WO2012140304A2 - Souches de trichoderma utiles pour le traitement et/ou la prévention d'infection provoquées par des micro-organismes phytopathogènes - Google Patents

Souches de trichoderma utiles pour le traitement et/ou la prévention d'infection provoquées par des micro-organismes phytopathogènes Download PDF

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WO2012140304A2
WO2012140304A2 PCT/ES2012/070252 ES2012070252W WO2012140304A2 WO 2012140304 A2 WO2012140304 A2 WO 2012140304A2 ES 2012070252 W ES2012070252 W ES 2012070252W WO 2012140304 A2 WO2012140304 A2 WO 2012140304A2
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trichoderma
cect
strain
strains
products
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PCT/ES2012/070252
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Spanish (es)
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WO2012140304A3 (fr
Inventor
Juan Bautista BARROSO ALBARRACÍN
Alfonso CARRERAS EGAÑA
Raquel VALDERRAMA RODRÍGUEZ
Mounira CHAKI
Juan Carlos BEGARA MORALES
Jesús MERCADO BLANCO
Encarnación PÉREZ ARTÉS
Ana María RINCÓN ROMERO
Antonio CARBALLO CODÓN
Tahía BENÍTEZ FERNÁNDEZ
Antonio VALVERDE CORREDOR
Felipe GUEVARA PEZOA
María Jesús RODRÍGUEZ PALERO
Rafael DUEÑAS SÁNCHEZ
Jesús FIERRO RISCO
Antonio Alejandro LÓPEZ GARCÍA
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Universidad De Jaén
Nutesca, S. L.
Fundación Citoliva
Consejo Superior De Investigaciones Científicas
Universidad De Sevilla
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Priority claimed from ES201130606A external-priority patent/ES2391324B1/es
Priority claimed from ES201130609A external-priority patent/ES2393728B1/es
Priority claimed from ES201130608A external-priority patent/ES2395518B1/es
Priority claimed from ES201130605A external-priority patent/ES2390859B1/es
Priority claimed from ES201130607A external-priority patent/ES2393143B1/es
Application filed by Universidad De Jaén, Nutesca, S. L., Fundación Citoliva, Consejo Superior De Investigaciones Científicas, Universidad De Sevilla filed Critical Universidad De Jaén
Publication of WO2012140304A2 publication Critical patent/WO2012140304A2/fr
Publication of WO2012140304A3 publication Critical patent/WO2012140304A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38—Trichoderma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma

Definitions

  • the present invention falls within the field of agriculture, microbiology and phytosanitary products, and specifically refers to new fungal strains belonging to the Trichoderma atroviride and Trichoderma harzianum species resistant to copper capable of inhibiting the growth of other microorganisms, preferably fungi, phytopathogens.
  • the invention also relates to a composition comprising said strains, as well as the use of these strains and this composition as fertilizers and for the prevention and / or treatment of infections of plants and / or soils caused by microorganisms, preferably fungi, phytopathogens .
  • the present invention relates to the use of Trichoderma harzianum CECT 2413, Trichoderma harzianum PF1, Trichoderma atroviride IMI 206040 or any combination thereof, with each other or with the new previous strains, for the prevention and / or treatment of plant infections and / or soils caused by phytopathogenic fungi belonging to the genus Verticillium, more preferably by Verticillium dahliae, the causative agent of verticillosis.
  • Verticillosis or vascular wilt are diseases caused by the fungus Hyphomycete Verticillium dahliae Kleb. which can attack a wide range of crops, both woody and herbaceous, including fruit, 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.
  • Verticillium dahliae isolates that infect olive trees can be differentiated by their virulence on the plant and thus classified into defoliant (D) or non-defoliant (ND) patotypes by their ability or not to cause the generalized fall of the green leaves of infected branches respectively.
  • the VO can lead to the death of whole trees or the drying of branches causing a delay in growth and production losses.
  • verticillosis are diseases that are difficult to control, contributing to factors such as the pathogen's ability to survive long in the soil (more than 15 years), the wide range of plants susceptible to infection by it, the difficulty of to reach the fungus, since it is in the xylem of the plant and buried in the ground (between 10 and 30 cm deep), and the ineffectiveness of the chemical treatments so far explored to combat the fungus during its parasitic phase in the xylem
  • efficient control of VO requires the application of an integrated disease management strategy (López-Escudero and Mercado-Blanco, 201 1, Plant and Soil, 344, pp. 1 -50 ).
  • the control measures for fungal diseases of the olive tree have been in many cases indirect (elimination of diseased plants, use of potassium fertilizers, etc.).
  • indirect indirect (elimination of diseased plants, use of potassium fertilizers, etc.).
  • CBAs biological control agents
  • Biological control of phytopathogenic organisms is a more attractive alternative than that of chemical products, since these ACBs are less harmful to the environment than the commonly used chemical compounds.
  • the use of such ACBs has been limited by different causes, including inadequate identification of them.
  • Trichoderma filamentous fungus such as strains belonging to the Trichoderma harzianum species, have been used as biological control agents against plant pathogenic fungi, due to their protective capacity both against soil fungi that infect the roots and against which produce 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 fungi. Relations of mycoparasitism have been described between Trichoderma harzianum and Rhizoctonia solani, Sclerotium rolfsii or Pythium, where lithic enzymes play a primary role, and competitive relationships between Trichoderma harzianum and Botrytis cinerea (on vine) or Fusarium (on cotton). Thus, strains of Trichoderma harzianum, Trichoderma virens and Trichoderma viride (Hermosa et al., 2000, Applied and Environmental Microbiology, 66: 5, pp.
  • Trichoderma asperellum Trichoderma atroviride and Trichoderma inhamatum (ES2200602 A1 ) 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 collection 30595, 30596 and 30597) 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 (WO20071 10686 A2).
