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WO2006129998A1 - Souches ameliorees de trichoderma utilisees en tant qu’agents de lutte biologique, procede d’obtention de celles-ci et leur utilisation dans la lutte contre des maladies entrainees par les champignons phytopathogenes - Google Patents

Souches ameliorees de trichoderma utilisees en tant qu’agents de lutte biologique, procede d’obtention de celles-ci et leur utilisation dans la lutte contre des maladies entrainees par les champignons phytopathogenes Download PDF

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WO2006129998A1
WO2006129998A1 PCT/MX2005/000114 MX2005000114W WO2006129998A1 WO 2006129998 A1 WO2006129998 A1 WO 2006129998A1 MX 2005000114 W MX2005000114 W MX 2005000114W WO 2006129998 A1 WO2006129998 A1 WO 2006129998A1
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gene
trichoderma
strain
map kinase
strains
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PCT/MX2005/000114
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Spanish (es)
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Alfredo Herrera Estrella
Artemio Mendoza Mendoza
Carlos CORTÉS PENAGOS
Pedro MARTÍNEZ HERNÁNDEZ
Vianey Olmedo Monfil
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Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38—Trichoderma

Definitions

  • Trichoderma Improved strains of Trichoderma as biocontrol agents, methods for obtaining them and their use for the control of diseases caused by phytopathogenic fungi.
  • the present invention relates to the development of modified microorganisms useful as biocontrol agents, specifically to obtain improved Trichoderma strains useful for the treatment of diseases caused by phytopathogenic fungi.
  • Trichoderma hydrolytic enzymes such as chitinases, ⁇ -1-3 glucanases, ⁇ -1-6 glucanases, and proteases, facilitate host 1 penetration.
  • Trichoderma species have been used as potent biocontrol agents against a variety of phytopathogenic fungi 2 .
  • Trichoderma asperellum T34 (2) isolates obtained from composts prepared from the organic fraction of market waste, sewage sludge and garden waste 3 , improved strains such as T. viride Li 49 4 , T. have been used.
  • harzianum YC459 5 , T-1-R9 6 virid T, or mutants deficient in the production of viridiol but retaining its effectiveness as biocontrol agents, such as T. virens TV-111, TV-115 and TV-109 7 .
  • Trichoderma spores are known in the technical field, for example by fermentative culture in bioreactors, subsequent lyophilization and filtration of the material obtained in filters that only allow the spores to pass, or by inoculation of wheat flour with Trichoderma species and subsequent dilution using diatomaceous earth.
  • these methods are low productivity, mainly due to the low spore production characteristics of the species of
  • Tr ⁇ choderma used so far.
  • Trichoderma species have been obtained that can coexist in culture by transforming some of the strains by radiation of T. longibratum by cobalt 8 , biocontrol compositions comprising two or more Trichoderma strains of the species asperellum, atroviride and inhamatum 9 , or compositions with synergistic effects of chitin degradation containing Trichoderma strain proteins in conjunction with bacterial proteins, for example Streptomyces 10 .
  • Trichoderma induces the expression of proteins of interest, such as lytic enzymes that destroy the cell wall of pathogenic fungi under certain culture conditions, to subsequently isolate the genetic sequences encoding such proteins and use them to transform selected strains of Trichoderma through expression vectors.
  • proteins of interest such as lytic enzymes that destroy the cell wall of pathogenic fungi under certain culture conditions
  • Trichoderma's antagonistic effect is due to the joint expression of several lytic enzymes, encoded by several genes that have been designated as mycoparasitism-related genes (MRGs). Due to this effect, the genetic manipulation methods aimed at the isolation of lithic enzyme genes to subsequently obtain transformed strains of Trichoderma over-producing said enzymes, only achieve the increased expression of at least two isolated genes at the same time, with which obtaining improved strains of Trichoderma for use in the fight of plant pathogenic fungi, is quite limited.
  • MRGs mycoparasitism-related genes
  • FIG. 1 Southern blot analysis of the T. virens tvkl gene.
  • the restriction enzymes used are shown in the upper part of the figure.
  • the probe used was the BamHI fragment corresponding to 3.2 Kb and containing the entire open reading frame.
  • FIG. 1 Tvkl gene sequence The possible start and end of the translation is shown. The donor and acceptor sequences of introns are indicated with underlined letters.
  • Figure 3. Strategy for the interruption of the tvkl gene and the analysis of transformants.
  • A Schematic representation of the replacement of Tvkl. The thick arrows represent the coding regions of tvkl and arg2. The lines represent regions 5 1 and 3 1 of the tvkl gene. Cross-linking events are indicated by dotted lines.
  • EV EcoRV
  • B BamHI
  • S Sa / I
  • C C / al
  • A Apa ⁇ ).
  • MAPK is a recombinant MAP kinase used as a positive control.
  • FIG. 1 Expression of MRGs in simulated mycoparasitism.
  • TO Northern type analysis of the Tv-prb ⁇ , Tv-nag1, Tv-cht1 and Tv-bgn2 genes under carbon-limiting conditions with and without R. solani walls in The wild strain and the mutant ⁇ tvk24. The numbers indicate the induction time.
  • B Northern type analysis of Tv-prb1 under conditions of nitrogen limitation and simulated mycoparasitism. The symbols (+) and
  • (-) denote the presence and absence of R solani cell walls, respectively. Fifteen micrograms of total RNA were loaded in each lane. The 28S ribosomal gene was used as a control.
  • FIG. 6 Expression of mycoparasitism-related genes (MRGs) in a confrontation with R solani.
  • MRGs mycoparasitism-related genes
  • A Schematic representation of the interaction between T. virens and R. solani. T indicates the strain of T. virens growing alone in boxes with VMS, TR is the strain of T. virens growing in the presence of R solani, and the interaction zone is indicated by the color black.
  • B Expression of MRGs in strains of T. virens. The samples were collected when the T. virens strains overgrown the colony of R solani. The probes used for the Northern type analysis were the same indicated in Figure 5. 15 ⁇ g of RNA were loaded in each lane.
  • FIG. 7 Biocontroller activity of wild strains, ⁇ tvk24 and ⁇ tvk133.
  • TO Disease index in the root system of cotton plants infected with R solani.
  • B Percentage of surviving cotton seedlings challenged with P. ultimum. The columns that appear with
  • FIG. 8 Effect of Tvk1 on the enzymatic activity produced in supernatants of wild T. virens (wt) and ⁇ tvk24 under simulated mycoparasitism.
