ES2395518B1 - USEFUL TRICHODERMA CEPA FOR THE TREATMENT AND / OR PREVENTION OF INFECTIONS CAUSED BY FUNGES BELONGING TO THE VERTICILLIUM GENDER. - Google Patents

Abstract

Trichoderma strain useful for the treatment and / or prevention of fungal infections belonging to the genus Verticillium. # The invention relates to the use of the Trichoderma atroviride IMI 206040 fungal strain for the prevention and / or treatment of plant infections and / or or soils caused by phytopathogenic fungi belonging to the genus Verticillium, preferably by the defoliant or non-defoliant patotypes of Verticillium dahliae, the causative agent of olive verticillosis. In addition, the invention proposes the use of this fungal strain in combination with other fungi of the genus Trichoderma.

Description

  USEFUL TRICHODERMA CEPA FOR THE YIO TREATMENT PREVENTION OF INFECTIONS CAUSED BY FUNGES BELONGING TO THE VERTICILLUM GENDER

The present invention falls within the field of agriculture, microbiology and phytosanitary products, and specifically refers to the use of the Trichoderma atroviride IMI 206040 fungal strain for the prevention and / or treatment of plant and / or soil infections.

caused by phytopathogenic fungi belonging to the genus

Verticillium, more preferably by Verticillium dahliae, causative agent

of verticillosis.

STATE OF THE PREVIOUS TECHNIQUE

Verticillosis or vascular wilt is a disease caused by

Hypomycete fungus Verticiflium dahliae Kleb. that can attack a wide

range of crops, both woody and herbaceous, including fruit trees,

legumes, horticultural, forestry, ornamental and weeds. Thus, attacks of verticilosis constitute a serious problem in various crops such as artichoke, cotton, melon, tomato, potato or olive.

Currently, in crops such as the olive tree, verticillosis is considered one of the most serious and threatening phytopathological problems,

causing significant economic losses. Olive verticillosis (VO) is a disease in worrying expansion in countries where the

Olive grove is a relevant crop, severely affecting both adult olive groves and young plantations, possibly as a result, among others, of the establishment of intensive plantations

in soils infested by the pathogen and / or the use of infected planting material. Verticillium dahliae isolates that infect

Olive trees can be differentiated by their virulence on the plant and

thus be classified in the defoliant (O) or non-defoliant (NO) patotypes by their ability or not to cause the generalized fall of the green leaves of infected branches, respectively. The va can produce the

death of whole trees or dry branches causing a delay in growth and production losses.

In general, verticillosis are diseases that are difficult to control, to which

contributing factors such as the ability of the pathogen to survive long in the soil (up to 15 years), the wide range of plants susceptible to infection by it, the difficulty of reaching the fungus, since it is in the xi motto of the plant and buried in the ground (between 10 and 30 cm deep), and the inefficiency of treatments

Chemicals so far explored to fight the fungus during its parasitic phase in the xi motto. As with other verticillosis of woody plants, efficient control of VO requires the application of a

integrated disease management strategy.

Control measures for fungal diseases of the olive tree have been

in many indirect cases (elimination of diseased plants, utilization

of potassium fertilizers, etc.). However, from the perspective of sustainable agriculture and an integrated disease control strategy, one of the possible control measures to be applied is the use

of microbial antagonists with potential as control agents

Biological (ACBs) of the disease. Biological control of phytopathogenic organisms is a more attractive alternative than that of chemical products, since these ACBs are less harmful to

environment than the chemical compounds commonly used.

However, the use of such ACBs has been limited by different

causes, among them, an inadequate identification of them.

Some strains of the Trichoderma filamentous fungus, such as

strains belonging to the species Trichoderma harzianum, have been

used as biological control agents against plant pathogenic fungi, due to their protective ability against both soil fungi

that infect the roots as against those that cause diseases in the aerial parts. Some of these strains also have a fertilizing effect on plants, stimulating their vigor, growth and defense mechanisms.

Trichoderma strains use different mechanisms to attack

Mushrooms Relationships of myco parasitism have been described between

Trichoderma harzianum and Rhizoctonia salani, Sclerotium rolfsii or Pythium,

where lithic enzymes play a primary role, and relationships of

competition between Trichoderma harzianum and Botrytis cinerea (on vine) or Fusarium (on cotton). Thus, strains of Trichoderma harzianum,

Trichoderma virens and Trichoderma viride (Beautiful on al., 2000, Applied and Environmenla / Microbi% gy, 66: 5, pp. 1890--1898), and others of

Trichoderma asperellum, Trichoderma atroviride and Trichoderma inhamalum (ES2200602 A 1), have been used in combination as ACBs in compositions against different plant pathogenic fungi. An example of a synergistic composition of this type is one that comprises different strains of Trichoderma harzianum (those with access number in the NRRL 30595, 30596 and 30597 collection) that has a phytopathogenic fungus controlling capacity, is tolerant of abiotic stress, stimulates growth of the plant as well as its phenol content and induces systemic resistance to diseases caused by phytopathogenic organisms (W0200711 0686 A2).

In the case of the Verticillium dahliae pathogen, a wide range of antagonists have been characterized, among which are

different strains of Trichoderma (Berg el al., 2005, Applied and Environmenlal Microbiology, 71: 8, pp. 4203-4213). In fact, one of the proposed methods for the biological control of this phytopathogenic fungus

in the olive tree it is based on the application of spores of Trichoderma

harzianum, which also favors biofertilization (W02009083819 A1).