  • Trichoderma strains that provide an improved palliative effect against infection by microorganisms, preferably fungi, phytopathogens, specifically against the ND and D patotypes of Verticillium dahliae in plant varieties susceptible to being infected by it, such as the olive tree.
  • microorganisms preferably fungi, phytopathogens
  • these strains with activity as ACBs have a better capacity to adapt to the environment, so that they can successfully face the biological competence that takes place in the environment to be treated.
  • these strains allowed a combined treatment with fungicides of a chemical nature.
  • the present invention provides new fungal strains, belonging to the species Trichoderma atroviride and Trichoderma harzianum, capable of inhibiting the growth of other microorganisms, preferably fungi, phytopathogens. Therefore, its use is proposed as ACBs against them, preferably against Verticillium, more preferably against the D and ND patotypes of Verticillium dahliae, the agent causing verticillosis.
  • the present invention provides the use of other fungal strains such as Trichoderma harzianum PF1, Trichoderma atroviride IMI 206040 or Trichoderma harzianum CECT 2413 for the prevention and / or treatment of plant and / or soil infections caused by phytopathogenic fungi belonging to the genus Verticillium, more preferably by the D or ND patotypes of Verticillium dahliae, the causative agent of verticillosis.
  • other fungal strains such as Trichoderma harzianum PF1, Trichoderma atroviride IMI 206040 or Trichoderma harzianum CECT 2413 for the prevention and / or treatment of plant and / or soil infections caused by phytopathogenic fungi belonging to the genus Verticillium, more preferably by the D or ND patotypes of Verticillium dahliae, the causative agent of verticillosis.
  • the examples of the present invention demonstrate that these new strains, belonging to Trichoderma atroviride and Trichoderma harzianum, have an ability to inhibit the growth of the phytopathogenic fungus Verticillium dahliae improved with respect to other strains of the same genus.
  • this antifungal ability is observed in pathogen isolates, both sensitive and resistant, to fungi of the genus Trichoderma. Therefore, the strains described in the present invention have a high palliative value against infection of plants and / or soils caused by microorganisms, preferably fungi, phytopathogens, more preferably by Verticillium, even more preferably by Verticillium dahliae, being applicable both in nursery conditions like field.
  • the strains described in the present invention are resistant to copper, so they have the advantage that, compared to other strains of the same genus and species, it has a better ability to adapt to media characterized by the presence of metal contaminants .
  • metal pollutants are commonly used as antifungals and, among them, copper is widely used in the olive grove sector.
  • the copper resistance of these strains allows their use in combination with chemical antifungal agents, which results in better control and greater protection of the plant and / or soil against microorganisms, preferably fungi, phytopathogens compared to The simple chemical or biological treatment.
  • Trichoderma harzianum PF1 strain has an ability to inhibit the growth of the enhanced phytopathogenic fungus Verticillium dahliae with respect to the strain from which it originates, Trichoderma harzianum CECT 2413, in pathogen isolates both sensitive and resistant to fungi of the genus Trichoderma.
  • This capacity is due to its superexcretory activity of hydrolytic enzymes, such as, but not limited to, glucanases, proteases and / or chitinases.
  • the Trichoderma harzianum PF1 fungal strain 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 in countryside.
  • Trichoderma harzianum PF1 hydrolytic enzymes gives this strain the advantage that, compared to other strains of the same genus and species, it can better cope with the biological competence that takes place in the plant's root environment. try.
  • the examples of the present invention demonstrate that the Trichoderma atroviride IMI 206040 strain has an ability to inhibit the growth of the phytopathogenic fungus Verticillium dahliae improved with respect to other strains of the same genus, and this inhibition is observed both in isolated from pathogen D pathogen and ND pathotype, and both sensitive and fungal resistant of the genus Trichoderma. 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
  • the examples of the present invention demonstrate that the strain Trichoderma harzianum CECT 2413 is capable of inhibiting the growth of the phytopathogenic fungus Verticillium dahliae, and this inhibition is observed in both isolates of the pathogen of the D-type and the ND-pathogen, and both sensitive and resistant to fungi of the genus Trichoderma.
  • this strain has a preventive effect against the infection of plants by Verticillium dahliae improved with respect to other strains of the same genus.
  • the strain of fungi Trichoderma harzianum CECT 2413 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
  • one aspect of the invention relates to a Trichoderma strain selected from the strain of the Trichoderma atroviride species deposited in the Spanish Type Culture Collection (CECT) with deposit number 20755 or the strain of the Trichoderma species harzianum deposited in the Spanish Type Crops Collection (CECT) with deposit number 20756.
  • first strain of the invention Spanish Type Culture Collection (CECT) with deposit number 20755, hereafter "first strain of the invention” or “RCU1”, was deposited with the CECT on October 15, 2009. It is a spontaneous mutant (not genetically manipulated) resistant to copper, specifically, but not limited to 4 mM of CU2SO4, derived from Trichoderma atroviride IMI 206040.
  • This first strain of the invention shares its phenotypic characteristics with the strain from which it originates. It is able to grow in vitro in, for example, although without limiting the culture media CECT 54/68, at an incubation temperature of, for example, but not limited to 25 ° C, for example, but not limited to 5 days and in aerobic conditions.
  • This second strain of the invention shares its phenotypic characteristics with the strain from which it comes. It is capable of growing in vitro in, for example, but not limited to, CECT culture media 54/68, at an incubation temperature of, for example, but not limited to, 25 ° C, for example, but not limited to , 5 days and in aerobic conditions.
  • the strains of the invention are in a sporulated state.
  • "Sporulated state” means the stage of the fungus in which it is in the form of a spore.