  • the arrow indicates the activity of Prbl Total glucanase (C) and chitinase (D) activities were detected under simulated mycoparasitism in the absence of a carbon source.
  • FIG. 1 Direct confrontation of Trichoderma with various phytopathogenic fungi as hosts.
  • Phytophthora capsici A
  • Sclerotium rolfsii B
  • Rhizoctonia solani C
  • Colletotrichum lindemuthianum D
  • Tr ⁇ choderma overproducers of several lytic enzymes Provide strains of Tr ⁇ choderma overproducers of several lytic enzymes to be used as antagonists of phytopathogenic fungi
  • Tr ⁇ choderma overproducers of several lytic enzymes Provide strains of Tr ⁇ choderma overproducers of several lytic enzymes and at the same time with great capacity of spore production to be used as antagonists of phytopathogenic fungi,
  • Trichoderma genes coding for MAP kinases related to the establishment of the parasitic relationship of Trichoderma and its hosts,
  • - Provide a genetic construct for the transformation of fungal cells, which include the polynucleotide sequences described in this invention, - Provide proteins with MAP kinase activity related to the establishment of the parasitic relationship of Trichoderma and its hosts,
  • the parental strain T. virens Tv10.4 has been deposited in the American Type Culture Collection on December 20, 2004 with the number PTA-6474.
  • the strain T. virens ⁇ tvk24 was deposited in the American Type Culture Collection on December 20, 2004 with the number PTA-6474 and the strain T. virens ⁇ tvk133 was deposited in the American Type Culture Collection on December 20, 2004 with the number PTA-6474.
  • the units, prefixes and symbols may be mentioned using their abbreviation accepted by Sl.
  • the nucleic acid sequence is written from left to right in the 5 'to 3' orientation;
  • the amino acid sequence is written from left to right in the orientation of the amino edge to the carboxy terminal edge.
  • the numerical ranges are inclusive of the numbers that define the range and include each of the whole numbers that define the range.
  • Amino acids can be designated either by their known three-letter symbols or by their single-letter symbols that have been recommended by Ia
  • nucleotides can be designated by their commonly accepted unique letter code.
  • the terms of software, electrical and electronic are used as defined in the New IEEE Dictionary for Electrical and Electronic Terms 12 .
  • amplification is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the sequence of a nucleic acid using at least one nucleic acid sequence as the initial reference frame.
  • the amplification systems include the polymerase chain reaction (PCR), the ligase dependent chain reaction (LCR), the nucleic acid sequence dependent amplification (NASBA, Canteen, Mississauga , Ontario), the Q-Beta Replicase system, the Transcription-dependent Amplification System (TAS), and the Strand Displacement Amplification -SDA, for its acronym in English) 13 .
  • PCR polymerase chain reaction
  • LCR ligase dependent chain reaction
  • NASBA nucleic acid sequence dependent amplification
  • TAS Transcription-dependent Amplification System
  • SDA Strand Displacement Amplification -SDA
  • antisense orientation refers to a double stranded polynucleotide sequence that is functionally linked to a promoter in an orientation in which the antisense orientation of said molecule is transcribed.
  • the antisense strand is sufficiently complementary to an endogenous transcription product so that the translation of the endogenous transcript product is inhibited.
  • 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.
  • conservatively modified variants refers to those nucleic acids encoding identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any type of protein. For example, the codons GCA, GCC, GCG and GCU encode the amino acid alanine. These nucleic acid variations are silent variations and represent conservatively modified variants. By Likewise, any nucleic acid sequence encoding a polypeptide herein describes all possible silent variations of the nucleic acid. In the same way, any silent variation of a nucleic acid sequence encoding a polypeptide is implicitly included in each polypeptide sequence of this document.
  • amino acid sequences it is recognized that individual substitutions, deletions or additions to a nucleic acid, a peptide, a polypeptide or a protein sequence that alters, adds or eliminates a single amino acid or a small percentage of amino acids in Ia
  • the encoded sequence is also a "conservatively modified variant" in which the alteration results in the substitution of an amino acid with a biochemically similar amino acid. Therefore, any number of amino acid residues selected from the number of intakes ranging from 1 to 15 can be altered in the manner described above. For example, 1, 2, 3, 4, 5, 7 or 10 alterations can be created.
  • Conservatively modified variants generally have a biological activity similar to that of the unmodified polypeptide sequence from which they were derived. For example, the specificity of the substrate, the enzymatic activity, or the binding properties between a receptor and its elicitor are generally from 30% to 90% of the activity of the native protein and its native substrate. Conservative substitution tables are widely known in the area.
  • nucleic encoding a protein may include untranslated sequences (introns, for example) contained within translated regions of the nucleic acid, or it may not include such sequences (as in a cDNA, for example).
  • the information of the encoded protein is specified in the use of codons.
  • the universal genetic code is used to determine the translation code of a nucleic acid sequence into an amino acid sequence.
  • the universal code in the genetic information contained within the mitochondria of some plants, animals or fungi, of the bacteroide organism Micoplasma capricolum, the ciliated organism Macronucleus can be included within the corresponding organism.
  • the codon preferences of the host organism in which the nucleic acid is intended to be expressed can be exploited.
  • the nucleotide sequences of the invention described herein can be expressed in Trichoderma species, the sequences can be modified taking into account codon preferences and GC content preference that may occur between species of the Trichoderma genus.
  • full-length sequence (of the English “full-length sequence") of a specific polynucleotide or its encoded protein refers to the complete sequence of the amino acid chain of a native (non-synthetic) endogenous protein and in its biologically active form.
  • the methods used to determine if a sequence is full length are widely known and as an example we can mention “Northern” or “Western” type hybridizations, primer extension, or ribonuclease protection 14 .
  • homologous full length sequences orthologs or paralogs
  • the consensus sequences generally present at the 3 'or 5' edge of the untranslated regions of a messenger RNA molecule (mRNA) help to identify the full length sequence of a polynucleotide.
  • mRNA messenger RNA molecule
  • the consensus sequence ANNNNAUGG 1 in which the underlined codon represents the methionine present in the N-terminal edge helps to determine if the polynucleotide has a 5 'terminal edge full.
  • Consensus sequences at the 3 'edge such as polyadenylation sequences, help determine if the 3' terminal edge is complete.
  • heterologous refers here to a nucleic acid that derives from a different species or, if derived from the same species, a nucleic acid that is substantially modified in its native form.
  • 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.
  • 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.
