 2 to 3 months. This pre-culturing allows the propagules to be reactivated and increases the number of propagules usable for the process according to the invention. According to one embodiment, the pre-culturing corresponds to the process covered by MYCOPHYTO patent EP3883364B1; the soil sample used in the subsequent stages of the process according to the invention is then the inoculum obtained by the process covered by patent EP3883364B1.

[0061] One of the advantages of the invention is that the process begins with a target sample, equivalent to a soil sample, preferably comprising several strains of environmental AMF, since they originate from the natural environment, unlike known processes that use exogenous strains from banks or laboratory cultures. By way of non-limiting example, at least one strain of arbuscular mycorrhizal fungi present in the soil sample belongs to the phylum Glomeromycota, which includes genera such as: Glomus, Gigaspora, Funneliformis, Rhizophagus, Rhizoglomus, Septoglomus, Claroideoglomus, Paraglomus, Pervetustus, Ambispora, Archaeospora, Diversispora, Acaulospora, Dentiscutata, Pacispora, Racocetra, Cetraspora, Scutellospora, Dominikia, Microdominikia, Oehlia, Sclerocystis, Kamienskia, and Entrophosphora (non-exhaustive list). A soil sample typically contains an average of 5 to 30 different species.

[0062] The process includes a first phase: the isolation phase.

[0063] This isolation phase is intended to isolate from the soil at least one arbuscular mycorrhizal fungus (AMF) advantageously of agronomic interest. Preferably, an AMF of agronomic interest is understood to be a strain of an environmental arbuscular mycorrhizal fungus. The AMF strain is advantageously in the form of a propagule.

[0064] The isolation phase includes a processing step for a soil sample of interest. The processing step advantageously includes separating the propagules from the soil sample and preferably concentrating them. For example, the processing step includes at least one sieving of the sample, preferably several successive sievings. Alternatively or cumulatively, the processing step includes separation using a sucrose gradient to concentrate the spores. Preferably, the processing includes several successive sievings with decreasing sieve sizes, i.e., progressively smaller ones. For example, the sieves are selected from the range of 800 µm to 20 µm, preferably from 500 µm to 30 µm; for example, a 500 µm sieve is used first, then a 250 µm sieve, and finally a 40 µm or even a 32 µm sieve. The sieving is advantageously carried out under a stream of water.The sieving stage removes a large portion of the soil or substrate and concentrates the propagules. The retained fractions, also called residues, are those from the 250 µm and 40 µm or 32 µm sieves; these fractions have higher propagule concentrations than the soil sample of interest.

[0065] According to one possibility, the processing step includes, prior to sieving, grinding the sample, possibly with the addition of water up to an equivalent volume to the sample volume. The grinding is intended to remove large particles and cut the roots. The grinding is carried out, for example, in a blender. As an example, the grinding is performed so that the root fragments are less than 1 cm in size, preferably on the order of 1 mm.

[0066] The isolation phase, following the processing step of the sample of interest, comprises an entrapment step. This entrapment step allows a propagule to be trapped within an alginate bead. Characteristically, each propagule Each individual propagule is trapped within an alginate bead. Thus, each alginate bead contains only one propagule.

[0067] Preferably, each propagule of the retained fractions from the sievings is taken and placed in an individual container. For example, the propagules are taken one by one using microforceps or a pipette under a stereomicroscope and placed individually into Eppendorf tubes. The alginate bead is formed in situ, ensuring the encapsulation of the propagule.

[0068] According to one possibility, each Eppendorf tube containing a propagule also contains a 2% alginate solution. The 2% alginate solution containing the propagule is deposited, advantageously using a pipette, into a 0.1 M calcium chloride solution. Upon contact with the calcium chloride, the alginate solidifies instantly, resulting in a small bead in which a single propagule is encapsulated. By way of example, the alginate bead has a diameter less than or equal to 1 cm, preferably less than or equal to 0.7 cm, and preferably between 0.2 and 0.7 cm.

[0069] The process according to the invention includes a step of culturing the propagule trapped in the alginate bead. This step ensures the individualized culture of each propagule. Preferably, the alginate bead containing the propagule obtained in the trapping step is placed in a solid substrate. The substrate is advantageously a neutral substrate comprising, for example, sand, perlite, or vermiculite, preferably in proportions of 70-15-15.

