MX2013014682A - LONGIBRACHIATUM THICA-4 TRICHODERMA CEPA WITH MYCOFUNGICIDE CAPACITY AND PROMOTER OF GROWTH IN AGRICULTURAL PLANTS OF INTEREST. - Google Patents

Abstract

The present invention relates to a purified strain of Trichoderma longibrachiatum identified as ThlCA-4 based on molecular analyzes with GenBank code: HQ667667.1 characterized in that it has the sequence SEQ ID: 1 and is deposited in the Agricultural Research Culture Collection (NRRL ) with the code: NRRL50762. The microorganism is a mycoparasite fungus with fungicidal properties of broad spectrum, and that produces plant growth promoting substances when inoculated in plants of horticultural interest mainly transgenic or conventional cotton, and cucumber. This organism is adapted to the environmental conditions of the Mexicali Valley, Baja California from where it was isolated and identified.

Description

i Cepa of Trichoderma longibrachiatum THICA-4 WITH MICOFUNGICIDE CAPACITY AND PROMOTER OF GROWTH IN PLANTS OF INTEREST AGRICULTURAL TECHNICAL FIELD The present invention corresponds to the technological area of biotechnology, but more specifically to the technology related to the field of agricultural biotechnology, since a new microorganism is described and claimed, specifically a new strain of the fungus Trichoderma longibrachiatum called THICA-4. which was isolated from wheat straw samples in the Mexicali BC valley. Said invention also claims its formulation with diatomaceous earth or any other substance that serves for the formulation of the product that is applied in powder form to the plants or soil. The use of this strain of Trichoderma longibrachiatum THICA-4 in plants stimulates vegetative growth and controls the development of phytopathogenic fungi that cause foliar and root diseases when applied to the soil.

BACKGROUND OF THE INVENTION In agriculture, a mycofungicide can be described as the use of live microorganisms (bacteria or fungi) and / or its products resulting from its metabolism, which affect the development and proliferation of soil or foliar fungi that cause diseases in plants. In particular, fungi of the genus Trichoderma are considered mycoparasite fungi due to their ability to inhibit the development of phytopathogenic fungi. The inhibition capacity of fungi of the genus Trichoderma is mainly due to the following mechanisms of action: a) direct competition for space or nutrients in the soil; b) production of antibiotic metabolites of a volatile or non-volatile nature; and c) direct parasitism of certain Trichoderma species on phytopathogenic fungi. In addition, the easy isolation and cultivation, the rapid growth in a large number of substrates, and the fact of not attacking the higher plants. It causes that some species of the fungus Trichoderma spp., Have been cataloged like excellent agents in the biological control of fungi causing diseases for different horticultural plants. Being, the strains of Trichoderma most marketed in the form of mycofungicides: T. viride, T. polysporum and T. harzianum. There are mainly two methods for the production of the inoculum that serves as the basis for the formulation of the product: a) solid fermentation and b) liquid fermentation. Where solid fermentation is the most common method for mass production of the fungus and involves the use of different products that can be used as economic substrates. The product obtained from the solid fermentation process based on fungi of the genus Trichoderma spp., Can be separated from the propagation medium and concentrated to be incorporated to the plants or to the soil in the form of talcs based on vermiculite and wheat fiber, granulated with wheat flour or mixed with Kaolin, and encapsulated in alginate. At present, the trend in the formulation of a mycofungicide from microorganisms is focused on the formulation of mycofungicides with strains adapted to the environmental conditions in which they wish to use. This is due to the fact that generally the micofungicides are elaborated with strains coming from other sites with different environmental conditions. Which can cause that the effect in the protection of the plants is not the awaited one since when not being adapted to the environmental conditions, it will require more time and quantity to be able to confront the conditions of the environment. The above implies greater spending of money and investment of time by the producer in achieving the benefits in the control of the pathogenic fungi that affect the plant.

Although there are several documents that describe the use of Trichoderma strains for the control of various phytopathogenic fungi, they do not affect the legal / patentability requirements of the present application and the most relevant documents related to the subject are listed below.

In the state of the technology there are the following registers: ES2188385, US20040136964, ES2101123, ES2109182, and ES2307870, which corresponds to the strains of Trichoderma asperellum, T. harzianun, viride and organic waste (compos-peat-vermiculite), liquid formulation and extracts of fungi, respectively. The records ES2188385, US20040136964, ES2101123, ES2109182, and ES2307870, indicate the use of the strains of Trichoderma spp., In the elaboration of products used for the control of a limited number of phytopathogenic fungi of the soil, mainly Fusarium oxisporum, F. lycopersici, and Rhizoctonia sotaní. However, the present invention corresponds to a completely different strain of Trichoderma longribachiatum.