  • the term “spore” refers to the reproductive cell produced in fungal sporangia, either asexually or as a result of a sexual reproduction process, usually haploid and unicellular, which at the same time allows 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, a first hypha arises from it, whose extension and branching constitutes a mycelium.
  • composition of the invention which comprises an element selected from the list:
  • the term "metabolism products” or “metabolites” refers to any molecule excreted by the strain or strains of the invention into the medium, preferably when said strains are growing in the presence of medium of PDA culture (Potato-Dextrose Agar).
  • PDA culture Paneto-Dextrose Agar
  • examples of these metabolites are, but are not limited to, amino acids, nucleotides, vitamins, organic acids, alcohols, such as polyols, sugars, such as polysaccharides, antibiotics, enzymes, such as, but not limited to, cell wall hydrolytic enzymes, etc.
  • the examples of the present invention demonstrate the efficacy, not only of the strains of the invention and its metabolism products, but also of other Trichoderma strains, in addition to Trichoderma atroviride CECT 20755 and Trichoderma harzianum CECT 20756, specifically Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 and Trichoderma harzianum PF1, and their metabolism products, to inhibit the growth of phytopathogenic microorganisms, such as, but not limited to, Verticillium dahliae.
  • the composition of the invention in addition to any of the strains of the invention comprises at least one strain of Trichoderma selected from the list: Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 or Trichoderma harzianum PF1.
  • the examples of the present invention demonstrate the efficacy of the composition of the invention comprising 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 microorganisms, such as, but not limited to, Verticillium dahliae. Therefore, in a more preferred embodiment, the composition of the invention comprises an element selected from the list: to. strains of the invention, that is, Trichoderma atroviride CECT 20755 and Trichoderma harzianum CECT 20756, preferably in a sporulated state,
  • the composition of the invention comprises: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 and Trichoderma harzianum PF1.
  • the proportion of each strain in the composition of the invention varies between 10% and 30% of the final volume of the composition (to total 100% with the five strains); more preferably the proportion of each strain in the composition of the invention is 20% of the final volume of said composition, that is, the five strains are found in the composition of the invention in the same proportion (1: 1: 1: eleven ).
  • Trichoderma strains Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 and / or Trichoderma harzianum PF1 are in a sporulated state in the composition of the invention.
  • composition of the invention further comprises products of the metabolism of at least one strain of Trichoderma selected from the list: Trichoderma harzianum CECT 20756, Trichoderma atroviride CECT 20755, Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413 or Trichoderma harzianum PF1.
  • Trichoderma atroviride IMI 206040, Trichoderma harzianum CECT 2413, and Trichoderma harzianum PF1 strains are accessible to one skilled in the art.
  • IMI 206040 are identifiable both for their 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, ITS1 e genes ITS2.
  • the trichoderma harzianum derivative CECT 2413, Trichoderma harzianum PF1, induced with nitrosoguanidine is 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.
  • the composition of the invention further comprises elements necessary for the growth and development of the strains of the invention and of the Trichoderma strains mentioned in the preceding paragraph.
  • said composition may comprise, but is not limited to, carbon sources, mineral salts and / or organic and inorganic nutrients.
  • composition of the invention may be presented in any form suitable for administration or application, for example, but not limited to, in solid or liquid form.
  • liquid presentation forms are suitable for spraying on the ground, the plant or the plant material, or for creating a solution in which the plants or plant material are submerged, so that preferably the composition of the invention is presented in liquid form
  • the examples of the present invention demonstrate that when phytopathogenic microorganisms are inoculated such as, for example, but not limited to, Verticillium dahliae, in a culture medium where at least one of the strains of the invention has previously grown and excreted all kinds of metabolites before if removed from said medium, there is inhibition of the growth of the phytopathogenic microorganism.
  • phytopathogenic microorganisms such as, for example, but not limited to, Verticillium dahliae
  • Trichoderma atroviride CECT 20755 Trichoderma harzianum CECT 20756
  • Trichoderma atroviride IMI 206040 Trichoderma harzianum CECT 2413
  • the inventors show that both the mycelium of the strains of the invention and of these other strains of Trichoderma, such as the metabolites excreted to the medium by them, are capable of inhibiting the growth of microorganisms, preferably fungi, phytopathogens such as for example, but not limited to, Verticillium dahliae. Therefore, another aspect of the invention relates to the use of the strains of the invention, preferably in a sporulated state, and / or of the products of its metabolism, or of the composition of the invention, for the prevention and / or treatment of infections caused by phytopathogenic microorganisms.
  • the metabolism products of the strains of the invention can be isolated and applied, alone or in combination with said strains, directly to the plant, both the roots and the aerial part, the plant material, including but not limited to seeds, leaves or fruits, or to the soil.
  • the mode of administration can be simultaneous or sequential.
  • the strains 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 they produce said metabolic products in situ. .
  • treatment refers to combating the effects caused as a result of an infection caused by microorganisms, preferably fungi, phytopathogens, more preferably by Verticillium, even more preferably by Verticillium dahliae, in a plant, in a crop or in a soil.
  • microorganisms preferably fungi, phytopathogens, more preferably by Verticillium, even more preferably by Verticillium dahliae, in a plant, in a crop or in a soil.
  • prevention is to prevent the occurrence of damage caused by infections caused by microorganisms, preferably fungi, phytopathogens, more preferably by Verticillium, even more preferably by Verticillium dahliae; that is, to prevent infection from occurring 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, for example, but not limited to, a plant, by pathogens, which in the context of the present invention consist of microorganisms, preferably fungi, phytopathogens, which invade the organism and they multiply in it, producing toxic substances.
  • the colonizing organism is detrimental to the normal functioning and survival of the host, whereby the agent is qualified as a 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, within this term, soil infestations caused by microorganisms, preferably fungi, phytopathogens are also included.