[0070] The alginate bead is placed in the substrate in contact with a seedling of a trap plant. Preferably, the alginate bead, including the propagule and the seedling, is placed in a hole made in the substrate so that the alginate bead and the seedling are as close as possible to each other to facilitate their meeting. For example, a hole 2 cm in diameter is used.

[0071] The substrate is commonly placed in a container called an isolation container. A container is defined as a receptacle for the substrate. For example, the isolation container has a volume of 200 ml. Similarly, the substrate has a volume of 200 ml.

[0072] The trap plant is advantageously chosen from among herbaceous plants of the Fabaceae, Poaceae, Alliaceae, Solanaceae, Cucurbitaceae, Lamiaceae, Asteraceae, Plantaginaceae, or Apiaceae families. Alfalfa is advantageously chosen because of its mycorrhizotrophic potential, that is, its ability to accept many different species of arbuscular mycorrhizal fungi (AMF). The trap plant used is advantageously a seedling, that is, a seed that has begun to germinate. Preferably, a 3-day-old plant is used. The trap plant seedling is advantageously grown from a seed that has been scarified, for example, with sulfuric acid and sterilized, for example, with a calcium hypochlorite solution.

[0073] Cultivation is carried out under controlled conditions. For example, the pots are placed under a bell jar in a climate-controlled room with alternating day / night cycles (8 a.m. / 4 p.m.) and a temperature of 24°C. The substrate moisture is regularly monitored, and the plants are watered once a week with a low-phosphorus nutrient solution to facilitate mycorrhization.

[0074] The isolation phase, and more specifically the culturing stage of this isolation phase, extends over a period of 10 to 16 weeks, preferably about 12 weeks.

[0075] The roots of alfalfa seedlings can be analyzed under a stereomicroscope to check the progress of mycorrhization.

[0076] According to one embodiment, the process includes, after the isolation phase, an amplification phase.

[0077] The amplification phase allows the isolated strains to be amplified during the isolation phase.

[0078] The amplification phase makes it possible to obtain enough propagules to allow a preservation phase to be carried out with an in vitro step: because the success rate of the preservation phase depends on the absence of contamination, i.e. in particular on good sterilization and the germination rate, i.e. an active and undamaged spore, therefore requires a certain number of propagules to succeed.

[0079] The amplification phase includes a transplanting step for each seedling obtained in the isolation phase. For amplification, the mycorrhizal seedling obtained at the end of the isolation phase is transplanted into another substrate with seeds intended to be mycorrhized in turn.

[0080] Advantageously, the mycorrhizal seedling is placed in the center of the substrate surface and the seeds to be mycorrhized are placed around it; this arrangement ensures a very efficient centrifugal propagation of the mycorrhization.

[0081] Preferably, the seed density is chosen so that each seed is at a distance of 1 to 2 cm from another seed.

[0082] The substrate is advantageously placed in a pot, called the amplification pot, having a larger volume than the isolation pot of the isolation phase. The quantity of substrate used in this amplification phase is greater than the quantity of substrate used in the isolation phase.

[0083] The plant seeds are advantageously all from the same plant and preferably identical to the trap plant used in the isolation phase. Advantageously, the seeds used are alfalfa seeds.

[0084] The substrate for the amplification phase comprises a neutral substrate advantageously comprising sand, perlite, and preferably vermiculite in proportions of 70-15-15 and at least part of the substrate from the isolation phase, this substrate being neutral mycorrhizal substrate, that is to say neutral substrate in which the propagule and the plantlet from the isolation phase multiplied.

[0085] According to one possibility, the substrate of the amplification phase, i.e. the amplification pot, comprises a first layer, called the lower layer, comprising neutral substrate and advantageously representing 2 / 3 of the total height of the amplification substrate, a second layer, called the intermediate layer, comprising the substrate from the isolation phase and a third layer, called the upper layer, comprising neutral substrate.

[0086] The third layer is that which forms the surface of the amplification substrate in which the seeds are placed.

[0087] For example, the amplification pot has a volume of 3L. For example, the amplification substrate has a volume of 3L.

[0088] The amplification substrate and / or the isolation substrate may include other secondary constituents such as fibers, for example. Rock or coconut fiber may be added, in some cases providing a more fibrous texture. Depending on the possibility, at least one mycorrhization-stimulating agent is added. A mycorrhization-stimulating agent is defined as any natural compound, plant extract, insect cell wall, microorganism, or organic matter that promotes mycorrhization. Preferably, the secondary constituents represent less than one-third of the total volume of the substrate.