The criteria to be used for the selection of Trichoderma strains suitable for use as ACBs present as main

characteristics: 1) production of lithic enzymes and 2) good development in competitive conditions. The latter also depends on: 3) the production and preparation conditions of the inoculum and 4) environmental factors, including the presence of fungicides and / or pesticides. In this

sense, a mutant strain of Trichoderma harzianum has been developed

CECT 2413, the strain Trichoderma harzianum PF1, which has a

lithic enzyme production capacity superior to that of the strain

wild, and its effect on agent growth has been tested

phytopathogens such as Rhizoctonia solani or Botrytis cinerea, demonstrating that indeed its antifungal activity is increased

with respect to that of the strain from which it comes (Rey el al., 2001, Applied Microbiology and Biolechnology, 55, pp. 604-608).

Regarding the improvement of the competition capacity of the strains in the

environment, this is a complex process but in some experimental approaches we have tried to obtain improved strains, more adaptable to the environment (increased mechanisms of resistance to dryness, the presence of metals, the production of enzymes or

toxic substances by pathogens, ability to colonize soils with extreme pHs, etc.). Above all, strains would be of interest

resistant to fungicides, which would allow a better adaptation to an environment treated with these compounds and the possibility of a treatment

integrated that combines small doses of fungicides to which the pathogens are sensitive combined with resistant Trichoderma strains. The combined effect would allow better control and greater

protection than simple chemical or biological treatment. On the other hand, the

competition of Trichoderma strains in the soil could be optimized

optimally applying the biocontrol agent.

In summary, it would be desirable to have biological control agents that

provided an improved palliative effect against infection by

ND and D pathogens of the phytopathogenic fungus Verticilfium dahliae in

varieties of plants susceptible to being infected by it, such as

For example, the olive tree.

DESCRIPTION OF THE INVENTION

The present invention proposes the use of the Trichoderma atroviride IMI 206040 fungal strain for the prevention and / or treatment of plant and / or soil infections caused by phytopathogenic fungi belonging to the

Verticillium genus, more preferably due to the defoliant or non-defoliant patotypes of Verticillium dahliae, the causative agent of verticillosis.

The examples of the present invention demonstrate that Trichoderma atroviride IMI 206040 strain has an ability to inhibit the growth of the phytopathogenic fungus Verticilfium dahliae enhanced with

compared to other strains of the same genus, and this inhibition is observed

both in isolates of the pathogen with defoliant capacity and not

defoliant, and both sensitive and fungal resistant of the genus

Trichoderma (Figures 2 and 3). Therefore, the strain of fungi Trichoderma atroviride IMI 206040 has a high palliative and preventive value against infection of plants and / or soils caused by phytopathogenic fungi of the genus Verticillium, more preferably by Verticillium dahliae, being applicable both in nursery conditions and field

Therefore, one aspect of the invention relates to the use of the Trichoderma atroviride IMI 206040 fungal strain, and / or the products of its metabolism, for the prevention and / or treatment of infections caused by phytopathogenic fungi belonging to the genus

Poured / lium.

The strain Trichoderma atroviride IMI 206040 is known in the state of the

technique and therefore is accessible to an expert in the field. This strain is identifiable both for its morphological characteristics, which can be found described in databases for

Trichoderma such as for example, but not limited to, the collection on http://www.isth.info, as for certain molecular markers, such as, but not limited to, the sequence of internal spacers of the rRNA genes, ITS1 and ITS2.

As used in the present invention, the term "products of the

metabolism "or" metabolites "refers to any excreted molecule

by the fungal strain Trichoderma atroviride IMI 206040, preferably

when said strain is growing in the presence of medium of

PDA culture (Potato-Dextrose Agar). Examples of these metabolites are,

although not limited, amino acids, nucleotides, vitamins, acids

organic, alcohols, such as polyols, sugars, such as polysaccharides,

antibiotics, enzymes, such as, for example, but not limited to, cell wall hydrolytic enzymes, etc.

Once excreted, the metabolism products of the Trichoderma atroviride IMI 206040 strain can be isolated and applied alone.

or in combination with said strain, directly to the plant, both to the roots and the aerial part, to the plant material, including but not

limit ourselves seeds, leaves or fruits, or to the ground. When the products of

metabolism are administered in combination with the Trichoderma strain

atroviride IMI 206040, the mode of administration can be simultaneous or sequential. Or, the Trichoderma atroviride IMI 206040 strain can be

administered alone to both the roots and the aerial part of the plant, to the plant material, including but not limited to seeds, leaves or fruits, or

to the soil, so that it produces these metabolism products in

if you.

Trichoderma atroviride IMI 206040 strain, and / or its products

metabolism, can be administered to, for example but not limited to, a plant, both the roots and the aerial part, the plant material, including but not limited to seeds, leaves or fruits, or to the soil, in a

effective amount for the treatment and / or prevention of infections

caused by phytopathogenic fungi belonging to the genus Verticillium. In the sense used in this description, the term "effective amount" refers to the sufficient amount of the Trichoderma atroviride IMI 206040 strain, and that of the products of its

metabolism, which allows to obtain the desired results. Bliss

effective amount can be administered at one time or in several administrations. In terms of treatment and prevention, a "sufficient amount" is the amount sufficient to avoid, alleviate, improve, stabilize, reverse, delay or delay the effects of infections.

caused by phytopathogenic fungi belonging to the genus Verticilfium. Preferably, the effective amount to be administered of the Trichoderma atroviride IMI 206040 strain is between 10 'and 10' spores / ml of irrigation water, and more preferably 105 spores / mi (from 1.4 x 10 "forming units of colonies / g).

