WO2021255267A1

WO2021255267A1 - Novel biocontrol agent and use thereof for controlling fungal diseases of plants

Info

Publication number: WO2021255267A1
Application number: PCT/EP2021/066674
Authority: WO
Inventors: Sophie LEPORINI; Christian BELLOY; Najat NASSR; Aude LANGENFELD NARDIN
Original assignee: Agro Industrie Recherches Et Developpements A.R.D.; Rittmo Agroenvironnement
Priority date: 2020-06-18
Filing date: 2021-06-18
Publication date: 2021-12-23
Also published as: FR3111521B1; EP4167744A1; FR3111521A1

Abstract

The present invention relates to a biocontrol agent and to the use thereof for controlling fungal diseases of plants.

Description

NEW BIOCONTROL AGENT AND ITS USE FOR THE CONTROL OF FUNGAL DISEASES OF PLANTS

FIELD OF THE INVENTION

The present invention relates to a novel biocontrol agent and its use for the control of fungal diseases of plants.

PRIOR ART

Damping-off (also known as root rot) is a plant disease caused by the pathogen Pythium spp. (mushroom). One of the main visible symptoms is rotting of young shoots during germination. By acting on the emergence of the plant, this complex disease allows the establishment of other phytopathogenic fungi such as Phytophthora spp. or Rhizoctonia spp., themselves responsible for damping-off. Damping-off, which affects among others the cultivation of tomato (Solanaceae family), potato (Solanaceae family), beet (Amaranthaceae family) and soybean (Fabaceae family), constitutes therefore a major agronomic problem both in greenhouse cultivation and in the field, with high economic consequences.

The genus Pythium spp. is represented by more than 130 species among which can be cited: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. deharyanum, P. elongatum, P. gracile, P. imperfectum, P. inflatum, P intermedium, P. irregulare, P. marinum, P. maritimum, P. middletoni, P. monospermum, P. myriotylum, P. ostracodes, P. pulchrum, P. salinum, P. thallassium and P. ultimum. The genus Rhizoctonia spp., Also responsible for rhizoctonia rhizoctonia, is represented by more than twenty species among which can be cited: R. alpine, R. anomala, R. callae, R. dichotoma, R. ferruginea, R. floccosa, R. fragariae, R. fraxini, R. fumigata, R. lanuginosa, R. leguminicola, R. menthae, R. mucoroides, R. muneratii, R. oryzae-sativae, R. pini-insignis, R. quercus, R. rubi, R. rubiginosa, R. silvestris, R. solani, R. stahlii, R. tuliparum and R. versicolor.

These two genera (Pythium and Rhizctonia) have very varied habitats (terrestrial or aquatic environments, cultivated or fallow soils, etc.) and have a high diversity of hosts. This is why they are known worldwide to be general pathogens that can cause Extensive and devastating damage to root systems and are known to be extremely difficult to control and eradicate.

Although fungicides have shown positive results in controlling damping-off caused by Pythium spp. and / or Rhizoctonia spp. , the phytotoxicity of these products and the possible residues are real problems inducing an environmental impact and risks for human health. The development of resistance to these molecules is also a problem.

Gray rot is a disease caused by the pathogen Botrytis cinerea. This fungus is able to develop on a very large number of host plants including the tomato (Solanaceae family), both in the open field and under shelter. Due to the high humidity in greenhouses, a factor favorable to the development of the pathogen, severe attacks will affect the crops. B. cinerea can infect seeds as well as several organs, namely the stem, leaves, flowers as well as fruits, making it a pathogen of great economic importance. Whatever parts of the plant are attacked by the pathogen, the tissues become covered with a gray felting, characteristic of an attack by B. cinerea. The main means of controlling gray rot remains, as with damping-off, the use of fungicides. However, strains have developed, following repeated use of phytosanitary products, resistance to these products.

Out of concern for the environment, natural biological control means were developed using microorganisms. The latter, also called direct microbiological control, consists of introducing specific microbial antagonists into the soil or into the plant material. These antagonists, also called biological control agents or BCA (Biological Control Agent), can interfere with the growth and / or survival of pathogens, thus making it possible to control them. To do this, biological control agents or BCA can control the target pathogens through one or more modes of action:

- the competition which may take place between the BCA and the pathogen for oxygen, for space, or for nutrients;

- antibiosis. which corresponds to interactions involving at least one diffusible low molecular weight compound or an antibiotic produced by a microorganism which inhibits the growth of another microorganism; - parasitism and the production of hydrolytic enzymes and / or antibiotic metabolites to the detriment of another host organism; and or

- the induction of resistance in the host plant ("elicitation") like the bacteria of the rhizosphere, known under the name of PGPR (Plant Growth Promoting Rhizobacteria), which are capable of inducing systemic resistance in the plant Induced Systemic Resistance (ISR) which increases resistance to pathogens thanks to a phenomenon of potentiation (or priming), which corresponds to a waking state allowing a faster and more intense immune response of the plant.

Since the genus Trichoderma comprises various species of fungi capable of being biocontrol agents, their mode of action has been the subject of much work. Since then, the Trichoderma genus has been known for its biocontrol activity against telluric pathogens (living in the soil) but with low competitiveness at the level of the rhizosphere of plants (soil-root zone).

BRIEF OVERVIEW

In this context of combating fungal diseases, such as damping-off and / or rhizoctonia root rot and gray rot, a first aim of the invention therefore consists in providing an effective strain and its use as a biological control agent (or biocontrol agent). A second aim of the invention is to provide phytosanitary compositions for the prevention and / or treatment of fungal diseases of plants. A third aim of the invention is also to provide phytosanitary coating compositions where, in association with a plant seed, the strain of the invention is used as an agent for accelerating the germination of said seed. Another object of the invention is to provide methods for manufacturing said compositions and for implementing the prevention and / or treatment of fungal diseases of plants, and the acceleration of germination.

DETAILED DESCRIPTION

According to a first aspect of the invention, the latter relates to the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the National Collection of Cultures of Microorganisms (CNCM; Institut Pasteur, 25-28 rue du Docteur Roux , F-75724 PARIS CEDEX 15) under the number CNCM 1-5333 and / or one of its mutants. Surprisingly, this strain of Trichoderma atroviride TAL-17 developed by the inventors has excellent growth stimulating properties, antagonistic properties useful in agriculture as well as great stability in terms of viability. In fact, it constitutes an ideal biocontrol agent for effectively combating fungal diseases of plants such as damping-off and / or rhizoctonia.

By "isolated strain" is meant the culture of a single microorganism which has been isolated from various microorganisms present on and / or in the tissues of a fragment of a wheat leaf grown in fields.

By “one of its mutants” is meant functional mutant strains obtained by genetic mutations or manipulations of a reference strain, which in the context of the invention is the isolated strain of Trichoderma atroviride TAL-17 filed on July 03, 2018 at the CNCM under number CNCM 1-5333. The term “functional mutant strains” is understood to mean that these mutants retain the same physiological properties as the reference strain (ie. TAL-17) or even may exhibit improved physiological properties, in particular for mutants overexpressing genes involved in the synthesis of metabolites. 'interests (eg improvement of biocontrol capacities). Also, one of the objects of the invention relates to an isolated strain of Trichoderma atroviride, said isolated strain being a mutant strain of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under number CNCM 1-5333. One of the other subjects of the invention also relates to the overproduction of metabolites in the production matrix by an isolated strain of Trichoderma atroviride whose metabolite biosynthetic potential has been improved by genetic manipulation, said isolated strain being a mutant strain of the strain. isolated from Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under number CNCM 1-5333.

By “one of its mutants” is also meant, alternatively or cumulatively, functional mutant strains (see above) which have a sequence identity of at least 98% with the sequence SEQ ID NO: 5 of the gene. ech42 of the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333. By "a sequence identity of at least 98% with the sequence SEQ ID NO: 5" is meant a sequence identity of at least 98.5%, at least 99% or even at least 99, 5% with the sequence SEQ ID NO: 5 represented by the following nucleic acid sequence:

knowing that within the meaning of the invention, the sequence identity is measured by conventional tools for comparison of sequences known to those skilled in the art such as the algorithms of the BLAST platform or the MatGat program (Campanella, Bitincka and Smalley , 2003).

Moreover and for the purposes of the invention, it is understood that the expressions “the strain of the invention”, “the isolated strain of the invention”, “the isolated strain of Trichoderma atroviride of the invention”, “the isolated strain TAL-17 and / or one of its mutants "," the strain of the invention and / or one of its mutants "and" the isolated strain of Trichoderma atroviride of the invention and / or one of its mutants "are interchangeable and all denote the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants. In addition, by "the isolated strain of Trichoderma atroviride TAL-17 [...] and / or one of its mutants", is meant that the invention relates either to the isolated strain TAL-17, or one of its mutants, or the TAL-17 strain in combination with one of its mutants. All of these strains (ie TAL-17 and its mutants) can moreover be easily identified via the so-called real-time Polymerase Chain Reaction (qPCR) method. The inventors have developed specific thereof namely tools, TaqMan ^® probe GS285741-P1 and a pair of nucleotide primers associated GS285741-F1 and GS285741-R1 (Table 1), which can amplify a sequence specific to the strains of the invention {cf. Table 2).

Table 1: Nucleotide primers (Fl and RI) and TaqMan ^® probe (PI)

FAM: 6-carboxyfluorescein, MGB: minor groove binder

Table 2: Target sequence of oligonucleotides and of the TaqMan ^® probe in the TAL-17 strain and / or its mutants

According to this same aspect, a subject of the invention is also the isolated strain of Trichoderma atroviride of the invention as described above and / or one of its mutants in the form of spores, said spores being produced by fermentation in solid or liquid medium and which may be in purified form or in the production matrix, said production matrix being solid or liquid.

By “in the form of spores” is meant in the form of conidiospores or chlamydospores (more particularly conidiospores) ensuring the function of asexual multiplication in the fungus.

By "fermentation in a solid medium (FMS)" is meant any process allowing the development of the microorganism on the surface and / or inside a solid and humidified porous production matrix, in the absence of free water.

The term “liquid fermentation” is understood to mean any process allowing the development of the microorganism using a production matrix with the presence of free water. By “production matrix” is meant any natural and / or synthetic substrate, which allows the development of the microorganism and induces the production of biomass, and / or enzymes, and / or primary and / or secondary metabolites ( eg pyrone 6-pentyl-2H-pyran-2-one). Examples of enzyme or enzyme families produced by the fungus are proteases, chitinases and beta-1,3-glucanase.