In contrast to the records in question, our invention Trichoderma longribachiatum with the key deposit: NRRL 50762 presents the ability to inhibit the following pathogens of the root and leaf tissue under in vitro conditions: Fusarium moniliforme, Aspergillus flavus, Phymatotrichum omnivorum, Colletotrichum spp., Botrytis cinerrea, Penicillium spp., and Macrophomina phaseolina. In addition, the strain described in the present invention is totally different from the strains of Trichoderma spp described in the records in question.

Other strains of Trichoderma spp., Different from our present invention have been used in the form of a hydroalcoholic extract in the invention number ES2307870. However, our invention differs from the previous one since we propose the use of spores and mycelium of a strain of Trichoderma longribachiatum with the key of deposit: NRRL 50762 which differs morphologically and molecularly from the invention number ES2307870.

There are other inventions such as MXPA04012451 and MX2008005371, in which products formulated based on other genetically transformed strains of Trichoderma sp are described, where said formulations have application to solanaceous plants. Although the present invention, in a further embodiment / proposes a formulation, differs from the commented inventions since the formulation of the present application contains spores of Trichoderma longibrachiatum THICA-4 obtained through a fermentation in the solid state using organic substrates and inorganic, where said formulation is applied to plants or soil.

Other inventions such as the one described in document US5922603, propose the generation of methodologies for obtaining enzymes (proteinases) from the overexpression of the gene related to their production. This differs from the present invention since our proposal consists in the formulation of a product from a solid fermentation that does not require sophisticated equipment for its elaboration, allowing the obtaining of mycelium and spores from a strain of Trichoderma different from those mentioned in said document.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Histogram showing the capacity of inhibition of strain THICA-4 (3) compared to two commercial strains called Trichoderma viride Q07 (1) and Trichoderma harzianum T22 (2) against phytopathogenic fungi, which were Macrophomina phaseolina (bar) dark gray color), and Colletotrichum spp., (light gray bar). To calculate the inhibition capacity of strains THICA-4 (3) and Trichoderma viride Q07 (1) and Trichoderma harzianum T22, dual cultures were performed which consisted in placing at each end of a petri dish prepared with fungal culture medium, a 5 mm diameter sample of each strain of ThlCA-4 and Trichoderma viride Q07 and Trichoderma harzianum T22; and at the other end of the box a 5 mm diameter sample of the pathogenic fungi to be evaluated. Said operation was performed for each strain of Trichoderma with each of the pathogenic fungi, the boxes of culture medium containing the fungi was incubated for three days at 32 ° C and at a relative humidity of 50%. After three days of growth, the growth radii of the pathogen (RCP) and the Trichoderma strains (RCT) were measured with the help of a vernier. The percentage of inhibition of radial growth (PICR), was calculated using the formula PICR = (R1 - R2) / R1 x 100, where R1 is the radius of the control pathogen and R2 is the radius of the pathogen in confrontation with the strains of Trichoderma spp., respectively.

In Figure 1, it is observed in said histogram on the X axis, the numbering (1), (2) and (3) that corresponds to the strains of Trichoderma viride Q07 (1); Trichoderma harzianum 22 (2) and Trichoderma longibrachiatum THICA-4 (3), respectively, and their effect on phytopathogenic fungi, Macrophomina phaseolina (dark gray bar), and Colletotrichum spp., (Light gray bar). The percentage of inhibition of the phytopathogenic fungi Macrophomina phaseolina (dark gray bar) and Colletotrichum spp. (Light gray bar) is shown on the Y axis of the histogram. In this histogram it is observed that the strain T. longibrachiatum THICA-4 (3) with the registry key NRRL 50762 was superior in the capacity of inhibition to the two commercial strains (1) and (2) on the third day under in vitro conditions , for pathogens. Where the strain of 7. longibrachiatum ThlCA-4 (3) inhibited 70% to Colletotrichum spp., And 90% to Macrophomina phaseolina. In contrast, the commercial strain Trichoderma viride Q07 (1) only inhibited Macrophomina phaseolina by 50% and showed no inhibitory effect on Colletotrichum. which consisted of placing at each end of a petri dish prepared with culture medium for fungi, a 5 mm diameter sample of each strain of ThlCA-4 and Trichoderma viride Q07 and Trichoderma harzianum T22; and at the other end of the box a 5 mm diameter sample of the pathogenic fungi to be evaluated. Said operation was performed for each strain of Trichoderma with each of the pathogenic fungi, the boxes of culture medium containing the fungi was incubated for three days at 32 ° C and at a relative humidity of 50%. After three days of growth, the growth radii of the pathogen (RCP) and the Trichoderma strains (RCT) were measured with the help of a vernier. The percentage of inhibition of radial growth (PICR), was calculated using the formula PICR = (R1 - R2) / R1 x 100, where R1 is the radius of the control pathogen and R2 is the radius of the pathogen in confrontation with the strains of Trichoderma spp., respectively.