The mode of administration of the Trichoderma atroviride IMI 206040 strain, and / or the products of its metabolism, can be carried out, for example, but not limited to, by irrigation water, spraying, spraying, coating, spraying, impregnation, mixing with the substrate, immersion of the root of the plant in a solution comprising the strain

Trichoderma atroviride IMI 206040, or spores thereof, and / or the products of its metabolism.

"Phytopathogenic fungi" are those organisms belonging to the

Fungi kingdom capable of causing infections, and sometimes diseases, in plants through alterations in their metabolism, for example, but not limited to, the secretion of enzymes, toxins, phytoregulators and other substances and, in addition,

by absorbing nutrients from the plant for its own

increase. The phytopathogenic fungi of the present invention are

those belonging to the genus Verticillium.

The genus of fungi Verticillium belongs to the division or edge Ascomicota,

Plectosphaereflaceae family and includes various groups that comprise

plant parasites and saprophytes, insects, nematodes, eggs of

mollusks and other fungi, so the genus has a wide range of

taxonomic groups, containing about 51 species, among which are, but not limited to, Verticillium dahliae, Verticillium alboatrum, Verticillium nubilium, Verticillium nigrescens or Verticillium tricorpus. Species of this genus are the causes of diseases known as verticillosis.

The presence of phytopathogenic fungi of the genus Verticiflium in plants or soil can be detected by biology techniques

molecular known in the state of the art to carry out said

identification, for example, by amplifying, for example, but not limited to, polymerase chain reaction (PCR), real time polymerase chain reaction (RT-PCR) or O-PCR, a region of the genama of the Phytopathogenic fungus present in a biological sample isolated from the soil or plant and comparing the product of

amplification obtained with the gene sequences available in bases

of data such as, for example, but not limited to, GenBank, specific organisms of the genus Vertici / lium, to determine the type

of pathogen present in the sample. The presence of fungi

Phytopathogens of the genus Vertici / lium in plants or soil as well

could be carried out by immunohistochemical techniques, such as by

example, but not limited to, ELlSA; or by growing in vi / ro

of the organisms contained in the biological sample isolated from soil or

of the plant in specific selective culture media of pathogens

belonging to the genus Verticillium. In this sense, the simplest method to identify pathogens of this genus through cultivation in a selective medium is, for example, but not limited to, cultivation in

PDA medium (Potato-Dextrose Agar) at room temperature, where the colonies grow white and the color of the bottom of the plates is between colorless and yellowish. Some crops may have a low profile with

Rounded edges and forming curves. The species Verticillium alboatrum, Verticillium dahliae and Verticillium tricorpus have colonies

prostrate and of an orange-yellow color in PDA culture medium, the

Verticillium nubilium species forms clamidospores globies and Verticillium nigrescens forms abundant clamidospores.

In a preferred embodiment, the phytopathogenic fungus is Verticilfium dahliae.

The Verticillium dahliae species is characterized by the production of

conidiophors with fiálidas arranged in verticil and the formation of

numerous microsclerocios, resistance structures. The fungus can be found in the soil in the form of mycelium and conidia, or in the form of microsclerotia. Some cultures infected by this pathogen may be asymptomatic, but in general two types of syndromes are distinguished

derived from this infection that causes the disease called

"verticilosis": stroke and slow decay. The initial symptoms of the first syndrome consist of the loss of color of the leaves, later there is a rapid drying of shoots and branches that usually begins

from the tip and that can cause the death of the tree. The bark of the affected branches may be purple or purple and sometimes a brown color is also observed in the tissues of the motto. The

most typical symptoms of slow decay are necrosis and mummified inflorescences while the leaves are usually

they come off, except those at the end. In the context of this

invention, are included within the term "Verticillium dahliae" both its

defoliant pathotype (D) as its non-defoliant pathotype (ND). The patotype D

Verticillium dahliae is characterized by causing widespread fall

of the green leaves of infected branches, unlike the ND patotype. The diagnosis of infection by one or another duck type of Verticilfium

dahliae could be carried out using visualization techniques

phenotypic based on the symptomatic characteristic above

mentioned, or by molecular techniques of characterization of

both patotypes, such as, but not limited to, by PCR (Mercado-Blanco et al., 2002, Olivae: official magazine of the International Olive Council, 94: 41-46).

Examples of plants susceptible to infection by phytopathogenic fungi belonging to the genus Verticillium, and preferably by Verticillium dahliae, are, but are not limited to, artichoke, cotton, melon, tomato, potato or olive. Therefore, in another preferred embodiment, the plant affected by the phytopathogenic fungus is selected from the list comprising: artichoke, cotton, melon, tomato, potato or olive. In a more preferred embodiment, the plant is the olive tree. When Verticilfium dahliae infects the olive tree

it causes what is called "olive verticillosis or VO". The present invention relates both to olive varieties that are very susceptible to

Patotype D, as for example, but not limited to, Picualo Arbequina, as the varieties susceptible to the ND patotype.