By "spores [...] in purified form", one understands the concentrated spores following the elimination of the major part of the remainder of the matrix after production not having been used for the formation of the spores, with the help of 'a sieving process for example.

By "spores [...] in the production matrix" is meant the spores that remain in the production matrix after the manufacturing process, without a purification step.

According to another embodiment, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent.

By “biological control agent” is meant that a strain of Trichoderma atroviride according to the invention can interfere with the growth and / or the survival of pathogens, thus making it possible to control them, via one or more of the methods. action described above (eg via the development of its biomass and / or its production of enzymes and / or secondary metabolites). For example, one of the means of demonstrating that a strain of Trichoderma atroviride according to the invention is a biological control agent is by carrying out a Dual culture test on culture medium in a Petri dish. More precisely by "Dual culture test" means a test on a Petri dish in the laboratory on culture medium, where the strain of the microorganism, biological control agent, and the strain of the pathogen are cultured on the same dish in order to '' observe the effect of one strain on another over time and vice versa.

According to another embodiment, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as an antifungal agent for plants. By “plant antifungal agent” is meant that a strain of Trichoderma atroviride according to the invention is capable of combating phytopathogenic fungi. For example, one of the means of demonstrating that a strain of Trichoderma atroviride according to the invention is an antifungal agent for plants is by carrying out a Dual culture test.

It should in particular be noted that among the phytopathogenic fungi against which a strain of Trichoderma atroviride according to the invention has a real inhibitory effect, we find:

- various telluric fungi responsible for damping off seedlings such as fungi of the genera Pythium (eg P. ultimum), Rhizoctonia (eg R. solani), Alternaria (eg A. dauci especially in carrots), Fusarium (eg F. culmorum in particular in cereals and F. oxysporum in particular in tomato) and Phoma (eg P. exigua in particular in potato); and

- the pathogen Botrytis (e.g. B. cinerea), responsible for gray rot in tomatoes in particular.

According to another embodiment, the subject of the invention is therefore the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a pathogenic fungus for plants chosen from fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis.

According to another embodiment, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a pathogenic fungus for plants:

- of the genus Pythium spp., said pathogenic fungus belonging in particular to a species chosen from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. deharyanum, P. elongatum, P. gracile, P. imperfectum, P. inflatum, P. intermedium, P. irregulare, P. marinum, P. maritimum, P. middletoni, P. monospermum, P. myriotylum, P. ostracodes, P. pulchrum, P. salinum, P. thallassium and P. ultimum, and in particular said pathogenic fungus being P. ultimum; and or

- of the genus Rhizoctonia spp. , said pathogenic fungus belonging in particular to a species chosen from: R. alpine, R. anomala, R callae, R. dichotoma, R. ferruginea, R floccosa, //. fragariae, //. fraxini, //. fumigata, //. lanuginosa, R. leguminicola, //. menthae, //. mucoroides, //. muneratii, R oryzae-sativae, R. pini-insignis, //. quercus, //. rubi, //. rubiginosa, R silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular said pathogenic fungus being //. solani; and or

- of the genus Botrytis spp., and in particular the said pathogenic fungus being

cinerea.

According to another embodiment, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a plant pathogenic fungus of the genus Pythium spp. , said pathogenic fungus belonging in particular to a species chosen from: I \ acanthicum, I \ afertile, I \ aphanidermatum, I \ aquatile, I \ debaryanum, I \ elongatum, I \ gracile, I \ imperfectum, I \ inflatum, I \ intermedium, I \ irregulare, I \ marinum, I \ maritimum, P. middletoni, I \ monospermum, I \ myriotylum, I \ ostracodes, P. pulchrum, I \ salinum, I \ thallassium and // ultimum, and in particular the said pathogenic fungus being / ¹ , ultimum.

According to another embodiment, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a pathogenic fungus of the genus Rhizoctonia spp. , said pathogenic fungus belonging in particular to a species chosen from: //. alpine, //. anomala, //. callae, //. dichotoma, //. ferruginea, //. floccosa, //. fragariae, //. fraxini, //. fumigata, //. lanuginosa, //. leguminicola, //. menthae, //. mucoroides, //. muneratii, //. oryzae- sativae, //. pini-insignis, //. quercus, //. rubi, //. rubiginosa, //. silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular said pathogenic fungus being //. solani. According to another embodiment, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a pathogenic fungus of the genus Botrytis spp., in particular the said pathogenic fungus being B. cinerea.

- of the genus Pythium spp., said pathogenic fungus being P. ultimum; and or

- of the genus Rhizoctonia spp., said pathogenic fungus being R. solani; and or

- of the genus Botrytis spp., the said pathogenic fungus being B. cinerea.

Advantageously, the subject of the invention is the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a pathogenic fungus for plants. of the genus Pythium spp., said pathogenic fungus being P. ultimum.

Advantageously, a subject of the invention is also the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a fungus pathogenic for plants of the genus Rhizoctonia spp., said pathogenic fungus being / ?, solani.

Advantageously, a subject of the invention is also the isolated strain of Trichoderma atroviride of the invention as described above as a biological control agent, in particular as an antifungal agent for plants directed against a fungus pathogenic for plants of the genus Botrytis spp., said pathogenic fungus being B. cinerea .. According to a second aspect of the invention, the latter relates to the use of an isolated strain of Trichoderma atroviride in the prevention and / or treatment of infection in a plant caused by a pathogenic fungus chosen from among the fungi of genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis, said isolated strain of Trichoderma atroviride being the TAL-17 strain deposited on July 3, 2018 at the CNCM under number CNCM 1-5333 and / or one of its mutants.

In particular, the subject of the invention is the use of an isolated strain of Trichoderma atroviride in the prevention and / or treatment of infection in a plant caused by a pathogenic fungus of the genus Pythium spp. and / or of the genus Rhizoctonia spp. and / or of the genus Botrytis spp. , said isolated strain of Trichoderma atroviride being the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants.

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention (ie the TAL-17 strain deposited on July 3, 2018 at the CNCM under the CNCM number 1-5333 and / or one of its mutants), in which the said pathogenic fungus belongs to:

- a species chosen from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. deharyanum, P. elongatum, P. gracile, P. imperfectum, P. inflatum, P. intermedium, P. irregulare, P. marinum, P. maritimum, P. middletoni, P. monospermum, P. myriotylum, P. ostracodes, P. pulchrum, P. salinum, P. thallassium and P. ultimum, and in particular belongs to the species P. ultimum; and or

- a species chosen from: R. alpine, R. anomala, R. callae, R. dichotoma, R. ferruginea, R. floccosa, R. fragariae, R. fraxini, R. fumigata, R. lanuginosa, R. leguminicola, //. menthae, //. mucoroides, //. muneratii, R oryzae-sativae, R. pini-insignis, //. quercus, //. rubi, //. rubiginosa, R silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular belongs to the species i ?. solani; and / or the species B. cinerea. According to another embodiment, the subject of the invention is the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus belongs to the genus Pythium spp. and to a species selected from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. debaryanum, P. elongatum, P. gracile, P. imperfection, P. inflatum, P intermedium, P irregulare, P marinum, P. maritimum, P middletoni, P monospermum, P myriotylum, P ostracodes, P. pulchrum, P salinum, P thallassium and P '. ultimum, and in particular said pathogenic fungus is P. ultimum.

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus belongs to the genus Rhizoctonia spp. and to a species chosen from: R. alpine, R. anomala, R. callae, R. dichotoma, R. ferruginea, R. floccosa, R. fragariae, R. fraxini, R. fumigata, R. lanuginosa, R. leguminicola , R. menthae, R mucoroides, R. muneratii, R. oryzae-sativae, R. pini-insignis, R. quercus, R. rubi, R. rubiginosa, R. silvestris, R. solani, R. stahlii, R. tuliparum and R. versicolor, and in particular said pathogenic fungus is R solani.

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus belongs to the genus Botrytis spp. and in particular said pathogenic fungus is B. cinerea.

According to another embodiment, the subject of the invention is the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea.

Advantageously, the subject of the invention is the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus is P. ultimum. Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus is R. solani. Advantageously, the subject of the invention is also use as described above of the isolated strain of Trichoderma atroviride of the invention, wherein said pathogenic fungus is B. cinerea.

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Rosaceae such as Pyrus spp. (pear), Prunus spp. (cherry), Fragaria spp. (strawberry), or Posa spp. (pink) ;

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper) ; and

^■ Valerianaceae, in particular said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper), and preferably said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Glycine max (soybean).

^■ Amaranthaceae such as Beta vulgaris (beet); ^■ Fabaceae such as Glycine max (soybean); and

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper), and in particular said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Glycine max (soybean).

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato) , Solanum tuberosum (potato) and / or Glycine max (soybean).

Advantageously, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant is Beta vulgaris (beet). Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant is Solanum lycopersicum (tomato). Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant is Solanum tuberosum (potato). Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant is Glycine max (soybean).

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea and said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

According to another embodiment, the subject of the invention is the use as described above of the isolated strain of Trichoderma atroviride of the invention, wherein said pathogenic fungus is P. ultimum and said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

According to another embodiment, the invention relates to the reuse as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus is R. solani and said plant is Beta vulgaris (beet). , Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said pathogenic fungus is B. cinerea and said plant is Beta vulgaris ( beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

Advantageously, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which:

- said pathogenic fungus is P. ultimum and said plant is Beta vulgaris (beet); Where

- said pathogenic fungus is P. ultimum and said plant is Solanum lycopersicum (tomato); Where

- said pathogenic fungus is P. ultimum and said plant is Solanum tuberosum (potato); Where

- said pathogenic fungus is P. ultimum and said plant is and / or Glycine max (soybean); Where

- said pathogenic fungus is R. solani and said plant is Beta vulgaris (beet); Where

- said pathogenic fungus is R. solani and said plant is Solanum lycopersicum (tomato); Where

- said pathogenic fungus is R. solani and said plant is Solanum tuberosum (potato); Where - said pathogenic fungus is R. solani and said plant is Glycine max (soya); Where

- said pathogenic fungus is B. cinerea and said plant is éla vulgaris (beet); Where

- said pathogenic fungus is B. cinerea and said plant is Solanum lycopersicum (tomato); Where

- said pathogenic fungus is B. cinerea and said plant is Solanum tuberosum (potato); Where

- said pathogenic fungus is B. cinerea and said plant is Glycine max (soya).