In Figure 1, it is observed in said histogram on the X axis, the numbering (1), (2) and (3) that corresponds to the strains of Trichoderma viride Q07 (1); Trichoderma harzianum 22 (2) and Trichoderma longibrachiatum THICA-4 (3), respectively, and their effect on phytopathogenic fungi, Macrophomina phaseolina (dark gray bar), and Colletotrichum spp., (Light gray bar). The percentage of inhibition of the phytopathogenic fungi Macrophomina phaseolina (dark gray bar) and Colletotrichum spp. (Light gray bar) is shown on the Y axis of the histogram. In this histogram it is observed that the strain T. longibrachiatum THICA-4 (3) with the registry key NRRL 50762 was superior in the capacity of inhibition to the two commercial strains (1) and (2) on the third day under in vitro conditions , for pathogens. Where the strain of T. longibrachiatum ThlCA-4 (3) inhibited 70% to Colletotrichum spp., And 90% to Macrophomina phaseolina. In contrast, the commercial strain Trichoderma viride Q07 (1) only inhibited Macrophomina phaseolina by 50% and showed no inhibitory effect on Colletotrichum. spp. The second commercial strain Trichoderma harzianum T22 (2) showed an inhibitory effect of 10% for Colletotrichum spp., And 70% on Macrophomina phaseolina, respectively, being lower compared to the strain T. longibrachiatum THICA-4 (3).

BRIEF DESCRIPTION OF THE INVENTION The present invention describes and claims a strain of Trichoderma longibrachiatum called THICA-4 characterized by having the deposit key: NRRL 50762, also because it has the ability to control the development of fungal phytopathogenic fungi foliar diseases and root. This strain THICA-4 was shown to have an effect on the control in the following phytopathogenic fungi: Fusarium moniliforme, Aspergillus flavus, Phymatotrichum omnivorum, Colletotrichum spp., Botrytis cinerrea, Penicillium spp., And Macrophomina phaseolina under in vitro conditions. Additionally, the use of said strain Trichoderma longibrachiatum ThlCA-4 is described and claimed to prepare an agronomic formulation to promote and promote the vegetative development in plants favoring a greater absorption of nutrients that is reflected in plants of greater height and vigor. As well as to inhibit the growth of phytopathogenic fungi of plants. The form of application of this strain is with its spores, which are obtained from a solid fermentation that does not require sophisticated equipment for its elaboration. Therefore, in another aspect of the present application, a method of producing said strain is described. Said method consists of cultivating the ThlCA-4 strain in a moist substrate, preferably sterilized rice, fermenting for seven days at 33 ± 2 ° C in a growth chamber with 60% relative humidity and obtaining the cultured spores with sterile distilled water. said strain where said spore solution is used to make an agronomic formulation myofungicide.

It is a further embodiment of the present invention to describe and claim an agronomic formulation comprising a strain of Trichoderma spp., Such as the aforementioned, diatomaceous earth, disaccharides and an adherent, generating at the end a formulation with a ratio of 1.5 X 107. spores per gram. The aforementioned formulation has the ability to promote the vegetative development in transgenic cotton plants, and cucumber favoring a greater absorption of nutrients that is reflected in plants of greater height and vigor.

In another additional embodiment, the addition of the strain Trichoderma longibrachiatum ThlCA-4 to the plant root system favors vegetative growth by the production of growth hormones in plants during the first days of germination. Therefore, a further aspect of the present application describes a method for promoting growth in plants.

DETAILED DESCRIPTION OF THE INVENTION.

Isolation and characterization of the strain Trichoderma spp.