The term "treatment", as understood in the present invention, refers to combating the effects caused as a result of a

infection caused by phytopathogenic fungi belonging to the genus Verticilfium, preferably by Verticillium dahliae, in a plant, in a crop or in a soil. This term includes:

(i) inhibit infection, that is, stop its development;

(ii) relieve the effects caused by the infection, that is, cause its regression; (jii) stabilize the effects caused by the infection.

The term "prevention", as understood in the present invention, is to prevent the occurrence of damage caused by infections.

caused by phytopathogenic fungi belonging to the genus Verticillium, preferably by Verticillium dahliae; that is, prevent it from

cause infection in a plant, in a crop or in a soil, in particular, when said plant, crop or soil is predisposed to infection but has not yet been diagnosed to have it.

"Infection" means the colonization of a host organism, by

example, but not limited to, a plant, by pathogens, which in the context of the present invention consist of fungi

phytopathogens belonging to the genus Verticillium, which invade the

organism and multiply in it, producing toxic substances. The colonizing organism is detrimental to normal functioning and

host survival, so the agent is rated as

pathogen or, in the context of the present invention, as a phytopathogen. The term "infection" includes both localized infections that affect a single area of the host organism, and generalized infections that affect the entire organism; and both symptomatic and asymptomatic infections. Likewise, such term also includes infections of fungal soils

phytopathogens belonging to the genus Verticillium, preferably by Verticillium dahliae.

The examples of the present invention demonstrate the efficacy, not only of the Trichoderma atroviride IMI 206040 strain and its products of

metabolism, but also of other strains of Trichoderma, specifically

of Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT

20756, Trichoderma harzianum PF1 and Trichoderma harzianum CECT

2413, and the products of their metabolism, to inhibit the

growth of phytopathogenic fungi belonging to the genus Verticillium,

such as, but not limited to, Verticillium dahliae. Thus,

S

Another preferred embodiment of this aspect of the invention relates to the use

of the fungus strain Trichoderma atroviride IMI 206040, and / or of the

products of your metabolism, in combination with at least one strain of

Trichoderma selected from the list comprising: Trichoderma

atroviride CECT 20755, Trichoderma harzianum CECT 20756,

10

Trichoderma harzianum PF1 or Trichoderma harzianum CECT 2413, for

the prevention and / or treatment of fungal infections

Phytopathogens belonging to the genus Verticillium.

The examples of the present invention also demonstrate the

fifteen

efficacy of the combination of the five strains of Trichoderma:

Trichoderma atroviride IMI 206040, Trichoderma atroviride CECT 20755,

Trichoderma harzianum CECT 2413, Trichoderma harzianum CECT

20756 and Trichoderma harzianum PF1, to inhibit the growth of

phytopathogenic fungi belonging to the genus Verticillium, as per

twenty

example, but not limited to, of Verticiflium dahliae. Therefore, a

most preferred embodiment refers to the use of the fungal strain

Trichoderma atroviride IMI 206040, and / or its products

metabolism, in combination with Trichoderma strains: Trichoderma

atroviride CECT 20755, Trichoderma harzianum CECT 2413, Trichoderma

25

harzianum CECT 20756 and Trichoderma harzianum PF1, for the

prevention and / or treatment of fungal infections

Phytopathogens belonging to the genus Verticiflium.

Trichoderma harzianum PF1, Trichoderma atroviride CECT strains

30

20755, Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT

2413 and Trichoderma harzianum CECT 20756 can be administered at a

plant, both the roots and the aerial part, the plant material,

including but not limited to seeds, leaves or fruits, or to the soil, for the treatment and / or prevention of infections caused by phytopathogenic fungi belonging to the genus Verticillium, preferably by Verticillium dahliae, simultaneously or sequentially. In addition, these strains can be administered in combination with elements necessary for their growth and development, such as, but not limited to, carbon sources, mineral salts and / or organic and inorganic nutrients.

In an even more preferred embodiment the Trichoderma strains

Trichoderma harzianum PF1, Trichoderma atroviride CECT 20755, Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 and / or Trichoderma harzianum CECT 20756 are in sporulated state.

"Sporulated state" means the stage of the fungus in which it

It is in the form of spore. The term "spore" refers to the cell

reproductive produced in fungal sporangia, either asexually or as a result of a process of sexual reproduction, usually haploid and unicellular, which at the same time allows the

dispersion and survival for a long time (dormancy) in adverse conditions. The spore produces a new organism by dividing by mitosis without fusion with another cell, producing a multicellular gametophyte. When

the spore germinates from it a first thread, by whose extension and

branching is becoming a mycelium.

The examples of the present invention demonstrate that when phytopathogenic fungi belonging to the genus Verticillium are inoculated, such

as for example, although without limiting ourselves, Verticilfium dahliae, in a culture medium where the Trichoderma atroviride IMI 206040 strain has previously grown and excreted all kinds of metabolites before being removed from said medium, there is inhibition of the growth of the lithopathogenic fungus.