According to a third aspect, the invention relates to G use of a phytosanitary composition in the prevention and / or treatment in a plant of infection caused by a pathogenic fungus chosen from fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium , Phoma and Botrytis, said phytosanitary composition comprising:

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants as active principle; Where

- the metabolites emitted during the production of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants as active principle; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during its production as an active principle; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants in combination with at least one other biological control agent as active principle; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during the production of the strain in association with at least one other biological control agent as an active principle. In particular, the subject of the invention is the use of a phytosanitary composition in the prevention and / or treatment in a plant of infection caused by a pathogenic fungus of the genus Pythium spp. and / or of the genus Rhizoctonia spp. and / or of the genus Botrytis spp., said phytosanitary composition comprising:

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during the production of the strain in association with at least one other biological control agent as an active principle.

By “metabolites emitted during the production of the strain” or “its metabolites emitted during its production” is meant any molecule produced in the production matrix by the strain of the invention and / or one of its mutants during its production. its development in the presence (co-culture) or not of the pathogenic fungus of the genus Pythium spp. and / or of the genus Rhizoctonia spp. and / or of the genus Botrytis spp. For example and without limitation, such metabolites can be:

- enzymes produced by the fungus such as proteases, chitinases, èéto-l, 3-glucanase; Where

- pyrone 6-pentyl-2H-pyran-2-one (6PP).

By “biological control agent” is meant a microorganism capable of interfering with the growth and / or survival of pathogens, thus making it possible to control them. The strain of the TAL-17 invention (filed on July 03, 2018 at the CNCM under the number CNCM 1-5333) is an example thereof, but within the meaning of the invention it is a question here of combining the strain of the invention and / or one of its mutants to at least one other biological control agent, which is therefore different from the strain of the invention and / or one of its mutants.

By “at least one other biological control agent” is meant that the plant protection composition of the invention can contain 2 biological control agents (that of the invention and another), just as it can contain 3, 4 or more. 5 (that of the invention and 2, 3 or 4 others).

According to another embodiment, the subject of the invention is the use as described above of the aforementioned phytosanitary composition, said phytosanitary composition being at the base (on purchase) in the form of a solid composition. or concentrated liquid. Such a solid or concentrated liquid composition can be, for example, obtained by means of a particular formulation of the spores of the isolated strain of Trichoderma atroviride of the invention and / or one of its mutants produced by fermentation in solid or liquid medium, said spores. which may be in purified form or in the production matrix, said production matrix being solid or liquid.

By "concentrated composition" is meant a composition in which the concentration of the active principle (ie the strain of the invention and / or one of its mutants, which may be in the form of spores) is greater than one. effective amount of 10 ⁷ spores / g of concentrated solid or liquid composition, ie 10 ⁷ CFU / g (CFU = colony-forming unit) of concentrated solid or liquid composition. Also, another particular object of the invention relates to the use as described above of the aforementioned phytosanitary composition, said phytosanitary composition being in the form of a solid or concentrated liquid composition in which the concentration of the active principle is greater than an effective amount of 10 ⁷ spores / g.

By "greater than an effective amount of 10 ⁷ spores / g" is also meant greater than an effective amount of 10 ⁸ spores / g, greater than an effective amount 10 ⁹ spores / g, greater than an effective amount 10 ¹⁰ spores / g. g, greater than an effective amount of 10 ¹¹ spores / g or greater than an effective amount of 10 ¹² spores / g. The user of the aforesaid composition must then, before its use, incorporate it into a solid medium (eg. Potting soil, etc.) or suspend it in an aqueous medium (eg water) to obtain a dilute composition comprising a quantity effective from 10 ⁴ to 10 ¹² spores of the Trichoderma atroviride strain of the invention and / or one of its mutants per gram of solid medium or of aqueous medium (spores / g). The composition, solid or liquid, diluted obtained can then be applied to plants for which the prevention and / or treatment of a fungal plant disease is desired.

Within the meaning of the invention, the spreading of the aforesaid composition comprises in particular the conventional spreading techniques known to those skilled in the art, which are used to spread solid materials (eg sewage sludge, manure over an area to be treated). , etc.) or liquid (eg pesticides, etc.) of agronomic interest.

According to another embodiment, the subject of the invention is the use as described above of the aforementioned phytosanitary composition, said phytosanitary composition comprising an effective amount of 10 ⁴ to 10 ¹² spores / g (of phytosanitary composition), more particularly from 10 ⁷ to 10 ⁹ spores / g (of phytosanitary composition), said phytosanitary composition optionally being obtained after a preparation step (eg dilution by suspension or mixing) making it ready for spreading.

By “effective amount of spores / g” is meant the number of spores capable of forming a colony on culture medium per g (gram) of product. In addition, the expression "from 10 ⁴ to 10 ¹² spores / g" can also mean from 10 ⁴ to 10 ⁵ spores / g, from 10 ⁴ to 10 ⁶ spores / g, from 10 ⁴ to 10 ⁷ spores / g, from 10 ⁴ to 10 ⁸ spores / g, from 10 ⁴ to 10 ⁹ spores / g, from 10 ⁴ to 10 ¹⁰ spores / g, from 10 ⁴ to 10 ¹¹ spores / g, from 10 ⁵ to 10 ⁶ spores / g, from 10 ⁵ to 10 ⁷ spores / g, from 10 ⁵ to 10 ⁸ spores / g, from 10 ⁵ to 10 ⁹ spores / g, from 10 ⁵ to 10 ¹⁰ spores / g, from 10 ⁵ to 10 ¹¹ spores / g, from 10 ⁵ to 10 ¹² spores / g, from 10 ⁶ to 10 ⁷ spores / g, from 10 ⁶ to 10 ⁸ spores / g, from 10 ⁶ to 10 ⁹ spores / g, from 10 ⁶ to 10 ¹⁰ spores / g, from 10 ⁶ to 10 ¹¹ spores / g, from 10 ⁶ to 10 ¹² spores / g, from 10 ⁷ to 10 ⁸ spores / g, from 10 ⁷ to 10 ⁹ spores / g, from 10 ⁷ to 10 ¹⁰ spores / g, from 10 ⁷ to 10 ¹¹ spores / g, from 10 ⁷ to 10 ¹² spores / g, from 10 ⁸ to 10 ⁹ spores / g, from 10 ⁸ to 10 ¹⁰ spores / g, from 10 ⁸ to 10 ¹¹ spores / g, from 10 ⁸ to 10 ¹² spores / g, from 10 ⁹ to 10 ¹⁰ spores / g, from 10 ⁹ to 10 ¹¹ spores / g, from 10 ⁹ to 10 ¹² spores / g, from 10 ¹⁰ to 10 ¹¹ spores / g, from 10 ¹⁰ to 10 ¹² spores / g or from 10 ¹¹ to 10 ¹² spores / g. According to another embodiment, the use as described above of the above-mentioned phytosanitary composition, in which said pathogenic fungus belongs to:

- a species chosen from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. debaryanum, P. elongatum, P. gracile, P imperfection, P. inflatum, P intermedium, P. irregulare, P marinum , P maritimum, P middletoni, P monospermum, P myriotylum, P ostracodes, P. pulchrum, P salinum, P thallassium and P '. ultimum, and in particular belongs to species I \ ultimum; and or

- a species chosen from: //. alpine, //. anomala, //. callae, //. dichotoma, R. ferruginea, R. floccosa, //. fragariae, //. fraxini, //. fumigata, //. lanuginosa, R. leguminicola, //. menthae, //. mucoroides, //. muneratii, R oryzae-sativae, R. pini-insignis, //. quercus, //. rubi, //. rubiginosa, R silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular belongs to the species //. solani; and or

- the cinerea species.

According to another embodiment, the subject of the invention is the use as described above of the above-mentioned phytosanitary composition, in which said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Rosaceae such as Pyrus spp. (pear), Prunus spp. (cherry), Fragaria spp. (strawberry), or // asc / .s ////, (pink);

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper) ; and ^■ Valerianaceae, in particular said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

According to another embodiment, the invention relates to the use as described above of the above-mentioned phytosanitary composition, in which said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea, and said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

According to another embodiment, the invention relates to the use as described above of the above-mentioned phytosanitary composition, in which said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea, and said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

Advantageously, the invention relates to the use as described above of the above-mentioned phytosanitary composition, in which:

- said pathogenic fungus is P. ultimum and said plant is Solanum lycopersicum (tomato); Where - said pathogenic fungus is P. ultimum and said plant is Solarium tuberosum (potato); Where

- said pathogenic fungus is R. solani and said plant is Solanum tuberosum (potato); Where

- said pathogenic fungus is R. solani and said plant is Glycine max (soya); Where

- said pathogenic fungus is B. cinerea and said plant is Beta vulgaris (beet); Where

- said pathogenic fungus is B. cinerea and said plant is Glycine max (soya).

Alternatively, the subject of the invention is the phytosanitary composition as described above for its use in the prevention and / or treatment of infection in a plant caused by a pathogenic fungus of the genus Pythium spp. and / or Rhizoctonia spp and / or Botrytis spp. In this regard, all of the specific uses cited above apply to this alternative.

According to a fourth aspect, the subject of the invention is therefore the use of an isolated strain of Trichoderma atroviride to promote the germination of a plant seed, said isolated strain of Trichoderma atroviride being the TAL-17 strain deposited on July 03. 2018 at the CNCM under number CNCM 1-5333 and / or one of its mutants and in which said plant seed belongs to a plant of a family chosen from: ■ Amaranthaceae such as Beta vulgaris (beet); ^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Valerianaceae, in particular said plant seed belongs to a plant of a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

According to another embodiment, the invention relates to the use as described above of the isolated strain of Trichoderma atroviride of the invention (ie the TAL-17 strain deposited on July 3, 2018 at the CNCM under the CNCM number 1-5333 and / or one of its mutants), in which said plant seed belongs to a plant of a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper), and in particular said plant is Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Glycine max (soybean). Advantageously, the subject of the invention is the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant seed belongs to the plant Beta vulgaris (beet). Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant seed belongs to the plant Solarium lycopersicum (tomato). Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant seed belongs to the plant Solarium tuherosum (potato). Advantageously, a subject of the invention is also the use as described above of the isolated strain of Trichoderma atroviride of the invention, in which said plant seed belongs to the plant Glycine max (soybean).

