WO2009136049A1

WO2009136049A1 - Method for obtaining tuber magnatum mycelium and mycelium obtained by means of the method

Info

Publication number: WO2009136049A1
Application number: PCT/FR2009/050582
Authority: WO
Inventors: Marc Buee; Francis Martin
Original assignee: Institut National De La Recherche Agronomique - Inra
Priority date: 2008-04-07
Filing date: 2009-04-06
Publication date: 2009-11-12
Also published as: FR2929489A1; EP2268790A1; FR2929489B1

Abstract

The subject matter of the present invention is a method for obtaining Tuber magnatum mycelium, comprising the following steps: a) culturing, in vitro, one or more fragments of Tuber magnatum tissue in the presence of plant roots, and b) collecting the Tuber magnatum mycelium obtained at the end of step a).

Description

TITLE OF THE INVENTION

Process for obtaining mycelium of Tuber magnatum and mycelium obtained by the process

FIELD OF THE INVENTION

The present invention relates to the field of production of Tuber magnatum, commonly referred to as white truffle, which is an edible mushroom with great commercial value.

PRIOR ART

Tuber magnatum, also known as Piedmont white truffle or Alba truffle, is a rare edible mushroom that is very popular with consumers. Tuber magnatum is an ectomycorrhizal fungus that grows and matures from October to December in the clay-limestone soils of northern Italy, usually at a depth of 5 to 50 centimeters. This fungus lives in symbiosis preferentially with poplars (black poplar, white poplar, aspen, etc.). Tuber magnatum can also live in symbiosis with other tree species such as oaks, linden trees and hazel trees.

Because Tuber magnatum is rare in nature but is highly valued by consumers, there is a need for the development of techniques to make this fungus available to the public in the desired quantity and quality. In the first place, there is a need for the development of techniques for obtaining, in the desired quantity and quality, Tuber magnatum mycelium, in order to achieve the mycorrhization of compatible species, then to cultivate mycorrhizal species for the production of these mushrooms. To the knowledge of the applicant, there is currently no technique that has allowed the production of Tuber magnatum mycelium under conditions allowing the mycorrhization of compatible species, despite the many attempts that have been made in the state of the art.

To the applicant's knowledge, the only scientific article describing the in vitro production of T. magnatum mycelium is the article by Fontana et al. (Mycological Research, 1993, Vol 97 (N ^O I): 40-44). From morphological analyzes, Fontana et al. thought they had obtained T. magnatum mycelium from mycorrhizae. However, a few years later, the same authors showed by genomic analyzes that this mycelium did not belong to the species T. magnatum but to the species T. melanosporum (MeIIo et al., 2001, Microbiological Research, Vol. 2001 (155), 279-284).

In addition, in a review article on edible fungi cultivation methods, Hall et al. had observed that production techniques had been developed for a variety of fungi, such as Lactarius deliciosus, Lyophyllum shimeji, Rhizopogon rubescens, Suillus granulatus, Terfezia, as well as a wide variety of Tuber (Hall et al., 2003, Trends in Biotechnology, Vol 21 (N ^o 10): 433-438). Among the Tuber for which culture techniques had been developed, these authors cite the examples of Tuber borchii, Tuber melanosporum and Tuber uncinatum. But Hall et al. (2003, cited above) also pointed out that no technique could be developed for growing Tuber magnatum.

With the aim of subsequently defining techniques for the controlled production of Tuber magnatum, various scientific works have been carried out to better understand the physiology of this fungus and in particular the nature of the interactions of this fungus with the surrounding ecosystem.

Illustratively, Murat et al. had confirmed that no technique for the controlled production of Tuber magnatum was possible in 2005, while such techniques were fully available for Tuber borchii and Tuber uncinatum and Tuber melanosporum (Murat et al., 2005, FEMS Microbiology Letters, Vl. 245: 307-313). As a result, these authors focused on studying the interactions of Tuber magnatum with its ecosystem and the results obtained concluded that additional analyzes were needed to improve knowledge of the Tuber magnatum ecosystem and thus increase the chances of success. of success for a future development of an experimental production of this mushroom. Also, Barbieri et al. had observed that, despite the success of many edible truffles, such as Tuber melanosporum, Tuber aestivum and Tuber borchii, there had been no development of a controlled production technique of Tuber magnatum (Barbieri et al., 2007, Environmental Microbiology, Vol 9 (No. 9): 2234-2246). These authors, who had studied the diversity of microbial communities associated with Tuber magnatum, had concluded that potentially nitrogen-fixing bacteria were likely to contribute to the development and maturation of these truffles' ascomas.

Thus, to the knowledge of the applicant, the need for a controlled production process of Tuber magnatum has still not been satisfied to date.

SUMMARY OF THE INVENTION

The present invention relates to a method for obtaining Tuber magnatum mycelium comprising the following steps: a) cultivating in vitro one or more fragments of Tuber magnatum tissue in the presence of plant roots, and b) collecting the Tuber magnatum mycelium. obtained at the end of step a), associated or not associated with the roots of plants.

