MXPA04012451A - Improved trichoderma strains as biocontrol agents, method for obtaining the same and use thereof for controlling diseases caused by phytopathogen fungi. - Google Patents

Abstract

The present invention describes the obtention of Trichoderma strains over-producing various lytic enzymes acting as phytopathogen fungi antagonists due to their high mycoparasitic activity. A coding Trichoderma virens gene was clonned for a mitogen-activated protein kinase (MAP kinase), said gene being related to the mycoparasitic response, conidiation and biocontrol observed in Trichoderma. The Trichoderma strains having a suppressed expression of the isolated MAP kinase gene showed an apparent increase in the expression level of genes related to mycoparasitism (MRGs) under mycoparasitism simulated conditions and while brought into direct contact with the pathogen of Rhizoctonia solani plants. The biocontrol assays consistently showed that the inventive strains were substantially more effective in the control of diseases than wild strains or a chemical fungicide. Furthermore, the inventive strains abundantly sporulated in submerged cultures, a condition which normally does not lead t o the formation of spores in a 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 the obtaining of improved strains of Trichoderma useful for the treatment of diseases caused by phytopathogenic fungi.

BACKGROUND OF THE INVENTION Agriculture is one of the most important commercial activities in terms of the production of food or inputs, both for man and for livestock, so that the control and effective combat of diseases that affect plants is 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 occurs, caused mainly by fungi such as Fusarium oxysporum, Verticillium spp. and Rhizoctonia solani among others. The death of plants caused by this last species of fungus is considered one of the most harmful and important effects worldwide affecting 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 is sometimes irreversible.

Due to the above, some specific antagonistic microorganisms have been used, especially within the genera Pseudomona and Tríchoderma as an alternative for combating 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 of Trichoderma species against pathogenic fungi. In this sense, the hydrolytic enzymes of Trichoderma, such as chitinases, β-1-3 glucanases, β-1-6 glucanases, and proteases, facilitate the penetration of the host.

Many species of Trichoderma have been used as powerful biocontrol agents against a variety of phytopathogenic fungi. For example Trichoderma asperellum T34 (2) isolates obtained from composts prepared from the organic fraction of market waste, sludge from wastewater treatment plant and garden remains3, improved strains such as T. vi'ride Li 49 4, 7. harzianum YC459 5, T. viride T-1-R9 6, or mutants deficient in viridiol production but retaining their effectiveness as agents of biocontrol, such as T. virens TV-111, TV-115 and TV-1097.

Despite the above developments, in practice it has been shown that it is very difficult to ensure that these antagonistic microorganisms are 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 passage of the spores, or by inoculation of wheat flour with Trichoderma species and subsequent dilution using diatomaceous earth. However, these methods are of low productivity, mainly due to the low spore production characteristics of the Trichoderma species used so far.

On the other hand, to try to eliminate the problem in obtaining low amounts of Trichoderma, we have obtained Trichoderma species that can coexist in culture by transforming some of the strains by radiation of T. longibratum through 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 proteins of Trichoderma strains in conjunction with bacterial proteins, for example from Streptomyces ™. This is intended to obtain synergistic effects of biocontrol, as well as a greater amount of spores, however the methods of cultivation of these microorganisms do not allow, given the antagonistic characteristics of the Trichoderma species, the production of two or more at the same time species together, and therefore lytic enzymes are not obtained, since they are only produced in the presence of other fungi.

Due to the above, several methodologies aimed at obtaining improved Trichoderma strains have continued to be generated. In this sense, the observation that transformed strains of Trichoderma that overexpress lytic enzymes show to be better biocontrol agents than the corresponding parental strains has acquired particular relevance.

In addition to the methodologies of isolation and selection of strains from natural substrates to obtain over-producing strains of lytic enzymes, classical strategies of genetic manipulation have been used to achieve this objective. In this sense, Trichoderma is induced in general 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 coding for such proteins and use them to transform selected strains of Trichoderma by means of expression vectors. This is achieved by increasing the expression capacity of the previously isolated gene, thus obtaining transformed strains of Trichoderma with better levels of production of the protein of interest compared with the parental strains. As examples of this, the increase in the expression of native extracellular enzymes of Trichoderma species in inducer or repressor means of such expression can be mentioned, in strains that have been previously transformed with at least one transgene under the control of an inducible 0 promoter, or by means of regulatory genes of T. viride, such as, for example, the cellulase cbhl 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, with which there may be problems associated with the non-recognition of such sequences, both regulatory and open reading frames, by the microorganism transformed due to multiple effects, including the characteristic use of body codons.

On the other hand, it has been observed that the antagonistic effect of Trichoderma 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, genetic manipulation methods aimed at the isolation of lytic enzyme genes to subsequently obtain transformed strains of Trichoderma over-producing said enzymes, only achieve the increased expression of at most two isolated genes at the same time, with which the obtaining of improved strains of Trichoderma to be used in the combat of pathogenic fungi of plants, is quite limited. This coupled with the fact of 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 lytic enzyme genes without it representing no problem for the strain of transformed Trichoderma, as for example toxic effects.

For the reasons previously explained, it is necessary to continue the development of methodologies that allow obtaining strains of over-producing Trichoderma in all or in the great majority of lytic enzymes, to be used as efficient antagonistic organisms for phytopathogenic fungi.

Prior to the present invention, there were no methodologies that allowed obtaining strains of Trichoderma over-producers 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 tvkl gene of T. virens. 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. Sequence of the Tvk1 gene. The possible start and end of the translation is shown. The donor and acceptor sequences of the introns are indicated with underlined letters. Figure 3. Strategy for the interruption of the tvkl gene and the analysis of the transformants. (A) Schematic representation of the replacement of Tvkl. The thick arrows represent the coding regions of tvkl and arg2. The lines represent the 5 'and 3' regions 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) Southern analysis of the mutants. Genomic DNA was digested with Bam. Lanes 2-5 represent four independent transformants; Lane 1 wild (WT). The membrane was hybridized with the probe indicated in A.

(C) Immunoblot analysis of the crude protein extracts of 7. virens (strains WT and Atvk24) 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 Atvkl mutants (Atvk24 and Atvk133) in liquid medium. The strains were incubated in PDB for 72 hours. (B &C) Microscopic analysis of the Atvk133 mutant strain. Figure 5. Expression of MRGs in simulated mycoparasitism. (TO). Northern analysis of the genes Tv-prb1, Tv-nag1, Tv-cht1 and Tv-bgn2 under conditions of carbon limitation with and without walls of R. solani in the wild strain and the mutant Atvk24. The numbers indicate the induction time. (B) Northern analysis of Tv-prb1 under conditions of limitation of nilrogen and simulated mycoparasitism. The symbols (+) and (-) denote the presence and absence of cell walls of R. solani, respectively. Fifteen micrograms of total RNA were loaded in each lane. The 28S ribosomal gene was used as a control. Figure 6. Expression of genes related to mycoparasiism (MRGs) in a confrontation with R. solani. (A) Schematic representation of the inirection 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 zone of iniration is indicated by the color black. (B) Expression of MRGs in strains of T. virens. The samples were collected when the strains of T. virens overgrowed the R. solani colony. The probes used for the Northern ipo analysis were the same as those indicated in Figure 5. ^ 5 μg of RNA were loaded in each lane. Figure 7. Bioconical activity of the silvesfre, Átvk24 and Atvk133 strains. (TO). Disease index in the root system of cotton plañís infected with R. solani. (B) Percentage of overvolved 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 level of significance 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 T. virens wild (wt) and Atvk24 under simulated mycoparasitism. Gel analysis of total protease activity under simulated conditions of mycoparasitism in the absence of a carbon source (A), or nitrogen (B). The arrow indicates the activity of Prb1. Total activities of glucanase (C) and chitinase (D) were detected under simulated mycoparasitism in the absence of a carbon source. Gel analysis of endochitinase activities under simulated mycoparasitism in the absence of a carbon source (E). The arrow indicates the presence of an additional endochitinase activity. The presence (+) or absence (-) of Rhizoctonia cell walls is indicated. 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 challenged on potato dextrose agar plates against Tríchoderma virens Gv29.8 (left), Atvk24 (center ) and Atvkl 33 (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 such 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 Trichoderma over-producers of various lytic enzymes to be used as antagonists of phytopathogenic fungi, - Provide strains of Trichoderma over-producers of various lytic enzymes and at the same time with high spore production capacity to be used as antagonists of phytopathogenic fungi , - Provide a method to obtain strains of Trichoderma over-producing several lytic enzymes by suppressing the expression of genes encoding MAP kinases in parental strains of Trichoderma, - Provide a method to increase the activity of lytic enzymes and mycoparasitic strains of Trichoderma by suppressing the expression of genes coding for MAP kinases in parental strains of Trichoderma, - Providing novel Trichoderma genes encoding MAP kinases, related to the establishment of the parasitic relationship of Trichoderma and its hosts, - Providing a const genetic transformation for the transformation of fungal cells, including the polynucleotide sequences that are described in this invention, - To provide proteins with MAP kinase activity related to the establishment of the parasitic relationship of Trichoderma and its hosts, - To provide a replacement vector gene useful for the suppression of the expression of genes encoding MAP kinases in parental strains of Trichoderma, and - Provide a method of biocontrol of phytopathogenic fungi, using strains of Trichoderma over-producing several lytic enzymes.