They also demonstrate such inhibition when the pathogen is inoculated into

a culture medium where the strains of

Trichoderma Trichoderma harzianum PF1, Trichoderma atroviride CECT

20755, Trichoderma harzianum CECT 2413 and / or Trichoderma harzianum CECT 20756 and excreted all kinds of metabolites before being removed

of said medium. Thus, the inventors show that both the

Mycelium of the strain Trichoderma atroviride IMI 206040 And of these other strains of Trichoderma, such as metabolites excreted to the environment by

they are capable of inhibiting the growth of phytopathogenic fungi belonging to the genus Verticillium, such as, for example, but not limited to, Verticillium dahliae. Therefore, another preferred embodiment relates to the use of the Trichoderma atroviride IMI 206040 fungal strain,

and / or the products of your metabolism, in combination with products

of the metabolism of at least one strain of Trichoderma selected from

the list comprising: Trichoderma harzianum PF1, Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 2413 or Trichoderma harzianum CECT 20756, for prevention and / or treatment

of infections caused by phytopathogenic fungi belonging to

Verticiflium genus. A more preferred embodiment relates to the use of the Trichoderma atroviride IMI 206040 fungal strain, and / or the products

of its metabolism, in combination with metabolic products of Trichoderma strains: Trichoderma harzianum PF1, Trichoderma

atroviride CECT 20755, Trichoderma harzianum CECT 2413 and Trichoderma harzianum CECT 20756, for the prevention and / or treatment of infections caused by phytopathogenic fungi belonging to the

Verticillium genus.

Trichoderma harzianum PF1, Trichoderma harzianum CECT strains

2413, and the Trichoderma atroviride CECT 20755 and Trichoderma harzianum CECT 20756 mutants, are accessible to an expert in

matter. Trichoderma harzianum PF1 is a mutant strain derived from Trichoderma harzianum CECT 2413 induced with nitrosoguanidine,

characterized by its superexcretory capacity of hydrolytic enzymes

(glucanases, proteases and / or chitinases). This strain shares the

morphological characteristics of the strain from which it comes. Strain

Trichoderma harzianum CECT 2413 is identifiable both by its

morphological characteristics, which can be found described in

databases for Trichoderma such as, but not limited to, the collection at http://www.isth.info. as for certain molecular markers, such as, but not limited to, the sequence of the internal spacers of the rRNA, ITS 1 and ITS2 genes. The mutant derived from Trichoderma atroviride IMI 206040, Trichoderma atroviride CECT 20755 or RCU1, and the mutant derived from

Trichoderma harzianum CECT 2413, Trichoderma harzianum CECT

20756 or RCU2, both resistant to copper, share the morphological characteristics of the strains from which they originate.

Trichoderma atroviride CECT 20755 or RCUl and Trichoderma harzianum CECT 20756 or RCU2 strains are resistant to copper, metal used

as a fungicide mainly in the olive grove sector. Therefore, this

capacity allows the use of them in combination with others

antifungal agents in a method of treatment and / or prevention of

infections caused by phytopathogenic fungi belonging to

Verticillium genus. Therefore, another preferred embodiment relates to the use of

the strain of fungi Trichoderma atroviride IMI 206040, and / or of the

products of its metabolism, in combination with other antifungal agents, for the prevention and / or treatment of infections caused by phytopathogenic fungi belonging to the genus

Verticilfium When the Trichoderma atroviride IMI 206040 strain, and / or the

products of your metabolism, it is administered in combination with one of

The RCUl or RCU2 strains, or with both, can be used in

combination with other antifungal agents of chemical nature.

The term "antifungal agent" refers to those substances, both biological and chemical in nature, which are used to inhibit growth (fungistatics) or to kill (fungicides) fungi capable of causing infections in plants and / or soils, preferably to

Verticillium, more preferably Verticillium dahliae. The term refers to both protective or contact antifungal agents, which are applied before the infection occurs, as well as to eradicating or systematic antifungal agents, which are applied once the infection has occurred, as well as to antifungal agents used in coatings. of seeds, for use in soil disinfection, for application on plants, etc. An example of an antifungal agent of a biological nature is microorganisms that act as biological control agents of phytopathogenic fungi, such as, but not limited to, fungi of the genus Trichoderma other than those described in the present invention, fungi belonging to other genera or bacteria , and their mixtures, as long as these are not incompatible with the

Strains Trichoderma harzianum PF1, Trichoderma atroviride CECT 20755, Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 and Trichoderma harzianum CECT 20756. Some types of "agents

antifungals of a chemical nature "are, but are not limited to, those composed of copper chloride, copper oxychloride, cupric oxide,

"Bordeaux broth", S-copper quinolinolate, basic copper carbonate, copper naphthenate, copper sulfate, copper chromate or copper oleate.

On the other hand, fungi belonging to the genus Trichoderma with

They often act as fertilizing agents. For this reason, and why

In addition, the Trichoderma atroviride IMI 206040 strain has the capacity to

inhibit the growth of phytopathogenic fungi belonging to the genus

Verticilfium, which can compromise the viability and development of the plant to which they infect or the suitability of the crop soil, another application of the Trichoderma atroviride IMI 206040 strain and / or the products of its metabolism, is its use as a fertilizer , preferably in olive plants. A preferred embodiment of this aspect of the

The invention relates to the use of the Trichoderma atroviride IMI 206040 fungal strain, and / or the products of its metabolism, in combination with at least one Trichoderma strain selected from the list that

comprises: Trichoderma harzianum PF1, Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 2413 or Trichoderma harzianum CECT 20756, as fertilizer. A more preferred embodiment relates to the use of the Trichoderma atroviride IMI 206040 fungal strain, and / or of the

products of your metabolism, in combination with the strains of

Trichoderma: Trichoderma harzianum PF1, Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 2413 and Trichoderma harzianum CECT 20756, as fertilizer.