In some embodiments of the method, the plant roots consist of roots transformed with Agrobacterium rhizogenes. In some embodiments of the method, the roots are from plants of a genus selected from Pinus, Picea, Abies, Corylus, Castanea, Tilia, Cedrus, Populus, Quercus, Cistus and Daucus.

In some embodiments of the method, step a) of in vitro culture is carried out in a culture medium comprising one or more strains of bacteria naturally associated with fungi of the genus Tuber, including bacteria of the species Stenotrophomonas maltophilia.

In some embodiments of the method, said method comprises the following additional step: c) transferring the mycelium collected in step b) into a root-free culture medium.

In some embodiments of the method, said method, after step c), comprises the following additional step: d) culturing in said root-free culture medium, the mycelium transferred to step c).

The present invention also relates to a Tuber magnatum mycelium characterized in that it is obtained by the process defined above.

The present invention also relates to a method of plant mycorrhization by Tuber magnatum comprising the following steps: a) inoculation by contacting the roots of seedlings or cuttings with a Tuber magnatum mycelium obtained by the method or with a mycelium of Tuber magnatum as defined above; b) in vitro culture of the seedlings or inoculated cuttings obtained in step a) until the appearance of mycorrhizae.

DESCRIPTION OF THE FIGURES Figure 1 illustrates the growth curve of Tuber magnatum in the presence of poplar roots transformed with Agrobacterium rhizogenes. On the ordinate: size of the Tuber magnatum mycelium, expressed in millimeters. On the abscissa, time elapsed from the incubation of the carpophore fragment with the poplar roots, expressed in days.

Figure 2 illustrates a comparison of the growth level of Tuber magnatum mycelium, respectively in the absence (left part of Figure 2) or in the presence (right part of Figure 2) of Ciste roots transformed with Agrobacterium rhizogenes. On the ordinate: production of mycelium cultures from a piece of Tuber magnatum (percent).

Figure 3 shows a snapshot of an agar explant containing Tuber magnatum mycelium in the absence of plant root.

Figure 4 shows a snapshot of an agar explant containing Tuber magnatum mycelium in the presence of Populus roots transformed with Agrobacterium rhizogenes, 6 weeks after the start of incubation. Fig. 5 is a snapshot of an agar explant containing Tuber magnatum mycelium in the presence of Daucus carota roots transformed with Agrobacterium rhizogenes, 6 weeks after the start of incubation.

Figure 6 is a snapshot of an agar explant containing Tuber magnatum mycelium in the presence of Cistus roots transformed with Agrobacterium rhizogenes, 6 weeks after the start of incubation.

Figure 7 shows a snapshot of seedlings (cuttings) of Populus mycorrhizae by Tuber magnatum.

FIG. 8 illustrates the comparison of the growth level of the Tuber melanosporum mycelium, respectively in the absence (Mel-28) or in the presence (Mel-28 + cistus) of Ciste roots transformed with Agrobacterium rhizogenes, after 9 months of incubation. On the ordinate: percentage of production of mycelium cultures from T. melanosporum mycelium fragments.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors have shown that Tuber magnatum mycelium can be obtained in a simple manner by cultivating in vitro, under controlled conditions, a fragment of Tuber magnatum tissue in the presence of plant roots. Thus, the present invention relates to a process for obtaining mycelium from

Tuber magnatum comprising the following steps: a) cultivating in vitro one or more fragments of Tuber magnatum tissue in the presence of plant roots, and b) collecting the Tuber magnatum mycelium obtained at the end of step a), in association or not with plant roots.

As shown in the examples, a single fragment of Tuber magnatum tissue is sufficient in step a) to obtain mycelium at the end of step a). However, two tissue fragments or more than two fragments of Tuber magnatum tissue can be cultured in vitro in step a) without particular disadvantage. In some embodiments illustrated in the examples, in step a) one or more Tuber magnatum carpophore fragments are used. However, the process according to the invention can also be carried out using, in step a), fragments of other types of Tuber magnatum tissue, such as mycorrhizal fragments or fragments of mycelium. It should be noted that the fragments of carpophore include, inter alia, spores and / or gleba. Gleba is condensed mycelium. Thus in some embodiments, the method of producing mycelium can be implemented using directly gleba or spores from the carpophore, gleba and spores being considered fragments of carpophore.

It will generally be preferred to use one or more Tuber magnatum carpophore fragments, because the carpophore is an organ for which the morphological characteristics of Tuber magnatum are most readily recognizable. Tuber magnatum mycelium grown in vitro obtained by said method will also be used.

It is preferred to use Tuber magnatum fragments collected in the internal tissues of the carpophore to avoid introducing undesirable soil microorganisms into subsequent cultures. These undesirable microorganisms are located on the outer surface of the carpophores, in contact with the soil.

In some embodiments of the method, the Tuber magnatum tissue fragment (s) and the plant roots are contacted with each other during step a).

However, as illustrated in the examples, Tuber magnatum mycelium can be produced during step a) of in vitro culture without close physical contact between the Tuber magnatum tissue fragment and the plant roots.