  • phytopathogenic microorganisms means those microorganisms belonging to the Fungi kingdom or to the Oomycetes class, capable of causing infections, and sometimes diseases, in plants.
  • Examples of phytopathogenic microorganisms belonging to the Oomycetes class are, but not limited to, those belonging to the genera Phytophthora, Plasmopara, Pseudoperonospora or Pythium.
  • Plant pathogenic 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 growth.
  • phytopathogenic fungi that cause diseases to grow and multiply in the xylem and / or phloem of the plant and, therefore, to block the transport of water and nutrients from the root to the leaves or the flow of sap from the leaves to the rest of the plant
  • Some examples of phytopathogenic fungi are, but not limited to, those belonging to the genera Armillaria, Botrytis, Chondrostereum, Colletotrichum, Cladosporium, Fusarium, Monilia, Nectria, Rhizoctonia, Rhizopus, Sclerotinia, Sclerotium or Verticillium.
  • the presence of phytopathogenic microorganisms in plants or in the soil can be detected by molecular biology techniques known in the state of the art to carry out said identification, such as, for example, by amplifying, for example, but not limited to, chain reaction. of polymerase (PCR), polymerase chain reaction in real time (RT-PCR) or Q-PCR, a region of the genome of the phytopathogenic microorganism present in a sample isolated from the soil or plant and comparing the amplification product obtained with the gene sequences available in databases such as, for example, but not limited to GenBank, organism specific to determine the type of pathogen present in the sample.
  • PCR polymerase
  • RT-PCR polymerase chain reaction in real time
  • Q-PCR Q-PCR
  • phytopathogenic microorganisms in plants or soil could also be carried out by immunohistochemical techniques, such as, but not limited to, ELISA; or by in vitro growth of the organisms contained in the isolated soil or plant sample in specific pathogen-specific culture media known in the state of the art.
  • strains belonging to the genus Trichoderma are capable of exerting growth inhibition not only on a single type of phytopathogenic microorganism but on a wide variety of them, such as strains belonging to the Trichoderma atroviride or Trichoderma harzianum species. Therefore, although the examples of the present invention demonstrate the effect of the mentioned strains and the composition of the invention on the inhibition of the growth of the phytopathogenic fungus Verticillium dahliae, said strains and composition are also useful in the prevention and / or treatment of infections caused by other microorganisms Phytopathogens that can be controlled by strains belonging to the Trichoderma atroviride or Trichoderma harzianum species.
  • the phytopathogenic microorganism belongs to a genus selected from the list comprising: Armillaria, Botrytis, Chondrostereum, Colletotrichum, Cladosporium, Fusarium, Monilia, Nectria, Phytophthora, Plasmopara, Pseudoperonospora, Pythium , Rhizoctonia, Rhizopus, Sclerotinia, Sclerotium or Verticillium.
  • the phytopathogenic microorganism belongs to the genus Verticillium.
  • the phytopathogenic microorganism is Verticillium dahliae.
  • the genus of fungi Verticillium belongs to the Ascomicota division or phylum, Plectosphaerellaceae family and includes various groups comprising parasites and saprophytes of plants, insects, nematodes, mollusc eggs 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 simplest method to identify pathogens of the genus Verticillium is, for example, but not limited to, cultivation in a selective medium, such as, but not limited to, in PDA medium (Potato-Dextrose Agar) at room temperature, where colonies grow white and the background color of the plates is between colorless and yellowish. Some crops may have a low profile with rounded edges and forming curves.
  • PDA medium Panato-Dextrose Agar
  • the species Verticillium alboatrum, Verticillium dahliae and Verticillium tricorpus have prostrate and orange-yellow colonies in PDA culture medium, the species Verticillium nubilium forms gloomy clamidospores and Verticillium nigrescens forms abundant clamidospores.
  • Verticillium dahliae species is characterized by the production of conidiophores with phyllides 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, although it is preferably found in form of microsclerocios.
  • Some cultures infected by this pathogen may be asymptomatic.
  • stroke and slow decay In the olive grove there are two types of syndromes derived from this infection that causes the disease called "verticillosis": stroke and slow decay.
  • the initial symptoms of the first syndrome consist of the loss of coloration of the leaves, later there is a rapid drying of buds 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 seen in the tissues of the xylem.
  • the most typical symptoms of slow decay are necrosis and mummification of the inflorescences while the leaves generally come off, except those at the end.
  • both its defoliant (D) and its non-defoliant (ND) patotype are included within Verticillium dahliae.
  • the Verticillium dahliae patotype D is characterized by causing, in olive groves and cotton, the generalized fall of the green leaves of infected branches, unlike the ND patotype.
  • the diagnosis of infection by one or another V is characterized by causing, in olive groves and cotton, the generalized fall of the green leaves of infected branches, unlike the ND patotype.
  • dahliae pathotype could be carried out by means of phenotypic visualization techniques based on the symptomatic characteristic mentioned above, or by molecular techniques of characterization of both patotypes, such as, but not limited to , by PCR (Mercado-Blanco et al., 2003, Plant Disease, 87, pp. 1487-1494).
  • Examples of plants susceptible to infection with Verticillium dahliae are, but not limited to, artichoke, cotton, melon, tomato, potato or olive. Therefore, in another preferred embodiment, the plant affected by the phytopathogenic microorganism 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 Verticillium 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, such as, but not limited to, Picual or Arbequina, as well as to varieties susceptible to ND patotype.
  • the strains of the invention are resistant to copper, a metal used as a fungicide mainly in the olive grove sector. Therefore, this ability of the strains of the invention allows their use, individually or together, in a method of treatment and / or prevention of infections caused by microorganisms, preferably fungi, phytopathogens in combination with other antifungal agents. Therefore, another preferred embodiment relates to the use of the strains of the invention, and / or of the products of its metabolism, or of the composition of the invention, in combination with other antifungal agents.