Regarding this particular aspect, it should be noted that the germination aid advantageously and at the same time allows the prevention and / or treatment of fungal diseases at the time of sowing. The development of a disease after sowing is thus avoided and the yield is optimized via the biostimulant aspect of the strain of the invention.

Advantageously, this aspect also makes it possible to avoid a significant slowdown in the development of the crop if a period of drought occurs some time after sowing (which can happen in spring) and therefore to avoid loss of yields at harvest. Likewise, the biocontrol effect of the strain of the invention (ie. TAL-17 strain and / or one of its mutants) by allowing the plant to germinate rapidly and without its metabolism being affected by the defense against pathogenic, its resources are entirely dedicated to its growth. Physiologically, the plant is thus advantageously more resistant to attacks by subsequent (phyto-) pathogens.

Consequently, it is understood that one of the subjects of the invention relates to a phytosanitary coating composition comprising the isolated strain of Trichoderma atroviride of the invention and / or one of its mutants, and optionally at least one binding agent, said phytosanitary coating composition making it possible to produce a coated seed. A subject of the invention is therefore also a phytosanitary coating composition comprising the isolated strain of Trichoderma atroviride of the invention and / or one of its mutants, said phytosanitary coating composition making it possible to produce a coated seed; a phytosanitary coating composition comprising the isolated strain of Trichoderma atroviride of the invention and / or one of its mutants, and at least one fixing agent, said phytosanitary coating composition making it possible to produce a coated seed.

More broadly, said phytosanitary composition as described above or below, or said phytosanitary coating composition as described above or below comprises:

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants as active principle, and optionally at least one binding agent; Where

- the metabolites emitted during the production of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants as active principle, and possibly at least a fixing agent; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during its production as an active principle, and possibly at least a fixing agent, and optionally at least one fixing agent; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants in combination with at least one other biological control agent as active principle, and optionally at least one fixing agent; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during the production of the strain in association with at least one other biological control agent as active principle, and optionally at least one fixing agent.

It is also understood that another of the objects of the invention relates to the use of a phytosanitary (coating) composition for promoting the germination of a plant seed. In particular, the invention relates to the use as described above of one of the phytosanitary compositions of the invention (see above). Alternatively, this object of the invention relates to a phytosanitary (coating) composition for promoting the germination of a plant seed.

According to another embodiment, the subject of the invention is also a coated seed comprising a plant seed, in which said plant seed is coated with a phytosanitary coating composition and belongs to a plant of a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Valerianaceae, and in which said phytosanitary coating composition comprises:

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during its production as an active principle, and possibly at least a fixing agent, and optionally at least one fixing agent; Where - the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants in combination with at least one other biological control agent as active principle, and optionally at least one fixing agent; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during the production of the strain in association with at least one other biological control agent as active principle, and optionally at least one fixing agent, in particular said plant belonging to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

By "coated seed" is meant a seed selected for sowing, said seed having undergone a particular treatment which has enabled it to be coated in a phytosanitary coating composition.

According to another embodiment, the invention relates to the coated seed as described above, said coated seed comprising an effective amount of 10 ⁵ to 10 ⁸ (or 10 ⁶ to 10 ⁷ ) spores / coated seed, preferably 10 ⁶ (or 10 ⁷ ) spores / coated seed.

According to another embodiment, the invention relates to the coated seed as described above, in which said plant seed belongs to a plant of a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

Advantageously, the subject of the invention is the coated seed as described above, in which said plant seed belongs to the plant Beta vulgaris (beet). Advantageously, a subject of the invention is also the coated seed as described above, in which said plant seed belongs to the plant Solanum lycopersicum (tomato). Advantageously, a subject of the invention is also the coated seed as described above, in which said plant seed belongs to the plant Solanum tuberosum (potato). Advantageously, a subject of the invention is also the coated seed as described above, in which said plant seed belongs to the plant Glycine max (soybean).

According to another aspect of the invention, the subject of the invention is a process for the protection and / or treatment of a plant against a disease caused by a pathogenic fungus chosen from fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis; in particular a pathogenic fungus of the genus Pythium spp. and / or of the genus Rhizoctonia spp. and / or of the genus Botrytis spp. ; comprising the application of a phytosanitary composition to at least part of said plant or of the soil near said plant. In particular, the subject of the invention is the method as described above, in which said at least part of said plant is a leaf, a fruit or a seed.

According to another embodiment, the subject of the invention is a method of protecting and / or treating a plant against a disease caused by a pathogenic fungus chosen from fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis comprising the application of a phytosanitary composition to at least part of said plant or of the soil near said plant, said at least part of said plant being in particular a leaf, a fruit, a seed, said phytosanitary composition comprising: - the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants as active principle; Where

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and its metabolites emitted during the production of the strain in association with at least one other biological control agent as active principle, and said plant belonging in particular to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Rosaceae such as Pyrus spp. (pear), Prunus spp. (cherry), Fragaria spp. (strawberry), or Rosa spp. (pink) ;

^■ Valerianaceae, in particular said plant belonging to a family chosen from: ^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper), and preferably said plant being Beta vulgaris (beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Glycine max (soybean).

In particular, the subject of the invention is a process for the protection and / or treatment of a plant against a disease caused by a pathogenic fungus of the genus Pythium spp. and / or of the genus Rhizoctonia spp. and / or Botrytis spp. comprising the application of a phytosanitary composition to at least part of said plant or of the soil near said plant, said at least part of said plant being in particular a leaf, a fruit, a seed, said phytosanitary composition comprising:

- the metabolites emitted during the production of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under number CNCM 1-5333 and / or one of its mutants as active principle; Where

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot); ^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Valerianaceae, in particular said plant belonging to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

Insofar as this aspect includes the application of a phytosanitary composition, which has moreover been described previously through a use thereof, it is important to note that all of the aforementioned particular characteristics apply. to this aspect. In fact, the invention has in particular for the process as described above, in which said phytosanitary composition comprises an effective amount of 10 ⁴ to 10 ¹² spores / g (of phytosanitary composition), more particularly 10 ⁷ to 10 ⁹ spores / g (of phytosanitary composition), said phytosanitary composition optionally being obtained after a preparation step (eg dilution by suspending or mixing) making it ready for application (eg spreading). According to another embodiment, the subject of the invention is therefore the process as described above, in which said phytosanitary composition is spread at a rate of 10 to 500 L / ha, in particular 50 to 250 L / ha, especially for aerial spraying; or in an effective amount of 10 ¹⁰ to 10 ¹⁴ spores / ha, in particular 10 ¹⁰ to 10 ¹² spores / ha, in particular for aerial application of the foliar spray type.

The expression "10 to 500 L / ha (liter / hectare)" also means 10 to 400 L / ha, 10 to 300 L / ha, 10 to 200 L / ha, 10 to 100 L / ha , 100 to 500 L / ha, 200 to 500 L / ha, 300 to 500 L / ha, 400 to 500 L / ha, 50 to 500 L / ha, 50 to 250 L / ha or 250 to 500 L / ha. The expression "from 10 ¹⁰ to 10 ¹⁴ spores / ha" also means from 10 ¹⁰ to 10 ¹¹ spores / ha, from 10 ¹⁰ to 10 ¹² spores / ha, from 10 ¹⁰ to 10 ¹³ spores / ha, from 10 ¹¹ to 10 ¹² spores / ha, 10 ¹¹ to 10 ¹³ spores / ha, 10 ¹¹ to 10 ¹⁴ spores / ha, 10 ¹² to 10 ¹³ spores / ha, 10 ¹² to 10 ¹⁴ spores / ha or 10 ¹³ to 10 ¹⁴ spores / ha.

According to another embodiment, the subject of the invention is the method as described above, in which said pathogenic fungus belongs to:

- a species chosen from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. debaryanum, P. elongatum, P. gracile, P. imperfection, P. inflatum, P. intermedium, P. irregulare, P. marinum, P. maritimum, P. middletoni, P. monospermum, P. myriotylum, P. ostracodes, P. pulchrum, P. salinum, P. thallassium and P. ultimum, and in particular belongs to the species P. ultimum; and or

- a species chosen from: R. alpine, R. anomala, R. callae, R. dichotoma, R. ferruginea, R. floccosa, R. fragariae, R. fraxini, R. fumigata, //. lanuginosa, R. leguminicola, //. menthae, //. mucoroides, //. muneratii, R oryzae-sativae, R. pini-insignis, //. quercus, //. rubi, //. rubiginosa, R silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular belongs to the species //. solani; and / or the species B. cinerea. By "P. ultimum" is meant the pathogen responsible for damping-off also called root rot or kPythium rot. By "R. solani" is meant the pathogen responsible for damping-off and / or rhizoctonia. By "B. cinerea" is meant the pathogen responsible for gray rot.

According to another embodiment, the subject of the invention is the process as described above, in which said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Rosaceae such as Pyrus spp. (pear), Prunus spp. (cherry), Fragaria spp. (strawberry), or Rasa spp. (pink) ;

^■ Valerianaceae, in particular said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

According to another embodiment, the subject of the invention is the method as described above, in which said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea and said plant belongs to a family chosen from: ^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

According to another embodiment, the subject of the invention is the method as described above, in which said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea and said plant is Beta vulgaris ( beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

Advantageously, the subject of the invention is the process as described above, in which:

- said pathogenic fungus is A. solani and said plant is Glycine max (soya); Where

- said pathogenic fungus is B. cinerea and said plant is Solanum lycopersicum (tomato); Where - said pathogenic fungus is B. cinerea and said plant is Solanum tuberosum (potato); Where

- said pathogenic fungus is B. cinerea and said plant is Glycine max (soya).

Advantageously, a subject of the invention is also the method as described above, in which said disease is damping-off and / or rhizoctonia and / or gray rot. In particular, the invention also relates to the method as described above, in which said disease is damping-off, also called kPythium rot. In particular, a subject of the invention is also the method as described above, in which said disease is rhizoctonia rhizoctonia. In particular, the invention also relates to the method as described above, in which said disease is gray rot.