Without wishing to be bound by any theory, the inventors believe that the roots of growing plants that are present in step a) of the process produce or release one or more metabolites that are necessary for the development or growth of the mycelium. Thus, in some other embodiments of the method, the Tuber magnatum tissue fragment (s) are simply incubated together in step a), the important characteristic being the presence of plant roots. This joint incubation allows metabolites released by the plant roots into the culture medium to diffuse into the culture medium to the Tuber magnatum tissue fragment (s). It should be noted that the root introduced into the environment is free of all mycorrhizae.

It should also be noted that under the conditions of the process according to the invention, the plant root introduced into the culture medium is not mycorrhized by the mycelium of T magnatum, growing even after physical contact with each other. During the production process of the mycelium according to the present invention, therefore, there is no close physical interaction (i.e., symbiotic association) between the growing mycelium and the plant root.

It has been shown according to the invention that step a) can be performed with a wide variety of plant roots.

In some embodiments, isolated root tissue that has been previously prepared from plants is used in step a). Preferably, the isolated root tissue is subjected to one or more intense washing steps, for example with sterile water or with culture medium, in order to eliminate almost all or all the microorganisms likely to be therein. initially associated. Thus, plant roots isolated from a whole plant and having been subjected to one or more appropriate washing steps can be used successfully in step a) of the process.

However, roots isolated from whole plants have reduced longevity. Such roots have in particular the disadvantage of developing and differentiating in the culture medium and thus giving birth to seedlings.

This is why step a) of the method is carried out preferentially with root tissue whose ability of the constituent cells to differentiate into tissues other than root tissue has been blocked. Preferably, plant roots are used which have been transformed with

Agrobacterium rhizogenes. The roots of plants transformed with Agrobacterium rhizogenes are transformed into "hairy roots" which have the advantage of developing exclusively into a root tissue that can be grown in the long term and transplanted, without requiring a new recourse to a stage of isolation of roots from an entire plant. In addition, without wishing to be bound by any theory, the inventors believe that the roots transformed by Agrobacterium rhizogenes, also called "hairy roots" or "root organ cultures" (ROC), produce during their growth important secondary metabolites which contribute to the development of Tuber magnatum mycelium, in step a) of the process of the invention. The root tissue transformation techniques with Agrobacterium rhizogenes are well known to those skilled in the art. By way of illustration, the plant roots used in step a) of the process may be derived from a transformation with different bacterial strains, such as strains 15384, 1724, 8196 or 2659 of Agrobacterium rhizogenes.

In general, as illustrated in the examples, roots from a wide variety of plants can be used in step a) since tuber magnatum mycelium is produced with roots of plants as far apart as the species. ligneous and carrots.

In some embodiments of the method according to the invention, in step a), roots of a plant belonging to a woody species are used. In a general way, in step a) of the method, roots of host plants of

Tuber magnatum.

In certain embodiments of the method according to the invention, roots originating from plants of a genus chosen from among Pinus, Picea, Abies, Salix, Ostrya, Corylus, Castanea, Tilia, Cedrus, Populus, Quercus, Cistus and Daucus are used. where appropriate in the form of plants or hybrid varieties.

Populus nigra, Populus alba, Populus trichocarpa, Populus deltoids, Populus simonii, Populus tremula, Populus yunnannensis, Populus canescens and Populus canescens may be used as roots of the genus Populus. Populus lasiocarpa, as well as poplar hybrids, including poplar hybrids that have become wild-type and poplar hybrids that form cultivars artificially created by humans.

Among the roots of plants of the genus Cistus, it is possible to use the roots of the following species of cistus: Cistus albidus, Cistus crispus, Cistus creticus, Cistus incanus, Cistus monseliensis, Cistus ladaniferus, Cistus laurifolius, Cistus populifolius, Cistus salvifolius, Cistus clusii , Cistus inflatus and Cistus pouzolzii.

Among the roots of plants of the genus Qercus, the roots of the following species of oaks may be used: Quercus rubra, Quercus agrifolia, Quercus alba, Quercus coccifera, Quercus faginea, Quercus nigra, Quercus robur, (= pedunculata) Quercus petraea, Quercus // ex, Quercus libani, Quercus pyrenaica and Quercus pubescens.

Corylus maxima, Corylus colurna, Corylus chinensis, Corylus americana, Corylus avellana, Corylus cornuta, Corylus ferox, Corylus heterophylla, Corylus jacquemontii, Corylus sieboldiana may be used as roots of plants of the genus Corylus. and Corylus tibetica.

Tilia platyphyllos, Tilia americana, Tilia amurensis, Tilia chinensis, Tilia cordata, Tilia dasystyla, Tilia xeuchlora, Tilia xeuropacea, Tilia xflavescens, Tilia henryana, and Tilia platyphyllos, Tilia americana, Tilia , Tilia insularis, Tilia intonsa, Tilia japonica, Tilia kiusiana, Tilia mandshurica and Tilia maximaowicziana.

Among the roots of plants of the genus Salix, it is possible to use roots from the following willow species: Salix caprea and Salix viminalis.