  • 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 to 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 antifungal agent of a biological nature is microorganisms that act as ACBs of phytopathogenic fungi, such as, but not limited to, fungi of the genus Trichoderma other than those included in the composition of the invention, fungi belonging to other genera or bacteria, and mixtures thereof, as long as these are not incompatible with the strains of the invention and with the fungi included in the composition of the invention.
  • Some types of antifungal agents that could be used in combination with the strain of the invention, and / or with the products of its metabolism, or with the composition of the invention are, but not limited to, those composed of copper chloride, oxychloride.
  • another aspect of the invention relates to the use of one of the strains of the invention or both, and / or of the products of its metabolism, or of the composition of the invention, as fertilizer.
  • a preferred embodiment of this aspect of the invention relates to the use of one of the strains of the invention or both, and / or of the products of its metabolism, or of the composition of the invention, as fertilizer in olive plants.
  • 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
  • Trichoderma strains mentioned herein, and / or the products of their metabolism, or the composition of the invention can be administered to a plant, soil or plant material, including but not limited to seeds, leaves or fruits, in combination with one or more antifungal agents and / or fertilizers simultaneously or sequentially.
  • the strains mentioned herein, and / or the products of their metabolism, as well as the composition of the invention 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 an amount effective for the treatment and / or prevention of infections caused by microorganisms, preferably fungi, phytopathogens and to cause fertilization.
  • the term "effective amount” refers to the sufficient amount of the strains, and / or the products of their metabolism, or of the composition of the invention, which allows to obtain the desired results. Said effective amount can be administered at one time or in several administrations.
  • a "sufficient amount” is the amount sufficient to avoid, alleviate, improve, stabilize, reverse, retard or delay the effects of infections caused by microorganisms, preferably fungi, plant pathogens, or to Stimulate their growth.
  • the effective amount to be administered from the strains in a sporulated state is between 10 4 and 10 6 spores / ml of irrigation water, and more preferably 10 5 spores / ml (from 1, 4 x 10 11 forming units of colonies / g).
  • the mode of administration of the strains, and / or the products of their metabolism, as well as of the composition of the invention can be carried out, for example, but not limited to, by irrigation, spraying, spraying, coating, fumigation water , impregnation, mixing with the substrate, immersion of the root of the plant in a solution comprising the strains, or spores thereof, and / or the products of their metabolism, or the composition of the invention.
  • 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.
  • Another aspect of the invention relates to the use of Trichoderma harzianum PF1 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.
  • a preferred embodiment of this aspect of the invention relates to said use in combination with at least one strain of Trichoderma selected from the list comprising: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 or Trichoderma harzianum CECT 2413.
  • said use is in combination with the Trichoderma Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 and Trichoderma harzianum CECT 2413 strains.
  • said Trichoderma strains are in a sporulated state.
  • Another preferred embodiment of this aspect of the invention relates to said use in combination with metabolism products of at least one strain of Trichoderma selected from the list comprising: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 or Trichoderma harzianum CECT 2413.
  • a more preferred embodiment refers to said use in combination with metabolic products of Trichoderma strains: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 and Trichoderma harzianum CECT 2413.
  • Another aspect of the invention relates to the 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.
  • a preferred embodiment of this aspect of the invention relates to said use 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.
  • said use is in combination with the Trichoderma Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma harzianum PF1 and Trichoderma harzianum CECT 2413 strains.
  • said Trichoderma strains are in sporulated state. .
  • Another preferred embodiment of this aspect of the invention relates to said use in combination with metabolic products of 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.
  • a more preferred embodiment refers to said use in combination with metabolism products of Trichoderma strains: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma harzianum PF1 and Trichoderma harzianum CECT 2413.
  • Trichoderma harzianum CECT 2413 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.
  • a preferred embodiment of this aspect of the invention relates to said use in combination with at least one strain of Trichoderma selected from the list comprising: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 or Trichoderma harzianum PF1.
  • said use is in combination with the strains of Trichoderma Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 and Trichoderma harzianum PF1.
  • said Trichoderma strains are in a sporulated state.
  • Another preferred embodiment of this aspect of the invention relates to said use in combination with metabolism products of at least one strain of Trichoderma selected from the list comprising: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 or Trichoderma harzianum PF1.
  • a more preferred embodiment relates to said use in combination with metabolic products of Trichoderma strains: Trichoderma atroviride CECT 20755, Trichoderma harzianum CECT 20756, Trichoderma atroviride IMI 206040 and Trichoderma harzianum PF1.
  • the phytopathogenic fungus is Verticillium dahliae.
  • the plant affected by the phytopathogenic fungus is selected from the list comprising: artichoke, cotton, melon, tomato, potato or olive. More preferably the plant is the olive tree.
  • Fig. 1 Shows the results of the direct confrontation tests between the Trichoderma strains and two isolates of Verticillium dahliae.
  • A Petri dishes in which the Trichoderma atroviride IMI 206040, Trichoderma atroviride RCU1 or Trichoderma harzianum CECT 2413 strains are growing at one end, and the Verticillium dahliae 1A-2 or 2A-12 isolates at the opposite end.
  • Control Petri dishes in which only Verticillium dahliae 1A-2 or 2A-12 was inoculated. The images were taken after 18 days of growth.
  • Fig. 2 It shows the growth of two isolates of Verticillium dahliae, 1A-2 (A) and 2A-12 (B), in media where the Trichoderma, Trichoderma atroviride IMI 206040 (7. atroviride), Trichoderma, Trichoderma strains had previously grown atroviride RCU1, Trichoderma harzianum CECT 2413 (7. harzianum), Trichoderma harzianum RCU2 or Trichoderma harzianum PF1, or a combination of the five strains formed by 1/5 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 dahliae 1A-2 (A) or 2A-12 (B) in Petri dishes in which no strain of Trichoderma had previously grown.
