WO2006129998A1 - Improved strains of trichoderma as biocontrol agents, methods of obtaining same and use thereof for controlling diseases caused by phytopathogenic fungi - Google Patents

Abstract

The invention relates to a method of obtaining strains of Trichoderma that overproduce various lytic enzymes for use as antagonists of phytopathogenic fungi owing to the increased mycoparasitic activity thereof. According to the invention, a gene of Trichoderma virens that codes for a mitogen-activated kinase protein (MAP kinase) was cloned, said gene being related to the mycoparasitic response, conidiation and observed biocontrol of Trichoderma. Strains of Trichoderma with suppressed expression of the isolated MAP kinase gene demonstrated a clear increase in the expression level of mycoparasitism-related genes (MRGs) under simulated mycoparasitism conditions during direct confrontation with the plant pathogen Rhizoctonia solani. Biocontrol assays consistently demonstrate that the inventive strains are considerably more effective at controlling diseases than the wild strain or a chemical fungicide. In addition, the inventive strains sporulate abundantly in submerged cultures, which does not normally lead to sporulation in the wild strain.

Description

 Improved strains of Trichoderma as biocontrol agents, methods for obtaining them and their use for the control of diseases caused by phytopathogenic fungi.

Field of the invention.

The present invention relates to the development of modified microorganisms useful as biocontrol agents, specifically to obtain improved Trichoderma strains useful for the treatment of diseases caused by phytopathogenic fungi.

Background of the invention.

Agriculture is one of the most important commercial activities in what refers to the production of food or inputs, both for man and for livestock, so that the effective control and control of diseases that affect plants is of Vital importance.

Of all the diseases that affect the plants, the two most frequent are those that cause wilting and those in which the death of the same is caused, mainly caused by fungi such as Fusaríum oxysporum, Verticillium spp. and Rhizoctonia solani among others. The death of plants caused by the latter species of fungus is considered as one of the most harmful and important effects worldwide that affect agricultural production.

Due to the great losses caused by the diseases caused by these microorganisms, both chemical substances and pesticides have been used to date, as well as antagonistic microorganisms for their combat.

Currently, soil treatments are based on the use of pesticides to eliminate pathogenic fungi. Unfortunately, these compounds are often toxic to humans and animals and can potentially contaminate water deposits or rivers, thereby causing damage to the environment that are sometimes irreversible. Due to the above, some specific antagonistic microorganisms have been used, especially within the genus Pseudomona and Trichoderma as an alternative to combat the diseases mentioned above.

In the case of Trichoderma, the antagonistic effect that is triggered in response to the presence of a potential Trichoderma host, translates among other effects, in the production of antibiotics, the formation of specialized structures and the degradation of the host cell wall followed by the assimilation of its cellular content, a process called mycoparasitism. It has been proposed that this last phenomenon is the main mechanism responsible for the antagonistic activity presented by Trichoderma species to pathogenic fungi. In this sense, Trichoderma hydrolytic enzymes, such as chitinases, β-1-3 glucanases, β-1-6 glucanases, and proteases, facilitate host ¹ penetration.

Many Trichoderma species have been used as potent biocontrol agents against a variety of phytopathogenic fungi ² . For example, Trichoderma asperellum T34 (2) isolates obtained from composts prepared from the organic fraction of market waste, sewage sludge and garden waste ³ , improved strains such as T. viride Li 49 ⁴ , T. have been used. harzianum YC459 ⁵ , T-1-R9 ⁶ virid T, or mutants deficient in the production of viridiol but retaining its effectiveness as biocontrol agents, such as T. virens TV-111, TV-115 and TV-109 ⁷ .

Despite the previous developments, in practice it has been shown that it is very difficult to ensure that these antagonistic microorganisms are found in the substrate applied in sufficient quantities to achieve effective control of plant diseases caused by pathogenic fungi. As alternative solutions to this, different ways of obtaining Trichoderma spores are known in the technical field, for example by fermentative culture in bioreactors, subsequent lyophilization and filtration of the material obtained in filters that only allow the spores to pass, or by inoculation of wheat flour with Trichoderma species and subsequent dilution using diatomaceous earth. However, these methods are low productivity, mainly due to the low spore production characteristics of the species of

Tríchoderma used so far.

On the other hand, to try to eliminate the problem in obtaining low amounts of Tríchoderma, Trichoderma species have been obtained that can coexist in culture by transforming some of the strains by radiation of T. longibratum by cobalt ⁸ , biocontrol compositions comprising two or more Trichoderma strains of the species asperellum, atroviride and inhamatum ⁹ , or compositions with synergistic effects of chitin degradation containing Trichoderma strain proteins in conjunction with bacterial proteins, for example Streptomyces ¹⁰ . This is intended to obtain synergistic effects of biocontrol, as well as a greater amount of spores, however the methods of culture of these microorganisms do not allow, given the characteristics of antagonism of Trichoderma species, the production at the same time of two or more species together, and therefore no lytic enzymes are obtained, since these are only produced in the presence of other fungi.

Due to the above, several methodologies have been generated to obtain improved Trichoderma strains. In this sense, the observation that transformed strains of Trichoderma that overexpress lithic enzymes have shown to be better biocontrol agents than the corresponding parental strains ¹ has acquired particular relevance.

In addition to the isolation and selection methodologies of strains from natural substrates to obtain over-producing strains of lithic enzymes, classical genetic manipulation strategies have been used to achieve this objective. In this sense, in general Trichoderma induces the expression of proteins of interest, such as lytic enzymes that destroy the cell wall of pathogenic fungi under certain culture conditions, to subsequently isolate the genetic sequences encoding such proteins and use them to transform selected strains of Trichoderma through expression vectors. With this, it is possible to increase the expression capacity of the previously isolated gene, thus obtaining transformed Trichoderma strains with better production levels of the protein of interest compared to the parental strains. How Examples of this can be mentioned the increase in the expression of native extracellular enzymes of Trichoderma species in inducer or repressor media of such expression, in strains that have been previously transformed with at least one transgene under the control of an inducible promoter ¹⁰ , or either by T. viride regulatory genes such as the cellulase cbh1 gene, which allow a high expression of proteins of interest, especially cellulase ¹¹ .

However, these methods use genetic sequences from other organisms that are not part of the genome of the microorganism, so there may be problems associated with the non-recognition of such sequences, both regulatory and open reading frames, by the transformed microorganism due to multiple effects, including the characteristic use of organism codons.

On the other hand, it has been observed that Trichoderma's antagonistic effect is due to the joint expression of several lytic enzymes, encoded by several genes that have been designated as mycoparasitism-related genes (MRGs). Due to this effect, the genetic manipulation methods aimed at the isolation of lithic enzyme genes to subsequently obtain transformed strains of Trichoderma over-producing said enzymes, only achieve the increased expression of at least two isolated genes at the same time, with which obtaining improved strains of Trichoderma for use in the fight of plant pathogenic fungi, is quite limited.

This coupled with the fact that the problems involved in obtaining several suitable expression vectors that could coexist in the same transformed strain of Trichoderma, to allow and regulate the expression of several lithic enzyme genes without representing any problem for the strain of transformed Trichoderma, such as toxic effects.

For the reasons stated above, it is necessary to continue the development of methodologies that allow obtaining over-producing Trichoderma strains in all or in the great majority of lytic enzymes, to be used as efficient antagonistic organisms for phytopathogenic fungi. Until before the present invention, there were no methodologies that allowed obtaining strains of Trichoderma overproducers of several lytic enzymes using the suppression of the expression of genes coding for mitogen activated protein kinases (MAP kinases) in parental strains.

Brief description of the figures.

Figure 1. Southern blot analysis of the T. virens tvkl gene. The restriction enzymes used are shown in the upper part of the figure. The probe used was the BamHI fragment corresponding to 3.2 Kb and containing the entire open reading frame.

Figure 2. Tvkl gene sequence The possible start and end of the translation is shown. The donor and acceptor sequences of introns are indicated with underlined letters. Figure 3. Strategy for the interruption of the tvkl gene and the analysis of transformants. (A) Schematic representation of the replacement of Tvkl. The thick arrows represent the coding regions of tvkl and arg2. The lines represent regions 5 ¹ and 3 ¹ of the tvkl gene. Cross-linking events are indicated by dotted lines. EV (EcoRV); B (BamHI); S (Sa / I); C (C / al) and A (Apa \). (B). Type analysis

Southern of the mutants. Genomic DNA was digested with BamH. Lanes 2-5 represent four independent transformants; wild lane 1 (WT). The membrane was hybridized with the probe indicated in A. (C) Immunoblot analysis of the crude protein extracts of T. virens (strains WT and Δtvk24) using a polyclonal antibody specific for ERK1 / ERK2. The arrow indicates the signal corresponding to Tvkl p42

MAPK is a recombinant MAP kinase used as a positive control.

Figure 4. Conidiation in submerged culture. (A) Conidiation of the Δtvki mutants

(Δtvk24 and Δtvk133) in liquid medium. The strains were incubated in PDB for 72 hours. (B & C) Microscopic analysis of the mutant strain

Δtvk133.

Figure 5. Expression of MRGs in simulated mycoparasitism. (TO). Northern type analysis of the Tv-prbí, Tv-nag1, Tv-cht1 and Tv-bgn2 genes under carbon-limiting conditions with and without R. solani walls in The wild strain and the mutant Δtvk24. The numbers indicate the induction time. (B) Northern type analysis of Tv-prb1 under conditions of nitrogen limitation and simulated mycoparasitism. The symbols (+) and

(-) denote the presence and absence of R solani cell walls, respectively. Fifteen micrograms of total RNA were loaded in each lane. The 28S ribosomal gene was used as a control.

Figure 6. Expression of mycoparasitism-related genes (MRGs) in a confrontation with R solani. (A) Schematic representation of the interaction between T. virens and R. solani. T indicates the strain of T. virens growing alone in boxes with VMS, TR is the strain of T. virens growing in the presence of R solani, and the interaction zone is indicated by the color black. (B) Expression of MRGs in strains of T. virens. The samples were collected when the T. virens strains overgrown the colony of R solani. The probes used for the Northern type analysis were the same indicated in Figure 5. 15 μg of RNA were loaded in each lane.