Among the roots of plants of the genus Ostrya, we can use the roots of hops-charm (Ostrya carpinifolia). Among the roots of plants of the genus Daucus, one can use the roots from any of the commonly found carrot species for human food or livestock.

Preferentially, roots from plants selected from poplar, rockrose and carrot are used, as illustrated in the examples. In general, the process for obtaining Tuber magnatum mycelium according to the invention is carried out under so-called axenic conditions, ie under controlled conditions in which the culture medium of step a) does not comprise undesirable microorganisms, for example microorganisms resulting from undesirable contamination of the plant roots used or undesirable contamination of the culture medium used.

First, as with any cell culture, a sterile culture medium is used.

The sterility of the roots or fragments of Tuber magnatum tissue used can be obtained according to conventional techniques well known to those skilled in the art, such as that the elimination of microorganisms by one or more washing steps, and / or prior steps of incubation in a salt buffer, for example a culture medium, comprising antibiotics. For example, it is possible to use a liquid or solid medium comprising one or more antibiotics chosen from cefotaxime sodium, disodium carboline, vancomycin, ampicillin sodium, claforan, streptomycin sulfate or tetracycline, for example at a concentration which may be go from 100 to 500 μg / mL

In some embodiments of the method of the invention, prior decontamination of the plant roots is performed and the Tuber magnatum tissue fragment (s) are not decontaminated, such that the tissue fragments of Tuber magnatum are removed. are likely to contain cells of one or more bacterial species that are naturally present in the environment of this fungus when found in the ground.

In other embodiments of the process of the invention, said process is carried out under completely axenic conditions, with prior decontamination of both (i) the Tuber magnatum fragment (s) and (ii) plant, and use of a sterile culture medium.

According to a particular aspect of implementation of the method of the invention, step a) of in vitro culture is carried out in a culture medium comprising one or more strains of bacteria naturally associated with fungi of the genus Tuber. In a first embodiment of this particular aspect of the process, step a) of culture is carried out with fragments of Tuber magnatum tissue which have not undergone a decontamination step by elimination of the associated microorganisms.

In a second embodiment of this particular aspect of the process, step a) of culture is carried out under axenic conditions and naturally occurring bacteria associated with fungi of the genus Tuber are added to the culture medium at the beginning of step a) .

In general, bacteria naturally associated with fungi of the genus

Tuber, and particularly bacteria associated with Tuber magnatum, may be selected from the bacteria described by Barbieri et al. (2007, Environmental Microbiology, Vol 9 (No. 9):

2234-2246). Preferably, bacteria of the species Stenotrophomonas maltophilia are added.

By way of illustration, it is possible to use one or more strains of Stenotrophomonas maltophilia chosen from the group consisting of the strains deposited at the ATCC and referenced under the numbers

BAA-902, 13637D-5, 13270, 13636, 13637, 13843, 14535, 15099, 17445, 17666, 17671,

17672, 17673, 17674, 17675, 17676, 17677, 17678, 17679 and 17806. Illustrative embodiments of the process of the invention in which the tissue fragments of Tuber magnatum are cultured in step a). plant roots in the presence of bacteria associated with fungi of the genus Tuber, and more particularly in presence of bacteria of the species Stenotrophomonas maltophilia are described in the examples.

When the process of the invention is carried out under axenic conditions with addition of exogenous bacteria naturally associated with the fungus of the genus Tuber, at the beginning of step a) the amount of bacteria appropriate to obtain a final concentration of at least 10 and at most 10 ⁹ bacterial cells per ml of culture medium at the time of their addition.

The examples show that in step a), the growth of the bacteria is very moderate.

Without wishing to be bound by any theory, the inventors believe that the presence of bacteria naturally associated with fungi of the genus Tuber promotes the development and growth of the Tuber magnatum mycelium.

Thus, in preferred embodiments of the method of the invention, a ternary combination of (i) fragment (s) of Tuber magnatum tissue, (ii) plant roots and (iii) naturally occurring bacteria is cultured in vitro. associated with fungi of the genus Tuber, and more particularly bacteria associated with Tuber magnatum.

Preferably, step a) of in vitro culture is carried out at a temperature ranging from 1 0 ⁰ C to 30 ⁰ C and very preferably from 15 ⁰ C to 25 ⁰ C.

Preferably, step a) of in vitro culture is carried out in the dark.

The culture medium used in step a) may be any type of suitable culture medium, known to those skilled in the art.

By way of illustration, use is made of minimum medium, also referred to as "medium M", the composition of which is described in particular by Bécart et al. (1988, New Phytol., Vol 108: 21-218).

Preferentially, a culture medium comprising a gelling agent is used.

For example, agar may be used as a gelling agent to form an agar medium. It is also possible to use a gelling agent chosen from agar and phytogel. For example, the gelling agent sold under the name Phytagel® may be used by Sigma-Aldrich.

The amount of gelling agent present in the culture medium can be easily adapted by those skilled in the art. The amount of gelling agent depends on the gelling agent used and the final viscosity desired for the culture medium used in step a) of the process.