According to another embodiment, the invention relates to the method as described above, in which the protection of said plant comprises a step of coating the seed with said phytosanitary composition; and / or the protection of said plant comprises a step of spraying said phytosanitary composition at the time of sowing or at the germination of said plant; and / or the protection of said plant comprises either a preventive application at soil level before sowing said phytosanitary composition with the contribution of organic amendment, or a preventive application at soil level before sowing said phytosanitary composition on the crop former ; and / or the treatment of said plant comprises a step of spraying said phytosanitary composition during different phenological stages of the plant (to prevent a risk of infection of said pathogenic fungus of the whole or part of the plant).

By "different phenological stages" is meant from seed to flowering (BBCH000 to BBCH619 according to the BBCH scale of phenological stages of vegetables in the Solanaceae family (Feller et al., 1995 - Phànologische entwicklungsstadien von gemüsepflanzen: II . Fruchtgemüse und hülsenfrüchte; Nachrichtenbl. Deut. Pflanzenschutzd)). In all respects, it should be noted that the different aspects of the invention, as well as the different embodiments thereof, are interdependent. The latter can therefore be combined in abundance to obtain aspects and / or preferred embodiments of the invention not explicitly described. This is also valid for all the definitions provided in the present description, which applies to all aspects of the invention and its embodiments.

Further, the present invention is illustrated, but not limited to, by the following Figures and Examples.

LIST OF FIGURES

Figure 1 shows an illustration of the so-called Dual-culture test (A; BCA = Biological Control Agent) and the measurement of the growth diameter of P. ulltimum in the presence (bottom gray line) or not (top black line) of the strain TAL-17 (abbreviated TAL) (B).

FIG. 2 represents the measurement of the growth diameter of R. solani in the presence (bottom gray line) or not (top black line) of the TAL-17 strain (abbreviated TAL).

Figure 3 shows photographs of tomato seedlings according to the classification of symptoms due to the pathogen P. ultimum on a scale of 0 to 5. Plants rated 0 are healthy, plants rated 5 are dead, intermediate ratings reflect the appearance of symptoms: leaves yellowed, crown attacked, seedling lying down.

Figure 4 shows the observation of symptoms of infection of tomato seedlings by Pythium ultimum after 21 days of culture in the presence or absence of TAL-17 (abbreviated TAL) and in comparison to uninfected controls.

FIG. 5 represents the observation of the germination of tomato seeds in the presence or not of TAL-17 and in the presence or not of Pythium ultimum. FIG. 6 represents the measurement of the growth diameter of A. dauci in the presence (bottom gray line) or not (top black line) of the TAL-17 strain.

FIG. 7 represents the measurement of the growth diameter of F. culmorum in the presence (bottom gray line) or not (top black line) of the TAL-17 strain.

Figure 8 represents the measurement of the growth diameter of F. oxysporum f. sp. lycopersici in the presence (bottom gray line) or not (top black line) of the TAL-17 strain.

FIG. 9 represents the measurement of the growth diameter of P. exigua in the presence (bottom gray line) or not (top black line) of the TAL-17 strain.

Figure 10 represents the percentage inhibition of the growth of P. ultimum in the presence of the TAL-17 strain, of two strains of Trichoderma atroviride derived from commercial products (Vintec ^® and Esquive ^® WP) and of a strain of Trichoderma harzanium (ARD collection). The results were analyzed according to a Student's test with a significance level of 5% compared to the TAL-17 strain (* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001).

Figure 11 represents the percentage inhibition of the growth of R. solani in the presence of the TAL-17 strain, of two strains of Trichoderma atroviride derived from commercial products (Vintec ^® and Esquive ^® WP) and of a strain of Trichoderma harzanium (ARD collection). The results were analyzed according to a Student's test with a significance level of 5% compared to the TAL-17 strain (* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001).

Figure 12 represents the percentage inhibition of the growth of F. oxysporum f. sp. lycopersici in the presence of the TAL-17 strain, of two strains of Trichoderma atroviride derived from commercial products (Vintec ^® and Esquive ^® WP) and of a strain of Trichoderma harzanium (ARD collection). The results were analyzed according to a Student test with a significance level of 5% compared to the TAL-17 strain (* = p ≤ 0.05; ** = p ≤ 0.01; ***

= p ≤ 0.001). Figure 13 represents a photograph of the development of the TAL-17 strain, of two strains of T. atroviride derived from commercial products (Vintec ^® and Esquive ^® WP) as well as a strain of T. harzanium (isolated by ARD according to the same process as the TAL-17 strain) after 48 h (broken lines) and 72 h (continuous lines) of growth on PDA medium. From left to right: Vintec ^® , Esquive ^® WP, T. harzanium and TAL-17.

Figure 14 represents the measurement of the growth diameter of TAL-17, of two strains of T. atroviride derived from commercial products (Vintec ^® and Esquive ^® WP) as well as a strain of T. harzanium (isolated by ARD according to the same process than the TAL-17 strain).

FIG. 15 represents histograms representing the effect of the strain TAL-17 (abbreviated TaL) on the development of gray rot induced by the pathogen Botrytis cinerea Bel. The TAL-17 strain arriving at the stage of sporulation on a dish was placed in liquid culture with gentle stirring at 1.10 ⁷ sp / mL in ½ PDB. The liquid culture of the TAL-17 strain was sprayed / sprayed on the tomato leaves at D0 (immediate effect - Figure 15A) and after 2 days of agitation (effect after 2 days - Figure 15B) 3 hours before the inoculation of the leaves by a deposit of 10 μL of a solution of Bel spores at 1.10 ⁶ spores / mL. The size of the necrotic lesions was measured on each leaflet from 2 or 3 days after inoculation (dpi for day post inoculation) and this up to a maximum of 7 days. The values represent the mean size of necrotic lesions in 12 leaflets per treatment modality ± standard deviation.

Figure 16 is a photograph showing the absence and presence of necrotic lesions induced by B. cinerea Bel on single tomato leaflets (white arrows). The left Petri dish corresponds to the TAL-17 modality (abbreviated TaL) + Bel at 3 dpi and the right dish corresponds to the Bel modality.

Figure 17 shows histograms representing the severity of gray rot in whole tomato plants 3 days after inoculation. The TAL-17 strain (abbreviated TaL) arriving at the stage of sporulation on a dish was placed in liquid culture for 2 days with stirring at 1.10 ⁷ sp / mL in ½ PDB. The liquid culture of the TAL-17 strain was sprayed / sprayed on whole plants approximately 16 hours before inoculation of the leaves by a deposit of 10 μl of a solution of Bel spores at 1.10 ⁶ spores / mL. The size of the necrotic lesions was measured on each leaflet from 3 days after inoculation. The values represent the mean size of the necrotic lesions of 4 tomato pots (2 inoculated leaf stages) per treatment modality ± the standard deviation.

EXAMPLES

(I) Isolation of Trichoderma atroviride strain TAL-17

Isolated stump of a wheat leaf grown in fields.

The leaf sample from the wheat crop was ground and then brought into contact with an infinitely mixed PDA (Potato Dextrose Agar) agar at 50 ° C which contains spores of Fusarium graminearum. The dish was cultured at 25 ° C. under a light hood. Thus only the strains being able to reproduce in the presence of Fusarium graminearum developed on the dish. Among these strains TAL-17 was present. A specific sample from an area of interest in the dish and several successive subcultures made it possible to isolate the TAL-17 strain, which was then deposited on July 3, 2018 at the National Collection of Cultures of Microorganisms (CNCM; Institut Pasteur , 25-28 rue du Docteur Roux, F-75724 PARIS CEDEX 15) under number CNCM 1-5333.

(II) In vitro study by dual-culture test of the efficacy of the TAL-17 strain on the pythopathogen Pythium ultimum Protocol

Several dual-culture tests (FIG. 1A) with the TAL-17 vs P. ultimum strain were carried out in proximity on PDA medium (Potato Dextrose Agar). 30 μL containing 1.10 ⁴ spores / mL (sp / mL) of P. ultimum are inoculated in the center of a Petri dish 8.5 cm in diameter, containing PDA medium, and 30 μL containing 1.10 ⁶ spores / mL of strain TAL-17 2 cm from the left and right end of the Petri dish (see Figure IA). The Petri dishes were incubated at 23-27 ° C under artificial light (24 hours a day) (fluorescent tube F36WT8 / 2084 BriteGro ^® ). Macroscopic observations were made every day after inoculation and the growth diameter of P. ultimum is measured daily for 5 days. Controls with P. ultimum alone were also carried out. Results

It is observed that the presence of the TAL-17 strain leads to an inhibition of the growth of the pathogen P. ultimum of the order of 50% compared to the control after 5 days of growth (see Figure IB).

(III) In vitro study by dual-culture test of the effectiveness of the TAL-17 strain on the pythopathogen Rhizoctania solani Protocol

Several dual-culture tests (FIG. 2) with the TAL-17 vs R. solani strain were carried out in proximity on PDA medium (Potato Dextrose Agar). 30 μL containing 1.10 ⁴ spores / mL (sp / mL) of R. solani are inoculated in the center of a Petri dish 8.5 cm in diameter, containing PDA medium, and 30 μL containing 1.10 ⁶ spores / mL of strain TAL-17 2 cm from the left and right end of the Petri dish. The Petri dishes were incubated at 23-27 ° C under artificial light (24 hours a day) (fluorescent tube F36WT8 / 2084 BriteGro ^® ). Macroscopic observations were made every day after inoculation and the growth diameter of R. solani is measured daily for 5 days. Witnesses with R. solani alone were also made.

Results

It is observed that the presence of the TAL-17 strain leads to an inhibition of the growth of the pathogen R. solani of the order of 35% compared to the control after 5 days of growth (see Figure 2).

(IV) Test conducted to study the biocontrol effect of the TAL-17 strain on the patho-system

Pythium ulümum / tomatt on growing medium

The ability of the TAL-17 strain to limit the contamination of tomato plants by Pythium ultimum (P. ultimum) and the appearance of associated symptoms was verified by a cultivation test under controlled conditions. Cultivation of Pythium ultimum

The strain was cultured on millet for 4 days at 25 ° C. The strain originates from the laboratory: Eidgenôssisches Departement für Wirtschaft, Bildung und Forschung WBF / Agroscope / Müller-Thurgau-Strasse 29, 8820 Wàdenswil.

Culture of the TAL-17 strain

The strain was cultured on millet for 4 days at 25 ° C. 250 g of millet were placed in 100 mL of water. The concentration was measured by observation on a Malassez cell. The concentration was adjusted to 10 ⁸ spores / mL.