  • Fig. 3 Shows the results of the direct confrontation tests between the Trichoderma atroviride IMI 206040 strain, or a combination of Trichoderma atroviride IMI 206040 and Trichoderma harzianum CECT 2413, and various isolates of Verticillium dahliae.
  • Fig. 4 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.
  • IMI 206040 Trichoderma atroviride IMI 206040
  • Fig. 5 It shows the growth inhibition (%) in ten-day cultures of the different Verticillium isolates (1A2, 1A3, 1A4, 2A12, 2A16, 2A17, 4B31, 4B34, 4B35) by the metabolites of T strains.
  • harzianum CECT 2413 2413
  • IMI T. atroviride IMI 206040
  • VIRIDE T. viride CECT 2423
  • Fig. 6. It shows the results of the direct confrontation tests between the Trichoderma harzianum CECT 2413 strain, or a combination of Trichoderma harzianum CECT 2413 and Trichoderma atroviride IMI 206040, and different isolates of Verticillium dahliae.
  • Fig. 7 It shows the evolution of the total number of leaves over time in olive plants inoculated with V. dahliae (vert.) And previously treated with strains of T. harzianum CECT 2413 (T. Harz.), T. asperellum (T. Asp.) or mix both strains. Cont. + Vert .: Control, olive plants treated with V. dahliae but not with Trichoderma.
  • Fig. 8 It shows the analysis of the symptomatology of the disease by means of the scale of severity of damage over time in olive plants previously treated with strains of T. harzianum CECT 2413 (T. Harz.), T. asperellum ( T. Asp.) Or with the mixture of both strains, during treatment with V. dahliae (vert.). Cont. + Vert .: Control, olive plants treated with V. dahliae but not with Trichoderma.
  • two types of antagonism tests were carried out: one of direct confrontation and the other of growth capacity 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).
  • the trials included direct confrontation in solid medium and cellophane tests to see the effect of the substances secreted by Trichoderma, following usual protocols in the laboratory for Rhizoctonia and Fusarium tests.
  • -Trichoderma atroviride RCU1 (deposit number in CECT 20755), derived from Trichoderma atroviride IMI 206040, or first strain of the invention.
  • -Trichoderma harzianum RCU2 (deposit number in CECT 20756), derived from Trichoderma harzianum CECT 2413, or second strain of the invention.
  • Trichoderma harzianum CECT 2413 Trichoderma atroviride IMI 206040 and Trichoderma harzianum PF1.
  • This last strain is 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).
  • isolate 1A-2 representative of pathotype D
  • isolate 2A-12 more sensitive
  • the direct confrontation tests consisted of inoculating a portion of mycelium of the different isolates of V. dahliae to be tested (1A-2 or 2A-12) at one end of Petri dishes containing PDA nutritive culture medium (2% mashed potatoes , 2% glucose, 2% agar). The boxes were incubated at 22 ° C for 4 days to allow the growth of the pathogen and subsequently inoculated, at the opposite end of the boxes, 20 ⁇ of spores of the different Trichoderma strains at a concentration of 10 5 spores / ml. The boxes continued to incubate at 22 ° C for 18 days. As a control, boxes with PDA nutrient medium were inoculated with the different isolates of V. dahliae in which Trichoderma was not inoculated.
  • the fungi came into contact and there could be two different situations: that both stopped growing in the contact area or that one of them caused a lysis of the mycelium of the other and ended up growing above it.
  • the antagonistic effect was verified by seeing the overgrowth of Trichoderma and the possible death of the pathogen under a microscope and by seeding in a selective medium for the pathogen (growth in the presence of toxins in which Trichoderma could not grow but at which the pathogen was resistant ).
  • Figure 1 shows the results of the confrontations with the different Verticillium isolates. It was observed that the Trichoderma strains used overgrow the pathogen, reducing the area occupied by it. As can be seen in this figure, the composition of the five strains of Trichoderma (formulation) is more effective against the two isolates of Verticillium dahliae tested than most of the strains that compose it separately, except in the case of Trichoderma atroviride IMI 206040 and its derived strain, RCU1, which have a similar capacity.
  • two types of antagonism tests were carried out: one of direct confrontation and another of growth capacity 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 ).
  • the trials included direct confrontation in solid medium and cellophane tests to see the effect of the substances secreted by Trichoderma, following usual protocols in the laboratory for Rhizoctonia and Fusarium tests.
  • 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, and T harzianum CECT 2413 + T viride CECT 2423, and in combination with Verticillium isolates 1A-2, 1A-3, 1A-4, characterized as pathotype D, and 2A-12, 2A-16 , 2A-17,4B-31, 4B-34, 4B-35, of the ND patotype.
  • -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.
  • 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), was also included in the assays of the present invention.
  • Trichoderma atroviride IMI 206040 Trichoderma atroviride IMI 206040, Trichoderma atroviride RCU1, Trichoderma harzianum CECT 2413, Trichoderma harzianum RCU2 and Trichoderma harzianum PF1.
  • the direct confrontation assays consisted of inoculating a portion of mycelium of the different isolates of V. dahliae to be tested in one end of Petri dishes containing PDA nutritive culture medium (2% mashed potatoes, 2% glucose, 2% agar) .
  • PDA nutritive culture medium 2% mashed potatoes, 2% glucose, 2% agar
  • the boxes were incubated at 22 ° C for 4 days to allow the growth of the pathogen and subsequently inoculated, at the opposite end of the boxes, 20 ⁇ of spores of the different Trichoderma strains at a concentration of 10 5 spores / ml.