[0089] The amplification phase advantageously includes a cultivation step preferably under controlled conditions advantageously for a minimum duration intended to reduce the risk of damping-off.

[0090] The amplification phase advantageously includes a step of maintaining the mycorrhizal plants in vivo. The mycorrhizal plants are kept growing and the amplification phase can be repeated; one or more seedlings are transplanted with seeds to achieve centrifugal propagation of the mycorrhization again.

[0091] The mycorrhizal plants obtained at the end of the amplification phase constitute a collection of monospecific inoculum of environmental strains of AMF.

[0092] According to one embodiment, the process according to the invention comprises an in vitro preservation phase in the form of a biobank comprising an in vitro plant / AMC pair system. The objective of this phase is to preserve the isolated propagules, also called isolates, in vitro and to develop a monospecific in vitro biobank.

[0093] The preservation phase includes collecting propagules obtained at the end of the amplification phase. The propagules are advantageously collected using microforceps under a stereomicroscope. The preservation phase preferably includes sterilization of the collected propagules. For example, a sterilization cycle comprising seven successive 5-minute steps, namely: 1) sterile purified water, 2) 2% chloramine + 0.05% Tween, 3) sterile mQ water, 4) 2% chloramine + 0.05% Tween, 5) sterile purified water, 6) sterile purified water, 7) antibiotic solution, preferably 0.02% Streptomycin + 0.01% Gentamicin.

[0094] The preservation phase comprises the establishment of a whole trap plant culture in vitro. This establishment advantageously includes the sterilization and pre-germination of trap plant seeds. The sterilization and pre-germination in this phase are preferably identical to those in the isolation phase. This establishment then comprises the aseptic placement of the trap plant seedlings in an in vitro culture dish containing a nutrient agar medium that is preferably low in phosphate. Characteristically, the seedling is positioned in the in vitro culture dish with the root apex in contact with the agar medium and the aerial part of the seedling outside the culture dish. The dish includes a hole through which the aerial part of the seedling passes. The hole is advantageously coated with silicone to maintain a sterile environment in the culture dish.

[0095] The advantage of whole-plant culture compared to root hair culture, which is usually used, is that it ensures conditions closer to the plant's natural environment, respecting the importance of photosynthesis and the metabolites supplied to the arbuscular mycorrhizal fungus (AMF) via root exudates. The number of species compatible with this whole-plant culture is also greater.

[0096] As an example, the trap plant is chosen from the families Fabaceae, Poaceae, Solanaceae, Lamiaceae, Asteraceae, preferentially the trap plant is alfalfa.

[0097] The preservation phase includes the introduction into the culture dish in contact with the agar medium near the roots of propagules taken at the end of the amplification phase and then sterilized.

[0098] Each culture box includes a seedling and at least one propagule and advantageously several propagules; this is possible since the propagules all come from the same strain of AMF.

[0099] In vitro culture dishes comprising the propagule and the plantlet, whose symbiosis has developed advantageously without contamination, i.e., only the propagule and the plantlet have developed, constitute a collection or monospecific biobank of environmental strains of AMF in vitro on whole plant.

[0100] According to a preferred embodiment of the invention, the in vitro culture dish used differs from a Petri dish. The in vitro culture dish used in the present process has a height of 4 cm, which is greater than that of a Petri dish. The height of the dish allows for optimal observation of the development of the mycorrhizal symbiosis under a stereomicroscope. The dish has the advantage of reducing the risk of contamination while being easily refilled with nutrient solution and therefore has a long lifespan. In addition, the collection of the inoculum is facilitated.

[0101] The method according to the invention makes it possible to collect numerous environmental strains from a soil sample in order to better study them and understand their potential as biostimulating agents. The method makes it possible to obtain pure AMF strains, that is, without contamination by other microorganisms, for easier characterization. Ultimately, it will be possible to create customized mixes by selecting the species best suited to each context. Examples

[0102] Example 1: Isolation phase illustrated in [Fig.1]