The search for a strain of Trichoderma spp., With the capacity to function as a microorganism for the biological control of phytopathogenic fungi, was carried out in a site that presented bales of rotting wheat straw. The collection procedure consisted of random sampling on different points of the wheat bales in decomposition. From each point, 300 g of straw were collected. Samples from each selected point were placed in bags and transported at room temperature to the laboratory. For the selection and isolation of the strain of Trichoderma spp., Of other fungi and bacteria, serial dilutions were carried out by diluting 1 gram of straw in 10 ml of sterile water (dilution 1), then, from dilution 1, 1 ml was taken. of the water and straw solution and placed in another tube with 9 ml of sterile water (dilution 2), this operation was repeated again until obtaining the dilution 3.

From each dilution, 100 ml of samples were taken and placed in a fungal growth medium consisting of agar, potato and dextrose (PDA) at a pH of 4.0, eliminating the presence of bacteria in the medium. cultivation and the growth of the fungus that could be present in the processed straw sample was guaranteed. Subsequently, the samples were incubated at 32 ° C for 7 days on average and, once the growth of the fungus was observed, each fungus was individually isolated in petri dishes with the PDA medium, allowing them to grow for three days at 32 °. C, discarding those fungi that did not present the characteristics of the Trichoderma spp. Genus, such as the color of the later white mycelium developing to a dark green color after sporulation. This resulted in the selection of a strain with characteristics of the genus Trichoderma spp.

Molecular identification of the microorganism.

Confirmation of the species was molecularly characterized, first by purifying the DNA and then amplifying by PCR using the primers designed to identify species of the genus Trichoderma, uTforward (5'-AACGTTACCAAACTGTTG-3 ') and uTreverse (5'-AAGTTCAGCGGGTATTCCT-3') . The forward primer comprises the last nine base pairs of the 18S rRNA and the first nine base pairs of the ITS1 region while the reverse primer comprises from position 48 to 57 of the 28S rDNA of the Trichoderma sp. Sequence, which amplify a segment of 540 base pairs. The fragments of the sequences obtained from each strain were analyzed in the GenBank database with the BLAST package (httb: WWW.INCB.BLAST).

Deposit of the stock The institution of the deposit, address, date of deposit and accession number of the sample of the biological materials of the present invention, as well as the nomenclature by classification of the biological material is listed in Table 1.

Table 1. Deposit and Classification of the Trichoderma spp., THICA-4 strain in the Agricultural Research Service (ARS) Patent Culture Collection from the United States Department of Agriculture (USDA) Ability to inhibit the growth of phytopathogenic fungi and comparison of antagonism of the Trichoderma longibrachiatum ThlCA-4 strain with respect to other species of the genus Trichoderma spp.

The ability to inhibit the growth of phytopathogenic fungi of the THICA-4 strain with the registry key NRRL 50762 was compared with different species of the genus Trichoderma: an isolated strain of a commercial product called Trichoderma viride Q07, and a second commercial strain called Trichoderma harzianum T22. For To calculate the capacity of inhibition of the strains THICA-4, Trichoderma viride Q07 and Trichoderma harzianum T22, dual cultures were performed consisting of placing a sample of 5 mm in diameter of each strain: THICA-4, Trichoderma viride Q07 and Trichoderma harzianum T22; and at the other end of the box a 5 mm diameter sample of the pathogenic fungi to be evaluated in a petri dish prepared with 25 ml of Papa Dextrose Agar (APD) which is a synthetic medium for the cultivation of fungi. The selected pathogenic fungi were Macrophomina phaseolina, and Colletotrichum spp., Which were isolated from vegetable producing areas and identified taxonomically for their use. The decision to use Macrophomina phaseolina as a pathogenic fungus in the study was based on the assumption that Macrophomina phaseolina is a fungus that affects the root tissue of plants of agricultural importance. In addition, it has a wide morphological, physiological, pathogenic and genetic variability, which has allowed it to adapt to different environmental conditions and have a wide geographical distribution. On the other hand, the use of Colletotrichum spp., Which is a pathogenic fungus, was due to the fact that it causes a disease known as anthraenosis that consists of a black rot in the fruits of different plants of agricultural importance and that affects all stages of its development, mainly in tender tissues. After three days of the start of the pathogenic fungi challenge experiment with the strains THICA-4, Trichoderma viride Q07 and Trichoderma harzianum T22, the growth rays of the pathogen (RCP) and the Trichoderma strains (RCT) were measured. the help of a vernier. The percentage of inhibition of radial growth (PICR), was calculated using the formula PICR = (R1 - R2) / R1 x 100, where R1 is the radius of the control pathogen and R2 is the radius of the pathogen in confrontation with the strains of Trichoderma spp., respectively. The results showed that the strain of T. longibrachiatum THICA-4 NRRL 50762 was superior in the capacity of inhibition to the two commercial strains on the third day under in vitro conditions, for the pathogens Macrophomina phaseolina, and Colletotrichum spp. Where the strain of T. longibrachiatum THICA-4 showed an inhibition of 70% Colletotrichum spp., And of 90% of the growth for Macrophomina phaseolina. In contrast, the commercial strain Trichoderma viride Q07 only inhibited Macrophomina phaseolina 50% and had no inhibitory effect on Colletotrichum spp. With respect to the second commercial strain Trichoderma harzianum T22, it showed an inhibitory effect of 10% and 90% less for Colletotrichum spp. and Macrophomina phaseolina, respectively, compared to strain T. longibrachiatum THICA-4 (Figure 1).