Prior cultivation of the plant

The tomato seeds are placed to germinate in a growing medium for 3 weeks in a seedling plate.

Test modalities

Six modalities were studied:

- negative control (without pathogen and without the TAL-17 strain),

- positive control (without pathogen and with the TAL-17 strain), patho dose 1 (D1), patho dose 2 (D2), and patho dose 2 (D2) + the TAL-17 strain.

Each modality is repeated 4 times.

Launch of the trial

The culture medium used is Klassmann ^® peat. Each plant is placed in a 1.5 L pot. This contains 400 g of substrate (1 L).

The pathogen is mixed with the substrate, two doses are tested: 20 g and 80 g of millet with pathogen / L of substrate (respectively Patho Dl and Patho D2 modalities). For the modalities with the pathogen, the weight of millet added is subtracted from the quantity of substrate to be added. Pathogen-free modalities contain only substrate.

For each pot, the substrate / millet mixture is homogenized and then placed in the pot. Hydration is carried out by sub-irrigation. A planting hole is prepared in the center of the pot. The tomato seedling is taken out of the seedling plate and placed in the planting hole. For the modality concerned, the TAL-17 strain (= TAL) is added by adding the liquid solution containing 10 ⁸ spores / mL at the base of the plantlet (10 mL / pot).

The culture is carried out in a phytotron with the following instructions: 12 h in the dark at 20 ° C and 12 h in light at 23 ° C, the substrate is watered at 80% of the _max CR in water.

Symptom monitoring

Symptoms were rated on a scale of 0 to 5. Plants rated 0 are healthy, plants rated 5 are dead, intermediate ratings reflect the onset of symptoms: yellow leaves, crown attacked, seedling lying down (see Figure 3). .

Results

This observation of the degrees of infection was carried out after 21 days of culture (see Figure

4)

The control modality present in healthy seedlings, after 3 weeks only yellowing of the cotyledons is observed. The seedlings treated with strain TAL-17 (= TAL) only are healthy, with yellowing of the cotyledons as in the control modality. Adding the pathogen to the two doses tested causes the onset of strong symptoms, with dead seedlings in both doses tested. The dose effect appears to be limited. The modality treated with both the pathogen at dose 2 (D2) and with the TAL-17 strain shows healthier seedlings than the modality with the pathogen at dose 2 (D2) without the TAL-17 strain.

Infection grades were used for analysis of variance (95% ANOVA), which is presented in Table 3 above. A significant effect is observed (P-value = 0.0004), the modalities are as follows:

Table 3: Statistical analysis Modality Average Statistical group Conclusion

Treatment of the seedlings during repotting with a solution of the TAL-17 strain allowed a limitation of the appearance of symptoms of infection by Pythium ultimum in tomato seedlings.

IV) Test conducted to study the biocontrol effect of the TAL-17 strain on the patho-system

Pythium ultimum / tomato during seed germination Protocol

The objective of this trial is to evaluate the efficacy of strain TAL-17 in limiting damping-off in tomato seeds treated with Pythium ultimum.

The terms of the test are as follows:

1. Water + water: Healthy plants, "inoculated" with sterile water,

2. Water + Pu: Untreated plants inoculated with P. ultimum,

3. Fong + Pu: Plants treated with a fungicide and inoculated with P. ultimum,

4. 2Dla + Pu: Plants treated with the TAL-17 strain at 10 ⁶ CFU / mL then inoculated with P. ultimum, and

5. 2Dlb + Pu: Plants treated with the TAL-17 strain at 10 ⁷ CFU / mL then inoculated with P. ultimum.

The strains are cultured on PDA then the dishes are used to prepare spore solutions.

The seeds are soaked for 1 hour: in water for modalities 1 and 2, in solutions of spores of the TAL-17 strain (BCA 2D1) for modalities 4 and 5, in a fungicide solution for modality 3 (Previcur ENERGY ^® : mixture of fosetyl aluminum / propamocarb).

Next, the seeds are soaked in a solution of Pythium ultimum spores at a concentration of 10 ⁷ spores / ml.

After treatment, the seeds are placed together in filter paper which is rolled up on itself. These rollers are placed in water to ensure good hydration of the seedlings throughout the test. Each modality is represented by 3 rolls of 5 seeds. After 15 days of culture, the rolls are unrolled for observation of the germination of the seeds. Results

After 15 days, the rolls are unwound for scoring of seed germination (% seed germinated) (see Figure 5).

Control plants treated with Pythium ultimum and water show lower germination than the control without P. ultimum. The fungicidal treatment has a positive effect on the germination of seeds. Treatment with the TAL-17 strain had a positive effect on germination at the two concentrations tested.

(VD Specificity test of the TaqMan ^® GS285741-P1 probe and the associated primer pair

GS285741-F1 and GS285741-R1 with respect to the strain of the invention (TAL-17 and / or one of its mutants)

Protocol

In order to test the specificity of the molecular tools (TaqMan ^® probe GS285741-P1 and the pair of associated primer GS285741-F1 and GS285741-R1), a classic analysis by Polymerase Chain Reaction in real time (qPCR) was carried out on 3 distinct strains, namely: the Trichoderma viride strain of the species T. asperellum (TV); the strain Trichoderma atroviride TAS; and the Trichoderma atroviride strain of the invention (TAL-17).

An internal control standard is also produced with genomic DNA extracted from the strain of the invention, which has been serially diluted.

All qPCR assays were performed using a CFX 96 touch thermal cycler from Biorad ^® . The amplification program used is that recommended by the supplier with a hybridization temperature of 60 ° C. For the TaqMan ^® method, the Luna ^® Universal Probe qPCR Master Mix (NEB ^® ) reaction mixture was used. The amplification program is 95 ° C 2 min followed by 40X 95 ° C 15 ”and 60 ° C 30”.

Results

The results of this analysis are presented in Table 4 below.

Table 4: Specific detection by qPCR of the strain of the invention

Conclusion

The TaqMan ^® probe GS285741-P1 and the associated primer pair GS285741-F1 and GS285741-R1 only made it possible to detect the TAL-17 strain. These tools therefore allow the detection and identification in a sample of the presence of the strain of the invention TAL-17 and / or one of its mutants. (VII) In vitro study by dual-culture test of the efficiency of the TAL-17 strain on other pythopathogens Protocol

Several Dual-culture tests with the TAL-17 strain us Alternaria dauci, Fusarium culmorum, Fusarium oxysporum f. sp. lycopersici and Phoma exigua were carried out in proximity on PDA medium (Potato Dextrose Agar). 30 μL containing 1.10 ⁴ spores / mL (sp / mL) of Alternaria dauci, Fusarium culmorum, Fusarium oxysporum f. sp. lycopersici or Phoma exigua in the center of a Petri dish 8.5 cm in diameter, containing PDA medium, and 30 pL containing 1.10 ⁶ spores / mL of the TAL-17 strain 2 cm from the left and right extremity of the Petri dish. The Petri dishes were incubated at 23-27 ° C under artificial light (24 hours a day) (fluorescent tube F36WT8 / 2084 BriteGro ^® ). Macroscopic observations were made daily after inoculation and growth diameter of Alternaria dauci, Fusarium culmorum, Fusarium oxysporum f. sp. lycopersici and Phoma exigua is measured daily. Witnesses with Alternaria dauci, Fusarium culmorum, Fusarium oxysporum f. sp. lycopersici and Phoma exigua alone have also been produced.

Results

It is observed that the presence of the TAL-17 strain leads to an inhibition of the growth of the pathogens Alternaria dauci, Fusarium culmorum, Fusarium oxysporum f. sp. lycopersici and Phoma demanded about 41%, 23%, 26% and 26% respectively from the control after 10 days of growth (see Figures 6 to 9).

(VIII) In vitro study by dual-culture test of the effectiveness of the TAL-17 strain on the pythopathogens P. ultimum, R. solani and Fusarium oxysporum f. sp. lycopersici compared to other Trichoderma spp.

The ability of strain TAL-17 to limit the development of three different phytopathogens (P. ultimum, R. solani and Fusarium oxysporum f. Sp. Lycopersici) was compared with that of other strains of Trichoderma spp. during a test under controlled conditions. Protocol

Several dual-culture tests with the TAL-17 strain, two strains of T. atroviride from commercial products (Vintec ^® and Esquive ^® WP) as well as a strain of T. harzanium (isolated by A RD according to the same method as strain TAL-17) us Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici were carried out in proximity on PDA medium (Potato Dextrose Agar). 30 μL containing 1.10 ⁴ spores / mL (sp / mL) of Pythium ultimum, Rhizoctonia solani or Fusarium oxysporum f. sp. lycopersici 1 cm from the right end of an 8.5 cm diameter Petri dish, containing PDA medium, and 30 μL containing 1.10 ⁶ spores / mL of the TAL-17 strain, of the two commercial strains or of the T. harzanium strain 1 cm from the left end of the Petri dish. The Petri dishes were incubated at 23-27 ° C under artificial light (16h / 8h) (fluorescent tube F36WT8 / 2084 BriteGro ^® ). Controls with Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici as well as with TAL-17, the two strains of T. atroviride derived from commercial products (Vintec ^® and Esquive ^® WP) and the T. harzanium strain alone were also produced. Macroscopic observations were made daily after inoculation and growth diameter of Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum f. sp. lycopersici is measured daily in order to express the results as a percentage inhibition of the growth of pathogens. The percentage inhibition of the growth of pathogens (PICP) was calculated as follows: PICP = (DI - D2) / D1 x 100, where DI corresponds to the diameter of the pathogen and D2 to that of the pathogen in the presence of l one of the strains of Trichoderma spp. studied. The results were analyzed according to a Student's test with a significance level of 5% compared to the TAL-17 strain (* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001).