Another preferred embodiment of this aspect of the invention relates to the use

of the fungus strain Trichoderma atroviride IMI 206040, and / or of the

products of its metabolism, in combination with products of the metabolism of at least one strain of Trichoderma selected from the list comprising: Trichoderma harzianum PF1, Trichoderma afroviride

CECT 20755, Trichoderma harzianum CECT 2413 or Trichoderma harzianum CECT 20756, as fertilizer. A more preferred embodiment relates to the use of the Trichoderma atroviride IMI 206040 fungal strain,

and / or the products of your metabolism, in combination with products

of the metabolism of Trichoderma strains: Trichoderma harzianum PF1, Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 2413 and Trichoderma harzianum CECT 20756, as fertilizer.

The term "fertilizer" refers to any substance or mixture of chemical and / or biological substances, of natural or synthetic origin, used to supply the inorganic or organic elements to the soil in order for the plant to absorb them and thus favor their growth or vigor

The term "genus" refers to the category of biological classification (taxonomic category) that is located between the family and the species and that includes one or more morphologically similar species phylogenetically related. "Taxonomic category" means the level of hierarchy used for the classification of organisms.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Shows the results of the direct confrontation tests between the Trichoderma afroviride IMI 206040 strain, or a combination of Trichoderma atroviride IMI 206040 and Trichoderma harzianum CECT 2413, and different isolates of Verticillium dahliae. Petri dishes containing PDA at different incubation times (3 and 15 days) at 22 'e in which they are growing: (A) different isolates of V. dahliae (control), (B) different isolates of V. dahliae in one end and Trichoderma atroviride IMI 206040 at another end, (e) different isolates of V. dahliae at one end and a combination of the Trichoderma atroviride IMI 206040 and Trichoderma harzianum eEeT 2413 strains at another end.

Fig. 2. It shows the growth of different Verticillium dahliae isolates during the direct confrontation tests with the Trichoderma harzianum CECT 2413 (2413), Trichoderma viride CECT 2423 (2423) or Trichoderma atroviride IMI 206040 (IMI) strains, or combinations of the same, at 3 (white), 6 (gray) and 15 (black) days of incubation. (A) Isolated from V. dahliae 1A-2. (B) Isolated from V. dahliae 1A-3. (e) Isolated from V. dahliae 1A-4. (D) Isolated from V. dahliae 2A-12. (E) Isolated from V. dahliae 2A-16. (F) Isolated from V. dahliae 2A-17.

(G) Isolated from V. dahliae 4B-31. (H) Isolated from V. dahliae 4B-34. (1) Isolated from V. dahliae 4B-35.

Fig. 3. It shows the results of the direct confrontation tests between the strains of Triehoderma Triehoderma alroviride IMI 206040, Triehoderma alroviride RCU1, Triehoderma harzianum CECT 2413, Triehoderma harzianum RCU2 and / or Triehoderma harzianum PF1, and two dahlia isolates. (A) Petri dishes in which

Trichoderma atroviride IMI strains are growing at one end

206040, Triehoderma atroviride Re U1 or Triehoderma harzianum and Ee T 2413, and at the opposite end the isolates of Vertieillium dahliae 1 A-2 or 2A-12. (B) Petri dishes in which they are growing in a

Extreme Trichoderma harzianum RCU2, Trichoderma harzianum PFl strains or a combination of the five Trichoderma afroviride IMI strains

206040, Triehoderma atroviride Re U1, Triehoderma harzianum and ECT 241 3, Triehoderma harzianum Re U2 and Triehoderma harzianum PF1, formed by 1/5 of each (formulation), and at the opposite end the isolates of Vertici / lium dahliae 1 A- 2 or 2A-12. Control: Petri dishes in which only Vertieil / ium dahliae 1 A-2 or 2A-12 was inoculated. The

Images were taken after 18 days of growth.

Fig. 4. It shows the growth inhibition (%) in ten-day cultures of the different Verlieillium isolates (1A2, 1A3, 1A4, 2A12, 2A16, 2A17, 4B31, 4B34, 4B35) by the metabolites of T strains. harzianum CECT 2413 (2413), T. atroviride IMI 206040 (IMI) and T. viride CECT 2423 (VIRIDE) or by the melabolils of combinations of

These strains with each other.

Fig. 5. Shows the growth of two isolates of Verticillium dahliae, lA-2 (A) and 2A-12 (B), in media where the Trichoderma, Trichoderma atroviride IMI 206040 (T. atroviride), Trichoderma strains had previously grown atroviride RCU1, Trichoderma harzianum CECT 2413 (T. harzianum), Trichoderma harzianum RCU2 or

Trichoderma harzianum PF1, or a combination of these five strains formed by 1 / S of each (formulation). The figure shows the growth of two isolates of the pathogen in the presence of metabolites excreted by the different strains of Trichoderma. Control: growth of Verticillium dah / iae 1A · 2 (A) or 2A · 12 (B) in Petri dishes in which no strain of Trichoderma had previously grown.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the strain

Trichoderma atroviride IMI 206040 in the inhibition of the growth of representative isolates of the defoliant and non-defoliant pathogens of the phytopathogenic fungus Verticillium dah / iae. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1. In vitro antagonism assays of different strains of Trichoderma and isolates of Verticillium dahliae.