Detailed description of the invention.

/. Deposit of microorganisms.

The strain T. virens Atvk24, biological material that will be deposited before an institution recognized by the Industrial Property Institute, a record that will be exhibited before this Institute once it is issued.

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

For purposes of the present invention, the units, prefixes and symbols may be mentioned using their abbreviation accepted by the SI. Unless explicitly indicated, the sequence of the nucleic acids is written from left to right in the orientation 5 'to 3'; 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 integral numbers that define the range. Amino acids can be designated either by their familiar three-letter symbols or by their single-letter symbols that have been recommended by the IUPAC-IUB Nomenclature Commission. In the same way, nucleotides can be designated by their commonly accepted unique letter code. Software, electrical and electronic terms 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 that is 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. 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 Transcriptional-dependent Amplification System (TAS), and the Strand Displacement Amplification System (SDA). For its acronym in English) 13. The product of amplification 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 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 refer to those nucleic acids that encode 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 GCA, GCC, GCG and GCU codons encode the amino acid alanine. These variations of nucleic acid are silent variations and represent conservatively modified variants. Therefore, any nucleic acid sequence encoding a polypeptide in this document describes all possible silent variations of the nucleic acid. In the same manner, any silent variation of a nucleic acid sequence encoding a polypeptide is implicitly included in each polypeptide sequence of this document.

As for the 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 the The encoded sequence is also a "conservatively modified variant" in which the alteration results in the substitution of an amino acid for a biochemically similar amino acid. Therefore, any number of amino acid residues selected from the number of integrants 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. The 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 30% to 90% of the activity of the native protein and its native substrate. The tables of conservative substitutions 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) Aspargin (N), Glutamine (Q) 4) Arginine (R), Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M) ), Valine (V) 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) The term "encoded" or "encoding" in reference to a specific nucleic acid refers to the translation information of the corresponding protein . A nucleic acid encoding a protein may include untranslated sequences (introns, for example) contained within translated regions of the nucleic acid, or 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 in 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, the bacterioid organism Micoplasma capricolum, the ciliated organism Macronucleus can be included within the corresponding organism.

When the nucleic acid is synthetically generated or 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 the codon preferences and the GC content preference that may occur between the Trichoderma species.

The term "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" hybridizations, extension of primers, or ribonuclease protection. 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' border of the untranslated regions of a messenger RNA molecule (mRNA) aid in the identification of the full length sequence of a polynucleotide. For example, the ANNNNAUGG consensus sequence, in which the underlined codon represents the methionine present at the N-terminal border helps determine if the polynucleotide has a complete 5 'terminal terminus. Consensus sequences at the 3 'border, such as the polyadenylation sequences, help determine if the terminal 3' border is complete.

The term "heterologous" refers herein to a nucleic acid derived from a different species or, if derived from the same species, a nucleic acid which is substantially modified from 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. The host cells can be prokaryotic cells such as those of E. coli, or eukaryotic cells such as those of yeast, insect, amphibian or mammalian cells. Preferably, the host cells are strains of Trichoderma.

The term "hybridization complex" refers to a double strand structure of nucleic acid 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 "transfect" or "transform" and includes the incorporation of nucleic acids into a eukaryotic or prokaryotic cell in which the nucleic acid can be incorporated into the cell. cell genome (in the DNA of a chromosome, 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 protein) that is: (1) substantially or completely free of the components that normally accompany it or interact with it in its natural form. The isolated material can 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 altered in a synthetic way by a deliberate human intervention that modifies its composition or allocates 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 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 to it. Nucleic acids that are "isolated" following this definition can be referred to as "heterologous" nucleic acids.

The term "nucleic acid" or "nucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in its single or double strand form and comprises those analogous molecules that have the essential nature of the natural nucleotides of being able to hybridize to strand nucleic acids simple in a way similar to that of natural nucleotides.

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

The term "polynucleotide" refers to a deoxyribonucleotide, a ribopolynucleotide, or its analogues having 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 the nucleotides. that occur naturally, or that allow translation in the same amino acids as natural nucleotides. A polynucleotide may 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 containing 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 tritylated bases to name but two examples, would also be considered "polynucleotides" for purposes of the present invention. It follows from this paragraph that a 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 forms of chemically-modified polynucleotides, enzymatically or metabolically, 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 herein interchangeably to designate a polymer of amino acids. 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 they are 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, glycolisation, lipid binding, sulfation, the carboxylation range of glutamic acid-containing residues, hydroxylation and ADP-ribosylation. mention that the polypeptides are not completely linear.For example, polypeptides can be branched as a result of ubiquitination, they can 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 completely synthetic processes.In addition, this invention also includes the terminal amino acid variants with or without methionine of each of the proteins relating to the present invention.

The term "promoter" refers to a region of DNA located in the direction 5 'of the start codon of transcription and that is involved in the recognition and binding of an RNA polymerase and other proteins necessary for the initiation of 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 stages include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots or seeds. This type of promoters are called "tissue-preferred". Those promoters that only initiate transcription in certain tissues are called "specific promoters". A specific promoter of "cell type" is a promoter that only directs the expression in certain types of cells located in one or several organs, such as 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. The tissue-specific promoters, the tissue-preferred promoters, the cell-type specific promoters and the inducible promoters constitute the class of "non-constitutive" promoters. The "constitutive" promoters are those that direct expression systemically and under most 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 in identical form within the native (non-recombinant) form of the cell or that express native genes that would otherwise be sub-expressed, expressed abnormally, or not expressed as a result of human intervention. The term "recombinant" does not include the alteration of the cell or the vector by events that occurred naturally (for example, spontaneous mutation, or transformation, transduction or transposition) as 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 the transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated in a plasmid, in a chromosome, in mitochondrial DNA, in plastid DNA, in a virus, or in a nucleic acid fragment. Typically, the recombinant expression cassette portion 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 can function in a manner similar to natural amino acids.

The term "hybrid selectively" refers to the hybridization (under stringent hybridization conditions) of a nucleic acid sequence to a specific nucleic acid target 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. 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 into which a polynucleotide can be inserted. Vectors are often replicons. Expression vectors allow the transcription of a nucleic acid that has been inserted into them.

The terms indicated below are used to describe the relationship of 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 a basis for the comparison of sequences. 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 range" 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 absent elements (eg. example, "gaps") when compared to the reference sequence (which has no additional or missing elements) so that the alignment of the two sequences is optimal. In general, the comparison interval is at least 20 contiguous nucleotides and can often include more than 100 nucleotides. Those technicians in the art who know how to perform this type of analysis understand that 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 methods of sequence alignment are well known. The optimal alignment of sequences 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 computerized implementation of these algorithms, including but not limited to: CLUSTAL in the Intelligenetics PC / Gene program, 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 the search of homologies between a protein sequence and a nucleotide sequence; and TBLASTX for a comparison of a nucleotide sequence with a set of nucleotide sequence databases27.

Unless explicitly stated otherwise, the values of identity or similarity of a sequence that are indicated in this document refer to the values obtained with the BLAST 2.0 version using the parameters "defaulf 28. The software to perform these analyzes It is public domain and can be accessed or obtained through the website of the National Center for Biotechnology Information of the United States of America (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, these words must be identical or very similar to that of a threshold of value T when align with a word of the same length contained in a database of the public domain.This initial similarity between two sequences leads to the beginning of a series of buses You are left to find sequences of greater length and high degree of similarity. A T is called the word neighborhood value threshold 28. 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 parameters M (value of reward for a pair of matching residues, always greater than 0) and (punishment value for non-coincident residues, always less than 0). For amino acid sequences, a matrix value is used to calculate the cumulative value. The length of the words found stops in each of the two directions when: a) the cumulative alignment value falls by the quantity 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 a negative value; or c) the end of each sequence is reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the sequence alignment. The BLASTN program (for nucleotide sequences) uses a word length W of 11 as a default value, an expectation value E of 10, a cut-off 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 BLAST algorithm 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 may occur at random.

BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences that may be enriched with one or more amino acids. Those regions of low complexity can be aligned from unrelated proteins although other regions of said proteins are completely different and do not show similarity. Some programs that function as low complexity filters can be used to reduce this type of low complexity alignments. For example, the program SEG 31 and XNU 32. 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 the residues in both sequences that are the same when aligned to find the greatest correspondence within a window specific comparison. When the percentage of sequence identity is used in reference to proteins it is recognized that the positions of the residues that are not identical often differ by substitutions of conserved amino acids, in which the amino acids are replaced by other amino acids that have similar biochemical properties (charge 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 upward 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 these kinds of adjustments is routine for those with skills in the art. It usually involves assessing a conservative substitution as a partial and non-complete sequence discrepancy, thus 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 algorithm of Meyers and Miller 33, as implemented in the PC / GENE program (Intellegentics, Mountain View, California, USA). (d) The term "identity sequence percentage" refers to the value determined from the comparison of two sequences optimally aligned from a specific comparative window, and in which the polynucleotide sequence portion in the comparison 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 the 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 comparison window and multiplying per 100 to obtain the identity sequence percentage. (e) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence having 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 degeneracy or amino acid similarity. For this purpose the substantial identity of amino acid sequences usually 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 each other under stringent hybridization conditions. However, nucleic acids that do not hybridize to one another 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 codon of degeneracy 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, optimal alignment is effected using the alignment algorithm of Needleman and Wunsch 21. An indication that two peptide sequences are substantially identical is that a peptide is immunologically identical to the second peptide. For example, two peptides will be substantially identical if they only differ by a conservative substitution.

///. Detailed description.

The present invention allows obtaining strains of Trichoderma over-producers of 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 occupied as antagonistic organisms for the effective control of plant diseases and used as a biocontrol mechanism. The Trichoderma strains of the invention preserve 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 adequate cellular response of Trichoderma. In this case, it is known that through the MAP kinase pathways a great variety of signals are transduced, including those associated with pathogenesis.

The parasitism exerted by Trichoderma, resembles in many aspects 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 grisea (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, homologs of the Kss1 MAP kinase of S. cerevisiae. c) In the induction of the pectate lyase (p / i) gene of F. Oxysporum, since in null mutants of fmkl this activity was abolished 35. d) In the positive control of the expression of those enzymes involved in penetration of the plant, for example of Bmp1 from B. cinerea m, e) In the regulation of transcription of the prb1 gene that encodes a protease produced in response to nitrogen limitation of Trichoderma atroviride (species closely related to Tríchoderma 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 coding genes 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, as for example of gene tvkl, causes a considerable increase in the synthesis of enzymes related to the observed mycoparasitic effect of strains of Trichoderma, so less with an increase of 10 times more 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 employed as efficient antagonistic organisms against multiple diseases caused by phytopathogenic fungi.

Likewise with the results observed in the behavior of the over-producing strains of enzymes 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, whereby the invention allows obtain strains of Trichoderma with greater capacity to control phytopathogenic fungi.

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

In contrast to that 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 low in the colonization of pathogenic fungi (R. solani), as well as a significant reduction in the antagonistic properties towards Sclerotium rolfsii in comparison with the wild strain 38, the methodology of the invention allows Trichoderma strains to be obtained with increased activities in their properties of mycoparasitism, as well as in the expression of multiple enzymes involved in the antagonist effect.

On the other hand, according to Mukherjee the abolition of tmkA gene expression results in much less effective Trichoderma strains to be used as 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 previous, the present invention demonstrates that the protein Tvk1 (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 strains of Trichoderma with the purpose of improving 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 field combat of plant diseases, the experimental data of Mukherjee do not show such effects.

In contrast, the present invention shows that in the transformed strains of Trichoderma, in which the expression of the tvkl gene was abolished, the activities of chitinase and protease were greater than in the wild, correlating with the high levels of the transcripts detected. In addition, the activity of certain enzymes is markedly increased in the strains of the invention compared with the wild-type strain, for example in the total activity of β-1,3-glucanase, indicating further that in the strains of the invention other glucanase genes they 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 of of 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 gene tmkA coding for a MAP kinase of Trichoderma, reduces the efficiency of biocontrol of T. virens 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 potato dextrose agar medium, 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 potato dextrose agar medium with several phytopathogenic fungi, including Sclerotium rolfsii and R. solani. The results obtained indicate that varying the environment of the strains of the invention in comparison with the parental strain, may present a decrease in the capacity for growth inhibition depending on the host phytopathogenic strain (Figure 9).

All strains of Trichoderma of the invention show a 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 being albino. In contrast, all the 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. lagenaríum 39.

Considering the observed increase in the production of lytic enzymes in the Trichoderma strains of the invention and the importance of these enzymes in the biocontrol 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 can be used which allows to obtain, by means of the teachings described herein, strains of Trichoderma over-producing lytic enzymes; likewise, any species of the genus Trichoderma can be occupied, although T. virens is even more preferred.

In order to protect or treat 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 prepared by widely known methods and can be made in any form of presentation, either in liquid or solid forms. The liquid forms are susceptible to be applied by aerosols in the soil or in the plant, or used for the elaboration of baths in which the plants or the material of these 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 tvkl gene of Trichoderma 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 Trichoderma, including growth, conidiación, expression of MRGs, secretion of enzymes that degrade cell wall and a biocontrol activity.

As shown in Figure 2, the tvkl gene contains three sequences of introns that have been reported for other fungal MAP kinase genes. The intron located between the nucleotide +854 and +907 is not present in its closest recently reported homolog, tmkA of strain IMI306092 of T. virens. Tvkl 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 preserve the activity of the Tvk1 protein. Following the teachings of the present invention, a person skilled in the technical field could easily predict the existence of such modified sequences and could easily produce them. The invention also comprises modified sequences of amino acids 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 capacity of negative regulation in the expression of MRGs of Tríchoderma.

As described herein, the suppression of the expression of MAP kinase genes encoding Trichoderma strains allows to increase the capacity of the Trichoderma strains of the invention to control other fungi that attack plant crops, together with the fact that due to this With the effect of suppression, lytic enzyme expression levels are present at high levels in most of the selected genes encoding lytic enzymes (MRGs) compared to wild-type strains.

In order to achieve the above, the inhibition of the function of a MAP kinase can be occupied by specific antagonist substances, or by suppression of the expression of the gene coding for it. The latter can be obtained by directed inhibition of gene expression using specific probes, by interfering RNA, antisense RNA expression, by inserting foreign polynucleotide sequences into 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 gene-homologous recombination techniques. As the invention shows, this suppression of the expression of genes coding for AP kinases, can be achieved using the gene replacement vector of the invention, which comprises: a) A replacement gene that allows the identification of transformants where a coding ungen is replaced for a MAP kinase of Trichoderma, b) A non-coding fragment that is naturally outside the coding region of the MAP kinase gene 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 MAP kinase gene 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 any strain of Trichoderma, although those genes that come from strains of Trichoderma of the same species of the strain to be transformed are preferred. As a replacement gene, any gene that allows partial or total removal of the gene encoding the MAP kinase can be used or that allows the interruption of the open reading frame of said gene, with the consequent suppression of the expression of the protein encoded by the protein. the same and that also allows the identification of the modified strains. With the above, 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 fragments of the MAP kinase gene that flank the replacement gene, are those fragments of any size that are found either before or after the beginning of the open reading frame of the gene as well as those that are before or after. 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 in order to produce specific genetic recombination events between the genome of the Trichoderma strain to be transformed in the region of the wild type MAP kinase gene and the replacement polynucleotide sequence contained in the gene replacement vector.

Preferably, the MAP kinase gene fragments that flank the replacement gene come from the tvkl gene and are: - For the 5 'end the smaller BamHI / EcoRV fragments shown in Figure 3A, although more preferably those fragments comprising the 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 the nucleotide sequence are preferred. 2692 to nucleotide 5892 of SEQ ID NO: 1.

Likewise, the function of the replacement gene can restore some deficiency in the Trichoderma strain to be transformed given by auxotrophy or allow the selection of transformants due to its tolerance to a chemical compound such as in the case of the use of antibiotics or herbicides or by to give it the ability to use alternate sources of carbon and / or nitrogen, or to be able to identify itself through an enzymatic activity encoded by it. For example, if the Trichoderma strain to be transformed is auxotrophic, the replacement gene must complement said auxotrophy. In this case it is preferred to use the arg2 gene of Trichoderma and auxotrophic strains to arginine. In the case of resistance to chemical compounds, the gene that confers resistance to hygromycin (hph or hpt) of Escherichia coli, or genes of resistance to the herbicide Basta can be used. In the case of the use of alternate 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 means of transformation the genes of interest that will later be useful for integrating the replacement genes into the genome of the strains of Trichoderma by means of an efficient recombination. It is important to note that the replacement vectors of the invention must contain characteristics that allow them to be amplified by conventional techniques (replication sites)., promoters, etc.) and endowed with unique cloning sites, both for the insertion of the replacement genes and for the subsequent linearization necessary to transform the Trichoderma strains. In this sense, vectors that allow the insertion of DNA fragments and that can be amplified in biological systems efficiently can be occupied. Likewise, in a preferred embodiment of the invention, the vectors of the invention use as a selection marker gene a replacement gene of the tvkl gene of Trichoderma with some associated function that may 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 comes preferentially from Trichoderma strains.

Using the gene replacement vector described above, the Trichoderma strains of the invention can be obtained by 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 prototrophic transformants generated in selective medium, and c) Corroborate the elimination of MAP kinase production.