  • host cell refers to a cell that contains a vector and that ensures the replication and / or expression of said vector.
  • Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells.
  • the host cells are Trichoderma strains.
  • hybridization complex refers to a double stranded nucleic acid structure formed by two single stranded nucleic acid sequences hybridized together selectively.
  • the term "introduced” in reference to the act of inserting a nucleic acid into a cell means “transfecting” or “transforming” and includes the incorporation of nucleic acids into a eukaryotic or prokaryotic cell in which the nucleic acid can be incorporated into the cell. genome of the cell (in the DNA of a chromosome, of a plasmid, a plastid or a mitochondrion), or it can be converted into an autonomous replicon, or expressed transiently.
  • isolated refers to a material (nucleic acid or a protein) that is: (1) substantially or completely free of the components that they usually accompany or interact with him in his natural form.
  • the isolated material may optionally comprise another material that is not associated with the isolate in its natural form; or (2) in case the material is in its natural environment, if the material has been synthetically altered by a deliberate human intervention that modifies its composition or assigns it to a specific place in the cell (for example, an organelle) different from the place where it is in its natural environment.
  • the alteration that gives rise to the synthetic form of the material can be directed to the material (nucleic acid and / or protein) or depend on a removal from the natural environment.
  • a natural nucleic acid can be isolated if it is altered or if it is transcribed from DNA that has been previously altered from a deliberate human intervention performed in the cell from which the nucleic acid originated 15 '16 .
  • a natural nucleic acid for example, a promoter
  • Nucleic acids that are "isolated” following this definition may be referred to as "heterologous" nucleic acids.
  • nucleic acid or “nucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in its single-stranded or double-stranded form and comprises those analogous molecules that have the essential nature of natural nucleotides to hybridize to stranded nucleic acids simple in a manner similar to that of natural nucleotides.
  • nucleic acid library refers to a collection of RNA or DNA molecules that substantially comprise and represent the integrity of the transcribed fraction of the genome of a specific organism.
  • EXAMPLES constructions libraries, either genomic or cDNA libraries libraries are described conventional molecular biology references 17 '18' 19.
  • polynucleotide refers to a deoxyribonucleotide, a ribopolinucleotide, or its analogues that have the essential properties of a natural ribonucleotide, such as the fact that they hybridize, under conditions of astringent hybridization, to essentially the same nucleotide sequences as nucleotides. that occur naturally, or that allow translation in the same amino acids as natural nucleotides.
  • a polynucleotide can be full length or a sequence segment of a structural gene or a native or heterologous regulatory gene. Unless explicitly mentioned, the term includes the specified sequence as well as its complementary sequence.
  • DNA or RNA molecules that contain segments that have been modified to increase their stability or for other reasons are also "polynucleotides” for purposes of the present invention.
  • DNA or RNA molecules that include infrequent nucleotide bases, such as inosine, or modified nucleotide bases, such as tritilated bases to cite just two examples would also be considered “polynucleotides” for purposes of the present invention. It follows from this paragraph that a wide variety of modifications have been made to DNA or RNA molecules, and that such modifications serve multiple purposes.
  • the term polynucleotide is used herein to designate chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and simple or complex cells.
  • polypeptide refers to amino acid polymers in which one or more of the amino acids is an analogue of a corresponding natural amino acid.
  • the essential nature of such analogs is that when incorporated into a protein, said protein can be specifically recognized by antibodies designed to recognize that same protein when it is composed exclusively of natural amino acids.
  • polypeptide ",” peptide ", and” protein also include, without limitation, glycolization, lipid binding, sulphation, carboxylation range of residues containing glutamic acid, hydroxylation and ribosylation of ADP.
  • polypeptides are not completely linear, for example, the polypeptides may be branched as a result of ubiquitination, they may be circular, with or without branching as a result of post-translational events such as natural processes and events caused by human manipulation that they do not occur naturally, circular and / or branched polypeptides can be synthesized by natural processes that do not depend on translation and by processes completely synthetic Additionally, this invention also includes terminal amino acid variants with or without methionine of each of the proteins related to the present invention.
  • promoter refers to a region of DNA located in the 5 'direction of the codon at the start of the transcription and which is involved in the recognition and binding of an RNA polymerase and other proteins necessary for the initiation of the transcription.
  • a "promoter” is a sequence derived from another organism but capable of initiating transcription in heterologous cells. Some examples of promoters include those obtained from the DNA of plant viruses and bacteria that contain genes that are expressed in plants such as Agrobacterium or Rhizobium. Examples of promoters that are under control of developmental states include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots or seeds. These types of promoters are called “tissue-preferred”.
  • a specific "cell type” promoter is a promoter that only directs expression in certain types of cells located in one or more organs, such as, for example, the cells of the vasculature in leaves and roots.
  • An "inducible” or “repressible” promoter is a promoter that responds to control signals that it emits from the environment. Some examples of environmental conditions that may have an effect on transcription dependent on inducible promoters are the presence of anaerobic conditions, or the effect of light.
  • Tissue-specific promoters, tissue-preferred promoters, specific cell-type promoters and inducible promoters constitute the class of "non-constitutive" promoters. "Constitutive" promoters are those that direct expression systemically and under most of the environmental conditions.
  • 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.
  • recombinant cells express genes that are not found identically within the native (non-recombinant) form of the cell or that express native genes that would otherwise be under-expressed, expressed abnormally, or not express as a result of human intervention.
  • the recombinant term does not include the alteration of the cell or of the vector by events that occur naturally (for example, spontaneous mutation, or transformation, transduction or transposition) like all those that occur without human intervention.
  • expression cassette refers to a nucleic acid construct (generated in recombinant or synthetic form) that contains a series of nucleic acid elements that allow transcription of a particular nucleic acid in a host cell.
  • the recombinant expression cassette can be incorporated into a plasmid, on a chromosome, in mitochondrial DNA, in plastidic DNA, in a virus, or in a nucleic acid fragment.
  • the portion of the recombinant expression cassette of an expression vector includes, among other sequences, the nucleic acid to be transcribed and a promoter.
  • amino acid residue or “amino acid residue” is used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide or a peptide.
  • the amino acid may be natural or may include synthetic amino acids analogous to natural amino acids that may function similarly to natural amino acids.
  • sequences that selectively hybridize typically have 90% shared sequence identity, and preferably 100% shared sequence identity.