Figure 7. Biocontroller activity of wild strains, Δtvk24 and Δtvk133. (TO). Disease index in the root system of cotton plants infected with R solani. (B) Percentage of surviving cotton seedlings challenged with P. ultimum. The columns that appear with

The same letter did not show significant differences according to the Fisher PLSD test at a significance level of 5%. (+) or (-) indicates

The presence or absence of the pathogen, respectively.

Figure 8. Effect of Tvk1 on the enzymatic activity produced in supernatants of wild T. virens (wt) and Δtvk24 under simulated mycoparasitism. Gel analysis of total protease activity under simulated conditions of mycoparasitism in the absence of a source of carbon (A), or nitrogen (B). The arrow indicates the activity of Prbl Total glucanase (C) and chitinase (D) activities were detected under simulated mycoparasitism in the absence of a carbon source.

Gel analysis of endocytinase activities under simulated mycoparasitism in the absence of a carbon source (E). The arrow indicates the presence of an additional endocytinase activity. Ia is indicated presence (+) or absence (-) of Rhizoctonia cell walls. All cultures were stored after 24 hrs.

Figure 9. Direct confrontation of Trichoderma with various phytopathogenic fungi as hosts. Phytophthora capsici (A), Sclerotium rolfsii (B), Rhizoctonia solani (C), Colletotrichum lindemuthianum (D) and

Phytophthora citricola (E) were confronted on papa dextrose agar plates against Tríchoderma virens Gv29.8 (left), Δtvk24 (center) and Δtvk133 (right).

Objectives of the invention.

There is a need in the field of agricultural biotechnology and molecular biology of fungi to generate improved strains as biological control agents that are more effective in said control and allow their use as an alternative or aid in the chemical control of plant diseases.

Therefore, the objectives of the invention presented here are:

- Provide strains of Tríchoderma overproducers of several lytic enzymes to be used as antagonists of phytopathogenic fungi,

- Provide strains of Tríchoderma overproducers of several lytic enzymes and at the same time with great capacity of spore production to be used as antagonists of phytopathogenic fungi,

- Provide a method to obtain Tripoderma strains overproducers of several lytic enzymes by suppressing the expression of genes coding for MAP kinases in parental strains of Tríchoderma, - Provide a method to increase the activity of lytic enzymes and mycoparasitic strains of Trichoderma by means of the suppression of the expression of genes coding for MAP kinases in parental strains of Trichoderma,

- Provide innovative Trichoderma genes coding for MAP kinases, related to the establishment of the parasitic relationship of Trichoderma and its hosts,

- Provide a genetic construct for the transformation of fungal cells, which include the polynucleotide sequences described in this invention, - Provide proteins with MAP kinase activity related to the establishment of the parasitic relationship of Trichoderma and its hosts,

- Provide a gene replacement vector useful for the suppression of the expression of genes coding for MAP kinases in parental Trichoderma strains, and - Provide a method of biocontrol of phytopathogenic fungi, using Trichoderma strains over-producing several lymphatic enzymes.

Detailed description of the invention.

/. Deposit of microorganisms.

The parental strain T. virens Tv10.4 has been deposited in the American Type Culture Collection on December 20, 2004 with the number PTA-6474. Likewise, the strain T. virens Δtvk24 was deposited in the American Type Culture Collection on December 20, 2004 with the number PTA-6474 and the strain T. virens Δtvk133 was deposited in the American Type Culture Collection on December 20, 2004 with the number PTA-6474.

//. Definition of the terms used in the invention.

For the purposes of the present invention, the units, prefixes and symbols may be mentioned using their abbreviation accepted by Sl. Unless explicitly indicated, the nucleic acid sequence is written from left to right in the 5 'to 3' orientation; The amino acid sequence is written from left to right in the orientation of the amino edge to the carboxy terminal edge. The numerical ranges are inclusive of the numbers that define the range and include each of the whole numbers that define the range. Amino acids can be designated either by their known three-letter symbols or by their single-letter symbols that have been recommended by Ia

Nomenclature Commission of the IUPAC-IUB. In the same way, nucleotides can be designated by their commonly accepted unique letter code. The terms of software, electrical and electronic are used as defined in the New IEEE Dictionary for Electrical and Electronic Terms ¹² .

Likewise, for the purposes of the present invention, the following terms will have the definition detailed below. By "amplification" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the sequence of a nucleic acid using at least one nucleic acid sequence as the initial reference frame. The amplification systems include the polymerase chain reaction (PCR), the ligase dependent chain reaction (LCR), the nucleic acid sequence dependent amplification (NASBA, Canteen, Mississauga , Ontario), the Q-Beta Replicase system, the Transcription-dependent Amplification System (TAS), and the Strand Displacement Amplification -SDA, for its acronym in English) ¹³ . The amplification product is called an amplicon.

The "antisense orientation" refers to a double stranded polynucleotide sequence that is functionally linked to a promoter in an orientation in which the antisense orientation of said molecule is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product so that the translation of the endogenous transcript product is inhibited.

A "chromosomal region" refers to the length of a chromosome that can be measured by reference to the linear segment of DNA that said region includes. The region can also be defined from two unique DNA sequences or molecular markers.

The term "conservatively modified variants" applies to both nucleotide sequences and amino acid sequences. Referring to specific nucleotide sequences, conservatively modified variants refers to those nucleic acids encoding identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any type of protein. For example, the codons GCA, GCC, GCG and GCU encode the amino acid alanine. These nucleic acid variations are silent variations and represent conservatively modified variants. By Likewise, any nucleic acid sequence encoding a polypeptide herein describes all possible silent variations of the nucleic acid. In the same way, any silent variation of a nucleic acid sequence encoding a polypeptide is implicitly included in each polypeptide sequence of this document.

As for amino acid sequences, it is recognized that individual substitutions, deletions or additions to a nucleic acid, a peptide, a polypeptide or a protein sequence that alters, adds or eliminates a single amino acid or a small percentage of amino acids in Ia The encoded sequence is also a "conservatively modified variant" in which the alteration results in the substitution of an amino acid with a biochemically similar amino acid. Therefore, any number of amino acid residues selected from the number of intakes ranging from 1 to 15 can be altered in the manner described above. For example, 1, 2, 3, 4, 5, 7 or 10 alterations can be created. Conservatively modified variants generally have a biological activity similar to that of the unmodified polypeptide sequence from which they were derived. For example, the specificity of the substrate, the enzymatic activity, or the binding properties between a receptor and its elicitor are generally from 30% to 90% of the activity of the native protein and its native substrate. Conservative substitution tables are widely known in the area.

The following 6 groups contain amino acids that represent conservative substitutions within each of the groups:

1) Alanine (A), Serine (S), Threonine (T).

2) Aspartic Acid (D), Glutamic Acid (E)

3) Aspargina (N), Glutamine (Q)

4) Arginine (R), Usina (K) 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V)

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

The term "encoded" or "encoded" in reference to a specific nucleic acid refers to the translation information of the corresponding protein. An acid Nucleic encoding a protein may include untranslated sequences (introns, for example) contained within translated regions of the nucleic acid, or it may not include such sequences (as in a cDNA, for example). The information of the encoded protein is specified in the use of codons. In general, the universal genetic code is used to determine the translation code of a nucleic acid sequence into an amino acid sequence. However, there are variants of the universal code in the genetic information contained within the mitochondria of some plants, animals or fungi, of the bacteroide organism Micoplasma capricolum, the ciliated organism Macronucleus can be included within the corresponding organism.

When the nucleic acid is generated or synthetically altered, the codon preferences of the host organism in which the nucleic acid is intended to be expressed can be exploited. For example, although the nucleotide sequences of the invention described herein can be expressed in Trichoderma species, the sequences can be modified taking into account codon preferences and GC content preference that may occur between species of the Trichoderma genus.

The term "full-length sequence" (of the English "full-length sequence") of a specific polynucleotide or its encoded protein refers to the complete sequence of the amino acid chain of a native (non-synthetic) endogenous protein and in its biologically active form. The methods used to determine if a sequence is full length are widely known and as an example we can mention "Northern" or "Western" type hybridizations, primer extension, or ribonuclease protection ¹⁴ . The comparison with homologous full length sequences (orthologs or paralogs) can also be used to identify the full length sequence of sequences included in the invention described herein. The consensus sequences generally present at the 3 'or 5' edge of the untranslated regions of a messenger RNA molecule (mRNA) help to identify the full length sequence of a polynucleotide. For example, the consensus sequence ANNNNAUGG ₁ in which the underlined codon represents the methionine present in the N-terminal edge helps to determine if the polynucleotide has a 5 'terminal edge full. Consensus sequences at the 3 'edge, such as polyadenylation sequences, help determine if the 3' terminal edge is complete.

The term "heterologous" refers here to a nucleic acid that derives from a different species or, if derived from the same species, a nucleic acid that is substantially modified in its native form. For example, a promoter that is operatively linked to a heterologous structural gene belongs to a different species from which the structural gene was originally obtained as long as it originates from a deliberate human intervention. In case of belonging to the same species, one to several heterologous genes must be substantially modified from their original form. A heterologous protein can originate from a different species, or from the same species as long as it originates from a deliberate human intervention.

The term "host cell" refers to a cell that contains a vector and that ensures the replication and / or expression of said vector. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. Preferably, the host cells are Trichoderma strains.

The term "hybridization complex" refers to a double stranded nucleic acid structure formed by two single stranded nucleic acid sequences hybridized together selectively.

The term "introduced" in reference to the act of inserting a nucleic acid into a cell, means "transfecting" or "transforming" and includes the incorporation of nucleic acids into a eukaryotic or prokaryotic cell in which the nucleic acid can be incorporated into the cell. genome of the cell (in the DNA of a chromosome, of a plasmid, a plastid or a mitochondrion), or it can be converted into an autonomous replicon, or expressed transiently.

The term "isolated" refers to a material (nucleic acid or a protein) that is: (1) substantially or completely free of the components that they usually accompany or interact with him in his natural form. The isolated material may optionally comprise another material that is not associated with the isolate in its natural form; or (2) in case the material is in its natural environment, if the material has been synthetically altered by a deliberate human intervention that modifies its composition or assigns it to a specific place in the cell (for example, an organelle) different from the place where it is in its natural environment. The alteration that gives rise to the synthetic form of the material can be directed to the material (nucleic acid and / or protein) or depend on a removal from the natural environment. For example, a natural nucleic acid can be isolated if it is altered or if it is transcribed from DNA that has been previously altered from a deliberate human intervention performed in the cell from which the nucleic acid originated ^{15 '16} . In the same way, a natural nucleic acid (for example, a promoter) is isolated if it is introduced by unnatural means into a locus of the genome that is not native. Nucleic acids that are "isolated" following this definition may be referred to as "heterologous" nucleic acids.