As for 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 tvkl gene, coding for the MAP kinase Tvk1, to occupy as a replacement gene the Trichoderma gene arg2. and use auxotrophic strains to arginine.

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

The selection of the transformant 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 as the sole carbon source is preferred.

To confirm the elimination of MAP kinase synthesis in the selected transformants, this can be done by means of Western blot assays using antibodies specific for the protein and the elimination of the coding MAP kinase gene can be confirmed by means of Southern blot assays, using specific probes .

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 targeted manner, generating strains of Trichoderma with improved enzymatic characteristics that can be occupied as antagonistic organisms of Trichoderma. 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 the MAP kinases, related substances specific to these could be obtained in order to block their function in Trichoderma strains, thereby increasing the expression of lytic enzymes and thus increasing the mycoparasitic function of the strain of interest. To this end, known techniques of adsorption of biological materials using MAP kinases isolated and placed in fixed supports to capture specific related substances that can be subsequently isolated by conventional biochemical methods of purification and characterization could be employed. The use of these MAP kinase function inhibitors could be useful for obtaining strains of Trichoderma with increased antagonist 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 combating and / or preventing infections or diseases caused by phytopathogenic fungi, using multiple and diverse known techniques for its application.

As a way to illustrate 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 1. The phytopathogens R. solani AG-4 and Pythium ultimum isolated from diseased roots and seedlings were used as guests. All fungal strains were maintained on Papa dextrose agar (PDA, Difco), unless otherwise specified.

Example 2. Bacterial strains and plasmids.

Strains of Escherichia coli DH5a (Bethesda Research Laboratories) and JM103 (Invitrogen) were used for all DNA manipulations. The plasmids used were pBluescript (Stratagene) and pCB1004 (Fungal Genetics Stock Center) All the PCR products were cloned in the vector pCR2.1 (Invitrogen) The probes used for the Northern blot analysis were obtained in the following manner: Hind fragment IBamH 1.3 Kb of Tv-prb1 cDNA was obtained from plasmid pPOE A HindUUXbal fragment of 1.4 Kb of Tv-cht1 was obtained from plasmid pCOE 42. From the plasmid pSZD2 a fragment Pst was obtained / Xho \ 0.52 Kb corresponding to the Tv-bgn2 gene Two fragments corresponding to Tv-cht2 (from position 988 to 1417) and to Tv-nag1 (from position 513 to 1608) were obtained from T genomic DNA virens by PCR using oligonucleotides designed based on the sequs reported by Kim43.

Example 3. Manipulation of DNA and RNA.

The plasmid DNA was isolated using a commercial system (Qiagen). Trichoderma DNA was obtained according to a previously described procedure. 4 Total RNA was isolated using extractions with phenol-chloroform according to the procedure described by Jones 45. Southern and Northern analyzes were carried out using Hybond N + membranes (Amersham) according to the manufacturer's recommendations.

Example 4. Immunodetection.

The 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 Schgger buffer 2X 46 and boiled after adding β-mercaptoethanol (5%). The proteins were separated by SDS-PAGE in 10% gels according to Schágger and von Jagow 6. The proteins were transferred to Hybond ™ -C extra membranes (Amersham) and the detection was carried out following the instructions of the Phospho Plus ® system p42 / p44 MAP kinase (Thr / Tyr) Antibody kit (Cell Signaling Technology, Inc).

Example 5. Cloning and sequng of tvkl.

Genome DNA of T. virens Gv29-8 was used as annealed in PCR amplification reactions using primers previously described 47. The PCR product was cloned and sequd later using the Sanger 48 method with the Sequenase system (version 2.0, US Biochemichals). A fragment that showed high similarity to the pmkl gene of M. grissay 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 sequng. A Southern analysis allowed the identification of a SamHI fragment of 3.3 Kb containing the tvkl gene (see figure 1), said fragment was subcloned into the Bam site of plasmid pCB1004 (pDXG35). This fragment was sequd in its entirety and subsequently analyzed by the BLAST program using the DNA-protein data bank.

Using DNA from T. virens as annealed and the degenerate oligonucleotides reported by Xu and Hamer 47 ', several PCR amplification products were obtained. These products were subcloned into the vector pCR2.1 and sequd. After performing a computational analysis using the BlastX algorithm, one of these presented a high similarity to the pmkl gene of M. grisea. This amplification product was radioactively labeled and used in a genomic library of T. virens. Three clones were obtained that gave positive signal and one of them was selected, subcloned and sequd. A Southern blot analysis using T. Virens DNA and the tvkl gene obtained, suggests that this gene is found as a single copy in the genome of Trichoderma (figure 1).

The tvkl gene contains four exons interrupted by three introns as has been reported for other genes in this same family of kinases (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 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 mating factor ??? - 1-Mc.

The protein sequence deduced from tvkl is 360 amino acids corresponding to a theoretical molecular mass of 41.6 KDal 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. grisea, and F. oxysporum, respectively, have 95.8% identity with Tvk1, and Kss1 of S. cerevisae have 54.7%. The region between residues 58 to 160 in Tvk1 contains the sequence observed in several members of the family of MAP kinases: F-x (10) -R-E-x (72.86) -R-D-x-K-x (9) -C 49 '. This protein also contains the phosphorylated residues [T (184) -E-Y (186)] required for its phosphorylation and activation by MAP kinase 50.

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

To construct the tvkl gene replacement vector, a 1.48 Kb EcoRV / Sa / l fragment containing most of the coding region of tvkl (from amino acid 88 to the last amino acid of the protein) was replaced by a Smal fragment / 3.2 Kb EcoRV of the arg2 gene of T. virens 4. A BamHI / EcoRV fragment from pDGX35 was subcloned into the SamHI / EcoRV sites of pBluescript SK (-) to generate the pAM1 plasmid. Plasmid pAM1 was digested with EcoRV and ligated to an EcoRM / Sma1 fragment of the gene arg2 gene (pAM2). In order to obtain the C-terminal region of Tvkl, a 0.4 Kb Sa / l / BamHI 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. Transformation of fungi.

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

Example 8. Generation of null mutants of tvkl.

The interruption of tvkl was evaluated in 20 stable mutants by means of Southern analysis using as probe a BamHI / EcoRV fragment of 1.4 Kb of the plasmid pDGX35. Expecting to hybridize a 3.3 Kb fragment 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 selected transformants showed additional bands to the one corresponding to the replacement event. The transformants designated Atvk24 and Atvk133 were chosen arbitrarily for phenotypic analysis and physiological studies.

To verify that Tvkl was not produced in the mutants, total protein extracts of the wild strain of T. virens and strain Atvk24 were analyzed using a polyclonal antibody that recognizes MAP p42 / p44 kinases from animal cells (Figure 3C). As expected, two proteins of 42 KDa and 44 KDa were detected in the wild strain (Figure 3C, lane 1). In contrast, only one signal corresponding to MAP kinase of 44kDa MAP was detected in strain Atvk24 (figure 3C, lane 2). These results demonstrated that strain Atvk24 did not produce the homologue of MAP p42 kinase. Similar results were obtained when analyzing other mutants.Example 9. Analysis of submerged cultures.

Spores (1x106 spores / ml) of the wild-type strain and the mutants Atvk24 and Atvk133 were inoculated in PDB (Dextrose Potato Broth, Difco), Vogel's medium (VMS) or minimal medium (MM) 51 and incubated for 72 hours at 28 ° C. The 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 Atvkl mutants showed a reduction in their colonial growth rate and in the development of aerial hyphae in solid medium. Suspensions of conidia of the wild strain showed an intense green coloration, compared with the pale green of the conidial suspensions of the mutants. Additionally, the null mutants produced twice as many conidia as the wild strain when grown in PDA medium without significant changes in the conidiophore morphology. When the growth in liquid medium was analyzed, all the Atvkl mutants formed smaller "pellets" than those formed by the wild type strain. Surprisingly, the Atvkl mutants conidiated massively in late phases in submerged cultures (72 h), whereas 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), Atvk24 and Atvk133 mutants (two central panels) and the parental strain (Tv10.4) (right panel) after 72 hours of incubation in VMS medium. The ability to conidiate in liquid medium of the mutants seems to be independent of the culture medium used, since conidiation was observed in both VMS and PDB. Microscopic observations of samples of sporulating liquid cultures of Atvk133 showed a normal development of the conidiophore, similar to those produced in aerial hyphae (Figure 4B-C).

Example 10. Trials of simulated mycoparasitism.

Trichoderma spores (1x 106 spores / ml) were germinated and grown for 48 h in VMS. The mycelium was then collected and transferred to fresh medium. Vogel's minimum medium without carbon or nitrogen source (VM or VM-N, respectively) was used to evaluate the effect of nutrient limitation; VMS containing 0.5% cell walls of R. solani (VMSR), and Vogel's medium without nitrogen or carbon source added with 0.5% R. solani cell walls (VM-NR or VMR, respectively) was used to similar mycoparasitism conditions. 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 -70 ° C until used. For the analysis of the enzymatic activities, the culture filtrate was recovered, frozen at -20 ° C and stored at the same temperature until its use.