Formulations containing the strain Trichoderma longibrachiatum ThlCA-4.

Once the strain Trichoderma longibrachiatum THICA-4 was identified and deposited with the deposit No. NRRL 50762, the appropriate culture medium was determined for the growth of said organism and the optimum conditions for its growth. In a further embodiment, the strain of the present invention can be used in spores, since they have the ability to last longer than when using the active cells. The application form of this strain Trichoderma longibrachiatum THICA-4, but more specifically of its spores, is made by the formulation of a product from a solid fermentation that does not require sophisticated equipment for its preparation. Later, the water with the spores (suspension of spores), is mixed with diatomaceous earth. The excess moisture in said mixture can be removed by drying. Subsequently, a formulation is made that includes a diatomaceous earth base with the spores of the THICA-4 strain, disaccharides as carbon source and an adherent, generating at the end an agronomic formulation with a 1.5 X ratio 107 spores per gram of the mixture described above. Said formulation can be prepared in granules or powders.

Use and application of the Trichoderma longibrachiatum strain ThlCA-4 for resistance to phytopathogens.

The ability to inhibit the growth of phytopathogenic fungi of strain ThlCA-4 with the registry key NRRL 50762, was compared with the following phytopathogenic fungi under in vitro conditions: Fusarum moniliforme, Aspergillus flavus, Phymatotrichum omnivorum, Colletotrichum spp., Botrytis cinerrea , Penicillium spp., And Macrophomina phaseolina. Where the criterion for their selection was that all phytopathogenic fungi had a wide geographical distribution and infection to agricultural crops. As well as the potential risk that these mushrooms could represent to the producers. For this, the following methodology was carried out: petri dishes were prepared with potato dextrose agar, where a 5 mm diameter disk of a culture of the phytopathogenic fungus was placed. Subsequently, we wait for the necessary time for this to start its mycelial growth. After the conditioning period of the phytopathogenic fungus, a 5 mm diameter disc of the culture of Trichoderma longibrachiatum THICA-4 was seeded on the opposite side, at a distance of 6 cm. For each phytopathogenic fungus, three replicates were prepared and left in incubation for three days under conditions of 33 + 2 ° C. As a control, a mycelial disk of the pathogen without the Trichoderma longibrachiatum strain THICA-4 is used. After sowing the strain Trichoderma longibrachiatum THICA-4, the antagonistic activity was determined according to the degree of mycoparasitism with a scale of 1 to 5 in which 1 is 100% of mycoparasitism and 5 is 0% of mycoparasitism (Table 2) . It was observed that the strain Trichoderma longibrachiatum THICA-4 presented an antagonistic capacity between 1 and 2, depending on the Phytopathogenic fungus. This allows to classify the strain Trichoderma longibrachiatum THICA-4 as highly inhibitory of the growth of the phytopathogenic fungi evaluated.

Use and application of Trichoderma longibrachiatum ThlCA-4 strain to stimulate plant growth.

On the other hand, to evaluate the effect that the present invention has on the ability to stimulate growth in plants of agricultural interest. The following experiment was performed. Two economically important crops were selected for the northwest region, such as cotton and cucumber. On plants of transgenic cotton and cucumber with 15 days after having germinated, the application of the present invention was made called ThlCA-4 with registration key NRRL 50762, by applying a concentration of 1.5 X 107 spores per gram of the product formulated with diatomaceous earth to the root system of each of the plant species evaluated. This concentration of spores was defined on the basis of which higher doses for example doses of 1 X 1012 or low 1 x 102 spores per gram of product can cause a variable effect in the plants. Once inoculated, the seedlings were kept in a growth chamber with a temperature range between 30 and 35 ° C during the day, and between 26 and 28 ° C during the night.