The term "nucleic acid" or "nucleotide" refers to a deoxyribonucleotide or ribonucleotide polymer in its single-stranded or double-stranded form and comprises those analogous molecules that have the essential nature of natural nucleotides to hybridize to stranded nucleic acids simple in a manner similar to that of natural nucleotides.

The term "nucleic acid library" refers to a collection of RNA or DNA molecules that substantially comprise and represent the integrity of the transcribed fraction of the genome of a specific organism. EXAMPLES constructions libraries, either genomic or cDNA libraries libraries are described conventional molecular biology references ^{17 '18' 19.}

The term "polynucleotide" refers to a deoxyribonucleotide, a ribopolinucleotide, or its analogues that have the essential properties of a natural ribonucleotide, such as the fact that they hybridize, under conditions of astringent hybridization, to essentially the same nucleotide sequences as nucleotides. that occur naturally, or that allow translation in the same amino acids as natural nucleotides. A polynucleotide can be full length or a sequence segment of a structural gene or a native or heterologous regulatory gene. Unless explicitly mentioned, the term includes the specified sequence as well as its complementary sequence. Therefore, DNA or RNA molecules that contain segments that have been modified to increase their stability or for other reasons are also "polynucleotides" for purposes of the present invention. Additionally, DNA or RNA molecules that include infrequent nucleotide bases, such as inosine, or modified nucleotide bases, such as tritilated bases to cite just two examples, would also be considered "polynucleotides" for purposes of the present invention. It follows from this paragraph that a wide variety of modifications have been made to DNA or RNA molecules, and that such modifications serve multiple purposes. The term polynucleotide is used herein to designate chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and simple or complex cells.

The term "polypeptide", "peptide", and "protein" are used interchangeably herein to designate an amino acid polymer. The terms refer to amino acid polymers in which one or more of the amino acids is an analogue of a corresponding natural amino acid. The essential nature of such analogs is that when incorporated into a protein, said protein can be specifically recognized by antibodies designed to recognize that same protein when it is composed exclusively of natural amino acids. The terms polypeptide "," peptide ", and" protein "also include, without limitation, glycolization, lipid binding, sulphation, carboxylation range of residues containing glutamic acid, hydroxylation and ribosylation of ADP. It is important mention that the polypeptides are not completely linear, for example, the polypeptides may be branched as a result of ubiquitination, they may be circular, with or without branching as a result of post-translational events such as natural processes and events caused by human manipulation that they do not occur naturally, circular and / or branched polypeptides can be synthesized by natural processes that do not depend on translation and by processes completely synthetic Additionally, this invention also includes terminal amino acid variants with or without methionine of each of the proteins related to the present invention.

The term "promoter" refers to a region of DNA located in the 5 'direction of the codon at the start of the transcription and which is involved in the recognition and binding of an RNA polymerase and other proteins necessary for the initiation of the transcription. A "promoter" is a sequence derived from another organism but capable of initiating transcription in heterologous cells. Some examples of promoters include those obtained from the DNA of plant viruses and bacteria that contain genes that are expressed in plants such as Agrobacterium or Rhizobium. Examples of promoters that are under control of developmental states include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots or seeds. These types of promoters are called "tissue-preferred". Those promoters that only initiate transcription in certain tissues are called "specific promoters". A specific "cell type" promoter is a promoter that only directs expression in certain types of cells located in one or more organs, such as, for example, the cells of the vasculature in leaves and roots. An "inducible" or "repressible" promoter is a promoter that responds to control signals that it emits from the environment. Some examples of environmental conditions that may have an effect on transcription dependent on inducible promoters are the presence of anaerobic conditions, or the effect of light. Tissue-specific promoters, tissue-preferred promoters, specific cell-type promoters and inducible promoters constitute the class of "non-constitutive" promoters. "Constitutive" promoters are those that direct expression systemically and under most of the environmental conditions.

The term "recombinant" refers to a cell or a vector that has been modified by the introduction of nucleic acid or that a cell is derived from another cell that was modified by said introduction. For example, recombinant cells express genes that are not found identically within the native (non-recombinant) form of the cell or that express native genes that would otherwise be under-expressed, expressed abnormally, or not express as a result of human intervention. The recombinant term does not include the alteration of the cell or of the vector by events that occur naturally (for example, spontaneous mutation, or transformation, transduction or transposition) like all those that occur without human intervention.

The term "expression cassette" refers to a nucleic acid construct (generated in recombinant or synthetic form) that contains a series of nucleic acid elements that allow transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, on a chromosome, in mitochondrial DNA, in plastidic DNA, in a virus, or in a nucleic acid fragment. Typically, the portion of the recombinant expression cassette of an expression vector includes, among other sequences, the nucleic acid to be transcribed and a promoter.

The term "residue" or "amino acid residue" is used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide or a peptide. The amino acid may be natural or may include synthetic amino acids analogous to natural amino acids that may function similarly to natural amino acids.

The term "selectively hybrid" refers to the hybridization (under astringent hybridization conditions) of a nucleic acid sequence to a specific white nucleic acid sequence that can be distinguished by detection of its hybridization to a non-white nucleic acid sequence , Which serves to substantially exclude non-white nucleic acids. Sequences that selectively hybridize typically have 90% shared sequence identity, and preferably 100% shared sequence identity.

The term "transgenic strain" or "transformant" or "transformed strain" refers to a well characterized isolate of the fungus that contains in its genome a polynucleotide introduced into the laboratory by transformation techniques known in the art. Generally said polynucleotide is stably integrated and is transmitted to the offspring of said fungus. The polynucleotide can be integrated into the genome in isolation or as part of a recombination vector. He The term "transgenic" includes here any cell or cell line, for which the genotype has been altered by the introduction of a nucleic acid, including those transgenic elements that were created by transformation. The term "vector" refers to a nucleic acid used for the transformation of a host cell in which a polynucleotide can be inserted. Vectors are often replicons. Expression vectors allow the transcription of a nucleic acid that has been inserted. The terms indicated below are used to describe the relationship between the sequences of two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison interval", (c) "identity of The sequence ", (d) percentage of identity of the sequence" and (e) "substantial identity".

(a) The term "reference sequence" is a defined sequence that is used as the basis for sequence comparison. A reference sequence may be a portion or a complete sequence; for example, a segment of a full-length cDNA, or the full length of the cDNA or an entire gene.

(b) The term "comparison interval" refers to a specific and contiguous segment of a polynucleotide sequence that can be compared to a reference sequence and for which the comparative portion of the polynucleotide sequence may include additional or missing elements (by example, "gaps") when compared with the reference sequence (which has no additional or missing elements) so that the alignment of the two sequences is optimal. In general, the comparison range is at least 20 contiguous nucleotides and can often include more than 100 nucleotides. Those skilled in the art who know how to perform this type of analysis understand that in order to avoid a high similarity with the reference sequence due to "gaps" in the polynucleotide sequence, penalty values for the "gap" can be used. Several sequence alignment methods are well known.

The optimal sequence alignment for comparison can be obtained from the local homology algorithm of Smith and Waterman ²⁰ , by the algorithm of Needlman and Wunsch ²¹ , by the similarity search method of Pearson and Lipman ²² , by the computerized implementation of These algorithms, including but not limited to: CLUSTAL in the program

PC / Gene from Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA and TFASTA in the package called Wisconsin Genetics Software, Genetics Computer Group (GCG), 575 Sciende Dr., Madison Wisconsin, USA; the CLUSTAL program is described in detail in Higgins and

Sharp ^{23 24} ' ²⁵ ' ²⁶ . The family of BLAST programs that can be used for comparative sequence searches includes: BLASTN for comparative search of nucleotide sequences compared to nucleotide sequences contained in public domain databases; BLASTP for comparative search of protein sequences compared to protein sequences contained in public domain databases; TBLASTN for finding homologies between a protein sequence and a nucleotide sequence; and TBLASTX for a comparison of a nucleotide sequence with a set of nucleotide sequence databases ²⁷ .

Unless explicitly stated otherwise, the identity or similarity values of a sequence indicated in this document refer to the values obtained with the BLAST 2.0 version using the "default" parameters ²⁸ . The software to perform these analyzes is in the public domain and can be accessed or obtained through the Center's website

National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The algorithm begins by identifying pairs of sequences with a high degree of similarity from the identification of short words with a length of W in the search sequence; said words must be identical or very similar to that of a threshold of value T when aligned with a word of the same length contained in a public domain database. This initial similarity between two sequences results in the beginning of a series of searches to find sequences of greater length and a high degree of similarity. AT is called the word neighborhood value threshold ²⁸ . The words found extend in both directions for each of the sequences as long as the cumulative alignment value continues to grow. For nucleotide sequences the cumulative values are calculated using the M parameters (prize value for a pair of matching waste; it will always be greater than

O) and (penalty value for mismatched waste; it will always be less than 0). For amino acid sequences, a matrix value is used to calculate the cumulative value. The extension of the words found stops in each of the two directions when: a) the cumulative alignment value falls by the amount X less than the maximum value obtained; b) the cumulative value becomes less than or equal to zero due to the accumulation of one or more alignment residues with negative value; or c) the end of each sequence is reached. Parameters W, T and X of the BLAST algorithm determine the sensitivity and speed of sequence alignment. The BLASTN program (for nucleotide sequences) uses as a "default" value a word length W of 11, an audience value E of 10, a cut-off threshold of 100, M = 5, N = -4, and makes the comparison of both strands of nucleotide sequences ²⁹ . In addition to calculating the percentage of sequence identity, the algorithm

BLAST also performs a statistical analysis of the similarity between 2 sequences ³⁰ . A measure of similarity provided by the BLAST algorithm is the minimum probability of sum (P (N)), which provides an indication of the probability with which the agreement between two nucleotide or amino acid sequences can occur at random.

BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of non-random sequences that may be enriched with one or more amino acids. These regions of low complexity can be aligned from unrelated proteins although other regions of said proteins are completely different and have no similarity. Some programs that function as low complexity filters can be used to reduce such low complexity alignments. For example, the SEG ³¹ and XNLJ ^{32 program} . This type of filters can be used individually or in combinations.

(c) The terms "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to residues in both sequences that are the same when they are aligned to look for the greatest correspondence within a window. comparative specific. When the percentage of the sequence identity is used in reference to proteins, it is recognized that the positions of residues that are not identical often differ by conserved amino acid substitutions, in which the amino acids are replaced by other amino acids that have similar biochemical properties. (load or hydrophobicity) and therefore does not change the functional properties of the molecule. If the sequences differ by the conservative nature of the substitution, the percentage of sequence identity can be adjusted upwards to correct the effect of the conservative nature of the substitution. It is said that those sequences that differ in conservative substitutions have "sequence similarity" or "similarity". The task of making such adjustments is routine for those with art skills. It generally involves assessing a conservative substitution as a partial and non-complete sequence discrepancy, thereby increasing the percentage of sequence identity. For example, when an identical amino acid is assigned a value of 1 and a non-conservative substitution is assigned a value of zero, a conservative substitution is assigned a value between zero and 1. The value of the substitutions is calculated using the Meyers and Miller ³³ algorithm, as implemented in the PC / GENE program (Intellegentics, Mountain View, California, USA).

(d) The term "percentage of identity sequence" refers to the value determined from the comparison of two optimally aligned sequences from a specific comparative window, and in which the portion of the polynucleotide sequence in the comparative window may include additions or deletions (ie absences) when compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of two sequences. The percentage is calculated by determining the number of positions from which the nucleotide base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing said number by the total number of positions in the comparative window and multiplying per 100 to obtain the percentage of identity sequence.

(e) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, preferably at least 80%, even more preferably 90% and ideally

95% sequence identity when compared to a reference sequence using one of the aforementioned alignment programs. It is recognized that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by 2 nucleotide sequences and taking into account codon degeneration or amino acid similarity. For this purpose the substantial identity of amino acid sequences normally means an identity of at least 60%, or preferably 70%, 80%, 90% and ideally 95%.

Another indication that nucleotide sequences are substantially identical is the fact that two molecules hybridize with one another under astringent hybridization conditions. However, nucleic acids that do not hybridize with each other under astringent conditions remain substantially identical if the polypeptides they encode are substantially identical. This can occur when, for example, a copy of the nucleic acid is created using the maximum degeneracy codon allowed by the genetic code. An indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded in the first nucleic acid is immunologically identical to the polypeptide encoded in the second nucleic acid.

The term "substantial identity" in reference to peptides indicates that a peptide comprises a sequence with at least 70%, preferably 80% or 85%, even more preferably 90%, and ideally 95% sequence identity with a reference sequence. in a specific comparative window. Optionally, the optimal alignment is performed using the alignment algorithm of Needleman and Wunsch ²¹ . An indication that two peptide sequences are substantially identical is that a peptide be immunologically identical to the second peptide. For example, two peptides will be substantially identical if they differ only by a conservative substitution. ///. Detailed description.

The present invention allows the obtaining of Trichoderma strains that overproduce several lytic enzymes to be used as antagonists of phytopathogenic fungi. The strains obtained are more efficient in the production of such enzymes and in the establishment of the mycoparasitic effect against phytopathogenic fungi. These characteristics allow the strains obtained to be used as antagonistic organisms for the effective control of plant diseases and used as a biocontrol mechanism. The Trichoderma strains of the invention retain the ability to detect and respond to different environmental conditions, including the presence of a potential host, essential factors for the successful colonization of soil, organic matter and the root system of plants.

The present invention takes advantage of the fact that the detection of such environmental conditions can occur through a series of transduction pathways, which determine the appropriate cellular response of Trichoderma. In this case, it is known that a wide variety of signals are transduced by the MAP kinase pathways, including those associated with pathogenesis.

The parasitism exerted by Trichoderma, resembles in many ways the interaction of a phytopathogenic fungus with its host. In this sense, MAP kinases have been directly implicated in several phenomena, such as:

a) Pathogenicity in Magnaporthe grísea (Pmk1, Pms1), Botrytis cinerea (Bmp1), Fusarium oxysporum (Fmk1), Cochliobolus heterostrophus (Cmk1) and

Ustilago maydis (Ubc3 / Kpp2) ³⁴ , b) In the expression of enzymes that degrade cell wall in several fungal systems, including phytopathogenic fungi, homologues of the MAP kinase Kss1 of S. cerevisiae. c) In the induction of the pectate lyase gene (pl1) of F. Oxysporum, since in null mutants of fmk1 this activity was abolished ³⁵ . d) In the positive control of the expression of those enzymes involved in the penetration of the plant, for example Bmp1 of B. cinérea ³⁶ , and e) In the regulation of the transcription of the prb1 gene that encodes a protease produced in response to nitrogen limitation of Trichoderma atroviride (species closely related to Trichoderma virens), which was blocked by the addition of a specific inhibitor of MAP kinases ³⁷ .

The present invention explores the relationship between the function of genes coding for MAP kinases with the mycoparasitic function observed in Trichoderma. Unexpectedly as the invention shows, the abolition in the expression of a gene coding for a MAP kinase, such as the tvk1 gene, causes a considerable increase in the synthesis of enzymes related to the observed mycoparasitic effect of Trichoderma strains, by less with a 10-fold increase in activity compared to the parental strain or with wild strains.

Due to the above, the mycoparasitic activity of the Trichoderma strains of the invention is considerably increased with respect to wild strains, whereby the strains of the invention can be used as efficient antagonistic organisms against multiple diseases caused by phytopathogenic fungi.

Likewise, with the results observed in the behavior of the enzyme-producing strains of the invention, it can be established that these MAP kinases are directly involved in the establishment of the parasitic relationship of Trichoderma and its hosts, with which the invention allows Obtain Trichoderma strains with greater capacity to control phytopathogenic fungi.

The Trichoderma strains of the invention show a clear increase in the level of expression of mycoparasitism-related genes (MRGs) under conditions of simulated mycoparasitism and during direct confrontation with plant pathogens, such as Rhizoctonia solani. Likewise, they also show an increase in protein secretion measured as the production of lithic enzymes in the culture supernatant of these strains, compared with that of the wild strain. Consistently in biocontrol trials, the strains are considerably more effective in the control of plant diseases than the wild strain or even chemical fungicides. Additionally, the strains of the invention sporulate abundantly in submerged cultures, a condition that normally does not lead to sporulation in the wild strain.

Unlike the one reported by Mukherjee, where mutations directed to the inactivation of the tmkA gene coding for a MAP kinase, cause in mutant strains of T. virens IMI304061 an important decrease in the colonization of pathogenic fungi (R. solaní), as well as a significant decrease in the antagonistic properties towards Sclerotium rolfsii in comparison with the wild strain ³⁸ , the methodology of the invention allows to obtain Trichoderma strains with increased activities in their mycoparasitism properties, as well as in the expression of multiple enzymes involved in the antagonistic effect.

On the other hand, according to Mukherjee, the abolition of the expression of the tmkA gene results in generating much less effective Trichoderma strains to be used as a potential biocontrol against pathogenic fungi and in apparent inactivation of genes that code for one or more enzymes responsible for degradation. of the host. In contrast to the above, the present invention demonstrates that the Tvk1 protein (MAP kinase) plays an important role in the regulation of the expression of the genes responsible for the production of lytic enzymes, with which it is possible to manipulate the expression of this protein in Trichoderma strains in order to improve the antagonistic properties associated with it.

While the present invention presents evidence of the increased expression of genes coding for lytic enzymes, as well as the effectiveness of the strains of the invention for effective combat in the field of plant diseases, the Mukherjee experimental data does not show such effects.

In contrast, the present invention shows that in the transformed strains of Tríchoderma, in which the expression of the tvk1 gene was abolished, the chitinase and protease activities were greater than in the wild, correlating with the high levels of the transcripts detected. In addition, the activity of certain enzymes is greatly increased in the strains of the invention. compared to the wild strain, for example in the total activity of β-1, 3-glucanase, further indicating that in the strains of the invention other glucanase genes could be under negative modulation by MAP kinases. Likewise, the increase obtained in the production of enzymes in the Trichoderma strains of the invention is also observed in a considerable increase in the secretion of proteins in liquid medium, seven times more when the strain grows without any source of nitrogen in the presence cell walls of the phytopathogenic fungus, or four times more when the strain grows without any carbon source in the presence of cell walls of the phytopathogenic fungus (Figure 8).

The results presented by Mukherjee, suggest that the deletion of the tmkA gene coding for a Trichoderma MAP kinase, reduces the efficiency of T. virens biocontrol based on tests of direct confrontation with phytopathogenic fungi, results that turn out to be completely opposite to what has been shown. in the present invention. This apparent contradiction can be explained by the fact that the experiments performed by Mukherjee were carried out in the middle of papa dextrose agar, where the expression of most of the MGR genes is repressed due to the high levels of glucose present in the medium. ⁴³ This statement is verified with the results obtained with the transformed Trichoderma strains of the invention in similar experiments using the papa dextrose agar medium with several phytopathogenic fungi, including Sclerotium rolfsii and R. solani. The results obtained indicate that by varying the environment of the strains of the invention compared to the parental strain, they may have a decrease in the capacity for growth inhibition depending on the host phytopathogenic strain (Figure 9).

All Trichoderma strains of the invention show reduced pigmentation with loss of the characteristic dark green color observed in the wild; the strains produce spores with reduced pigmentation in solid medium, without becoming albines. In contrast, all strains of the invention produce abundant conidia that show the characteristic dark green color of the wild in liquid cultures regardless of the medium used. These results suggest that MAP kinases could differentially regulate melanin biosynthesis as previously reported for C. lagenarium ³⁹ . Considering the increase observed in the production of lytic enzymes in the Trichoderma strains of the invention and the importance of these enzymes in the biocontrolling activity of this microorganism, the strains of the invention can be used as more effective biocontrol agents.