  • transgenic strain or “transformant” or “transformed strain” refers to a well characterized isolate of the fungus that contains in its genome a polynucleotide introduced into the laboratory by transformation techniques known in the art. Generally said polynucleotide is stably integrated and is transmitted to the offspring of said fungus. The polynucleotide can be integrated into the genome in isolation or as part of a recombination vector. He The term “transgenic” includes here any cell or cell line, for which the genotype has been altered by the introduction of a nucleic acid, including those transgenic elements that were created by transformation.
  • vector refers to a nucleic acid used for the transformation of a host cell in which a polynucleotide can be inserted. Vectors are often replicons. Expression vectors allow the transcription of a nucleic acid that has been inserted.
  • the terms indicated below are used to describe the relationship between the sequences of two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison interval”, (c) "identity of The sequence ", (d) percentage of identity of the sequence” and (e) "substantial identity”.
  • reference sequence is a defined sequence that is used as the basis for sequence comparison.
  • a reference sequence may be a portion or a complete sequence; for example, a segment of a full-length cDNA, or the full length of the cDNA or an entire gene.
  • comparison interval refers to a specific and contiguous segment of a polynucleotide sequence that can be compared to a reference sequence and for which the comparative portion of the polynucleotide sequence may include additional or missing elements (by example, "gaps") when compared with the reference sequence (which has no additional or missing elements) so that the alignment of the two sequences is optimal.
  • the comparison range is at least 20 contiguous nucleotides and can often include more than 100 nucleotides.
  • the optimal sequence alignment for comparison can be obtained from the local homology algorithm of Smith and Waterman 20 , by the algorithm of Needlman and Wunsch 21 , by the similarity search method of Pearson and Lipman 22 , by the computerized implementation of These algorithms, including but not limited to: CLUSTAL in the program
  • the family of BLAST programs that can be used for comparative sequence searches includes: BLASTN for comparative search of nucleotide sequences compared to nucleotide sequences contained in public domain databases; BLASTP for comparative search of protein sequences compared to protein sequences contained in public domain databases; TBLASTN for finding homologies between a protein sequence and a nucleotide sequence; and TBLASTX for a comparison of a nucleotide sequence with a set of nucleotide sequence databases 27 .
  • identity or similarity values of a sequence indicated in this document refer to the values obtained with the BLAST 2.0 version using the "default" parameters 28 .
  • the software to perform these analyzes is in the public domain and can be accessed or obtained through the Center's website
  • the algorithm begins by identifying pairs of sequences with a high degree of similarity from the identification of short words with a length of W in the search sequence; said words must be identical or very similar to that of a threshold of value T when aligned with a word of the same length contained in a public domain database.
  • This initial similarity between two sequences results in the beginning of a series of searches to find sequences of greater length and a high degree of similarity.
  • AT is called the word neighborhood value threshold 28 .
  • the words found extend in both directions for each of the sequences as long as the cumulative alignment value continues to grow. For nucleotide sequences the cumulative values are calculated using the M parameters (prize value for a pair of matching waste; it will always be greater than
  • BLAST also performs a statistical analysis of the similarity between 2 sequences 30 .
  • a measure of similarity provided by the BLAST algorithm is the minimum probability of sum (P (N)), which provides an indication of the probability with which the agreement between two nucleotide or amino acid sequences can occur at random.
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of non-random sequences that may be enriched with one or more amino acids. These regions of low complexity can be aligned from unrelated proteins although other regions of said proteins are completely different and have no similarity.
  • Some programs that function as low complexity filters can be used to reduce such low complexity alignments. For example, the SEG 31 and XNLJ 32 program . This type of filters can be used individually or in combinations.
  • sequence identity in the context of two nucleic acid or polypeptide sequences refers to residues in both sequences that are the same when they are aligned to look for the greatest correspondence within a window. comparative specific. When the percentage of the sequence identity is used in reference to proteins, it is recognized that the positions of residues that are not identical often differ by conserved amino acid substitutions, in which the amino acids are replaced by other amino acids that have similar biochemical properties. (load or hydrophobicity) and therefore does not change the functional properties of the molecule. If the sequences differ by the conservative nature of the substitution, the percentage of sequence identity can be adjusted upwards to correct the effect of the conservative nature of the substitution.
  • sequence similarity or “similarity”.
  • the task of making such adjustments is routine for those with art skills. It generally involves assessing a conservative substitution as a partial and non-complete sequence discrepancy, thereby increasing the percentage of sequence identity. For example, when an identical amino acid is assigned a value of 1 and a non-conservative substitution is assigned a value of zero, a conservative substitution is assigned a value between zero and 1. The value of the substitutions is calculated using the Meyers and Miller 33 algorithm, as implemented in the PC / GENE program (Intellegentics, Mountain View, California, USA).
  • the term "percentage of identity sequence” refers to the value determined from the comparison of two optimally aligned sequences from a specific comparative window, and in which the portion of the polynucleotide sequence in the comparative window may include additions or deletions (ie absences) when compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of two sequences. The percentage is calculated by determining the number of positions from which the nucleotide base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing said number by the total number of positions in the comparative window and multiplying per 100 to obtain the percentage of identity sequence.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, preferably at least 80%, even more preferably 90% and ideally
  • sequence identity when compared to a reference sequence using one of the aforementioned alignment programs. It is recognized that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by 2 nucleotide sequences and taking into account codon degeneration or amino acid similarity.
  • substantial identity of amino acid sequences normally means an identity of at least 60%, or preferably 70%, 80%, 90% and ideally 95%.
  • nucleotide sequences are substantially identical is the fact that two molecules hybridize with one another under astringent hybridization conditions. However, nucleic acids that do not hybridize with each other under astringent conditions remain substantially identical if the polypeptides they encode are substantially identical. This can occur when, for example, a copy of the nucleic acid is created using the maximum degeneracy codon allowed by the genetic code.
  • An indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded in the first nucleic acid is immunologically identical to the polypeptide encoded in the second nucleic acid.
  • substantially identical 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.
  • the optimal alignment is performed using the alignment algorithm of Needleman and Wunsch 21 .
  • An indication that two peptide sequences are substantially identical is that a peptide be immunologically identical to the second peptide. For example, two peptides will be substantially identical if they differ only by a conservative substitution. ///. Detailed description.
  • the present invention allows the obtaining of Trichoderma strains that overproduce several lytic enzymes to be used as antagonists of phytopathogenic fungi.
  • the strains obtained are more efficient in the production of such enzymes and in the establishment of the mycoparasitic effect against phytopathogenic fungi. These characteristics allow the strains obtained to be used as antagonistic organisms for the effective control of plant diseases and used as a biocontrol mechanism.