Results

It is observed that the presence of the TAL-17 strain leads to a rapid inhibition of the growth of the pathogen P. ultimum of the order of 20%, 80% and 95% after respectively 2, 4 and 6 days of growth (see Figure 10). The TAL-17 strain demonstrates superior efficacy against the pathogen P. ultimum. Indeed, the results obtained with the TAL-17 strain are statistically different from those of the three other strains of Trichoderma spp. tested (two strains of T. atroviride from the products Esquive ^® WP and Vintec ^® and the strain of T. harzanium). It is observed that the presence of the TAL-17 strain leads to an inhibition of the growth of the pathogen R. solani of the order of 20% from the second day of growth against less than 10% for the other strains of Trichoderma spp. (see Figure 11). After 4 days of growth, the TAL-17 strain leads to an inhibition of the pathogen R. solani of the order of 40%, a result statistically different only from the strain derived from the commercial product Esquive ^® WP. However, the TAL-17 strain exhibits at least 5% more efficacy than the other two strains of Trichoderma spp. tested (the T. atroviride strain from Vintec ^® products and the T. harzanium strain). After 6 days of growth, the percentage inhibition of the growth of the pathogen A. solani varies from 65 to 85% depending on the strains studied, the highest percentage of inhibition being that due to the TAL-17 strain.

It is observed that the presence of the TAL-17 strain leads to an inhibition of the growth of the pathogen F. oxysporum f. sp. lycopersici on the order of 15% more from the second day compared to the three other strains of Trichoderma spp. tested (see Figure 12). After 4 days, the TAL-17 strain demonstrates similar efficacy to the strain derived from the commercial product Esquive ^® WP while the other two strains of Trichoderma spp. (the T. atroviride strain from Vintec ^® products and the T. harzanium strain) show lower activity against the pathogen A. oxysporum f. sp. lycopersici of the order of 8 to 14%. After 6 days of growth, the presence of the TAL-17 strain leads to an inhibition of the growth of the pathogen of the order of 5 to 10% more depending on the strains of Trichoderma spp. studied. The percentages of inhibition obtained with the pathogen F. oxysporum f. sp. lycopersici are lower than with the pathogens P. ultimum and R. solani, as are the differences between the strains of Trichoderma spp. tested (the TAL-17 strain, the two strains of T. atroviride derived from the Esquive ^® WP and Vintec ^{® products} and the strain of T. harzanium), are less marked. The minimally invasive character of the pathogen F. oxysporum f. sp. lycopersici. can explain this.

It was also observed only for the TAL-17 strain, a green pigmentation of the spores, typical of the Trichoderma genus after 72 hours of growth (see Figure 13).

The development of the 4 strains of Trichoderma spp. tested alone was studied (see Figure 14). The strain of the invention, TAL-17, exhibits a development similar to the strain derived from the commercial product Vintec ^® while the growth of the other two strains of Trichoderma spp. (strain of T. atroviride derived from the product Esquive ^® WP and T. harzanium), the development of which does not differ, is much lower than that of the TAL-17 strain. Finally, it emerges from the tests carried out that the presence of the TAL-17 strain led to an inhibition of the growth of the pathogens P. ultimum, R. solani and Fusarium oxysporum f. sp. lycopersici respectively of the order of 97%, 86% and 20% relative to the control after 6 days of growth (see Figures 10 to 12). In addition, the strain of the invention has demonstrated greater efficacy than other Trichoderma spp. studied against the three pathogens P. ultimum, R. solani and F. oxysporum f. sp. lycopersici from the first days of growth, which was also observed throughout the duration of the tests carried out. During this study, a faster mycelial development as well as a sporulation (asexual reproduction) of the TAL-17 strain was also observed, which gives it a definite advantage in the fight against the three pathogens P. ultimum, R. solani and F. oxysporum f. sp. Lycopersici (space competition).

(IX) Study of the biocontrol effect of strain TAL-17 on gray rot in tomatoes

1. Materials and methods

1.1. Biological material

1.1.1. Solanum lycopersicum

The Clodano variety is a fast growing variety suitable for greenhouse cultivation. In addition, it is the crop variety most affected by gray rot.

The sowings were carried out in plates of 50 seeds in the substrate TS3 404, Klasman. The plates are placed in a climatic chamber at 25 ° C with a photoperiod of 16h until the seeds have germinated, then the seedlings were placed in a greenhouse under a natural photoperiod at 18 ° C at night and 23 ° C during the day. . When the first two leaves appeared the seedlings were repotted in 3L pots in the same potting soil and the same growing conditions as specified previously until they reached the 5/7 leaf developed stage (5-6 weeks maximum). Two to three times a week the seedlings and plants were watered with distilled water.

1.1.2. Strains

The plant pathogen: Botrytis cinerea Bel.

The Bel strain was cultured on Potato Dextrose Agar (PDA) at room temperature 20-23 ° C, under indirect ambient light until the stage of sporulation (for a maximum of 15 days). Two weeks before the start of the tests, the Bel strain was systematically returned to culture on PDA from cryostocks stored at -20 ° C (mixture of spores + mycelium in phosphate buffer, 20% glycerol). When the sporulation stage was reached, a spore solution was prepared in ¼ Potato Dextrose Broth (PDB) and the concentration adjusted to 1.10 ⁵ or 1.10 ⁶ spores / mL depending on the tests.

The biocontrol agent: Trichoderma atroviride Three strains of Trichoderma atroviride were tested, that of the TAL-17 invention and two others from commercial products (Esquive ^® and Vintec ^® ). They were cultured on PDA medium at 25 ° C. with a 16h day / 8h night cycle. They were subcultured every week by studs and / or freshly thawed from cryostocks stored at -20 ° C. (PDB mixture, 20% glycerol).

When the strains reach the sporulation stage (between 8-14 days max, dark green morphotype) a spore suspension was prepared in ½ PDB at a concentration of 1.10 ⁷ spores / mL.

The TAL-17 strain, if necessary, was also put back into liquid culture for 40-48 hours (mixture comprising the spores, the mycelium + a set of metabolites). To do this, from a dish arriving at the sporulation stage, a liquid culture of TAL-17 was prepared in an Erlenmeyer flask in PDB ½ at 1.10 ⁷ spores / mL and stirred gently at ambient temperature and indirect light for 40 at 48 h max (preparation of a culture of liquid TAL-17 with its metabolites).

1.2. Methods

1.2.1. Tomato leaf tests isolated

When the tomatoes reach the desired stage of development, the young leaf stages were cut in order to be treated with the Trichoderma strains (TAL-17, Esquive ^® and Vintec ^® ). Under PSM, each leaflet was sprayed with the Trichoderma solution at 1.10 ⁷ spores / mL using a small hand-held sprayer (1 spray on each side of the leaflet). The leaflets were then placed on 0.8% agar medium in a square Petri dish at the rate of 4 leaflets / Petri dish. The Petri dishes with the half-open lid were left under PSM for 3 hours before inoculation of the leaflets with the Bel strain. Each treatment modality comprises on average 8 to 12 leaflets or 2 to 3 Petri dishes. The pathogenic strain Bel was introduced in the form of a drop of 10 μL at 1.10 ³ , 1.10 ⁵ or 1.10 ⁶ spores / mL deposited on the upper surface of the leaf, at the rate of one drop per leaflet.

The Petri dishes were put in airtight and transparent plastic bags containing 1-2 cotton pads soaked in distilled water to maintain saturated humidity favoring the development of the pathogen. The whole was incubated flat in a Phytotron at 20 ° C with 12h day and 12h of darkness. The lesions resulting in necrosis on leaves (measurement of the diameter and / or length + width of the necrotic spot) were measured 3 days after inoculation (DPI = day post inoculation) and this for a maximum of 5 days (up to with complete disease development in inoculated, untreated controls).

1.2.2. Tests on whole tomatoes

The tomatoes grown in the greenhouse were placed approximately 7 hours before the treatment with the TAL-17 strain in climatic chambers at 23 ° C. and were slightly humidified. The methods tested were as follows:

Chamber 1: 4 control plants (untreated, uninoculated);

Chamber 2: 4 plants inoculated with the strain Botrytis cinerea Bel at 1.10 ⁶ spores / mL; Chamber 3: 4 plants treated with the TAL-17 strain at 1.10 ⁷ spores / mL (liquid culture of TAL-17 for 48 hours max); and

Chamber 4: 4 plants treated with TAL-17 at 1.10 ⁷ spores / mL and inoculated with the strain Botrytis cinerea Be 1 at 1.10 ⁶ spores / mL.

The liquid solution of TAL-17 (the entire liquid culture in Erlenmeyer flasks for 40-48 hours) was supplied by manual spraying of the aerial parts without going to the point of runoff. The tomatoes were incubated for 16h max before adding Bel at 23 ° C, 80-95% humidity, the incubation cycle was started with 8h of darkness. Control plants were sprayed with ½ PDB.

One night after the addition of the TAL-17 strain, the leaflets were inoculated with the Bel strain by adding 10 μL of a spore solution at 1.10 ⁶ sp / mL per leaflet, and this over 2 leaf stages ( inoculation of 5 leaflets / leaf stage). The tomatoes were incubated in humid rooms at 19 ° C. with 8 hours of darkness and 16 hours of daylight, in saturated humidity for 3 days. After 3 days the diameter of the necrotic lesions was measured daily and up to 5 days / mst-inoculation maximum. 2. Results

2.1. Tests on isolated tomato leaves: contribution of the TAL-17 strain in the form of a liquid culture - immediate effect and after 2 days of culture of the TAL-17 strain The objective of these in vitro tests was to measure a possible reduction / inhibition of the infection capacity of B. cinerea Bel and therefore of the development of gray rot (= necrotic lesions) induced by the TAL-17 strain on isolated tomato leaves.

Preliminary studies have shown an effect of the TAL-17 strain when cultured in ½ PDB with shaking at room temperature and light, compared to a TAL-17 inoculum prepared directly from a dish culture. petri dish arriving at the stage of sporulation.

In this test the strain TAL-17 was placed in liquid culture in PDB ½ at 1.10 ⁷ sp / mL in order to evaluate the effect of the latter on the development of gray rot immediately after cultivation (JO) and after 2 days of agitation. The mean size of the necrotic lesions on leaves was measured daily from 2 or 3 days after inoculation (dpi) for 7 days (Figures 15 and 16) and the percentage reduction of the disease was calculated (Table 5).

Table 5. Effect of strain TAL-17 on the development of gray mold induced by the pathogen Botrytis cinerea Bel

The method is the same as described in the legend of Figure 15. The values represent the mean size of the necrotic lesions measured on 12 leaflets by treatment modality ± the standard deviation and the percentage reduction of the disease. Dpi for for day post inoculation. A much stronger effect of the TAL-17 strain was observed after 2 days of culture on the development of the disease induced by Bel (Figure 15B) compared to the immediate effect of the strain (Figure 15 A). At D0 (immediate effect) the effect is also visible with a reduction in symptoms of 10.67% from 2 dpi (Table 1). This is also true at 7 dpi since a 58.2% reduction in symptoms was observed when the TAL-17 strain was cultured for 2 days compared to a 32% reduction when it was taken directly after. cultivation (Table 5).