In general, the culture medium used in step a) may comprise an amount of gelling agent of at least 3 percent by weight, based on the total weight of the culture medium, preferably at least 5 percent by weight. percent by weight, based on the total weight of the culture medium. Generally, the amount of gelling agent is at most 15 percent by weight, preferably at most 10 percent by weight, based on the total weight of the culture medium.

Step a) can be performed in various types of containers or culture dishes. Step a) is conventionally carried out in petri dishes. The mycelium, associated or not with the roots of plants, is collected in step b) at a time when the skilled person estimates that the amount of mycelium produced is sufficient for the intended use, for example to achieve a or several subcultures of mycelium by transplanting or to perform mycorhization of plants. As an illustration, the mycelium can be recovered in step b) when its size at the end of step a) reaches 50 millimeters or more, for example 80 millimeters or more.

In general, to collect the amount of mycelium sufficient to effect subcultures or mycorrhizations, the duration of step a) of the method is about at least three weeks, for example about four or more six weeks. The duration of step a) may be greater than six weeks but this is not generally necessary since the amount of mycelium produced is sufficient to carry out subcultures. For mass production of inoculum, for controlled mycorrhiza- tion, the cultures can be maintained for several months, for example for a period of two months or more and up to twelve months. As illustrated in the examples, the mycelium that is collected in step b) can then be used to perform subcultures.

Thus, thanks to the process of obtaining Tuber magnatum mycelium which is described above, one obtains mycelium whose culture can then be continued over a long period of time, without again requiring the use of the use of fragments. of Tuber magnatum tissue, other than the mycelium that is collected in step b).

In preferred embodiments, the process for obtaining Tuber magnatum mycelium comprises a step of transplanting the mycelium collected in step b), in the presence of plant roots, in order to achieve one or more mycelium subcultures. Tuber magnatum in the presence of new plant roots (ROC or hairy roots). In other preferred embodiments, the Tuber magnatum mycelium collected in step b) is subsequently used in a plant mycorrhizal method.

The production of mycelium subcultures or the realization of a mycorrhization process requires a step of transplanting the mycelium collected in step b) of the above process, by transfer into a suitable culture medium. Accordingly, in some embodiments, the method of the invention comprises the following additional step: c) transferring the mycelium collected in step b) into a culture medium.

In the embodiments of the method in which subcultures are carried out, said method comprises the following additional step: d) culturing in said root-free culture medium, the mycelium transferred to step c);

The mycelium may be cultured, or "subcultured" in vitro in step d) for several weeks, as long as the culture medium contains sufficient nutrients to maintain or grow the mycelium, without loss of viability. In general, it was shown in the examples that mycelium collected in step b) of the method could be subcultured and cultured in vitro for at least six weeks.

In general, the culture medium used in step d) may be the same as the culture medium used in step a). In some embodiments of steps c) and d), the mycelium is incubated in the culture medium in the absence of plant roots and without the addition of exogenous bacteria.

When no addition of exogenous bacteria is performed in steps c) and d), bacteria may nevertheless be present. Indeed, in the embodiments of step a) in which naturally occurring bacteria associated with fungi of the genus Tuber are present in the culture medium, these bacteria colonize the growing mycelium and can be transferred with the mycelium collected at the same time. step b) for the following process steps.

In certain other embodiments of steps c) and d), the mycelium is incubated in the culture medium in the absence of plant roots, but bacteria naturally associated with the fungi of the genus Tuber, in particular naturally associated bacteria, are added. Tuber magnatum, such as Stenotrophomonas maltophilia. In this case, the bacteria are added in the same quantity range as for step a) of the process.

In still other embodiments of steps c) and d), the mycelium is incubated in the culture medium in the presence of plant roots. Any of the types of plant roots that can be used to carry out step a) of the method can be used.

Thus, in certain embodiments of steps c) and d), the culture medium comprises (i) Tuber magnatum mycelium, (ii) plant roots and (iii) naturally occurring bacteria associated with Tuber fungi, especially bacteria associated with Tuber magnatum.

It follows from the foregoing that the present invention also relates to a method for subculture of Tuber magnatum mycelium, or for performing a subculture of Tuber magnatum mycelium, which comprises the following steps:

A) placing in a culture medium the Tuber magnatum mycelium obtained at the end of step b) of the production process described above, and

B) culturing in said culture medium the mycelium incubated in step A).

The characteristics of steps A) and B) of the above process are identical to the characteristics of steps c) and d) of the process for obtaining Tuber magnatum mycelium described above. The present invention also relates to a Tuber magnatum mycelium which has been collected in step b) of the production process described above.

The present invention also relates to a Tuber magnatum mycelium which has been obtained at the end of step d) of the production process described above. The subject of the invention is also a Tuber magnatum mycelium which has been obtained at the end of stage B) of the subculture process described above.

The Tuber magnatum mycelium that can be obtained (i) either in step a) of the process of obtaining above, (ii) in step d) of the obtaining process above, (iii) either in step B) of the above subculturing process, can then be used for the mycorrhization of plants to produce ready-to-eat white truffles.