  • the Trichoderma strains of the invention retain the ability to detect and respond to different environmental conditions, including the presence of a potential host, essential factors for the successful colonization of soil, organic matter and the root system of plants.
  • the present invention takes advantage of the fact that the detection of such environmental conditions can occur through a series of transduction pathways, which determine the appropriate cellular response of Trichoderma.
  • a wide variety of signals are transduced by the MAP kinase pathways, including those associated with pathogenesis.
  • MAP kinases have been directly implicated in several phenomena, such as:
  • Ustilago maydis (Ubc3 / Kpp2) 34 , b) In the expression of enzymes that degrade cell wall in several fungal systems, including phytopathogenic fungi, homologues of the MAP kinase Kss1 of S. cerevisiae. c) In the induction of the pectate lyase gene (pl1) of F. Oxysporum, since in null mutants of fmk1 this activity was abolished 35 . d) In the positive control of the expression of those enzymes involved in the penetration of the plant, for example Bmp1 of B.
  • the present invention explores the relationship between the function of genes coding for MAP kinases with the mycoparasitic function observed in Trichoderma.
  • the abolition in the expression of a gene coding for a MAP kinase, such as the tvk1 gene causes a considerable increase in the synthesis of enzymes related to the observed mycoparasitic effect of Trichoderma strains, by less with a 10-fold increase in activity compared to the parental strain or with wild strains.
  • the mycoparasitic activity of the Trichoderma strains of the invention is considerably increased with respect to wild strains, whereby the strains of the invention can be used as efficient antagonistic organisms against multiple diseases caused by phytopathogenic fungi.
  • the Trichoderma strains of the invention show a clear increase in the level of expression of mycoparasitism-related genes (MRGs) under conditions of simulated mycoparasitism and during direct confrontation with plant pathogens, such as Rhizoctonia solani. Likewise, they also show an increase in protein secretion measured as the production of lithic enzymes in the culture supernatant of these strains, compared with that of the wild strain. Consistently in biocontrol trials, the strains are considerably more effective in the control of plant diseases than the wild strain or even chemical fungicides. Additionally, the strains of the invention sporulate abundantly in submerged cultures, a condition that normally does not lead to sporulation in the wild strain.
  • MRGs mycoparasitism-related genes
  • the methodology of the invention allows to obtain Trichoderma strains with increased activities in their mycoparasitism properties, as well as in the expression of multiple enzymes involved in the antagonistic effect.
  • the abolition of the expression of the tmkA gene results in generating much less effective Trichoderma strains to be used as a potential biocontrol against pathogenic fungi and in apparent inactivation of genes that code for one or more enzymes responsible for degradation. of the host.
  • the present invention demonstrates that the Tvk1 protein (MAP kinase) plays an important role in the regulation of the expression of the genes responsible for the production of lytic enzymes, with which it is possible to manipulate the expression of this protein in Trichoderma strains in order to improve the antagonistic properties associated with it.
  • the present invention shows that in the transformed strains of Tr ⁇ choderma, in which the expression of the tvk1 gene was abolished, the chitinase and protease activities were greater than in the wild, correlating with the high levels of the transcripts detected.
  • the activity of certain enzymes is greatly increased in the strains of the invention. compared to the wild strain, for example in the total activity of ⁇ -1, 3-glucanase, further indicating that in the strains of the invention other glucanase genes could be under negative modulation by MAP kinases.
  • Trichoderma strains of the invention show reduced pigmentation with loss of the characteristic dark green color observed in the wild; the strains produce spores with reduced pigmentation in solid medium, without becoming albines. In contrast, all strains of the invention produce abundant conidia that show the characteristic dark green color of the wild in liquid cultures regardless of the medium used. These results suggest that MAP kinases could differentially regulate melanin biosynthesis as previously reported for C. lagenarium 39 . Considering the increase observed in the production of lytic enzymes in the Trichoderma strains of the invention and the importance of these enzymes in the biocontrolling activity of this microorganism, the strains of the invention can be used as more effective biocontrol agents.
  • the strains of the invention show a greater capacity than the wild strain to control and reduce the damage caused by R solani and P. ultimum.
  • T. virens has been used successfully in combination with several fungicides including metalaxyl (Apron FL) 40 .
  • 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.
  • any strain of Trichoderma that allows obtaining by means of the teachings described herein, Trichoderma strains overproducing lytic enzymes can be used; likewise, any species of the genus Trichoderma can be occupied, although T. virens is even more preferred.
  • the strains of the invention can be administered directly or through compatible compositions at the agronomic level, either in the soil, in the plant or in the material of the plant to be protected or treated.
  • the compositions can be made by widely known methods and can be made in any form of presentation, either in liquid or solid forms.
  • the liquid forms are capable of being applied by aerosols in the soil or in the plant, or used for the elaboration of baths in which the plants or their material are submerged.
  • the compositions containing the Trichoderma strains of the invention can be applied by conventional methods, either by aerosols or by immersion.
  • the present invention describes the isolation and characterization of the tvk1 gene of
  • Tr ⁇ choderma virens corresponding to SEQ ID NO: 2, which encodes the Tvk1 protein
  • MAP kinase mitogen activated protein kinase
  • SEQ ID NO: 3 which has an important role in several aspects of the life cycle of Tr ⁇ choderma, including growth, conidiation, expression of MRGs, secretion of enzymes that degrade cell wall and a biocontroller activity.
  • the tvk1 gene contains three intron sequences that have been reported for other fungal MAP kinase genes.
  • the intron located between nucleotide +854 and +907 is not present in its closest recently reported homologue, tmkA of strain IMI306092 of T. virens.
  • Tvk1 belongs to the family of kinases regulated by external signals (ERK), which is part of the superfamily of MAP kinases.
  • the signature sequence present in the Tvk1 protein indicates that it is related to the YERK1 family (ERK1 of yeasts and fungi) 38 .
  • the invention also comprises modified sequences of the DNA sequence shown in SEQ ID NO: 2, which code for amino acid sequences that retain the activity of the Tvk1 protein. Following the teachings of the present invention, a person versed in the technical field could easily predict the existence of such modified sequences and could easily produce them. Likewise, the invention comprises modified amino acid sequences derived from the sequence of the Tvk1 protein shown in SEQ ID NO: 3, which preserve and maintain the characteristics of the Tvk1 protein, mainly the enzymatic activity of MAP kinase as well as the ability to negative regulation in the expression of Trigoderma MRGs genes.