[0103] (1.1) A soil sample with a volume of 50 mL is taken and then mixed with an equivalent volume of water is added before being briefly blended to remove large particles and cut roots. (1.2) The sample is then passed through a set of sieves (500, 250, and 40 µm) under a stream of water. This sieving step removes much of the soil or substrate and concentrates the monospecific propagules (spores, spore clusters, mycorrhizal microscopic root fragments) in the fractions retained by the 40 and 250 µm sieves. (1.3) The propagules are individually collected using microforceps under a stereomicroscope and placed into Eppendorf tubes. (1.4) The propagules are then encapsulated in alginate beads before being (1.5) transferred to a 200 mL isolation jar. This pot contains neutral solid substrate based on sand, perlite and vermiculite (70-15-15) and a 3-day-old alfalfa plant.Alfalfa was chosen as a trap plant because of its mycorrhizotrophic potential, that is, its ability to accept many different species of arbuscular mycorrhizal fungi (AMF). The alginate pellet and the seedling are placed in a hole approximately 2 cm deep in the substrate, as close to each other as possible to facilitate their encounter. The alginate pellet prevents the propagule from being washed away. (1.6) The pots are placed under a cloche in a climate-controlled room with a day / night cycle (8 a.m. / 4 p.m.). and a temperature of 24°C. The substrate moisture is regularly monitored and the plants are watered once a week with a low-phosphorus nutrient solution to facilitate mycorrhization.

[0104] Example 2: amplification phase illustrated in [Fig.2]

[0105] (2.1) After 12 weeks, the roots of the alfalfa seedlings are analyzed under stereomicroscope to check the progress of mycorrhization. (2.2) The mycorrhizal plants are transferred into 3L "amplification pots". For the amplification stage, the pots are filled 2 / 3 with neutral substrate (sand-perlite-vermiculite), to which a layer containing the mycorrhizal substrate from the "isolation pot" is added, before filling the pot with neutral substrate. The mycorrhizal plant from the isolation phase is placed in the center of the pot and alfalfa seeds are sown over the entire surface of the pot. (2.3) The pots are placed in a climate-controlled room for 15 days for an incubation stage allowing the alfalfa seeds to germinate and overcome the risk of damping-off. (2.4) After this, the pots are transferred to a greenhouse where they are stored for amplification of the propagules and maintained in collection for many months to form a monospecific in vivo biobank, preferably in a greenhouse.

[0106] Example 3: in vitro conservation phase in the form of a biobank illustrated in [Fig.3].

[0107] (3.1) Initially, alfalfa seeds are sterilized with a A calcium hypochlorite-based solution is prepared and germinated for 3 days on agar medium. (3.2) In parallel, for each strain, propagules are collected using microforceps under a stereomicroscope and then sterilized in a cycle comprising seven successive 5-minute steps: sterile purified water, 2% chloramine + 0.05% Tween, sterile purified water, 2% chloramine + 0.05% Tween, sterile purified water, sterile water, and antibiotic solution (0.02% Streptomycin + 0.01% Gentamicin). (3.3) The whole-plant alfalfa in vitro system is then set up. The germinated seedlings are aseptically placed in an in vitro culture dish containing a low-phosphate agar nutrient medium. To do this, it is necessary to create a hole on the edge of the box halfway up, and insert the seedling in such a way that the apex of the root is in contact with the agar medium and the aerial part of the plant is outside the box.The hole into which the seedling was inserted must be sealed with silicone to maintain a sterile environment inside the dish. The sterilized propagules are then placed in contact with the agar near the roots. (3.4) The dishes are kept in a climate-controlled chamber with a day / night cycle (8 a.m. / 4 p.m.) and a temperature of 24°C for several months and constitute the in vitro monospecific biobank.

[0108] Example 4: percentage of successful isolation.

[0109] Propagules from a mix originating in the Alpes-Maritimes region underwent the isolation phase of the process according to the invention as described in Example 1. Of 50 seedlings placed in contact with an alginate bead containing a single propagule, 27 showed signs of mycorrhization after the 3-month isolation phase, i.e., a success rate of 54%, more than 50 times higher than what research laboratories usually obtain with the conventional method described in the prior art. The experiment was repeated on 11 different mixes, and the success rate ranged from 8% to 73%. Figure 4 illustrates a box plot (min to max) representing the distribution of successful isolation percentages for each mix tested so far (n=1). Each point indicates the percentage obtained for a mix. The intermediate line represents the median.The dotted line represents the average usually obtained with the conventional technique described in the prior art.

[0110] Example 5: Monitoring centrifugal dispersion

[0111] After 3 months of culture during the amplification phase carried out according to the invention, an intermediate quality control was carried out on 14 isolates from 3 different mixes to verify the validity of the centrifugal dispersion technique.

[0112] Figures 5A and 5B illustrate the sampling strategy for quality control (Fig. 5A) and the mycorrhization rate results obtained (Fig. 5B). For each sample, two seedlings are taken from each area, and the mycorrhization rate is checked.