Conventional mycorrhizal techniques well known to those skilled in the art that have already been applied to fungal species other than Tuber magnatum can be used. Thus, the present invention also relates to a method of plant mycorrhization by Tuber magnatum comprising the following steps: a) inoculation by contacting the roots of seedlings or cuttings with a Tuber magnatum mycelium, whether or not interacting with plant roots, obtained

by the process of obtaining described above, by the subculture process described above, or

with a Tuber magnatum mycelium as described above; b) in vitro culture of the inoculated seedlings obtained in step a) until the appearance of mycorrhizae.

For the production of inoculum and the realization of the mycorhization process above, the skilled person can refer to many publications or books describing them, for example to the article by Bokor (1954, Erdészeti Kutatàsok, Budapest, Vol 1, pp. 27-45), in the article by Le Tacon and Valdenaire (1980, Revue Forestière Française, Vol XXXII, no.

1664-1668), in the article by Le Tacon et al. (1983, Annales des Sciences Forestières, Vol.40, No. 2, pp. 165-176), in the article by Boutekrabt et al. (1990, Agronomy, Vol 2, pp. 127-132), in the article by Garbaye and Churin (1997, Forest Ecology and Management, Vol 98, pp. 221-228), in the article by Bécard and Piché (1989, Appl., Microbiol., Vol 55, pp. 2320-2325), in the article

Mortier et al. (1989, Ecosystem Agric., Environ 28, pp. 351-354), or to the article

Wenkart et al. (2001, Plant CeII Reports, Vol 20, pp. 369-373).

For the implementation of the above method of mycorrhization, seedlings of a genus chosen from among Pinus, Picea, Abies, Corylus, Castanea, Tilia, Cedrus, are preferably used.

Populus, Quercus, Cistus and Daucus, where appropriate in the form of hybrid plants or varieties.

Most preferably, seedlings or cuttings selected from Salix are used,

Ostrya, Populus, Quercus and Cistus. In some embodiments of the mycorrhizal method above, that comprises a step c) of transplanting the mycorrhizal seedlings obtained in step b) into the earth. In general, ready-to-eat white truffles are obtained after a period of at least about three years, sometimes at least about six years, following step c) transplanting in the ground.

The present invention is also illustrated by the following embodiments.

EXAMPLES

EXAMPLE 1 Process for producing Tuber magnatum mycelium

1-Preliminary work:

A) Root production of a Tuber magnatum host plant transformed by A. rhizogenes: Poplar (Populus tremula x Populus alba hybrid, INRA 717-1 B4 clone)

A1) Transformation of poplar cuttings (Populus hybrid tremula x Populus alba) INRA

717-1 B4 by different strains of A. rhizogenes.

Four bacterial strains were tested (strains provided by Yves Dessaux, CNRS DR,

Gif / Yvette. These strains belong to the public domain and are widely exploited in the scientific community).

These are the strains:

- 15834: Mannopine and agropine strain

- 8196: Mannopine strain

- 2659: Mannopine strain - 1724: Mannopine strain

At first, a classical production of roots transformed by A. rhizogenes was carried out. This requires the contact of bacterial strains with injured tissues of poplar (hybrid Populus tremula X Populus alba): fragments of leaves and stems from cuttings produced in axenic conditions at the INRA laboratory of the UMR

1 136.

Excellent transformation results were obtained with strains 15834 (100%) and 1724 (75%).

From these works, a selection of a root clone was performed. It is derived from the transformation of Poplar (Populus tremula x Populus alba INRA 717-1 B4 hybrid) by the strain 15834 of A. rhizogenes.

Transformation and maintenance of the rooted roots ("hairy roots" or "Root Organ Cultures" -ROC-) is done on the minimum medium, medium M (Bécard et al., 1988, New Phytol, 108: 21-218). It is important to note that the roots transformed with Agrobacterium rhizogenes strain 15834 are not Genetically Modified Organisms (GMOs). It is only a natural transformation generated in controlled and axenic conditions. 2-Cultivation of Tuber maanatum:

Contact of pieces of carpophore of T. magnatum (about 1 cm ³ ) with roots (hairy roots) of rockrose (Cistus incanus) and / or poplar (Populus tremula x Populus alba) on January 1, 2006 on medium M (see middle composition minimum, called "middle M"). Other host or possibly non-host species could be exploited for the synthesis of transformed roots in order to produce Tuber magnatum mycelium.

1. Biological material: Poplar roots (Populus tremula x Populus alba, INRA clone 717-1 B4) transformed with A rhizogenes strain 15834.

Cystic root (Cistus incanus) transformed by Agrobacterium rhizogenes.

Pieces of white truffle (Tuber magnatum) carpophores identified as belonging to strain Tm (520).