Northern blot analysis showed that in the wild strain the Tv-nag1 gene encoding an N-acetylglucosaminidase was expressed in carbon-free medium 3 h after the transfer and that the mRNA could not be detected at 6 h. While in the mutant the expression was detected after 3 h and this was increased at 6 h. In medium containing cell walls of R. solani 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 coding gene for analyzed kinase (Tv-cht1) was expressed in both strains in the absence of carbon but reached higher levels in Atvk24 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 carbon limitation conditions, except that this occurred before (Fig. 5A; Tv-cht1). The gene encoding the Tg-bgn2 glucanase was induced by cell walls at 6 h, but no differences were observed with the silvesire strain and the gene was not expressed in medium without carbon source (Fig. 5A; Tv-bgn2). The expression pattern of a third quifinase gene (Tv-cht2) under conditions of simulated parasiism was similar to that of TV-chtl, except that the maximum expression was reached after 24 hours. In a manner consistent with what was observed for other MRG analyzed, Atvk24 showed a much more pronounced induction for Tv-cht2 than the wild-type strain. Expression was determined under simulated conditions of mycoparasitism both in the absence and in the presence of an alternate source of carbon (Fig. 5A; Tv-prb1) or in VM-NR that does not contain ammonium (Fig. 5B; Tv-prb1) of the prb1 gene, which codes for a protease. Induction by cell walls 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 strain Atvk24 than for the wild strain. The Atvk24 mutant 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 in VM-N (Fig. 5B; Tv-prb1). In agreement with these observations, the analysis of the activities of protease gel, endochitinase, N-acetylglucosaminidase and glucanase of the culture filtrates showed up to ten times greater activity in the mutants than in the wild strain (see figure 8). Interestingly, it was observed the presence of an additional band of endochitinase activity in the null mutants under conditions of carbon limitation (see figure 8).

Example 11. Confrontation tests.

Trichoderma strains were subjected to contactless testing using R. solani as host 5. The confrontations were carried out in modified VMS-agar medium (mVMS) containing 0.75 g / l of sucrose and 0.45 g / l of NH4NO3. The mycelium of Trichoderma was collected from the zone of interaction between the fungi.

The analysis of the expression of the same set of genes was carried out in Atvk24 and wild-type strains during confrontation trials (Figure 6A). When Atvk24 overgrowed 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 to the control conditions where the mutant grew alone (T) (figure 6B). Interestingly, detectable levels of expression of all the genes used in the control (T) were observed. The accumulation of transcripts of all MRGs in Atvk24 was not only increased by the presence of the host, but reached much higher levels than for the wild strain. At the time of 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 on potato dextrose agar.

The Trichoderma strains of. The invention was subjected to direct confrontation trials using Phytophthora capsici, Sclerotium rolfsii, Rhizoctonia solani, Colletotrichum Hndemuthianum and Phytophthora citricola, as host. The confrontations were carried out in potato dextrose agar medium using the strains T. virens Gv29.8, Atvk24 and Atvk133 for 5 days under continuous light at 28 ° C. The results obtained are shown in Figure 9. As can be observed, there were no 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 assays.

The tests were carried out as previously described 42. Cotton seeds (cultivar Stoneville 112, Pedigree Seed Co) were covered with strains of Trichoderma and seeded in free medium of non-sterile soil (Metromix) infected with R. solani or P. ultimum Seeds planted in non-infected soil and seeds treated with the commercial fungicide Apron XL LS (active against P. ultimum) were used as positive controls. Surviving and healthy plants were counted at 10 days of incubation at 25 ° 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 of 0 (no symptoms) to 5 (the whole system discolored and decay) with a maximum value of 6 for dead seeds not germinated. Each treatment was performed in 6 replicates, with 10 seeds each, and the entire experiment was repeated twice.

The biocontrol capacity of Atvk24 and Atvk133 in vivo was evaluated 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 Atvk24 and Atvk133 was greater (mortalities 0% and 5%, respectively) than with the wild one (mortality 8%), the differences were not statistically significant. However, when the symptoms of disease were evaluated in the root system, significant differences were detected (Fig. 7A). The disease index was significantly lower for the mutant strains in Atvkl than in the 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 Atvk24 and Atvk133, 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 the treatment of the seeds with the commercial fungicide Apron (Fig 7B).