The strains of the invention show a greater capacity than the wild strain to control and reduce the damage caused by R solani and P. ultimum. T. virens has been used successfully in combination with several fungicides including metalaxyl (Apron FL) ⁴⁰ . Surprisingly, the strains of the invention were more effective than metalaxyl against P. ultimum (Fig. 7B). This increased biocontrol capacity seems to be associated with the expression of lytic enzymes observed in direct confrontation trials since both strains formed hooks and produced curls around R. solani and produced antibiotics at similar levels. The present invention shows that the deletion of a MAP kinase gene generates a more aggressive parasite and, consequently, a better biocontrol agent.

Within the scope of the invention, any strain of Trichoderma that allows obtaining by means of the teachings described herein, Trichoderma strains overproducing lytic enzymes can be used; likewise, any species of the genus Trichoderma can be occupied, although T. virens is even more preferred. With the purpose of protecting or treating plants or plant materials against infections or diseases caused by phytopathogenic fungi, the strains of the invention can be administered directly or through compatible compositions at the agronomic level, either in the soil, in the plant or in the material of the plant to be protected or treated. The compositions can be made by widely known methods and can be made in any form of presentation, either in liquid or solid forms. The liquid forms are capable of being applied by aerosols in the soil or in the plant, or used for the elaboration of baths in which the plants or their material are submerged. The compositions containing the Trichoderma strains of the invention can be applied by conventional methods, either by aerosols or by immersion. The present invention describes the isolation and characterization of the tvk1 gene of

Tríchoderma virens, corresponding to SEQ ID NO: 2, which encodes the Tvk1 protein, a mitogen activated protein kinase (MAP kinase) corresponding to SEQ ID NO: 3, which has an important role in several aspects of the life cycle of Tríchoderma, including growth, conidiation, expression of MRGs, secretion of enzymes that degrade cell wall and a biocontroller activity.

As shown in Figure 2, the tvk1 gene contains three intron sequences that have been reported for other fungal MAP kinase genes. The intron located between nucleotide +854 and +907 is not present in its closest recently reported homologue, tmkA of strain IMI306092 of T. virens. Tvk1 belongs to the family of kinases regulated by external signals (ERK), which is part of the superfamily of MAP kinases. The signature sequence present in the Tvk1 protein indicates that it is related to the YERK1 family (ERK1 of yeasts and fungi) ³⁸ .

The invention also comprises modified sequences of the DNA sequence shown in SEQ ID NO: 2, which code for amino acid sequences that retain the activity of the Tvk1 protein. Following the teachings of the present invention, a person versed in the technical field could easily predict the existence of such modified sequences and could easily produce them. Likewise, the invention comprises modified amino acid sequences derived from the sequence of the Tvk1 protein shown in SEQ ID NO: 3, which preserve and maintain the characteristics of the Tvk1 protein, mainly the enzymatic activity of MAP kinase as well as the ability to negative regulation in the expression of Trigoderma MRGs genes.

As described herein, the suppression of the expression of MAP kinase genes coding for Tríchoderma strains, allows to increase the capacity of the Tríchoderma strains of the invention to control other fungi that attack plant crops, coupled with the fact that due to this suppression effect, the expression levels of lytic enzymes are presented at high levels in most of the selected genes that encode lytic enzymes (MRGs) in comparison with wild strains. In order to achieve the above, the inhibition of the function of a MAP kinase by specific antagonistic substances, or by suppression of the expression of the gene coding for it can be used. The latter can be obtained by directed inhibition of the gene expression using specific probes, by interfering RNA, antisense RNA expression, by insertion of foreign polynucleotide sequences in the coding sequence of the MAP kinase gene or in its regulatory regions, by replacement of the wild gene by a mutated version, or, by eliminating the gene of interest from the genome of the organism through homologous gene recombination techniques. As the invention shows, this suppression of the expression of genes coding for MAP kinases can be achieved using the gene replacement vector of the invention, which comprises:

a) A replacement gene that allows the identification of the transformants where the coding anoint for a Trichoderma MAP kinase is replaced, b) A non-coding fragment that is naturally outside the coding region of the gene for MAP kinase at its 5 'end, flanking the 5 'end of the replacement gene, and. c) A non-coding fragment that is naturally outside the coding region of the gene for MAP kinase at its 3 'end, flanking the 3' end of the replacement gene.

For purposes of the invention, the replacement gene may or may not come from some Trichoderma strain, although those genes that come from Trichoderma strains of the same species of the strain to be transformed are preferred. As a replacement gene, any gene that allows the partial or total elimination of the gene coding for MAP kinase or that allows obtaining the interruption of the open reading frame of said gene can be used, with the consequent suppression of the expression of the protein encoded by the same and that also allows the identification of the modified strains. With the foregoing, the use of the vector of the invention avoids the use of sequences from other organisms that may cause undesirable or unexpected effects in the abolition of the expression of the gene coding for MAP kinase. On the other hand, the MAP kinase gene fragments that flank the replacement gene are those fragments of any size that are found either before or after the beginning of the gene's open reading frame as well as those that are found before or after the end of the open reading frame of the gene, as long as they come from the wild gene or from a gene with high similarity, this with the purpose of producing specific genetic recombination events between the genome of the Tríchoderma strain to be transformed in the region of the MAP wild kinase gene and the polynucleotide replacement sequence contained in the gene replacement vector.

Preferably the MAP kinase gene fragments that flank the replacement gene, come from the tvk1 gene and are:

- For the 5 'end, the smaller BamHI / EcoRV fragments shown in Figure 3A, although more preferably those fragments comprising nucleotide sequence no. 1 to nucleotide no. 1210 of SEQ ID NO: 1, and

- For the 3 'end, the Sall / Sall fragments shown in Figure 3A, although more preferably those fragments comprising nucleotide sequence no. 2692 to nucleotide 5892 of SEQ ID NO:!

Likewise, the function of the replacement gene can restore some deficiency in the strain of Tríchoderma to be transformed given by auxotrophy or allow the selection of transformants by their tolerance to a chemical compound such as in the case of the use of antibiotics or herbicides or by confer the ability to use alternative sources of carbon. and / or nitrogen, or because it can be identified through an enzymatic activity encoded by it. For example, if the Tríchoderma strain to be transformed is auxotrophic, the replacement gene should complement said auxotrophy. In this case, it is preferred to use the Tríderderma arg2 gene and auxotrophic strains to arginine. In the case of resistance to chemical compounds, the gene that confers hygromycin resistance (hph or hpt) of Escherichia coli, or Basta herbicide resistance genes can be used. In the case of the use of alternative sources of carbon or nitrogen, the amdS gene of Aspergillus nidulans can be used. To obtain the gene replacement vectors useful for obtaining the strains of the invention, vectors can be used to clone and introduce by transformation the genes of interest that will later be useful for integrating the replacement genes into the genome of the strains of Trichoderma through efficient recombination. It is important to note that the replacement vectors of the invention must contain characteristics that allow them to be amplified by means of conventional techniques (replication sites, promoters, etc.) and endowed with unique cloning sites, both for the insertion of the genes replacement as for the subsequent linearization necessary to transform the Trichoderma strains. In this sense, those vectors that allow the insertion of DNA fragments and that can be efficiently amplified in biological systems can be used. Likewise, in a preferred embodiment of the invention, the vectors of the invention use as a selection marker gene a replacement gene of the Trichoderma tvk1 gene with some associated function that can be identifiable, for example the synthesis of some protein that allows the growth of auxotrophic strains of Trichoderma in the appropriate culture medium (deficient in the essential nutrient) after its transformation with the vector. Likewise, the replacement gene preferably comes from Trichoderma strains.

Using the gene replacement vector described above, the Trichoderma strains of the invention can be obtained by means of the methodology comprising the following steps:

a) Suppress the ability of Trichoderma to produce at least one MAP kinase, by transforming a Trichoderma strain with the gene replacement vector of the invention, where the strain is auxotrophic to the nutrient for which the replacement gene contained in the vector confers prototrophy, b) Select the prototrophic transformants generated in selective medium, and c) Corroborate the elimination of the production of MAP kinase.

As with the replacement vector of the invention, in order to obtain the strains of the invention in this method it is preferred to suppress the expression of the gene tvk1, coding for the Tvk1 MAP kinase, use Trichoderma's arg2 gene as a replacement gene and use arginine auxotrophic strains.

For the transformation of the Trichoderma strain, it is preferable that the replacement vector is provided in a linear manner, this in order to provide the appropriate conditions for the genetic recombination process.

The selection of the transforming strains can be carried out with any selective means that allows the selection of the resulting prototrophic transformants, in direct relation with the function of the replacement gene; although Vogel minimum medium (VMS) containing sucrose is the only carbon source.

To corroborate in the selected transformants the elimination of the synthesis of the MAP kinase, this can be carried out by means of Western blot assays using specific antibodies for the protein and the elimination of the coding MAP kinase gene by means of Southern blot assays, using specific probes, is corroborated. .

On the other hand, by means of the methodology for obtaining the strains of the invention, the enzymatic activity related to the mycoparasitic activity of Trichoderma can be increased in a directed manner, generating Trichoderma strains with improved enzymatic characteristics that can be used as antagonistic organisms of phytopathogenic fungi.

Because MAP kinases are involved in the expression of MRG genes, they can be used as a regulatory factor for the expression of lytic enzymes in Trichoderma. Due to the function of MAP kinases, related substances specific to them could be obtained in order to block their function in Trichoderma strains, thereby increasing the expression of lithic enzymes and thereby increasing the mycoparasitic function of the strain of interest. For this purpose, known techniques of adsorption of biological materials could be used using MAP kinases isolated and placed on fixed supports to capture specific related substances that can subsequently be isolated by conventional biochemical methods of purification and characterization. The use of these inhibitors of the function of MAP kinases could be useful for obtaining strains of Trichoderma with an increased antagonistic activity. In this sense, the Tvk1 protein isolated in the present invention (Trichoderma MAP kinase) can be used for this purpose.

The Trichoderma strains of the invention, as well as the compositions containing them, can be used for the protection or treatment of plants or plant materials for the fight and / or prevention of infections or diseases caused by phytopathogenic fungi, using multiple and diverse Known techniques for its application.

As a way of illustrating the present invention, the following examples are presented, without limiting the scope thereof.

Example 1. Mushroom strains.