  • the boxes continued to incubate at 22 ° C for 18 days.
  • boxes with PDA nutrient medium were inoculated with the different isolates of V. dahliae in which Trichoderma was not inoculated.
  • the fungi came into contact and there could be two different situations: that both stopped growing in the contact area or that one of them caused a lysis of the mycelium of the other and ended up growing above it.
  • the antagonistic effect was verified by seeing the overgrowth of Trichoderma and the possible death of the pathogen under a microscope and by seeding in a 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 3 shows the results of the confrontations of the T. atroviride IMI 206040 strain (Fig. 3B), or of the combination of T. atroviride IMI 206040 and T harzianum CECT 2413 strains (Fig. 3C), with different isolates of Verticillium.
  • 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.
  • 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 4 A-1 at different incubation times (3, 6, and 15 days). It was observed that in most cases the Trichoderma strains used were overgrowth of 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 Verticillium 2A-17 a combination of T. atroviride IMI 206040 with T. viride CECT 2423 Improved IMI antagonistic capacity individually (Fig. 4).
  • Figure 1 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 strains, or a combination of these five strains (formulation), with Verticillium 1A-2 isolates, representative of the Patotype D, or 2A-12, isolated more sensitive to strains of the genus Trichoderma representative of the ND patotype.
  • composition of these five strains of Trichoderma was more effective against the two isolates of Verticillium dahliae tested than most of the strains that compose it separately, except in the case of Trichoderma atroviride IMI 206040 and of their derived strain, RCU1, which have a similar capacity.
  • EXAMPLE 3 In vitro antagonism assays of different strains of Trichoderma and isolates of Verticillium dahliae.
  • two types of antagonism tests were carried out: one of direct confrontation and the other of growth capacity 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).
  • the trials included direct confrontation in solid medium and cellophane tests to see the effect of the substances secreted by Trichoderma, following usual protocols in the laboratory for Rhizoctonia and Fusarium tests.
  • 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, and T harzianum CECT 2413 + T viride CECT 2423, and in combination with the isolates of Verticillium 1A-2, 1A-3, 1A-4, characterized as defoliant patotypes, and 2A-12, 2A-16, 2A-17,4B-31, 4B-34 and 4B-35, with non-defoliant capacity.
  • 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.
  • Trichoderma harzianum PF1 strain a nitrosoguanidine-induced mutant derived from Trichoderma harzianum CECT 2413 with a high capacity was also included in the assays of the present invention antifungal and characterized as a superexcretor of hydrolytic enzymes (glucanases, proteases and chitinases).
  • Trichoderma atroviride IMI 206040 Trichoderma atroviride IMI 206040, Trichoderma atroviride RCU1, Trichoderma harzianum CECT 2413, Trichoderma harzianum RCU2 and Trichoderma harzianum PF1.
  • the direct confrontation assays consisted of inoculating a portion of mycelium of the different isolates of V. dahliae to be tested in one end of Petri dishes containing PDA nutritive culture medium (2% mashed potatoes, 2% glucose, 2% agar) .
  • PDA nutritive culture medium 2% mashed potatoes, 2% glucose, 2% agar
  • the boxes were incubated at 22 ° C for 4 days to allow the growth of the pathogen and subsequently inoculated, at the opposite end of the boxes, 20 ⁇ of spores of the different Trichoderma strains at a concentration of 10 5 spores / ml.
  • the boxes continued to incubate at 22 ° C for 18 days.
  • boxes with PDA nutrient medium were inoculated with the different isolates of V. dahliae in which Trichoderma was not inoculated.
  • FIG. 6 shows the results of the confrontations of the Trichoderma harzianum CECT 2413 strain (Fig. 6B), or of the combination of T harzianum CECT 2413 and T atroviride IMI 206040 (Fig.
  • Figure 1 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 strains, or a combination of these five strains (formulation), with the isolates of Verticillium 1A-2, representative of patotype D, or 2A-12, isolated more sensitive to strains of the genus Trichoderma representative of the ND patotype. As can be seen, the composition of these five strains of Trichoderma (formulation) was more effective against the two isolates of Verticillium dahliae tested than most of the strains that compose it separately. 3.2.
  • EXAMPLE 4 In vitro trials of protection of olive plants against Verticillium dahliae infection by treatment with Trichoderma harzianum CECT 2413.
  • T harzianum CECT 2413 and T asperellum were used (the latter also called “olive strain” because it is native to the root of this plant), strains that have a high antagonism against the pathogen and also It was seen that they developed the greatest capacity to colonize the root of the olive tree among all the strains analyzed. The combination of both strains was also used.
  • Trichoderma preparations were made at a concentration of 10 5 spores strain / ml of irrigation water, and a volume of 35 ml per plant (determined previously, and that ensured a permanent contact without loss of the dissolution of Trichoderma with the substrate on which it is added) for a period of 32 days, and under experimental conditions based on a photoperiod of 12h / 12h, light intensity (fluorescent light) of 360 ⁇ m -2 - s -1 , temperature 22-24 ° C day / night and humidity 70-80%.
  • V. dahliae Plants inoculated with V. dahliae (VCG1A-3, pathotype D) (disease control), 6) Plants treated with Trichoderma harzianum CECT 2413 and inoculated with V. dahliae (VCG1A-3, patotype D) (biocontrol treatment of the disease),
  • V. dahliae Plants treated with Trichoderma asperellum and inoculated with V. dahliae (VCG1A-3, pathotype D) (biocontrol treatment of the disease), and
  • treatment n ° 1, n ° 2, n ° 3 and n ° 4 allowed the establishment of comparative effects among themselves, for example, possible stimulation of plant growth mediated by the Trichoderma strains (treatments n ° 2, n ° 3 and n ° 4) with respect to their absence (treatment n ° 1).