  • the suppression of the expression of MAP kinase genes coding for Tr ⁇ choderma strains allows to increase the capacity of the Tr ⁇ choderma strains of the invention to control other fungi that attack plant crops, coupled with the fact that due to this suppression effect, the expression levels of lytic enzymes are presented at high levels in most of the selected genes that encode lytic enzymes (MRGs) in comparison with wild strains.
  • MRGs lytic enzymes
  • the latter can be obtained by directed inhibition of the gene expression using specific probes, by interfering RNA, antisense RNA expression, by insertion of foreign polynucleotide sequences in the coding sequence of the MAP kinase gene or in its regulatory regions, by replacement of the wild gene by a mutated version, or, by eliminating the gene of interest from the genome of the organism through homologous gene recombination techniques.
  • this suppression of the expression of genes coding for MAP kinases can be achieved using the gene replacement vector of the invention, which comprises:
  • a replacement gene that allows the identification of the transformants where the coding anoint for a Trichoderma MAP kinase is replaced, b) A non-coding fragment that is naturally outside the coding region of the gene for MAP kinase at its 5 'end, flanking the 5 'end of the replacement gene, and. c) A non-coding fragment that is naturally outside the coding region of the gene for MAP kinase at its 3 'end, flanking the 3' end of the replacement gene.
  • the replacement gene may or may not come from some Trichoderma strain, although those genes that come from Trichoderma strains of the same species of the strain to be transformed are preferred.
  • any gene that allows the partial or total elimination of the gene coding for MAP kinase or that allows obtaining the interruption of the open reading frame of said gene can be used, with the consequent suppression of the expression of the protein encoded by the same and that also allows the identification of the modified strains.
  • 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.
  • the MAP kinase gene fragments that flank the replacement gene are those fragments of any size that are found either before or after the beginning of the gene's open reading frame as well as those that are found before or after the end of the open reading frame of the gene, as long as they come from the wild gene or from a gene with high similarity, this with the purpose of producing specific genetic recombination events between the genome of the Tr ⁇ choderma strain to be transformed in the region of the MAP wild kinase gene and the polynucleotide replacement sequence contained in the gene replacement vector.
  • MAP kinase gene fragments that flank the replacement gene come from the tvk1 gene and are:
  • the function of the replacement gene can restore some deficiency in the strain of Tr ⁇ choderma to be transformed given by auxotrophy or allow the selection of transformants by their tolerance to a chemical compound such as in the case of the use of antibiotics or herbicides or by confer the ability to use alternative sources of carbon. and / or nitrogen, or because it can be identified through an enzymatic activity encoded by it.
  • the Tr ⁇ choderma strain to be transformed is auxotrophic
  • the replacement gene should complement said auxotrophy. In this case, it is preferred to use the Tr ⁇ derderma arg2 gene and auxotrophic strains to arginine.
  • the gene that confers hygromycin resistance (hph or hpt) of Escherichia coli, or Basta herbicide resistance genes can be used.
  • the amdS gene of Aspergillus nidulans can be used.
  • vectors can be used to clone and introduce by transformation the genes of interest that will later be useful for integrating the replacement genes into the genome of the strains of Trichoderma through efficient recombination.
  • the replacement vectors of the invention must contain characteristics that allow them to be amplified by means of conventional techniques (replication sites, promoters, etc.) and endowed with unique cloning sites, both for the insertion of the genes replacement as for the subsequent linearization necessary to transform the Trichoderma strains. In this sense, those vectors that allow the insertion of DNA fragments and that can be efficiently amplified in biological systems can be used.
  • the vectors of the invention use as a selection marker gene a replacement gene of the Trichoderma tvk1 gene with some associated function that can be identifiable, for example the synthesis of some protein that allows the growth of auxotrophic strains of Trichoderma in the appropriate culture medium (deficient in the essential nutrient) after its transformation with the vector.
  • the replacement gene preferably comes from Trichoderma strains.
  • the Trichoderma strains of the invention can be obtained by means of the methodology comprising the following steps:
  • the replacement vector of the invention in order to obtain the strains of the invention in this method it is preferred to suppress the expression of the gene tvk1, coding for the Tvk1 MAP kinase, use Trichoderma's arg2 gene as a replacement gene and use arginine auxotrophic strains.
  • the replacement vector is provided in a linear manner, this in order to provide the appropriate conditions for the genetic recombination process.
  • the selection of the transforming strains can be carried out with any selective means that allows the selection of the resulting prototrophic transformants, in direct relation with the function of the replacement gene; although Vogel minimum medium (VMS) containing sucrose is the only carbon source.
  • VMS Vogel minimum medium
  • the enzymatic activity related to the mycoparasitic activity of Trichoderma can be increased in a directed manner, generating Trichoderma strains with improved enzymatic characteristics that can be used as antagonistic organisms of phytopathogenic fungi.
  • MAP kinases are involved in the expression of MRG genes, they can be used as a regulatory factor for the expression of lytic enzymes in Trichoderma. Due to the function of MAP kinases, related substances specific to them could be obtained in order to block their function in Trichoderma strains, thereby increasing the expression of lithic enzymes and thereby increasing the mycoparasitic function of the strain of interest. For this purpose, known techniques of adsorption of biological materials could be used using MAP kinases isolated and placed on fixed supports to capture specific related substances that can subsequently be isolated by conventional biochemical methods of purification and characterization. The use of these inhibitors of the function of MAP kinases could be useful for obtaining strains of Trichoderma with an increased antagonistic activity. In this sense, the Tvk1 protein isolated in the present invention (Trichoderma MAP kinase) can be used for this purpose.
  • Trichoderma strains of the invention can be used for the protection or treatment of plants or plant materials for the fight and / or prevention of infections or diseases caused by phytopathogenic fungi, using multiple and diverse Known techniques for its application.
  • Example 2 Bacterial strains and plasmids.
  • Escherichia coli DH5 ⁇ (Bethesda Research Laboratories) and JM103 (Invitrogen) strains were used for all DNA manipulations.
  • the plasmids used were pBluescript (Stratagene) and pCB1004 (Fungal Genetics Stock Centerj. All PCR products were cloned into the vector pCR2.1 (Invitrogen).
  • the probes used for Northern blot analysis were obtained as follows: a 1.3 kb Hind ⁇ / BamH ⁇ fragment of Tv-prb1 cDNA was obtained from plasmid pPOE.