The wild strain T. virens Gv29-8 and the auxotrophic mutant for arginine TV10.4 derived from it, were used in the present invention ⁴¹ . The phytopathogens R. solani AG-4 and Pythium ultimum isolated from diseased roots and seedlings were used as hosts. All fungal strains were maintained in Papa dextrose agar (PDA, Difco), unless otherwise specified.

Example 2. Bacterial strains and plasmids.

Escherichia coli DH5α (Bethesda Research Laboratories) and JM103 (Invitrogen) strains were used for all DNA manipulations. The plasmids used were pBluescript (Stratagene) and pCB1004 (Fungal Genetics Stock Centerj. All PCR products were cloned into the vector pCR2.1 (Invitrogen). The probes used for Northern blot analysis were obtained as follows: a 1.3 kb Hind \\\ / BamH \ fragment of Tv-prb1 cDNA was obtained from plasmid pPOE. A 1.4 Kb Hind \\\ / Xba \ fragment of Tv-cht1 was obtained from plasmid pCOE ^42. From plasmid pSZD2 a Pst \ / Xho \ 0.52 Kb fragment corresponding to the Tv-bgn2 gene was obtained, two fragments corresponding to Tv-cht2 (from position 988 to 1417) and Tv-nag1 (from position 513 to Ia 1608) were obtained from genomic DNA of T. virens by PCR using oligonucleotides designed based on the sequences reported by

Kim ⁴³ .

Example 3. DNA and RNA manipulation. Plasmid DNA was isolated using a commercial system (Qiagen). Trichoderma DNA was obtained according to a previously described procedure ⁴⁴ . Total RNA was isolated using phenol-chloroform extractions according to the procedure described by Jones ⁴⁵ . Southern and Northern type analyzes were carried out using Hybond N + membranes (Amersham) according to the manufacturer's recommendations.

Example 4. Immunodetection.

Protein extracts were prepared as previously described ³⁷ and the protein concentration was determined using the Bradford assay (Bio-Rad) with bovine serum albumin as standard. Equivalent amounts of protein (25 μg) of each sample were resuspended in Schágger 2X ⁴⁶ buffer and boiled after addition of β-mercaptoethanol (5%). The proteins were separated by SDS-PAGE in 10% gels according to Schágger and Von Jagow ⁴⁶ . The proteins were transferred to extra Hybond ™ -C membranes (Amersham) and the detection was performed following the instructions of the Phospho Plus ® p42 / p44 MAP kinase system (Thr / Tyr) Antibody kit (CeII Signaling Technology, Inc).

Example 5. Cloning and sequencing of tvk1.

Genomic DNA from T. virens Gv29-8 was used as tempering in PCR amplification reactions using the primers described previously ⁴⁷ . The PCR product was cloned and subsequently sequenced using the Sanger ⁴⁸ method with the Sequenase system (version 2.0, U. S Biochemichals). A fragment that showed high similarity with the M. grísea pmk1 gene was selected and used as a probe for the screening of a cosmid library of T. virens Gv28-9. Three clones were identified and one of them was selected for sequencing. A Southern type analysis allowed the identification of a 3.3 Kb BamH \ fragment containing the tvk1 gene (see figure 1), said fragment was subcloned into the BamH \ site of plasmid pCB1004 (pDXG35). This fragment was sequenced in its totally and subsequently analyzed by the BLAST program using the DNA-protein data bank.

Using T. virens DNA as temperate and degenerate oligonucleotides reported by Xu and Hamer ⁴⁷ , several PCR amplification products were obtained. These products were subcloned into the vector pCR2.1 and sequenced. After performing a computational analysis using the BlastX algorithm, one of these showed a high similarity with the pmk1 gene of M. grísea. This amplification product was radioactively labeled and used in a screening in a genomic library of T. virens. Three clones were obtained that gave a positive signal and one of them was selected, subcloned and sequenced. A Southern blot type analysis using T. Virens DNA and the tvk1 gene obtained, suggests that this gene is found as a single copy in the Trichoderma genome (Figure 1).

The tvk1 gene contains four exons interrupted by three introns as reported for other genes in this same kinase family (Figure 2).

Recently, a gene similar to the one reported here was cloned, however, unlike the genes of this family, the reported one does not present the third intron ³⁸ . A comparative analysis of the promoter region reveals the presence of possible response elements which are associated with the growth and conidiation processes, such as RAP-1, ABF-1, STUAP1 and three possible GCR1 binding sites, which is involved in the response to carbon limitation. In other systems it has been observed that the binding of RAP1 facilitates the binding of GCR1 to adjacent sites. Additionally, the promoter contains two STRE type binding sites and a possible site for the MAT-1-Mc mating factor.

The deduced protein sequence of tvk1 is 360 amino acids that correspond to a theoretical molecular mass of 41.6 KDaI and an isoelectric point of 6.44. An alignment analysis using the MegAlign-Clustal program indicates that Tvk1 and TmkA have a 99.4% similarity, while Tmk1 has a 97.7% similarity with Tvk1. The MAP kinases Cmk1, Pmk1 and Fmk1 of Colletotrichum lagenarium, M. grísea, and F. oxysporum, respectively, have a 95.8% identity with Tvk1, and S. cerevisae Kss1 has 54.7%. The region included among residues 58 to 160 in Tvk1 contains the sequence observed in several members of the family of MAP kinases: Fx (10) -REx (72,86) -RDxKx (9) -C ⁴⁹ . This protein also contains the phosphorylable residues [T (184) -EY (186)] required for its phosphorylation and activation by MAP kinase ⁵⁰ .

Example 6. Construction of the pTVK1 :: arg2 gene replacement vector.

To construct the replacement vector of the tvk1 gene, a £ coRV / Sa / l fragment of 1.48 Kb containing most of the coding region of tvk1 (from amino acid 88 to the last amino acid of the protein) was replaced by a fragment 3.2 Kb SmaVEcoRV of the arg2 gene from T. virens ⁴¹ . A SamHI / EcoRV fragment of pDGX35 was subcloned into the Ba / nHI / EcoRV sites of pBluescript SK (-) to generate plasmid pAM1. Plasmid pAM1 was digested with EcoRV and ligated to an EcoR \ // Sma \ fragment of the arg2 gene (pAM2) gene. With the purpose of obtaining the C-terminal region of Tvk1, a 0.4 Kb Sa / l / SamHI fragment of pDGX35 was used as a probe against cosmid DNA digested with several enzymes. A 3.2 Kb Sa / I fragment was cloned into pBluescript KS (+) which was then subcloned as a Cla \ / Apa \ fragment into pAM2. The resulting vector (pTVK1 :: arg2) was linearized and used to transform the auxotrophic strain TV10.4 of T. virens (see Figure 3A).

Example 7. Fungal transformation.

T. virens protoplasts were prepared and transformed according to a previously described method ⁴¹ . Prototrophic transformants were selected using Vogel minimum medium containing sucrose as the sole source of carbon (VMS). The interruption of the tvk1 gene in the selected transformants was confirmed by Southern and Western analysis.

Example 8. Generation of null mutants of tvk1.

The interruption of tvk1 was evaluated in 20 stable mutants by means of Southern type analysis using a 1.4 Kb βamHI / EcoRV fragment of plasmid pDGX35 as a probe. Waiting for a 3.3 Kb fragment to hybridize to the wild strain, a 4.8 Kb fragment in the null mutants and both bands in case of ectopic integrations were detected (Figure 3B). Seven transformants with the expected hybridization pattern were selected and analyzed to verify that no additional integration events had occurred. None of the transformants selected showed additional bands to the one corresponding to the replacement event. The transformants designated Atvk24 and Δtvk133 were arbitrarily chosen for phenotypic analysis and physiological studies. To verify that Tvk1 was not produced in the mutants, total protein extracts of the wild T. virens strain and of the Δtvk24 strain were analyzed using a polyclonal antibody that recognizes p42 / p44 MAP kinases of animal cells (Figure 3C). As expected, two proteins of 42 KDa and 44 KDa were detected in the wild strain (Figure 3C, lane 1). On the other hand, only a signal corresponding to the MAP kinase of 44kDa MAP was detected in the strain Δtvk24 (Figure 3C, lane 2). These results demonstrated that the Δtvk24 strain did not produce the MAP p42 kinase homologue. Similar results were obtained by analyzing other mutants.

Example 9. Analysis of submerged crops.

Spores (1x10 ⁶ spores / ml) of the wild strain and of the mutants Δtvk24 and Δtvk133 were inoculated in PDB (Potato Broth Dextrose, Difco), Vogel medium (VMS) or minimum medium (MM) ⁵¹ and incubated for 72 hours at 28 ⁰ C. Samples were analyzed using a light microscope (Olympus BX60). The images were captured and modified using the Image-Pro Plus 4.0 and Adobe Photoshop programs, respectively.

Colonies of the Δtvki mutants showed a reduction in their speed of colonial growth and in the development of aerial hyphae in solid medium. Conidia suspensions of the wild strain showed an intense green coloration, compared with the pale green of the conidia suspensions of the mutants. Additionally, the null mutants produced twice less conidia than the wild strain when they were grown in PDA medium without significant changes in the morphology of the conidophore. When the growth in liquid medium was analyzed, all Δivki mutants formed pellets smaller than those formed by the wild strain. Surprisingly, the Δtvki mutants massively conidia in late phases in submerged cultures (72 h), while no conidia were detected in the cultures of the parental strain even after seven days. Figure 4A shows liquid cultures of the wild strain of T. virens (Gv29-8) (left panel), mutants Δtvk24 and Δtvk133 (two central panels) and the parental strain (Tv10.4) (right panel) after 72 hours of incubation in VMS medium. The ability to conidia in liquid medium of the mutants seems to be independent of the culture medium used, since the conidiaclón was observed in both VMS and PDB. Microscopic observations of samples of sporulating liquid cultures of Δtvk133 showed a normal development of the conidiophore, resembling those produced in aerial hyphae (Figure 4B-C).

Example 10. Simulated mycoparasitism assays. Tríchoderma spores (1 x 10 ⁶ spores / ml) were germinated and grown for 48 h in VMS. The mycelium was then collected and transferred to fresh medium. Minimum Vogel medium without a carbon or nitrogen source (VM or VM-N, respectively) was used to assess the effect of nutrient limitation; VMS containing 0.5% of R. solani cell walls (VMSR), and Vogel medium without a source of nitrogen or carbon added with 0.5% of R solani cell walls (VM-NR or VMR, respectively) was used for similar conditions of mycoparasitism. VMS was used as a control. Samples were collected after 3, 6 and 24 h of incubation, these were frozen in liquid nitrogen and stored at -7O ⁰ C until used. For the analysis of the enzymatic activities, the filtrate was recovered from the cultures, frozen at -2O ⁰ C and stored at the same temperature until use.