A set of in vi / ro antagonism trials of different strains of Trichoderma were carried out. or combinations of strains. in order to determine its antifungal capacity against Vertici / lium dahliae isolates supplied by the Institute of Sustainable Agriculture

(IAS-CSIC-Córdoba). Thus, two types of antagonism trials were carried out: one of direct confrontation and another of the ability to

growth of different isolates of Verticillium dahliae in a medium

where Trichoderma had previously grown and excreted all kinds of metabolites (including antibiotics and cell wall hydrolytic enzymes).

As will be explained later, the trials included direct confrontation in solid medium and cellophane tests to see the effect of

substances secreted by Trichoderma, following usual protocols

in the laboratory for tests with Rhizoctonia and Fusarium.

The Trichoderma strains used in the trials were: Trichoderma

harzianum CECT 2413, Trichoderma viride CECT 2423 and Trichoderma atroviride IMI 206040, as well as the following combinations thereof: T atroviride IMI 206040 + T viride CECT 2423, T harzianum CECT 2413 + T atroviride IMI 206040, YT harzianum CECT 2413 + T viride CECT 2423, and in combination with Verticiflium 1 A-2, 1A-3, 1 A-4 isolates, characterized as defoliant patotypes, and 2A-12, 2A-1 6, 2A-17.4B-31, 4B- 34, 4B-35, with non-defoliant capacity.

In addition, we proceeded to the development and characterization of mutants

copper-resistant spontaneous (4 mM Cu, SO,) derived from

Trichoderma harzianum and Trichoderma atroviride, in order to have strains resistant to metal contaminants used as antifungals, among which copper is widely used in the olive grove sector. Specifically, these mutant strains are those that are detailed below and that are currently deposited in the Spanish Type Culture Collection (CECT):

-Trichoderma atroviride RCU1 (deposit number in CECT 20755), derived from Trichoderma atroviride IMI 206040. -Trichoderma harzianum RCU2 (deposit number in CECT 20756), derived from Trichoderma harzianum CECT 2413.

In addition, the Trichoderma harzianum PF1 strain, a nitrosoguanidine-induced mutant derived from Trichoderma harzianum CECT 2413 with a high antifungal capacity and characterized as a superexcretor of

Hydrolytic enzymes (glucanases, proteases and chitinases).

Trials have also been carried out to verify the in vitro efficacy of a composition comprising five strains of Trichoderma:

Trichoderma atroviride IMI 206040, Trichoderma atroviride RCU1, Trichoderma harzianum CECT 2413, Trichoderma harzianum RCU2 and Trichoderma harzianum PF1.

In the experiments it was intended, therefore, to establish the ability to

different strains of the Trichoderma fungus to control isolates of Verticillium dahliae that cause verticillosis in the olive tree.

1.1. Direct confrontation tests of different strains of

Trichoderma and isolates of VertiÓ / lium dahliae.

The direct confrontation assays consisted of inoculating a portion of mycelium of the different isolates of V. dahliae to be tested at one end.

of Petri dishes containing nutritive culture medium POA (puree of

2% potatoes, 2% glucose, 2% agar). The boxes were incubated at 22 2C

for 4 days to allow the growth of the pathogen and subsequently injected at the opposite end of the boxes, 20 fll of spores of the

different strains of Trichoderma at a concentration of 105 spores / ml. The boxes continued to incubate at 22 2C for 18 days. As control boxes were inoculated with POA nutritive medium with the different isolates of

V. dahliae in which Trichoderma was not inoculated.

After a while, the fungi came into contact and could occur two

different situations: that both stop their growth in the area of

contact or that one of them caused a lysis of the mycelium of the other and

I ended up growing above it. In fact, the antagonistic effect

it was verified seeing the overgrowth of Trichoderma and the possible

death of the pathogen under the microscope and by seeding in selective medium for

the pathogen (growth in the presence of toxins in which Trichoderma

could not grow but to which the pathogen was resistant).

Figure 1 shows the results of the confrontations of the strain T. atroviride IMI 206040 (Fig. 1 B), or the combination of strains T. atroviride IMI 206040 and T. harzianum CECT 2413 (Fig. 1C), with different isolates of Verticiflium. Thus, it was observed that the T. atroviride IMI 206040 strain and the combination of T. atroviride IMI 206040 and T. harzianum CECT 2413 strains overgrown the pathogen, reducing the area

occupied by it.

To quantitatively analyze the results obtained, the growth area of the different strains of Verticillium was measured in the different trials of confrontation with Trichoderma. The results obtained are represented in Figure 2 A-I at different incubation times (3, 6, and 15 days). It was observed that in most cases the strains of

Trichoderma used overgrowth the pathogen, although in some cases the strains only managed to stop their growth (see the case of

T. harzianum CECT 2413 against V. dahliae 1A-3 and 1A-4). The most efficient strain was T. atroviride IMI 206040, capable of reducing the growth of the pathogen in 100% of cases, and only against the strain of Verticilfium 2A-17 a combination of T. atroviride IMI 206040 with T. viride CECT 2423 IMI antagonist capacity was improved individually (Fig. 2).