  • Tv-cht1 A 1.4 Kb Hind ⁇ / Xba ⁇ fragment of Tv-cht1 was obtained from plasmid pCOE 42. From plasmid pSZD2 a Pst ⁇ / Xho ⁇ 0.52 Kb fragment corresponding to the Tv-bgn2 gene was obtained, two fragments corresponding to Tv-cht2 (from position 988 to 1417) and Tv-nag1 (from position 513 to Ia 1608) were obtained from genomic DNA of T. virens by PCR using oligonucleotides designed based on the sequences reported by
  • Example 3 DNA and RNA manipulation. Plasmid DNA was isolated using a commercial system (Qiagen). Trichoderma DNA was obtained according to a previously described procedure 44 . Total RNA was isolated using phenol-chloroform extractions according to the procedure described by Jones 45 . Southern and Northern type analyzes were carried out using Hybond N + membranes (Amersham) according to the manufacturer's recommendations.
  • 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 ⁇ g) of each sample were resuspended in Schágger 2X 46 buffer and boiled after addition of ⁇ -mercaptoethanol (5%). The proteins were separated by SDS-PAGE in 10% gels according to Schágger and Von Jagow 46 . The proteins were transferred to extra Hybond TM -C membranes (Amersham) and the detection was performed following the instructions of the Phospho Plus ® p42 / p44 MAP kinase system (Thr / Tyr) Antibody kit (CeII Signaling Technology, Inc).
  • Genomic DNA from T. virens Gv29-8 was used as tempering in PCR amplification reactions using the primers described previously 47 .
  • the PCR product was cloned and subsequently sequenced using the Sanger 48 method with the Sequenase system (version 2.0, U. S Biochemichals).
  • a fragment that showed high similarity with the M. gr ⁇ sea pmk1 gene was selected and used as a probe for the screening of a cosmid library of T. virens Gv28-9.
  • Three clones were identified and one of them was selected for sequencing.
  • a Southern type analysis allowed the identification of a 3.3 Kb BamH ⁇ fragment containing the tvk1 gene (see figure 1), said fragment was subcloned into the BamH ⁇ site of plasmid pCB1004 (pDXG35). This fragment was sequenced in its totally and subsequently analyzed by the BLAST program using the DNA-protein data bank.
  • T. virens DNA as temperate and degenerate oligonucleotides reported by Xu and Hamer 47 .
  • several PCR amplification products were obtained. These products were subcloned into the vector pCR2.1 and sequenced. After performing a computational analysis using the BlastX algorithm, one of these showed a high similarity with the pmk1 gene of M. gr ⁇ sea.
  • This amplification product was radioactively labeled and used in a screening in a genomic library of T. virens. Three clones were obtained that gave a positive signal and one of them was selected, subcloned and sequenced.
  • the tvk1 gene contains four exons interrupted by three introns as reported for other genes in this same kinase family ( Figure 2).
  • a gene similar to the one reported here was cloned, however, unlike the genes of this family, the reported one does not present the third intron 38 .
  • a comparative analysis of the promoter region reveals the presence of possible response elements which are associated with the growth and conidiation processes, such as RAP-1, ABF-1, STUAP1 and three possible GCR1 binding sites, which is involved in the response to carbon limitation. In other systems it has been observed that the binding of RAP1 facilitates the binding of GCR1 to adjacent sites. Additionally, the promoter contains two STRE type binding sites and a possible site for the MAT-1-Mc mating factor.
  • the deduced protein sequence of tvk1 is 360 amino acids that correspond to a theoretical molecular mass of 41.6 KDaI and an isoelectric point of 6.44.
  • An alignment analysis using the MegAlign-Clustal program indicates that Tvk1 and TmkA have a 99.4% similarity, while Tmk1 has a 97.7% similarity with Tvk1.
  • the region included among residues 58 to 160 in Tvk1 contains the sequence observed in several members of the family of MAP kinases: Fx (10) -REx (72,86) -RDxKx (9) -C 49 .
  • This protein also contains the phosphorylable residues [T (184) -EY (186)] required for its phosphorylation and activation by MAP kinase 50 .
  • Example 6 Construction of the pTVK1 :: arg2 gene replacement vector.
  • a £ coRV / Sa / l fragment of 1.48 Kb containing most of the coding region of tvk1 (from amino acid 88 to the last amino acid of the protein) was replaced by a fragment 3.2 Kb SmaVEcoRV of the arg2 gene from T. virens 41 .
  • a SamHI / EcoRV fragment of pDGX35 was subcloned into the Ba / nHI / EcoRV sites of pBluescript SK (-) to generate plasmid pAM1.
  • Plasmid pAM1 was digested with EcoRV and ligated to an EcoR ⁇ // Sma ⁇ fragment of the arg2 gene (pAM2) gene.
  • pAM2 EcoR ⁇ // Sma ⁇ fragment of the arg2 gene
  • T. virens protoplasts were prepared and transformed according to a previously described method 41 .
  • Prototrophic transformants were selected using Vogel minimum medium containing sucrose as the sole source of carbon (VMS). The interruption of the tvk1 gene in the selected transformants was confirmed by Southern and Western analysis.
  • Colonies of the ⁇ tvki mutants showed a reduction in their speed of colonial growth and in the development of aerial hyphae in solid medium.
  • Conidia suspensions of the wild strain showed an intense green coloration, compared with the pale green of the conidia suspensions of the mutants.
  • the null mutants produced twice less conidia than the wild strain when they were grown in PDA medium without significant changes in the morphology of the conidophore.
  • all ⁇ ivki mutants formed pellets smaller than those formed by the wild strain.
  • the ⁇ tvki mutants massively conidia in late phases in submerged cultures (72 h), while no conidia were detected in the cultures of the parental strain even after seven days.
  • Figure 4A shows liquid cultures of the wild strain of T. virens (Gv29-8) (left panel), mutants ⁇ tvk24 and ⁇ tvk133 (two central panels) and the parental strain (Tv10.4) (right panel) after 72 hours of incubation in VMS medium.
  • the ability to conidia in liquid medium of the mutants seems to be independent of the culture medium used, since the conidiaclón was observed in both VMS and PDB.
  • Microscopic observations of samples of sporulating liquid cultures of ⁇ tvk133 showed a normal development of the conidiophore, resembling those produced in aerial hyphae ( Figure 4B-C).