The effect of the TAL-17 strain after 2 days of culture is therefore greater, in particular due to its re-growth (production of spores) and the synthesis of metabolites.

2.2. Tests on isolated tomato leaves: effect of the TaL strain after 2 days of liquid culture in response to increasing concentrations of Botrytis cinerea Bel In this test was tested the effect of the TAL-17 strain at 1.10 ⁷ sp / mL after 2 days of liquid culture in ½ PDB in response to 3 Bel concentrations: 1.10 ³ , 1.10 ⁵ and 1.10 ⁷ sp / mL. The method is identical to the previous one and the results obtained are presented in the table below.

Table 6. Effect of strain TAL-17 on the development of gray rot in response to 3 concentrations of Botrytis cinerea Bel The strain TAL-17 arriving at the stage of sporulation on a Petri dish was placed in liquid culture at 1.10 ⁷ sp / mL in ½ PDB for 2 days. The whole culture of the TAL-17 strain was sprayed / sprayed on the tomato leaves 3 hours before inoculation of the leaves by a deposit of 10 μL of a solution of Bel spores at 1.10 ³ , at 1.10 ⁵ and 1.10 ⁷ spores / mL. The size of the necrotic lesions was measured on each leaflet from 3 days after inoculation (dpi) and this up to 7 days. The values represent the mean size of the necrotic lesions of 12 leaflets per treatment modality ± the standard deviation as well as the percentage reduction in symptoms on the leaf.

A maximum biocontrol effect of the TAL-17 strain was observed in response to a concentration of 1.10 ⁵ sp / mL in Bel. Indeed from 3 dpi was observed a reduction of 100% for the concentration at 1.10 ⁵ sp / mL followed by a reduction of the order of 66.67% for the concentration at 1.10 ³ sp / mL in Bel and 32 , 35% for the last concentration. This effect is retained for up to 7 days of reading.

2.3. Tests on single tomato leaves: comparison of the effect of 3 strains of Trichoderma atroviridae on the development of gray rot induced by the strain Bel

In this trial, the biocontrol effect of 3 strains of Trichoderma atroviride was tested: TAL-17, Esquive ^® and Vintec ^® , at 1.10 ⁷ sp / mL in response to a concentration of 1.10 ⁵ sp / mL in Bel. The method is identical to the previous one are presented in the table below.

The tomato leaves were treated with the TAL-17 strain after 2 days of liquid culture in Erlens and the inoculum of the other two strains was prepared directly from a Petri dish arriving at the sporulation stage (i.e. around 8 days). .

Table 7. Effect of strain TAL-17, Esquive ^® and Vintec ^® on the development of gray rot induced by the pathogen Botrytis cinerea Bel at 3 days after inoculation (dpi).

The values represent the mean size of the necrotic lesions of 12 leaflets by treatment modality ± the standard deviation as well as the percentage reduction in symptoms on the leaf.

Once again, a maximum biocontrol effect of the TAL-17 strain was observed with a reduction in symptoms at 3 dpi of the order of 68% compared to a reduction of 42% for the Esquive ^® strain and of 58% for the strain. Vintec ^® . 2.4. Whole plant test

A test was also conducted to evaluate the biocontrol effect of the TAL-17 strain, in liquid solution after 2 days of cultivation, on whole tomatoes under controlled conditions. At the end of this trial, a reduction of about 53.17% in symptoms was observed compared to untreated and inoculated plants. This effect was observed on 3 independent biological repeats (Figure 17).

Claims

1. Use of an isolated strain of Trichoderma atroviride in the prevention and / or treatment of infection in a plant caused by a pathogenic fungus selected from fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis, said strain isolated from Trichoderma atroviride being the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants.

2. Use of an isolated strain of Trichoderma atroviride according to claim 1, wherein said pathogenic fungus belongs to:

- a species chosen from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. deharyanum, P. elongatum, P. gracile, P. imperfectum, P. inflatum, P. intermedium, P. irregulare, P. marinum, P. maritimum, I \ middletoni, I \ monospermum, I \ myriotylum, I \ ostracodes, P. pulchrum, I \ salinum, I \ thallassium and I '. ultimum, and in particular belongs to species I \ ultimum; and or

- a species chosen from: //. alpine, //. anomala, //. callae, //. dichotoma, //. ferruginea, //. floccosa, //. fragariae, //. fraxini, //. fumigata, //. lanuginosa, R. leguminicola, //. menthae, //. mucoroides, //. muneratii, R. oryzae-sativae, R pini- insignis, //. quercus, //. rubi, //. rubiginosa, R. silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular belongs to the species //. solani; and or

- the cinerea species.

3. Use of an isolated strain of Trichoderma atroviride according to claim 1 or 2, wherein said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera); ^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Valerianaceae, in particular said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

4. Use of an isolated strain of Trichoderma atroviride according to any one of claims 1 to 3, wherein said pathogenic fungus is P. ultimum and / or R. solani and / or B. cinerea and said plant is Beta vulgaris ( beet), Solanum lycopersicum (tomato), Solanum tuberosum (potato) and / or Glycine max (soybean).

5. Use of a phytosanitary composition in the prevention and / or treatment in a plant of infection caused by a pathogenic fungus chosen from fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis, said phytosanitary composition comprising:

- the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants as active principle; Where - the metabolites emitted during the production of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under number CNCM I-5333 and / or one of its mutants as active principle; Where

6. Use of a phytosanitary composition according to claim 5, said phytosanitary composition comprising an effective amount of 10 ⁴ to 10 ¹² spores / g, more particularly 10 ⁷ to 10 ⁹ spores / g, said phytosanitary composition optionally being obtained after a preparation stage making it ready for spreading.

7. Use of an isolated strain of Trichoderma atroviride to promote the germination of a plant seed, said isolated strain of Trichoderma atroviride being the TAL-17 strain deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333 and / or one of its mutants and in which said plant seed belongs to a plant of a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber); ^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Valerianaceae, in particular said plant belongs to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

8. Coated seed comprising a plant seed, in which said plant seed is coated with a phytosanitary coating composition and belongs to a plant of a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae;

^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper) ; and ^■ Valerianaceae, and in which said phytosanitary coating composition comprises:

- the metabolites emitted during the production of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM I-5333 and / or one of its mutants as active principle, and possibly at least a fixing agent; Where

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

^■ Solanaceae such as Solanum lycopersicum (tomato), Solanum tuberosum (potato), Malus domestica (apple) or Capsicum spp. (pepper), and preferably said plant being Beta vulgaris (beet), Solanum lycopersicum

(tomato), Solanum tuberosum (potato), Glycine max (soybean).

9. Coated seed according to claim 8, said coated seed comprising an effective amount of 10 ⁵ to 10 ⁸ spores / coated seed, preferably 10 ⁶ spores / coated seed.

10. A method of protecting and / or treating a plant against a disease caused by a pathogenic fungus chosen from among the fungi of the genera Pythium, Rhizoctonia, Alternaria, Fusarium, Phoma and Botrytis comprising the application of a phytosanitary composition on to the at least part of said plant or of the soil near said plant, said at least part of said plant being in particular a leaf, a fruit, a seed, said phytosanitary composition comprising:

- the metabolites emitted during the production of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under number CNCM I-5333 and / or one of its mutants as active principle; Where

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Apiaceae such as Daucus carota (carrot);

^■ Asteraceae such as Lactuca sativa (lettuce) or Gerbera spp. (Gerbera);

^■ Brassicaceae;

^■ Chenopadiaceae; ^■ Cucurbits such as Cuccumis sativus (cucumber);

^■ Fabaceae such as Glycine max (soybean);

^■ Liliaceae;

^■ Primulaceae such as Cyclamen spp. (cyclamen);

^■ Valerianaceae, in particular said plant belonging to a family chosen from:

^■ Amaranthaceae such as Beta vulgaris (beet);

^■ Fabaceae such as Glycine max (soybean); and

11. The method of claim 10, wherein said phytosanitary composition is applied.

- at a rate of 10 to 500 L / ha, in particular from 50 to 250 L / ha, in particular for aerial spraying; Where

- at an effective amount of 10 ¹⁰ to 10 ¹⁴ spores / ha, in particular 10 ¹⁰ to 10 ¹² spores / ha, in particular for aerial application of the foliar spray type.

12. The method of claim 10 or 11, wherein said pathogenic fungus belongs to:

- a species chosen from: P. acanthicum, P. afertile, P. aphanidermatum, P. aquatile, P. debaryanum, P. elongatum, P. gracile, P. imperfectum, P. inflatum, P. intermedium, P. irregulare, P. marinum, P. maritimum, P. middletoni, P. monospermum, P. myriotylum, P. ostracodes, P. pulchrum, P. salinum, P. thallassium and P. ultimum, and in particular belongs to the species P. ultimum; and or

- a species chosen from: R. alpine, R. anomala, R. callae, R. dichotoma, //. ferruginea, //. floccosa, //. fragariae, //. fraxini, //. fumigata, //. lanuginosa, R. leguminicola, //. menthae, //. mucoroides, //. muneratii, R. oryzae-sativae, R pini- insignis, //. quercus, //. rubi, //. rubiginosa, R. silvestris, //. solani, //. stahlii, //. tuliparum and //. versicolor, and in particular belongs to the species //. solani; and or

- the cinerea species.

13. A method according to any one of claims 10 to 12, wherein

- the protection of said plant comprises a step of coating the seed with said phytosanitary composition; and or

the protection of said plant comprises a step of spraying said phytosanitary composition at the time of sowing or at the germination of said plant; and or

- the protection of said plant comprises either a preventive application at soil level before sowing said phytosanitary composition with the addition of organic amendment, or a preventive application at soil level before sowing said phytosanitary composition on the previous crop; and or

- The treatment of said plant comprises a step of spraying said phytosanitary composition during different phenological stages of the plant.

14. Isolated strain of Trichoderma atroviride, said isolated strain being a mutant strain of the isolated strain of Trichoderma atroviride TAL-17 deposited on July 3, 2018 at the CNCM under the number CNCM 1-5333.