[0113] Figure 5A illustrates a photograph of a pot showing optimal alfalfa density after 3 months in the amplification phase. The different circles represent the sampling zones (2 roots sampled per zone). Zone 1 is central (if recognizable, avoid sampling the initial plant), Zone 2 is intermediate, and Zone 3 is outer. Figure 5B illustrates a violin diagram representation of the mycorrhization rate results for the 14 samples tested. For each diagram, the upper and lower lines represent the 1st and 3rd quartiles, respectively, while the central line represents the median value. The width of the diagrams is proportional to the distribution of values.

[0114] The results obtained ([Fig.5B]) highlight a progressive dispersion of mycorrhization from the center of the pot towards the periphery, thus validating the amplification strategy developed

[0115] Example 6: monitoring of the in vitro preservation phase

[0116] At the end of the 6-month amplification phase, a new quality control check is carried out by taking 3 plants, one from each zone. For a transition to the next phase of conservation, the frequency of mycorrhization must be greater than 90% with a mycorrhization intensity of at least 15%.

[0117] The system described in [Fig. A] allows the aerial parts of the plant to develop outside the box and the sterile root parts inside the box. The height of the box allows for optimal observation of the mycorrhizal symbiosis development under a stereomicroscope. [Fig. B] and [Fig. C] illustrate the development of isolates in the in vitro system on alfalfa. The Rhizophagus irregularis - CSMO strain shown in [Fig. B] produces numerous spores, while the Rhizoglomus silesianum - MAL3 strain in [Fig. C] produces far fewer spores, but a much denser hyphal network. The development of symbiosis with several strains validates the process according to the invention, and in particular the in vitro culture.

Claims

Demands

1. A method for isolating environmental strains of arbuscular mycorrhizal fungi (AMF) comprising the following steps: an isolation phase comprising i. A step of processing a soil sample of interest to separate and concentrate propagules of arbuscular mycorrhizal fungi present in the soil sample of interest, ii. A step of encasing a propagule obtained at the end of the processing step in an alginate bead, iii. A step of culturing the propagule encased in an alginate bead in a solid substrate in contact with a seedling intended to be mycorrhized by the propagule.

2. A method according to the preceding claim wherein the processing step comprises a sieving comprising three successive sieves, selected between 800 µm and 20 µm.

3. A method according to any one of the preceding claims wherein the entrapment step comprises adding a propagule obtained at the end of the treatment step in an alginate bead in a 2% alginate solution and then adding this alginate solution comprising the propagule to a 0.1M calcium chloride (CaCl2) solution ensuring the instantaneous solidification of the alginate entrapping the propagule in the form of an alginate bead.

4. A method according to any one of the preceding claims wherein the solid substrate is a neutral substrate comprising sand, perlite, and vermiculite, preferably in proportions 70-15-15.

5. A method according to any one of the preceding claims wherein the seedling to be mycorrhized is selected from herbaceous plants of the families Fabaceae, Poaceae, Alliaceae, Solanaceae, Cucurbitaceae, Lamiaceae, Asteraceae, Plantaginaceae or Apiaceae.

6. A method according to any one of the preceding claims wherein the seedling to be mycorrhized is an alfalfa seedling.

7. A method according to any one of the preceding claims wherein the cultivation step lasts between 10 and 16 weeks, preferably 12 weeks under controlled conditions.

8. A method according to any one of the preceding claims comprising an amplification phase including transplantation of the mycorrhizal seedling, obtained at the end of the isolation phase, to the center of the surface of a pot of substrate covered with plant seeds to ensure centrifugal mycorrhization of the AMF and then cultivation under controlled conditions.

9. A method according to the preceding claim wherein for the amplification phase, the substrate pot is filled to 2 / 3 with a neutral substrate comprising sand, perlite and vermiculite, on which is added a layer containing the substrate of the isolation phase, before completing the pot with neutral substrate.

10. A method according to any one of the two preceding claims comprising an in vitro preservation phase in the form of a biobank comprising an in vitro plant / CMA pair system comprising a collection of propagules from the amplification phase, then sterilization of the collected propagules, in parallel plantlets are sterilized and cultured on an in vitro culture dish comprising an agar medium, the plantlet comprising an aerial part and a root comprising an apex disposed in contact with the agar medium and the aerial part of the plantlet being outside the dish, the sterilized propagules are deposited in contact with the agar medium near the roots constituting a monospecific in vitro biobank.

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