2. Initiation of Truffle Mycelium Cultivation (Tuber Magnatum)

After 2 to 3 weeks of cultivation at 25 ° C in the dark, there is a development of the mushroom, in mycelial form, from the pieces of carpophore in the presence of transformed roots of Cistus, but especially in the presence of poplar roots (hybrid Populus tremula X Populus alba) transformed by A. rhizogenes. This initiation of growth is induced without even contact between the transformed roots and the piece of carpophore, suggesting an induction of growth by compounds of root origin (present in transformed root exudates). It should be noted that, in these culture conditions, the introduced root is not mycorrhizal. From this first stage of cultivation, a third biological "partner" is identifiable:

The recurring presence of one or more bacterial species is noted. Molecular typing (16S DNA region) bacterial cultures, isolated from the 1 ^st experiment reveals that it is a strain of Stenotrophomonas maltophilia. This bacterium could also be involved in mycelial growth.

EXAMPLE 2 Method for transplanting, or production of subcultures, of the mycelium of tuber magna tu m

Transplanting of the whole system (root + mycelium + bacterium (s)) in a medium identical to that used in Example 1. There is very good growth and excellent recovery, indicating the possibility of carrying out cultures of this first mycelial culture of Tuber magnatum. From this new box, a new transplant is carried out on 9/02/2007. A good growth recovery of T magnatum is observed. This time, only the mycelium (possibly with the bacterial species) was removed and brought into contact with freshly transplanted poplar roots. Very good growth is observed in all the culture dishes. Figure 1 illustrates the growth of Tuber magnatum mycelium as a function of culture time. It can be noted that all the subcultures are carried out in the presence of new Poplar root (populus tremula X Populus alba hybrid), transformed by A. rhizogenes, in growth. The mycelium alone hardly grows when there is no root. There are simply some fungal hyphae very dispersed in "research" of the plant partner, followed by a rapid slowdown in growth. Roots, root exudates or metabolites from these roots seem to be essential for the growth of the fungus. The positive role (and therefore the presence) of certain bacteria, such as Stenotrophomonas maltophilia could also be decisive. It should be noted that in this interaction, at least tripartite, the bacterial development is very moderate, possibly regulated by the other elements present in the medium.

Example 3: Molecular type of the specificity of Tuber magnatum mycelium culture

The specificity of mycelial culture (Tuber magnatum) was verified by molecular typing. DNA extraction was performed and sequencing of the extracted DNAs was performed.

The maintenance of Tuber magnatum in axenic culture has been confirmed by comparison with the NCBI databases {h \ t2h {l ^^^ _. : Ω _. ΦλS} _. π \: B} h- £ 0lϋ sequences from the region

ITS of rDNA amplified from DNA extracted from mycelium neoformed in the presence of hairy roots (Rockrose and poplar) and pieces of Tuber magnatum carpophore, initially supplied (Tm 520): 98 to 100% homology after correction of raw sequences.

Example 4 Process for producing Tuber magnatum mycelium

Contacting different pieces of Tuber magnatum carpophore (from different truffles - carpophores -) with rockrose roots (Cistus incanus) transformed by A. rhizogenes.

Interaction of transformed roots and pieces of carpophores on M medium, 9/02/2007.

- Tm (614) + / - Cistus incanus (hairy root)

- Tm (616) + / - Cistus incanus (hairy root) - Tm (616) + / - Cistus incanus (hairy root)

- Tm (618) + / - Cistus incanus (hairy root)

- Tm (620) + / - Cistus incanus (hairy root)

- Tm (631) + / - Cistus incanus (hairy root) - Tm (645) + / - Cistus incanus (hairy root)

- Tm (650) + / - Cistus incanus (hairy root)

- Tm (653) + / - Cistus incanus (hairy root)

- Tm (659) + / - Cistus incanus (hairy root)

The experiments were made from pieces of white truffle (Tuber magnatum) from 9 different coniferous individuals taken in the open ground.

Some interaction experiments were also performed between poplar roots (Populus tremula X hybrid Populus alba) transformed by A. rhizogenes and T. magnatum.

Firstly, we observe that the growth of Tuber magnatum is initiated very rapidly in the presence of poplar roots (Populus tremula X hybrid Populus alba) transformed by A. rhizogenes. The growth of the fungus is obviously more important under these conditions than in the presence of roots of Cistus incanus transformed by A. rhizogenes.

In the experiment of comparing the growth of Tuber magnatum, from pieces of carpophores, in the presence and absence of root of Cistus incanus transformed by A. rhizogenes, one finds that for 60% (6/10) of the boxes containing the two biological partners (roots and fungus) the growth of Tuber magnatum is stimulated. In absence of root, no mycelial growth of Tuber magnatum is observable from pieces of carpophores in our growing conditions (medium M, 25 ⁰ C and darkness).

Figure 2 illustrates this stimulation of the mycelial growth of Tuber magnatum in the presence or absence of roots of Cistus incanus transformed by A. Rhizogenes, six weeks after the incubation of the pieces of t tamaran carpophores with the transformed roots of Cistus. incanus.