  • Treatment n ° 1 only received the manipulations that took place in the other treatments, for example, application of the same number of irrigations (volume, vehicle, etc.) that was performed for the Trichoderma treatments, as well as the manipulations that supported the plants of the rest of treatments (transplants, etc.).
  • Treatment No. 5 it was the reference for disease control and treatments No. 6, No. 7 and No. 8 were the problem treatments for disease biocontrol.
  • V. dahliae pathogen for the artificial inoculation of the V. dahliae pathogen in olive plants, conidia suspensions of the VCG1A-3 isolate (patotype D) obtained from cultures in dextrose potato broth (CPD) of 7 days of growth at 24 ° C, 125 were used. rpm in the dark These cultures were established from colonies of V. dahliae in PDA 2-3 weeks of growth at 24 ° C and darkness; three portions (-0.5 cm 2 ) taken from active growing areas of the colonies were deposited in flasks with 100 ml of CPD. The conidia suspension was obtained by filtration of the liquid cultures through sterile double gauze. The concentration of the inoculum used in the experiments was adjusted to 5 x 10 6 conidia-ml "1 by microscopic counting in Neubauer chamber.
  • Scalpel incisions were made in the four corners of the pots in order to break the root system to facilitate the penetration of the pathogen and avoid direct manipulation of the plant. Subsequently, the plants were inoculated by the direct addition to the soil of 35 ml of the conidia suspension. Finally, the pots were placed individually on Petri dishes in the culture chambers under controlled conditions of photoperiod, light intensity, temperature and humidity.
  • the evolution of the plants against the development of the disease was analyzed.
  • the total number of leaves was determined as an index of defoliation, both of the main stem and secondary, and a scale of severity of damage was applied in order to establish the symptomatology of the development of the disease.

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Abstract

L'invention concerne l'utilisation de souches de champignons Trichoderma pour prévenir et/ou traiter des infections dans des plantes et/ou des sols provoquées par des champignons phytopathogènes appartenant au genre Verticillium. En outre, l'invention concerne des nouvelles souches de champignons appartenant aux espèces Trichoderma harizanum et Trichoderma atroviride qui sont également résistantes au cuivre capables d'inhiber la croissance d'autres micro-organismes, de préférence, des champignons phytopathogènes. Ainsi, le mode de réalisation consiste à utiliser ces souches en tant qu'engrais et pour prévenir et/ou traiter des infections dans des plantes et/ou des sols provoquées par des micro-organismes phytopathogènes, de préférence, par des pathotypes défoliants ou non défoliants de Verticillium dahliae, un agent responsable de la verticilliose de l'olivier.
PCT/ES2012/070252 2011-04-15 2012-04-16 Souches de trichoderma utiles pour le traitement et/ou la prévention d'infection provoquées par des micro-organismes phytopathogènes WO2012140304A2 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
ESP201130609 2011-04-15
ESP201130606 2011-04-15
ESP201130605 2011-04-15
ES201130606A ES2391324B1 (es) 2011-04-15 2011-04-15 Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por microorganismos fitopatógenos
ES201130609A ES2393728B1 (es) 2011-04-15 2011-04-15 Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género verticillium
ESP201130608 2011-04-15
ES201130608A ES2395518B1 (es) 2011-04-15 2011-04-15 Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género verticillium.
ES201130605A ES2390859B1 (es) 2011-04-15 2011-04-15 Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por microorganismos fitopatógenos
ES201130607A ES2393143B1 (es) 2011-04-15 2011-04-15 Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género verticillium.
ESP201130607 2011-04-15

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WO2012140304A2 true WO2012140304A2 (fr) 2012-10-18
WO2012140304A3 WO2012140304A3 (fr) 2012-12-06

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EP3269828A1 (fr) * 2016-07-13 2018-01-17 EMPA Eidgenössische Materialprüfungs- und Forschungsanstalt Espèces supérieurs antagonistes de trichoderma capable de préserver du matériel végétal ou des produits à base de bois
CN108041079A (zh) * 2017-12-19 2018-05-18 北京科威拜沃生物技术有限公司 含哈茨木霉菌t-22和松脂酸铜的可湿性粉剂及其制备方法和应用
CN109716974A (zh) * 2018-12-10 2019-05-07 中国农业科学院棉花研究所 用于棉花黄萎病重病田的黄萎病防治方法
CN115232753A (zh) * 2022-06-06 2022-10-25 山东省科学院生物研究所 一种哈茨木霉zl-811、菌剂、其制备方法与应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3269828A1 (fr) * 2016-07-13 2018-01-17 EMPA Eidgenössische Materialprüfungs- und Forschungsanstalt Espèces supérieurs antagonistes de trichoderma capable de préserver du matériel végétal ou des produits à base de bois
WO2018011007A1 (fr) * 2016-07-13 2018-01-18 Empa Eidgenössische Materialprüfungs- Und Forschungsanstalt Espèces supérieures de trichoderma antagonistes pour la conservation de matière végétale et de produits du bois
CN108041079A (zh) * 2017-12-19 2018-05-18 北京科威拜沃生物技术有限公司 含哈茨木霉菌t-22和松脂酸铜的可湿性粉剂及其制备方法和应用
CN109716974A (zh) * 2018-12-10 2019-05-07 中国农业科学院棉花研究所 用于棉花黄萎病重病田的黄萎病防治方法
CN115232753A (zh) * 2022-06-06 2022-10-25 山东省科学院生物研究所 一种哈茨木霉zl-811、菌剂、其制备方法与应用
CN115232753B (zh) * 2022-06-06 2023-06-16 山东省科学院生物研究所 一种哈茨木霉zl-811、菌剂、其制备方法与应用

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