  • Example 10 Simulated mycoparasitism assays. Tr ⁇ choderma spores (1 x 10 6 spores / ml) were germinated and grown for 48 h in VMS. The mycelium was then collected and transferred to fresh medium. Minimum Vogel medium without a carbon or nitrogen source (VM or VM-N, respectively) was used to assess the effect of nutrient limitation; VMS containing 0.5% of R. solani cell walls (VMSR), and Vogel medium without a source of nitrogen or carbon added with 0.5% of R solani cell walls (VM-NR or VMR, respectively) was used for similar conditions of mycoparasitism. VMS was used as a control.
  • VMSR 0.5% of R. solani cell walls
  • VM-NR or VMR Vogel medium without a source of nitrogen or carbon added with 0.5% of R solani cell walls
  • Tv-cht1 was clearly induced by cell walls in the wild strain with a maximum level of expression at 6 h. In contrast, no obvious induction was observed in the mutant strain, which reached the same level observed under conditions of carbon limitation, except that this happened before (Fig. 5A; Tv-cht1).
  • the gene encoding the Tv-bgn2 glucanase was induced by cell walls at 6 h, but no differences were observed with the wild strain and the gene was not expressed in medium without a carbon source (Fig. 5A; Tv-bgn2).
  • the expression pattern of a third chitinase gene (Tv-cht2) under conditions of simulated parasitism was similar to that of Tv-cht1, except that the maximum expression was reached after 24 hours.
  • ⁇ tvk24 showed a much more pronounced induction for Tv-cht2 than the wild strain.
  • the expression was determined under simulated conditions of mycoparasitism both in the absence and in the presence of an alternate carbon source (Fig. 5A; Tv-prb1) or in VM-NR that does not contain ammonium (Fig. 5B; Tv-prb1) of the gene prb1, which codes for a protease.
  • Cell wall induction was observed for both strains, although higher levels of transcript were detected under conditions of carbon starvation than under conditions of nitrogen limitation. In both cases, the induction was much higher for the ⁇ tvk24 strain than for the wild strain.
  • the mutant ⁇ tvk24 showed the highest levels of expression after 6 h, between seven and ten times more than the expression in the wild strain.
  • the mutant strain showed detectable levels of Tv-prb1 expression after 6 h when grown on VM-N (Fig. 5B; Tv-prb1).
  • the analysis of the acivivities in proiease gel, endocytinase, N-acetylglucosaminidase and glucanase of the culture filtrates showed ten times greater activity in the muiahids than in the silves ⁇ re strain (see figure 8). It was also interesting to observe the presence of an additional band of endocytinase activity in null mutanids under conditions of carbon limitation (see figure 8).
  • Trichoderma strains were subjected to confrontational trials without coniacio using R solani as host 51 .
  • the confrontations were carried out in modified VMS-agar medium (mVMS) containing 0.75 g / l sucrose and 0.45g / l NH 4 NO 3 .
  • the mycelium of Trichoderma was collected from the zone of interaction between fungi.
  • Example 12 Direct confrontation assays in agarpapa dextrose.
  • the Trichoderma strains of the invention were subjected to direct confrontation assays using Phytophthora capsici, Sclerotium rolfsii, Rhizoctonia solani, Colletotrichum lindemuthianum and Phytophthora citricola, as host.
  • the confrontations were carried out in half papa dextrose agar using strains T. virens Gv29.8, ⁇ tvk24 and ⁇ tvk133 for 5 days under continuous light at 28 0 C. The results obtained are shown in Figure 9.
  • Cottonseed (cultivate Stoneville 112, Pedigree Seed Co) were covered with Trichoderma strains and planted in a medium free of non-sterile soil (Metromix) infected with R solani or P. ultimum. Seeds sown in uninfected soil and seeds treated with the commercial fungicide Apron XL LS (active against P. ultimum) were used as positive controls. Survivors and healthy plants were counted at 10 days of incubation at 25 0 C in a growth chamber.

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Abstract

L’invention concerne l’obtention de souches de Trichoderma qui surproduisent diverses enzymes lytiques afin de s'utiliser en tant qu’antagonistes de champignons phytopathogènes dufait de leur activité mycoparasitique accrue. Un gène de Trichoderma virens codifiant a été cloné pour une protéine cinase activée par mytogène (MAP cinase) lequel est associé à la réponse mycoparasitique, la cognidiation et la lutte biologique observée de Trichoderma. Les souches de Trichoderma avec l’expression supprimée du gène MAP cinase isolé, démontrent une claire augmentation dans le niveau d’expression de gènes associés au mycoparasitisme (MRG) dans des conditions de mycoparasitisme simulé et durant la confrontation directe avec le pathogène des plantes Rhizoctonia solani. De manière générale, des essais de lutte biologiques démontrent que les souches de l’invention ont été considérablement plus efficaces dans la lutte contre les maladies que la souche sauvage ou un fongicide chimique. En outre, les souches de l’invention se sont reproduites abondamment par sporulation dans des cultures immergées, une condition qui conduit normalement à la sporulation de la souche sauvage.
PCT/MX2005/000114 2004-12-10 2005-12-09 Souches ameliorees de trichoderma utilisees en tant qu’agents de lutte biologique, procede d’obtention de celles-ci et leur utilisation dans la lutte contre des maladies entrainees par les champignons phytopathogenes WO2006129998A1 (fr)

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WO2012001329A2 (fr) 2010-07-02 2012-01-05 Centre National De La Recherche Scientifique - Cnrs Utilisation d'un extrait naturel de marc de raisin pour stimuler les defenses naturelles de plantes

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US6808917B1 (en) * 2001-02-02 2004-10-26 Thomas D. Johnson Controlling plant pathogens with fungal/bacterial anatagonist combinations

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US6808917B1 (en) * 2001-02-02 2004-10-26 Thomas D. Johnson Controlling plant pathogens with fungal/bacterial anatagonist combinations

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Title
MENDOZA-MENDOZA A. ET AL.: "Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 100, no. 26, December 2003 (2003-12-01), pages 15965 - 15970, XP003004411 *
MUKHERJEE P.K. ET AL.: "TmkA, a mitogen-activated protein kinase of Trichoderma virens, is involved in biocontrol properties and repression of conidation in the dark", EUKARYOTIC CELL, vol. 2, no. 3, June 2003 (2003-06-01), pages 446 - 455, XP003004412 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012001329A2 (fr) 2010-07-02 2012-01-05 Centre National De La Recherche Scientifique - Cnrs Utilisation d'un extrait naturel de marc de raisin pour stimuler les defenses naturelles de plantes

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