Therefore, the analysis of these results showed an inhibitory effect of

growth, both of defoliant and non-defoliant Verticillium isolates, mainly in the case of Trichoderma atroviride IMI

206040, alone or in combination with T. viride CECT 2423 and T. harzianum CECT 2413, and with a clear trend that became apparent after 15 days

of incubation, completely canceling the growth of the pathogen

Verticillium

Figure 3 shows the results of the confrontations of the T. atroviride IMI 206040, T. atroviride RCU1, T. harzianum CECT 2413, T. harzianum RCU2, T. harzianum PF1, or a combination of these five strains (formulation ), with the isolates of Verticillium 1 A-2, representative of the defoliant pathotype, or 2A-12, isolated more sensitive to strains of the genus Trichoderma representative of the non-defoliant pathotype. As can be seen, the composition of these five strains of

Trichoderma (formulation) was more effective against the two isolates of

Verticillium dahliae tested that most of the strains that compose it separately, except in the case of Trichoderma atroviride IMI 206040 And of its derived strain, RCU 1, which have a similar capacity.

1.2. Growth capacity tests of different isolates of

Verticillium dahliae in the presence of metabolites excreted by

Trichoderma

Pathogen growth capacity tests were performed in

presence of metabolites excreted by Trichoderma by culturing the Trichoderma strains on a cellophane disk permeable to extracellular metabolites (hydrolases, antibiotics, etc.) placed in turn on the PDA culture medium of a Petri dish, at 22 ' C. To the

four days of growth, the cellophane was removed and with it the mycelium

developed and a piece of mycelium of the pathogen was placed in the center of the boxes. To quantitatively analyze the results obtained in

this cellophane test, after ten days the growth area was measured

of the different isolates of V. dahliae and the diameter of

pathogen growth in control boxes with growth in boxes

where Trichoderma had grown on a cellophane and that previously

It had been removed before inoculating the pathogen.

The results of growth inhibition obtained for pathogen isolates grown in media containing are shown in Figure 4.

metabolites of strains T. harzianum CECT 2413, T. viride CECT 2423,

T. atroviride IMI 206040, or metabolites secreted by combinations of these strains. As can be seen in this figure, all the Verticil / ium isolates tested showed high metabolite sensitivity

produced by these three strains of Trichoderma, their growth being inhibited by about 90%. Although the differences between strains

they were not very high, it seems that the T. harzianum CECT 2413 / T combination. atroviride IMI 206040 has a slight increase in the ability to inhibit the growth of all isolates of the pathogen in general.

The results obtained with the metabolites of the Trichoderma atroviride IMI 206040, Trichoderma atroviride CECT 20755 or RCU1, Trichoderma harzianum CECT 20756 or RCU2, Trichoderma harzianum PF1 or Trichoderma harzianum CECT 2413 strains are represented in Figure 5, or with

the metabolites secreted by a combination of these five strains

(formulation), on the growth of an isolate of the pathogen sensitive to Trichoderma strains (2A-12, non-defoliant pathotype) or of a more resistant isolate (IA-2, defoliant pathotype), at different incubation times (days). In this sense, in tests to determine the capacity of

inhibit the growth of V. dahliae that have the metabolites excreted by these strains of Trichoderma, results similar to those observed were observed.

previous although different according to the strain of the pathogen. The formulation (combination of these five strains) was as effective as the strains that made it up the most sensitive isolate of the pathogen (2A-12, non-defoliant patholipo) but was intermediate against the most resistant isolate (1 A-2, defoliant pathotype ). The latter may be due to a dilution effect of

the metabolites involved in this interaction because in the formulation the strains were in a proportion 1J5 each.

In summary, these results determine the specific ability of the Trichoderma strains proposed in the present invention to control and

prevent the development of the Verticiflium dahliae pathogen in vilro.

Claims (8)

1. Use of Trichoderma atroviride IMI 206040 strain and / or the products of its metabolism for the prevention and / or treatment of infections caused by phytopathogenic fungi belonging to the Verticillium genus. 2. Use of the strain and / or the products of its metabolism according to the claim 1 wherein the phytopathogenic fungus is Verticillium dahliae 3. Use of the strain and / or the products of its metabolism according to any of claims 1 or 2 wherein the affected plant Phytopathogenic fungus is selected from the list comprising: artichoke, cotton, melon, tomato, potato or olive. 4. Use of the strain and / or the products of its metabolism according to the claim 3 where the plant is the olive tree. 5. Use of the strain and / or products of its metabolism according to any one of claims 1 to 4 in combination with at least one strain of Trichoderma selected from the list comprising: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma harzianum PF1 or Trichoderma harzianum CECT 2413. 6. Use of the strain and / or the products of its metabolism according to the Claim 5 in combination with the Trichoderma strains: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma harzianum PF1 and Trichoderma harzianum CECT 2413.

7.

Use of the strain and / or the products of its metabolism according

anyone

from the claims one to 6 where the strains from

Trichoderma are in a sporulated state.

5

8. Use of the strain and / or the products of its metabolism according

anyone

from the claims one to 7 in combination with

metabolism products of at least one strain of Trichoderma

selected from the list comprising:

Atroviride trichoderma

CECT 20755,

Trichoderma harzianum CECT 20756, Trichoderma

10

harzianum PF1 or Trichoderma harzianum CECT 2413.

9.

Use of the strain and / or the products of its metabolism according to the

claim 8 in combination with metabolic products of

the strains of

Trichoderma: Trichoderma atroviride CECT 20755,

fifteen

Trichoderma harzianum CECT 20756, Trichoderma harzianum PF1

and Trichoderma harzianum CECT 2413.