Northern blot analysis showed that in the wild strain the Tv-nag1 gene encoding an N-acetylglucosaminidase was expressed in a carbon-free medium 3 h after transfer and that the mRNA could not be detected at 6 h. While in the mutant the expression was detected after 3 h and it increased at 6 h. In medium containing R. solani cell walls, it was expressed only after 6 h for both the wild strain and the mutant, but reached much higher levels in the mutant (Fig. 5A; Tv-nag1). The second chitinase coding gene analyzed (Jv-cht1) was expressed in both strains in the absence of carbon but reached higher levels in Δtvk24 at 6 h. Tv-cht1 was clearly induced by cell walls in the wild strain with a maximum level of expression at 6 h. In contrast, no obvious induction was observed in the mutant strain, which reached the same level observed under conditions of carbon limitation, except that this happened before (Fig. 5A; Tv-cht1). The gene encoding the Tv-bgn2 glucanase was induced by cell walls at 6 h, but no differences were observed with the wild strain and the gene was not expressed in medium without a carbon source (Fig. 5A; Tv-bgn2). The expression pattern of a third chitinase gene (Tv-cht2) under conditions of simulated parasitism was similar to that of Tv-cht1, except that the maximum expression was reached after 24 hours. Consistent with what was observed for other MRGs analyzed, Δtvk24 showed a much more pronounced induction for Tv-cht2 than the wild strain. The expression was determined under simulated conditions of mycoparasitism both in the absence and in the presence of an alternate carbon source (Fig. 5A; Tv-prb1) or in VM-NR that does not contain ammonium (Fig. 5B; Tv-prb1) of the gene prb1, which codes for a protease. Cell wall induction was observed for both strains, although higher levels of transcript were detected under conditions of carbon starvation than under conditions of nitrogen limitation. In both cases, the induction was much higher for the Δtvk24 strain than for the wild strain. The mutant Δtvk24 showed the highest levels of expression after 6 h, between seven and ten times more than the expression in the wild strain. The mutant strain showed detectable levels of Tv-prb1 expression after 6 h when grown on VM-N (Fig. 5B; Tv-prb1). According to these observations, the analysis of the acivivities in proiease gel, endocytinase, N-acetylglucosaminidase and glucanase of the culture filtrates showed ten times greater activity in the muiahids than in the silvesíre strain (see figure 8). It was also interesting to observe the presence of an additional band of endocytinase activity in null mutanids under conditions of carbon limitation (see figure 8).

Example 11. Confrontation trials.

Trichoderma strains were subjected to confrontational trials without coniacio using R solani as host ⁵¹ . The confrontations were carried out in modified VMS-agar medium (mVMS) containing 0.75 g / l sucrose and 0.45g / l NH ₄ NO ₃ . The mycelium of Trichoderma was collected from the zone of interaction between fungi.

The analysis of the expression of the same set of genes in the Atvk24 and wild strains was carried out during confrontational trials (Figure 6A). When Δtvk24 overgrowth in the presence of the R solani (TR) colony, a clear induction of Tv-prb1, Tv-nag1, Tv-cht1, and Tv-bgn2 was detected, when compared with the control conditions where the mutant grew alone (T) (figure 6B). It is interesting that detectable levels of expression of all genes used in the control (T) were observed. The accumulation of transcripts of all MRGs in Átvk24 was not only increased by the presence of the host but also reached much higher levels than for the wild strain. At the time of the sampling, no differences were observed in the expression of the MRGs when the wild strain was grown alone (T) or in the presence of R. solani (TR) (Figure 6B).

Example 12. Direct confrontation assays in agarpapa dextrose. The Trichoderma strains of the invention were subjected to direct confrontation assays using Phytophthora capsici, Sclerotium rolfsii, Rhizoctonia solani, Colletotrichum lindemuthianum and Phytophthora citricola, as host. The confrontations were carried out in half papa dextrose agar using strains T. virens Gv29.8, Δtvk24 and Λtvk133 for 5 days under continuous light at 28 ⁰ C. The results obtained are shown in Figure 9. As can be seen, they did not exist significant differences in the behavior of the strains of the invention with respect to the control strain, implying an inhibitory effect of the culture medium on the mycoparasitic behavior observed previously (see figures 6 and 7), according to what was reported by Kim ⁴³ .

Example 13. Biocontrol tests.

The tests were carried out as previously described ⁴² . Cottonseed (cultivate Stoneville 112, Pedigree Seed Co) were covered with Trichoderma strains and planted in a medium free of non-sterile soil (Metromix) infected with R solani or P. ultimum. Seeds sown in uninfected soil and seeds treated with the commercial fungicide Apron XL LS (active against P. ultimum) were used as positive controls. Survivors and healthy plants were counted at 10 days of incubation at 25 ⁰ C in a growth chamber. Additionally, the extension of symptoms of the disease in the root system was evaluated in plants infected with R solani using an arbitrary scale from 0 (no symptoms) to 5 (the entire discolored root system and decay) with a maximum value of 6 for seeds dead not germinated. Each treatment was performed in 6 replicates, with 10 seeds each, and the entire experiment was repeated twice.

The biocontrol capacity of Δtvk24 and Δtvk133 was evaluated in vivo against two root pathogens, R solani and P. ultimum. The mortality of the plants was very low in the treatments with R solani (10%). Although the protection by Δtvk24 and Δtvk133 was greater (mortality 0% and 5%, respectively) than with the wild (mortality 8%), the differences were not statistically significant. However, when disease symptoms were evaluated in the root system, significant differences were detected (Fig. 7A). The disease index was significantly lower for mutant strains in Δtvki than for wild. When cotton plants were infected with Pythium, only 10% of the untreated seeds survived. However, survival was increased to 70 and 80% in the treatments with Δtvk24 and Δtvk133, respectively, compared with only 20% for the wild strain. The level of protection achieved by the two mutant strains was comparable and even significantly higher than the protection obtained by treating the seeds with the commercial fungicide Apron (Fig 7B).

References. 1. Herrera-Estrella, A., & Chet, I. 2003. In Handbook of Fungal Biotechnology, ed. Arora, D. (Marcel Dekker, Inc., New York), In press.

2. Papavizas, G. C. 1985. Ann. Review Phytopathol. 23, 23-54.

3. Trillas Gay, María Isabel, et.al. 2003. Substrates containing a strain of Trichoderma asperellum for the biological control of Fusarium and Rhizoctonia. International Patent Application PCT / ES02 / 00311.

4. Muñoz, Antonio. 2003. Biological control of soil-born fungal pathogens. US Patent Application, US2003 / 0092574.

5. Chung, Young-Ryun. 2001. A microbial pesticide active against plant fungal pathogens and process for preparation thereof. PCT application, PCT / KR01 / 00616.

6. Papavizas, George C. 1984. Strain of Trichoderma viride to control Fusarium wilt. USPat 4489161. 7. Howell, Charles, R. 1999. Viridiol deficient mutant strains of the biocontrol agent Trichoderma virens, process of making and using as biocontrol agent. USPat 5882915.

8. Reyes Salinas, Mario. 2004. Bactericidal, bacteriostatic and fungicidal composition compressing two or more Uve species of Trichoderma. EP 1384405.

9. Beautiful Tight, Maria Rosa. 2004. Composition checking fungí of genus Trichoderma used as biological control agent and the applications thereof. EP 1279335.

10. Harman, Gary, E. 2002. Production and use of inducible enzymes from Trichoderma and bacteria for control of plant pests and for industrial processes.

PCT application, PCT / US01 / 25058.

11. Watanabe, Manabu., Et. to the. 2001. Regulatory sequence of cellulase cbh1 genes originating in Trichoderma viride and system for mass-producing proteins orpeptides therewith. USPat 6277596. 12. New IEEE Standard Dictionary of electrical and Electronic Terms. 5th edition, 1993.

13. Diagnostic Molecular Microbiology. Principies and Applications. 1993. D. H Persing et al., Ed., American Society for Microbiology, Washington D.C.

14. Plant Molecular Biology: A Laboratory Manual. 1997. Clark Ed., Springer-Verlag Berlin.

15. Kmiec. Compounds and Methods for Site Directed Mutagenesis in Eukaryotic CeIIs. U.S. Patent No. 5,565,350.

16. Zarling et al. In Vivo Homologous Sequence Targeting in Eukaryotic CeIIs. PCT / US93 / 03868. 17. Berger and Kimmel, Guide to Molecular Biology techniques, Methods in Enzymology, VoI. 152, Academic Press, Inc., San Diego CA (Berger).

18. Sambrok et al. 1999. Molecular Cloning. A Laboratory Manual, 2nd Ed., VoI 1- 3.

19. FM Ausubel et al. 1994. Current Protocols in Molecular Biology. Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John

Wiley and Sons, Inc.

20. Adv. Appl. Math 2: 482 (1981).

21. J. Mol. Biol. 48: 443 (1970).

22. Proc. Nati Acad. ScL USA 85: 2444 (1988). 23. CABIOS 5: 151-153 (1989).

24. Corpet et al. 1988. Nucleic Acids Research 16: 10881-90.

25. Pearson et al. 1994. Methods in Molecular Biology 24: 307-331.

26. Huang et al. 1992. Computer Applications in the Biosciences 8: 155-65. 27. Current Protocols in Molecular Biology. 1995. Chapt 19, Ausubel et al. Eds Grenne Publishing and Wiley-lnterscience, New York.

28. Altschul et al. 1997. Nucleic Acids Research 25: 3389-3402.

29. Henikoff and Henikoff. 1989. Proc. Nati Acad. Sci. USA 89: 10915.

30. Karlin and Altschul. 1993. Proc. Nati Acad. Sc. 90: 5787-5873. 31. Wooten and Federhen. 1993. Comput Chem., 17: 149-163.