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LIST OF SEQUENCES. 1. SEQ.ID. DO NOT. 1. (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3316 base pairs. (B) TYPE: DNA. (C) CHAIN TYPE: simple. (D) TOPOLOGY: linear. (ii) ORIGINAL SOURCE: (A) ORGANISM: Tríchoderma virens (B) CEPA: Tríchoderma virens Gv29-8 (iii) POSITION IN THE GENOME: (A) CHROMOSOME / SEGMENT: 3 Kb BamHI / BamHI fragment (iv) CHARACTERISTICS: (A) NAME / KEY: gen tvkl (B) LOCATION: (871) .... (2165) (D) OTHER INFORMATION: activity of protein kinase activated by mitogen (MAP kinase) in its coding product. SEQ.ID. DO NOT. 1. GGCCGCCGTC TCTTATAACT AGTGGGATCC GCGCGCCGCC TGAGCCCGTC CGGCACGAAC 60 CTGTAACTCC AGGCGGGCTT GCATGCATCT TGAAGCTCCA TCTAGACCTT GGACGAAGCT 120 CCGTGCCATC AGCGTCCAAC ATTAGCCCAA ATGCAGCGTG CCAGCCTCCA TCTCTTCCAG 180 GGCCTGCAGA CACCTGTCGA GCCCCAAGCA TAGGCTCTGC GCCCCCCCTT GCCCGGCCCT 240 CCTAGCCCGC CCCCGCTGGC CCTCCTGAAT TATTGCTCGT ACAAAGCACC AACTCGCTAA 300 AGCACCTCAG CTGAGCAGAT ACAAAGACCA TAGGCCACCC CCACTTGGCC AGCACGCACC 360 TTCGACCCCG CGCAGCCAGC GACTCGTGCT TCGTGAGTGA ACTCACCGAG ACTTCCATAT 420 CATTTGCCGG AATCCCAGCT GTCTTTGTCT CTGTCGTCTT TTTCTCGTCA TACATTGTTC 480 CTTATTCCGA GGTTGAGCGT ATTTCTCGTC TCTCCAACTT CTCTTCTTCG CCCCAAGAAC 540 AAGCGTCAGC AGCTTCTTCT GCATCTACGC AGCCTCCCAT TTACCCACCT CTGTTCGTAT 600 GCCGCGATTC CCCTCGTCTG CTGTGGGAAG AACCTCAAGC TGACTCCGTC TAGTTCGTGG 660 AAGTGGAGTT GCCGTTGATT GGGTTAACCG TATATTAGCC TGCGGGCATC GACGAGAATC 720 TCCAATCGCA TACGGAGAGG CCTTTGTTCA CCCTTTCCCG CCAGGTGCTG CGTTGCGTTT 780 CTCTACGCTC TCGAGCCCGC CATCGACATC CGCCCACAGC CACCTACCTC CCAGCCTCAA 840 GTCGCGCAAG AGCGCC CAAG CCCGACCATC ATGTCTCGGT CGAACCCCCC CAATAATGCG 900 TCGGCGTCGC GCAAAATCTC CTTCAACGTC AGCGAGCAGT ATGACATTCA GGATGTTGTT 960 GGTGAGGGAG CCTACGGAGT TGTTTGGTAA GTTTCTGGTT CCTGGGTCTA CCTACGTCTT 1020 AGTTGTAGCT GCACACGCCG TTTTCTAACA GCACTTGGGC GTCAACAGCT CTGCCATTCA 1080 CAAGCCGTCG GGACAAAAGG TTGCCATCAA GAAGATTACC CCCTTCGACC ACTCCATGTT 1140 CTGTCTGAGA ACTCTGCGAG AGATGAAGCT GCTGCGCTAT TTCAACCACG AAAACATCAT 1200 CTCCATCCTT GATATCCAGA AGCCCCGAAG CTACGATAGC TTCAACGAGG TCTATCTTAT 1260 TCAGGTACGT CATCCCACGG ACTCCACAAG CCCTCTCTAC CCGTCATCCG TACATGTGCT 1320 CACCGTAGAA CTTGCGTGTA GGAACTCATG GAGACGGACA TGCACAGAGT CATTCGAACC 1380 CAGGACCTCT CCGACGACCA CTGCCAGTAC TTCATCTACC AGACGCTGCG AGCGCTCAAG 1440 GCCATGCACT CGGCAAACGT TCTGCACCGA GATCTCAAGC CGTCCAACCT CTTGCTAAAT 1500 GCCAACTGTG ATCTGAAAGT CTGCGACTTC GGTCTGGCCC GATCTGCTGC TTCCCAGGAG 1560 GACAACTCAG GCTTCATGAC GGAATACGTT GCCGCGAGAT GGTACCGTGC TCCCGAGATC 1620 ATGCTGACCT TTAAGGAGTA CACCAAGGCC ATTGACGTCT GGTCTGTGGG CTGCATCCTG 1680 GCTGAGATGC TCAGCGGAAA GCCTCTGTTC CCCGGAAAGG ACTGTAAGTT GGCACTCACG 1740 CCCAAGCTCC ATGTACGCAA ATGAATGCTA ACGCAAGGAC CTTTCCAGAC CACCACCAGC 1800 TGACTCTGAT CCTTGATGTG CTCGGCACAC CGACCATGGA GGACTACTAT GGCATCAAGT 1860 CTCGACGAGC GAGAGAATAC ATCCGCTCGC TGCCCTTCAA GAAGAAGGTG CCATTCCGCA 1920 CCCTCTTCCC CAAGACTTCT GACTTGGCAT TGGACCTCTT GGAGAAACTG CTTGCGTTCA 1980 ACCCCGTAAA GCGAATCACG GTGGAGGATG CTCTCAAGCA CCCCTACCTC GAGCCATATC 2040 ACGACCCTGA TGACGAGCCA ACCGCGCCTC CGATCCCGGA GGAGTTCTTC GATTTCGACA 2100 AGCACAAAGA TACCCTGAGC AAGGAGCAGC TGAAGCAACT GATTTACCAA GAGATTATGC 2160 GGTAAAAGAT TGAGCTCGTG TTTTCAGAGC TCGATGAAGC ACATTGTTGG AGGAGATTGT 2220 AGCCATTTAT AAACAAGGTG AAGAAAAATG GAAGAATCTG TGGAGGATAT AGAGCAATGA 2280 GATTGGCAAA GAAAGGATAT TAGTCTTGGC CGGGCCAAGA TGAGCGGATA GCAAAGCAAG 2340 GAGGAACGAG ACGAGATATG AAACGAATAA ATACACAGGC CTCGGATGGC TCCTTTTTTT 2400 TCCTTTTCAC TTTGGTCACT TTTGCGGGGT TTCTTTAACT ACTTAGCCTG CGCATCTCAT 2460 TGAATACACG CACACATGGT CGATGATATA TATCCCTACA GAGTAGATTA TACTACGAGT 2520 ATATTTAGCA TCAAAAGGCT GGTCCCGGCC AAGGTGCGGG GCGAGTGTCG CGATCGACAT 2580 GGACGGGCTC TGTTTTGCAG TGGCCTGTTG CTCATAGGCC AAACCAATGG CAGCATGACT 2640 TGGCCCTGGC AAACCCCACC TCAGAGGTAA AGCATGGATC AACAGATACC TGTCGACAGC 2700 TAAATTGCTG CCCGTGACAG GCCGTTGCCG TTGTCCTTAG CACGTCGTTT TAGGTGTCTC 2760 GACTTGCGCA GGTACACGCA GCTTCTGCAC CTTGGATTTT CATTACGCCG CTGTTTTCTT 2820 TCTCTCACTC TTTCGGCGTC GCCCATCATC TCCGGCTTCT TTTTGCCCTA TTCCATGGCC 2880 GTCCAGCTTC CATCGCTCGG CTGGCGGGTC TCGTCTTTGG ATACATCGTA ACTGCTCGCG 2940 GCTGCTTACC GCCATACCGC AATTGAACAT TCTGCCAGCT CATACGTAGT ACTACCTACC 3000 TAGTAGTACA GAAGGATTAC CAACCTGCTG TGCCTGCGAC CTGCTGCATA CCTACTGGGT 3060 AATACTTCTA GGGACGGGCA AGATACTTGA CCTCCGGCTT ATACCACGAG CTGCAGGGCT 3120 GCTTACACGC CAGAAGCCAA CGGCAAAAAG CTGGTAGCAA TGTGGACGGG CCTGATGCGG 3180 CGCTTCTCGA CCGAAGAAAC GCGCGTTGGC AGCGAGTTTG ACGAAGCCGC CGACAGCCAC 3240 GGCCACCTCA AGGACGGCAT CAACGATGTC TACACGCCGA CGCTGCGCAC GCTGAGCCCC 3300 TTCCGGCCGC CGCCGC 3316 2. SEQ.ID. DO NOT. 2. (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3316 base pairs. (B) TYPE: DNA. (C) CHAIN TYPE: simple. (D) TOPOLOGY: linear. (ii) ORIGINAL SOURCE: (A) ORGANISM: Trichoderma virens (B) CEPA: Trichoderma virens Gv29-8 (iii) POSITION IN THE GENOME: (A) CHROMOSOME / SEGMENT: 3 Kb BamHI / BamHI fragment (iv) CHARACTERISTICS: (A) NAME / KEY: gen tvkl (B) LOCATION: (871) .... (2165) (D) OTHER INFORMATION: activity of protein kinase activated by mitogen (MAP kinase) in its coding product. (A) NAME / KEY: CDS (B) LOCATION: (871) .... (986) (A) NAME / KEY: Intron (B) LOCATION: (987) .... (1068) (A) NAME / KEY: CDS (B) LOCATION: (1069) .... (1264) (A) NAME / KEY: Intron (B) LOCATION: (1265) .... (1341) (A) NAME / KEY: CDS (B) LOCATION: (1342) .... (1724) (A) NAME / KEY: Intron (B) LOCATION: (1725) .... (1788) (A) NAME / KEY: CDS (B) LOCATION : (1789) .... (2165) (A) NAME / KEY: POLI-A (B) LOCATION: (2366) .... (2371) SEQ.ID. DO NOT. 2. GGCCGCCGTC TCTTATAACT AGTGGGATCC GCGCGCCGCC TGAGCCCGTC CGGCACGAAC 60 CTGTAACTCC AGGCGGGCTT GCATGCATCT TGAAGCTCCA TCTAGACCTT GGACGAAGCT 120 CCGTGCCATC AGCGTCCAAC ATTAGCCCAA ATGCAGCGTG CCAGCCTCCA TCTCTTCCAG 180 GGCCTGCAGA CACCTGTCGA GCCCCAAGCA TAGGCTCTGC GCCCCCCCTT GCCCGGCCCT 240 CCTAGCCCGC CCCCGCTGGC CCTCCTGAAT TATTGCTCGT ACAAAGCACC AACTCGCTAA 300 AGCACCTCAG CTGAGCAGAT ACAAAGACCA TAGGCCACCC CCACTTGGCC AGCACGCACC 360 TTCGACCCCG CGCAGCCAGC GACTCGTGCT TCGTGAGTGA ACTCACCGAG ACTTCCATAT 420 CATTTGCCGG AATCCCAGCT GTCTTTGTCT CTGTCGTCTT TTTCTCGTCA TACATTGTTC 480 CTTATTCCGA GGTTGAGCGT ATTTCTCGTC TCTCCAACTT CTCTTCTTCG CCCCAAGAAC 540 AAGCGTCAGC AGCTTCTTCT GCATCTACGC AGCCTCCCAT TTACCCACCT CTGTTCGTAT 600 GCCGCGATTC CCCTCGTCTG CTGTGGGAAG AACCTCAAGC TGACTCCGTC TAGTTCGTGG 660 AAGTGGAGTT GCCGTTGATT GGGTTAACCG TATATTAGCC TGCGGGCATC GACGAGAATC 720 TCCAATCGCA TACGGAGAGG CCTTTGTTCA CCCTTTCCCG CCAGGTGCTG CGTTGCGTTT 780 CTCTACGCTC TCGAGCCCGC CATCGACATC CGCCCACAGC CACCTACCTC CCAGCCTCAA 840 GTCGCGCAAG AGCGCCCAAG CCCGACCATC ATG TCT CGG TCG AAC CCC CCC 891 Met Ser Arg Ser Asn Pro Pro 1 5 AAT AAT GCG TCG GCG TCG CGC AAA ATC TCC TTC AAC GTC AGC GAG CAG 939 Asn Asn Wing Ser Wing Ser Arg Lys lie Ser Phe Asn Val Ser Glu Gln 10 15 20 TAT GAC ATT CAG GAT GTT GTG GGT GAG GGA GCC TAC GGA GTT TGT TG 986 Tyr Asp lie Gln Asp Val Val Gly Glu Gly Wing Tyr Gly Val Val Cys 25 30 35 GTAAGTTTCT GGTTCCTGGG TCTACCTACG TCTTAGTTGT AGCTGCACAC GCCGTTTTCT 1046 AACAGCACTT GGGCGTCAAC AG C TCT GCC ATT CAC AAG CCG TCG GGA CAA AAG 1099 Ser Ala lie His Lys Pro Ser Gly Gln Lys 40 45 GTT GCC ATC AAG AAG ATT ACC CCC TTC GAC CAC TCC ATG TTC TGT CTG 1147 Val Ala lie Lys Lys lie Thr Pro Phe Asp His Ser Met Phe Cys Leu 50 55 60 65 AGA ACT CTG CGA GAG ATG AAG CTG CTG CGC TAT TTC AAC CAC GAA AAC 1195 Arg Thr Leu Arg Glu Met Lys Leu Leu Arg Tyr Phe Asn His Glu Asn 70 75 80 ATC ATC TCC ATC CTT GAT ATC CAG AAG CCC CGA AGC TAC GAT AGC TTC 1243 lie lie Ser lie Leu Asp lie Gln Lys Pro Arg Ser Tyr Asp Ser Phe 85 90 95 AAC GAG GTC TAT CTT ATT CAG GTACGTCATC CCACGGACTC CACAAGCCCT 1294 Asn Glu Val Tyr Leu lie Gln 100 CTCTACCCGT CATCCGTACA TGTGCTCACC GTAGAACTTG CGTGTAG GAA CTC ATG 1350 Glu Leu Met 105 GAG ACG GAC ATG CAC AGA GTC ATT CGA ACC CAG GAC CTC TCC GAC GAC 1398 Glu Thr Asp Met His Arg Val lie Arg Thr Gln Asp Leu Ser Asp Asp 110 115 120 CAC TGC CAG TAC TTC ATC TAC CAG ACG CTG CGA GCG CTC AAG GCC ATG '1446 His Cys Gln Tyr Phe lie Tyr Gln Thr Leu Arg Ala Leu Lys Wing Met 125 130 135 CAC TCG GCA AAC GTT CTG CAC CGA GAT CTC AAG CCG TCC AAC CTC TTG 1494 His Ser Wing Asn Val Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu 140 145 150 155 CTA AAT GCC AAC TGT GAT CTG AAA GTC TGC GAC TTC GGT CTG GCC CGA 1542 Leu Asn Wing Asn Cys Asp Leu Lys Val Cys Asp Phe Gly Leu Wing Arg 160 165 170 T CT GCT GCT TCC CAG GAG GAC AAC TCA GGC TTC ATG ACG GAA TAC GTT 1590 Ser Wing Wing Gln Glu Asp Asn Ser Gly Phe Met Thr Glu Tyr Val 175 180 185 GCC GCG AGA TGG TAC CGT GCT CCC GAG ATC ATG CTG ACC TTT AAG GAG 1638 Ala Ala Arg Trp Tyr Arg Ala Pro Glu lie Met Leu Thr Phe Lys Glu 190 195 200 TAC ACC AAG GCC ATT GAC GTC TGG TCT GTG GGC TGC ATC CTG GCT GAG 1686 Tyr Thr Lys Ala lie Asp Val Trp Ser Val Gly Cys lie Leu Wing Glu 205 210 215 ATG CTC AGC GGA AAG CC CTG TTC CCC GGA AAG GAC T GTAAGTTGGC ACTC 1737 Met Leu Ser Gly Lys Pro Leu Phe Pro Gly Lys Asp Tyr 220 225 230 ACGCCCAAGC TCCATGTACG CAAATGAATG CTAACGCAAG GACCTTTCCA G AC CAC 1793 His CAC CAG CTG ACT CTG ATC CTT GAT GTG CTC GGC ACA CCG ACC ATG GAG 1841 His Gln Leu Thr Leu lie Leu Asp Val Leu Gly Thr Pro Thr Met Glu 235 240 245 GAC TAC TAT GGC ATC AAG TCT CGA CGA GCG AGA GAA TAC ATC CGC TCG 1889 Asp Tyr Tyr Tyr Gly lie Lys Ser Arg Arg Wing Arg Glu Tyr lie Arg Ser 250 255 260 265 CTG CCC TTC AAG AAG AAG GTG CCA TTC CGC ACC CTC TTC CCC AAG ACT 1937 Leu Pro Phe Lys Lys Lys Val Pro Phe Arg Thr Leu Phe Pro Lys Thr 270 275 280 TCT GAC TTG GCA TTG GAC CTC TTG GAG AAA CTG CTT GCG TTC AAC CCC 1985 Be Asp Leu Ala Leu Asp Leu Leu Glu Lys Leu Leu Ala Phe Asn Pro 285 290 295 GTA AAG CGA ATC ACG GTG GAG GAT GCT CTC AAG CAC CCC TAC CTC GAG 2033 Val Lys Arg lie Thr Val Glu Asp Ala Leu Lys His Pro Tyr Leu Glu 300 305 310 CCA TAT CAC GAC CCT GAT GAC GAG CCA ACC GCG CCT CCG ATC CCG GAG 2081 Pro Tyr His Asp Pro Asp Asp Glu Pro Thr Wing Pro Pro lie Glu 315 320 325 GAG TTC TTC GAT TAC GAC AAG CAC AAA GAT ACC CTG AGC AAG GAG CAG 2129 Glu Phe Phe Asp Phe Asp Lys His Lys Asp Thr Leu Ser Lys Glu Gln 330 335 340 345 CTG AAG CAA CTG ATT TAC CAA GAG ATT ATG CGG TAA AAGATTGAGC 2175 Leu Lys Gln Leu lie Tyr Gln Glu lie Met Arg 350 355 TCGTGTTTTC AGAGCTCGAT GAAGCACATT GTTGGAGGAG ATTGTAGCCA TTTATAAACA 2235 AGGTGAAGAA AAATGGAAGA ATCTGTGGAG GATATAGAGC AATGAGATTG GCAAAGAAAG 2295 GATATTAGTC TTGGCCGGGC CAAGATGAGC GGATAGCAAA GCAAGGAGGA ACGAGACGAG 2355 ATATGAAACG AATAAATACA CAGGCCTCGG ATGGCTCCTT TTTTTTTCCTT TTCACTTTGG 2415 TCACTTTTGC GGGGTTTCTT TAACTACTTA GCCTGCGCAT CTCATTGAAT ACACGCACAC 2475 ATGGTCGATG ATATATATCC CTACAGAGTA GATTATACTA CGAGTATATT TAGCATCAAA 2535 AGGCTGGTCC CGGCCAAGGT GCGGGGCGAG TGTCGCGATC GACATGGACG GGCTCTGTTT 2595 TGCAGTGGCC TGTTGCTCAT AGGCCAAACC AATGGCAGCA TGACTTGGCC CTGGCAAACC 2655 CCACCTCAGA GGTAAAGCAT GGATCAACAG ATACCTGTCG ACAGCTAAAT TGCTGCCCGT 2715 GACAGGCCGT TGCCGTTGTC CTTAGCACGT CGTTTTAGGT GTCTCGACTT GCGCAGGTAC 2775 ACGCAGCTTC TGCACCTTGG ATTTTCATTA CGCCGCTGTT TTCTTTCTCT CACTCTTTCG 2835 GCGTCGCCCA TCATCTCCGG CTTCTTTTTG CCCTATTCCA TGGCCGTCCA GCTTCCATCG 2895 CTCGGCTGGC GGGTCTCGTC TTTGGATACA TCGTAACTGC TCGCGGCTGC TTACCGCCAT 2995 ACCGCAATTG AACATTCTGC CAGCTCATAC GTAGTACTAC CTACCTAGTA GTACAGAAGG 3015 ATTACCAACC TGCTGTGCCT GCGACCTGCT GCATACCTAC TGGGTAATAC TTCTAGGGAC 3075 GGGCAAGATA CTTGACCTCC GGCTTATACC ACGAGCTGCA GGGCTGCTTA CACGCCAGAA 3135 GCCAACGGCA AAAAGCTGGT AGCAATGTGG ACGGGCCTGA TGCGGCGCTT CTCGACCGAA 3195 GAAACGCGCG TTGGCAGCGA GTTTGACGAA GCCGCCGACA GCCACGGCCA CCTCAAGGAC 3255 GGCATCAACG ATGTCTACAC GCCGACGCTG CGCACGCTGA GCCCCTTCCG GCCGCCGCCG 3315 C 3316 3. SEQ.ID. DO NOT. 3. (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 356 amino acids. (B) TYPE: amino acids (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: Protein (iii) ORIGINAL SOURCE: (A) ORGANISM: Trichoderma virens (B) CEPA: Trichoderma virens Gv29-8 (iv) CHARACTERISTICS: (A) ) NAME / KEY: Protein Tvk1 (D) OTHER INFORMATION: biological activity of protein kinase activated by mitogen (MAP kinase).