EXAMPLE 5 Process for producing subcultures of Tuber magna tu m mycelium in the presence of carota roots It is important to point out that carrot roots (clone DC1), transformed by A. rhizogenes (provided by Pr. G. Bécard), also stimulate the growth of Tuber magnatum, from cultures of T. magnatum obtained in interaction with the transformed roots of Cistus or Populus (strain T magnatum, Tm520). The growth stimulation of Tuber magnatum is less important than with the roots of Populus or Cistus. However, these results demonstrate that transformed roots of non-host plants have a positive effect on mycelial growth of this fungus (see Figures 3 to 6). Figures 3 to 6 show the Tm520 strain of Tuber magnatum in interaction or not with hairy roots (roots transformed by A. rhizogenes) of various plants, respectively Populus (Figure 4), Daucus carota (Figure 5) and Cistus (Figure 6). ). It should be noted that under these culture conditions, the root present in the culture medium is not mycorrhizal.

Example 6: Method of mycorrhization with the mycelium of Tuber magnatum.

Several processes of controlled poplar mycorhization by T. magnatum have been developed. Whatever the process, poplar cuttings (717-1 Populus tremula X Populus alba hybrid) are interacted with the Tuber magnatum mycelium from T. magnatum axenic cultures in interaction with the hairy roots (ROC) of poplar. Different systems have been developed.

According to a first process alternative, axenic tube cultures have been made. Fragments of sterile poplar cuttings are deposited on medium M poured sterilely into glass tubes. After development of the root system (2 weeks), an agar explant containing a high density of T. magantum mycelium (from an axenic culture ROC x T. magnatum) is deposited in the bottom of the tube.

According to a second process alternative, poplar root mycorrhizations were performed on square Petri dishes (Figure 7). An agar medium (medium M containing only 1 g / l of sucrose) is deposited on the lower half of the box and two pieces of poplar stem (cuttings) are introduced into the medium to generate new cuttings. The dishes are incubated at a temperature of 20 to 25 ^° C. with a day / night alternation (12h / 12h). The lower parts of the boxes are protected from light by black plastic pouches or aluminum. After initiation of the buds (upper part of the dish) and roots (in the agar medium, in the lower part), an agar explant of at least 2 to 3 cm ² and containing T. magnatum mycelium (from a axenic culture ROC x T. magnatum) is deposited on the agar part colonized by the roots. The first results showed an initiation of the growth of T. magnatum in the presence of the roots of poplar cuttings. After several weeks, or months, of contact, the formation of fungal mantles (similar to ectomycorrhizae) is observable on certain root tips.

Example 7 Application of the method of production of T. magnatum mycelium for T. melanosporum.

Mycelium fragments of T. melanosporum (Mel-28) were cultured in M medium, in the presence (+ cistus) and in the absence of rockrose (Cistus incanus) roots transformed by A. rhizogenes. Culture conditions similar to those of Example 5 were used. As shown in Figure 8, no significant difference in mycelium growth was observed in the presence and absence of transformed root at the end of the culture period. In addition, the growth rate of the mycelium was similar in the presence and absence of root throughout the incubation period.

These experiments therefore show that, under the culture conditions used, the growth of T. melanosporum mycelium is not induced by the presence of roots in the culture medium, contrary to what has been shown for T. magnatum. .

Claims

A method for obtaining Tuber magnatum mycelium comprising the steps of: a) growing in vitro one or more fragments of Tuber magnatum tissue in the presence of plant roots, and b) collecting the Tuber magnatum mycelium obtained at the plant. end of step a).

2. Method according to claim 1, characterized in that the Tuber magnatum tissue fragments are selected from carpophore fragments, mycorrhizal fragments and mycelium fragments.

3. Method according to one of claims 1 and 2, characterized in that the roots of plants consist of roots transformed by Agrobacterium rhizogenes.

4. Method according to one of claims 1 to 3, characterized in that the roots come from plants of a genus selected from among Pinus, Picea, Abies, Corylus, Castanea, Tilia, Cedrus, Populus, Quercus, Cistus and Daucus.

5. Method according to claim 4, characterized in that the roots come from plants selected from poplar, rockrose and carrot.

6. Method according to one of claims 1 to 5, characterized in that step a) in vitro culture is carried out in a culture medium comprising one or more strains of bacteria naturally associated with fungi of the genus Tuber.

7. Method according to claim 6, characterized in that the culture medium comprises bacteria of the species Stenotrophomonas maltophilia.

8. Method according to one of claims 1 to 7, characterized in that step a) is performed in the dark.

9. Method according to one of claims 1 to 8, characterized in that it comprises the following additional step: c) transfer the mycelium collected in step b) in a culture medium.

10. The method of claim 9, characterized in that it comprises the following additional step: d) cultivating in said root-free culture medium, the mycelium transferred to step c);

1. Method according to claim 10, characterized in that in step d) the mycelium is cultured for a period of up to 6 weeks or more.

Tuber magnatum mycelium characterized in that it is obtained by the method according to one of claims 1 to 1 1.

13. Process for plant mycorrhization by Tuber magnatum comprising the following steps: a) inoculation by contacting the roots of seedlings with a Tuber magnatum mycelium according to claim 12; b) in vitro culture of the inoculated seedlings obtained in step a) until the appearance of mycorrhizae.

14. The method of claim 13, characterized in that it comprises a step c) transplanting earth mycorhizal seedlings obtained in step b).