32. Claverie and States. 1993. Comput. Chem. 17: 191-201.

33. Comput. Appl. Biol. Sci. 4: 11-17 (1988).

34. Xu, J-R. 2000. Fungal Genet Biol. 31, 137-152.

35. Di Pietro, A., García-Maceira, F.I., Méglecz, E., & Roncero, I. G. 2001. Mol. Microbiol 39, 1140-1152.

36. Zheng, L., Campbell, M., Murphy, J., Lam, S., & Xu J-R. 2000. Mol. Plant- Microbe Internet 13, 724-732.

37. Olmedo-Monfil, V., Mendoza-Mendoza, A., Gómez, I., Cortés, C, & Herrera-Estrella, A. 2002. Mol. Gen. Genomics 267, 703-712. 38. Mukherjee, P.K., Latha, J., Hadar, R., & Horwitz, B.A. 2003. Eukaryotic CeII 2, 446-455.

39. Takano, Y. 2000. Mol. Plant-Microbe Interact. 13, 374-383.

40. Howell, C. R., Stipanovic, R. D, & Lumsden, R. D. 1993. Biocontrol Sci. And Tech. 3, 435-441. 41. Baek, J.-M., & Kenerley, CM. 1998. Fungal Genet. Biol. 23, 34-44.

42. Baek, J-M., Howell, C.R., & Kenerley, CM. 1999. Curr. Genet 35, 41-50.

43. Kim, D-J., Baek, J-M., Uribe, P., Kenerley, C, & Cook, D.R. 2002. Curr Genet 40, 374-384.

44. Reader, U., & Broda, P. 1985. Lett. Appl. Microbiol 1, 17-20. 45. Jones, J., Dunsmuir, P., & Bedbrook J. 1985. EMBO J. 4, 2411-2418.

46. Schágger, H & Von Jagow, G. 1987. Analytical Bioch. 166, 368-379.

47. Xu, J.-R., & Hamer, J. E. 1996. Genes Dev. 10, 2696-2706.

48. Sanger, F., Nicklen, S., & Coulson, AR 1977. Proc. Nati Academ Sci. USA 74, 5463-5467. 49. Dorin, D., Alano, P., Boccaccio, I., Cicero, L, Doerig, C ₁ Sulpice, R., Parzy,

D., & Doerig, D. 1999. J. Biol. Chem. 274, 29912-29920.

50. Kultz, D. 1998. J. Mol. Evol 46, 571-588.

51. Cortés, C, Gutiérrez, A., Olmedo, V., Chet, I., & Herrera-Estrella, A. 1998. Mol. Gen. Genet 260, 218-225.

Claims

Claims

1. A strain of Trichoderma over-producing lithic enzymes for the fight against phytopathogenic fungi, characterized in that it has lost the ability to produce at least one MAP kinase.

The strain of claim 1, characterized in that in its genome at least the function of a gene coding for a MAP kinase has been eliminated.

3. The strain of claim 2, characterized in that it is selected from the group comprising any species of Tríchoderma.

4. The strain of claim 3, characterized in that the strain is Trichoderma 10 virens.

5. The strain of claim 4, characterized in that the deleted gene coding for the MAP kinase has the DNA sequence corresponding to SEQ ID NO: 2.

6. The strain of claim 5, characterized in that it is the Trichoderma 15 virens Δtvk24 strain with the deposit number PTA-6474.

7. The strain of claim 5, characterized in that it is the strain Trichoderma virens Δtvk133 with the deposit number PTA-6474.

8. A fungicidal composition for the control and control of phytopathogenic fungi, characterized in that it comprises a Trichoderma strain selected from the

20 group comprising the strains of claim 1 to 7.

9. The composition of claim 8, characterized in that the strain of Trichoderma is the strain of claim 1.

10. The composition of claim 8, characterized in that the strain of Trichoderma is the strain of claim 6.

The composition of claim 8, characterized in that the strain of

Trichoderma is the strain of claim 7.

12. The composition of claim 8 to 11, characterized in that the Trichoderma strain is in any of its vegetative growth phases, mycelia and / or spores. - The composition of claim 12, characterized in that the strain of

Trichoderma is found as spores.

14. A Trichoderma MAP kinase, characterized in that it has the amino acid sequence corresponding to SEQ ID NO: 3.

15. A DNA molecule, characterized in that it codes for the MAP kinase of claim 14.

16. The DNA molecule of claim 15, characterized in that it has the sequence corresponding to SEQ ID NO: 2.

17. A recombinant vector characterized in that it contains the DNA molecule of claim 15.

18. A recombinant vector characterized in that it contains the DNA molecule of claim 16.

19. An organism transformed with the recombinant vector of claim 17.

20. An organism transformed with the recombinant vector of claim 18.

21. A method for the production of the protein of claim 14, characterized in that it comprises culturing the transformed organism of claim 19 to 20, producing and accumulating the protein and recovering it.

22. A gene replacement vector to suppress Trichoderma's ability to produce at least one MAP kinase, characterized in that it comprises: a) A replacement gene that identifies the transformants where a gene coding for a Trichoderma MAP kinase is replaced, b ) A non-coding fragment that is naturally outside the coding region of the gene for MAP kinase at its 5 'end, flanking the 5' end of the replacement gene, and. c) A non-coding fragment that is naturally outside the coding region of the gene for MAP kinase at its 3 ^! , flanking the 3 'end of the replacement gene.

23. The vector of claim 22, characterized in that the replacement gene comes from Trichoderma strains.

24. The vector of claim 22, characterized in that the replacement gene is selected from the group comprising the hph and hpt genes of E. coli, Basta herbicide resistance genes, the amdS gene of Aspergillus nidulans and the arg2 gene of Trichoderma .

25. The vector of claim 24, characterized in that the replacement gene is arg2.

26. The vector of claim 22 to 25, characterized in that the fragment of part b) comprises nucleotide sequence no. 1 to nucleotide no. 1210 of SEQ ID NO: 1.

27. The vector of claim 22 to 26, characterized in that the fragment of part c) comprises nucleotide sequence no. 2692 to nucleotide no. 5892 of SEQ ID NO: 1.

28. An organism transformed with the gene replacement vector of claim 22 to 27.

29. An organism transformed with the gene replacement vector of claim 25.

30. An organism transformed with the gene replacement vector of claim 27.

31. A method for obtaining strains of Tríchoderma overproducers of lytic enzymes for phytopathogenic fungi, characterized in that it comprises the steps of: a) Suppressing the ability of Tríchoderma to produce at least one MAP kinase, b) Select strains over- producers of lithic enzymes, and c) Corroborate the suppression of the production of MAP kinase.

32. The method of claim 31, characterized in that the stage of part a) is performed by eliminating the coding sequence for MAP kinase.

33. The method of claim 32, characterized in that the elimination of the sequence is performed by replacement by homologous recombination.

34. The method of claim 33, characterized in that the homologous recombination is carried out by means of the transformation of a Tríchoderma strain with the vector of claim 22 to 27.

35. The method of claim 34, characterized in that the vector is that described in claim 24.

36. The method of claim 34, characterized in that the vector is that described in claim 25.

37. The method of claim 32, characterized in that the coding sequence for MAP kinase is selected from the group comprising, the sequence of claim 15 and the gene corresponding to SEQ ID NO: 2.

38. The method of claim 37, characterized in that the gene coding for MAP kinase is the gene corresponding to SEQ ID NO: 2.

39. The method of claim 31 to 38, characterized in that the Tríchoderma strain is Tríchoderma virens.

40. The method of claim 31, characterized in that the stage of part b) is performed by selecting the transformants generated in selective medium.

41. The method of claim 40, characterized in that the selection of the prototrophic strains is carried out in a minimal Vogel medium.

42. The method of claim 31, characterized in that the suppression of the production of MAP kinase is corroborated using antibodies specific for MAP kinase by Western blot assays.

43. The method of claim 31, characterized in that the suppression of the production of MAP kinase is corroborated using specific probes for the gene coding for MAP kinase by Southern blot assays.

44. A method to increase the expression of Trichodθrma lytic enzymes for the control of phytopathogenic fungi, characterized in that it comprises the steps of: a) Suppressing the ability of Tríchoderma to produce at least one MAP kinase, b) Cultivate the organism obtained in a ) in a medium with low levels of carbon and / or nitrogen sources, and c) Corroborate the increase in the expression of lithic enzymes.

45. The method of claim 44, characterized in that the deletion is carried out by means of the elimination of the coding sequence for the MAP kinase by means of a homologous recombination replacement gene.

46. The method of claim 45, characterized in that the replacement gene comes from Tríchoderma strains.

47. The method of claim 45, characterized in that the replacement gene is selected from the group comprising the hph and hpt genes of E. coli, herbicide resistance genes Basta, the amdS gene of Aspergillus nidulans and the arg2 gene of Tríchoderma .

48. The method of claim 47, characterized in that the replacement gene is arg2.

49. The method of claim 44, characterized in that the coding sequence for MAP kinase is selected from the group comprising the sequence of claim 15 and the gene corresponding to SEQ ID NO: 2.

50. The method of claim 49, characterized in that the gene coding for MAP kinase is the gene corresponding to SEQ ID NO: 2.

51. The method of claim 47 to 50, characterized in that the strain of

Trichoderma is Trichoderma virens.

52. The method of claim 51, characterized in that, additionally, in step b) cell walls of phytopathogenic fungi are added.

53. The method of claim 47 to 52, characterized in that the increase in expression is corroborated using specific probes for the coding genes for lytic enzymes of Trichoderma by Northern blot assays.

54. A method for the protection or treatment of plants or plant materials against infections or diseases caused by phytopathogenic fungi, characterized in that it comprises the application of an effective amount of a Trichoderma strain selected from the group comprising the strains of claim 1 to 7.

55. The method of claim 54, characterized in that the Trichoderma strain is the strain of claim 1.

56. The method of claim 54, characterized in that the strain of Trichoderma is the strain of claim 6.

57. The method of claim 54, characterized in that the strain of Trichoderma is the strain of claim 7.

58. A method for the protection or treatment of plants or plant materials against infections or diseases caused by phytopathogenic fungi, characterized. because it comprises the application of an effective amount of a composition according to any of claims 8 to 13, in the soil or in the plant or in the material of the plant to be protected or treated.

59. The method of claim 58, characterized in that the composition is that described in claim 9.

60. The method of claim 58, characterized in that the composition is that described in claim 10.

61. The method of claim 58, characterized in that the composition is that described in claim 11.