SEQ.ID. DO NOT. 3. Met Being Arg Being Asn Pro Pro Asn Asn Wing Being Wing Being Arg Lys lie 1 5 10 15 Being Phe Asn Val Being Glu Gln Tyr Asp He Gln Asp val Val Gly Glu Gly Wing Tyr Gly Val Val Cys Ser Ala lie His Lys Pro Ser Gly Gln 35 40 45 Lys Val Ala lie Lys Lys lie Thr Pro Phe Asp His Ser Met Phe Cys 50 55 60 Leu Arg Thr Leu Arg Glu Met Lys Leu Leu Arg Tyr Phe Asn His Glu 65 70 75 80 Asn lie lie Be lie Leu Asp lie Gln Lys Pro Arg Ser Tyr Asp Ser 85 90 95 Phe Asn Glu Val Tyr Leu lie Gln Glu Leu Met Glu Thr Asp Met His 100 105 110 Arg Val lie Arg Thr Gln Asp Leu Ser Asp Asp His Cys Gln Tyr Phe 115 120 125 lie Tyr Gln Thr Leu Arg Ala Leu Lys Ala Met His Ser Ala Asn Val 130 135 140 Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Ala Asn Cys 145 150 155 160 Asp Leu Lys Val Cys Asp Phe Gly Leu Wing Arg Ser Wing Wing Ser Gln 165 170 175 Glu Asp Asn Ser Gly Phe Met Thr Glu Tyr Val Wing Wing Arg Trp Tyr 180 185 190 Arg Ala Pro Glu lie Met Leu Thr Phe Lys Glu Tyr Thr Lys Ala lie 195 200 205 Asp Val Trp Ser Val Gly Cys Lie Leu Ala Glu Met Leu Ser Gly Lys 210 215 220 Pro Leu Phe Pro Gly Lys Asp Tyr His His Gln Leu Thr Leu lie Leu 225 230 235 240 Asp Val Leu Gly Thr Pro Thr Met Glu Asp Tyr Tyr Gly lie Lys Ser 245 250 255 Arg Arg Ala Arg Glu Tyr lie Arg Ser Leu Pro Phe Lys Lys Lys Val 260 265 270 Pro Phe Arg Thr Leu Phe Pro Lys Thr Ser Asp Leu Ala Leu Asp Leu 275 280 285 Leu Glu Lys Leu Leu Wing Phe Asn Pro Val Lys Arg lie Thr Val Glu 290 295 300 Asp Ala Leu Lys His Pro Tyr Leu Glu Pro Tyr His Asp Pro Asp Asp 305 310 315 320 Glu Pro Thr Ala Pro Pro Lie Pro Glu Glu Phe Phe Asp Phe Asp Lys 325 330 335 His Lys Asp Thr Leu Ser Lys Glu Gln Leu Lys Gln Leu lie Tyr Gln 340 345 350 Glu lie Met Arg

Claims (60)

1. Claims 1. A strain of Trichoderma over-producing lytic enzymes for the fight against phytopathogenic fungi, characterized because it has lost the ability to produce at least one MAP kinase. 2. The strain of claim 1, characterized in that at least the function of a gene coding for a MAP kinase has been eliminated in its genome. 3. The strain of claim 2, characterized in that it is selected from the group comprising any Trichoderma species. 4. The strain of claim 3, characterized in that the strain is Trichoderma virens. 5. The strain of claim 4, characterized in that the deleted gene coding for MAP kinase has the DNA sequence corresponding to SEQ ID NO: 2. 6. The strain of claim 5, characterized in that it is the strain Trichoderma virens3tvk24. 7. A fungicidal composition for controlling and combating phytopathogenic fungi, characterized in that it comprises a Trichoderma strain selected from the group comprising the strains of claim 1 to 7. The composition of claim 8, characterized in that the Trichoderma strain is the strain of claim 1. 9. The composition of claim 8, characterized in that the Trichoderma strain is the strain of claim 6. 10. The composition of claim 8, characterized in that the Trichoderma strain is the strain of 7. The composition of claim 8 to 11, characterized in that the Trichoderma strain is in any of its vegetative growth stages, mycelia and / or spores. 12. The composition of claim 12, characterized in that the Trichoderma strain is found as spores. 13. A MAP kinase of Trichoderma, characterized in that it has the amino acid sequence corresponding to SEQ ID NO: 3. 14. A DNA molecule, characterized in that it encodes the MAP kinase of claim 14. 15. The DNA molecule of claim 15, characterized in that it has the sequence corresponding to SEQ ID NO: 2. 16. A recombinant vector characterized in that contains the DNA molecule of claim 15. 17. A recombinant vector characterized in that it contains the DNA molecule of claim 16. 18. An organism transformed with the recombinant vector of claim 17. 19. An organism transformed with the recombinant vector of claim 18. 20. 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 the same. 21. A gene replacement vector to suppress the ability of Trichoderma to produce at least one MAP kinase, characterized in that it comprises: a) A replacement gene that allows to identify transformants where a gene coding for a MAP kinase of Trichoderma is replaced, b) A non-coding fragment that is naturally outside the coding region of the MAP kinase gene 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 MAP kinase gene at its 3 'end, flanking the 3' end of the replacement gene. 22. The vector of claim 22, characterized in that the replacement gene comes from strains of Trichoderma. The vector of claim 22, characterized in that the replacement gene is selected from the group comprising the genes hph and hpt from E. coli, genes from resistance to the herbicide Basta, the amdS gene from Aspergillus nidulans and the arg2 gene from Trichoderma . 24. The vector of claim 24, characterized in that the replacement gene is arg2. 25. The vector of claim 22 to 25, characterized in that the fragment of part b) comprises the sequence of nucleotide no. 1 to nucleotide no. 1210 of SEQ ID NC. 26. The vector of claim 22 to 26, characterized in that the fragment of part c) comprises the sequence of nucleotide no. 2692 to nucleotide no. 5892 of SEQ ID NO: 1. 27. An organism transformed with the gene replacement vector of claim 22 to 27. 28. An organism transformed with the gene replacement vector of claim 25. 29. An organism transformed with the gene replacement vector of claim 27. 30. A method for obtaining strains of Trichoderma over-producing lytic enzymes for phytopathogenic fungi, characterized in that it comprises the steps of: a) Suppressing the ability of Trichoderma to produce at least one MAP kinase, b) Select over-producing strains of lytic enzymes, and c) Corroborate the suppression of MAP kinase production. 31. The method of claim 31, characterized in that the step of part a) is carried out by eliminating the coding sequence for MAP kinase. 32. The method of claim 32, characterized in that the deletion of the sequence is carried out by replacement by homologous recombination. 33. The method of claim 33, characterized in that the homologous recombination is performed by transforming a strain of Trichoderma with the vector of claim 22 to 27. 34. The method of claim 34, characterized in that the vector is that described in claim 24. 35. The method of claim 34, characterized in that the vector is that described in claim 25. 36. 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. 37. The method of claim 37, characterized in that the gene coding for MAP kinase is the gene corresponding to SEQ ID NO: 2. 38. The method of claim 31 to 38, characterized in that the Trichoderma strain is Trichoderma virens. 39. The method of claim 31, characterized in that the step of part b) is carried out by selecting the transformants generated in a selective medium. 40. The method of claim 40, characterized in that the selection of the prototrophic strains is carried out in a Vogel minimum medium. 41. The method of claim 31, characterized in that the suppression of MAP kinase production is corroborated using antibodies specific for MAP kinase by Western blot assays. 42. The method of claim 31, characterized in that the suppression of MAP kinase production is corroborated using probes specific for the gene encoding MAP kinase by Southern blot assays. 43. A method for increasing the expression of lytic enzymes of Trichoderma for the control of phytopathogenic fungi, characterized in that it comprises the steps of: a) Suppressing the ability of Trichoderma to produce at least one MAP kinase, b) Cultivating 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 lytic enzymes. 44. The method of claim 44, characterized in that the deletion is carried out by eliminating the coding sequence for the MAP kinase by a replacement gene by homologous recombination. 45. The method of claim 45, characterized in that the replacement gene comes from strains of Trichoderma. 46. The method of claim 45, characterized in that the replacement gene is selected from the group comprising the genes hph and hpt of E. coli, genes of resistance to the herbicide Basta, the amdS gene of Aspergillus nidulans and the arg2 gene of Trichoderma. 47. The method of claim 47, characterized in that the replacement gene is arg2. 48. 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. 49. The method of claim 49, characterized in that the gene coding for MAP kinase is the gene corresponding to SEQ ID NO: 2. 50. The method of claim 47 to 50, characterized in that the Trichoderma strain is Trichoderma virens. 51. The method of claim 51, characterized in that cell walls of phytopathogenic fungi are additionally added in step b). 52. The method of claim 47 to 52, characterized in that the increase in expression is corroborated using specific probes for the genes encoding Trichoderma lytic enzymes by Northern blot assays. 53. 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. 54. The method of claim 54, characterized in that the Trichoderma strain is the strain of claim 1. 55. The method of claim 54, characterized in that the Trichoderma strain is the strain of claim 6. 56 The method of claim 54, characterized in that the Trichoderma strain is the strain of claim 7. 57. 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 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. 58. The method of claim 58, characterized in that the composition is as described in claim 9. 59. The method of claim 58, characterized in that the composition is as described in claim 10. 60. The method of claim 58 , characterized in that the composition is that described in claim 11.