A photoperiod of 12-h light was used: darkness and 60% relative humidity. The plants established in the growth chamber were subjected to a daily irrigation regime with distilled water and fertilized weekly with a nutritious Hoagland's solution. 30 days after the inoculation, 10 inoculated and 10 non-inoculated seedlings with the THICA-4 strain with the registry key NRRL 50762 were randomly collected for physiological analysis. The growth parameters evaluated in the seedlings of Transgenic cotton and cucumber 30 days after inoculation were: relative growth range (RCR); duplication time (TD); height (H); number of leaves (Ho), and radical architecture (number of roots of 1o), 2nd and 3rd order). (Table 4 and 5). The relative growth range (RCR), which is the increase of the dry mass, per unit mass, per unit time (g / g day), was calculated with the following formula: RCR = (InMi-ln M2) / ( you). Where: M0 = the initial mass of the seedlings after 15 days of growth, M? = is the final mass after 30 days of growth, ti = days of growth. The TD was the time required by the plant to double its growth, expressed in days. This variable was calculated with the following formula: TD = In 2 / RCR. On the other hand, seedling height was determined by measuring the base of the stem at the apex of the youngest leaf using a 30 cm ruler. Finally, the evaluation of the architecture of the root was evaluated by counting the number of roots of 1st, 2nd and 4th order, using a stereomicroscope. The results showed that the inoculation of the present invention, the THICA-4 strain with the registration key NRRL 50762, favors a greater absorption of nutrients and water, which is reflected in plants with higher height, longer time of biomass duplication and rate of relative growth, number of leaves and root development, with respect to the plants that are not inoculated with our invention (Table 4 and 5).

EXAMPLES AND PREFERRED EMBODIMENT OF THE INVENTION.

The examples that follow are in no case to be considered as a limitation of the invention.

Example 1. Molecular characterization of Tríchoderma longibrachíatum ThlCA-4.

The identification and molecular confirmation of the strain of the present invention was carried out by means of the analysis of the partial gene of the 18S ribosomal DNA, for which it was amplified the gene by the PCR technique, was subsequently purified and sequenced, obtaining a nucleotide sequence corresponding to 534 base pairs. This sequence was analyzed in the GenBank database with the BLAST package (htt: WWW.INCB.BLAST), to confirm the identity of the microorganism and registered in the GenBank with the key: HQ667667.1. The fragments obtained were sequenced and the sequence obtained is that indicated in SEQ ID No. 1.

The analysis of the sequence of the strain showed a 90% homogeneity with Trichoderma longibrachlatum, so it was registered in the Gen-Bank database with the number HQ667667.

Example 2. Experiment to determine the antagonistic capacity (inhibition of growth) of the strain Trichoderma longibrachiatum ThlCA-4 on phytopathogenic fungi.

The antagonistic capacity or inhibition of the strain Trichoderma longibrachiatum THICA-4 on Fusarium moniliforme, Aspergillus flavus, Phymatotrichum omnivorum, Colletotrichum spp., Botrytis cinerrea, Penicillium spp., And Macrophomina phaseolina was carried out under in vitro conditions.

For this, the following methodology was carried out: petri dishes with Potato Dextrose Agar (PDA) were prepared in which a 5 mm diameter disc of a culture of the phytopathogenic fungus was placed. Subsequently, we wait for the necessary time for this to start its mycelial growth. After the conditioning period of the phytopathogenic fungus, a 5 mm diameter disc of the culture of Trichoderma longibrachiatum THICA-4 was seeded on the opposite side, at a distance of 6 cm. For each phytopathogenic fungus, three replications were prepared and left in incubation for three days under conditions of 33 ± 2 ° C. As a control, a mycelial disk of the pathogen without the Trichoderma longibrachiatum strain THICA-4 is used. After planting the Trichoderma strain longibrachiatum THICA-4 was determined, the antagonistic activity according to the degree of mycoparasitism with a scale of 1 to 5 in which 1 is 100% of mycoparasitism and 5 is 0% of mycoparasitism (Table 3). It was observed that the strain Trichoderma longibrachiatum THICA-4 presented an antagonistic capacity between 1 and 2, depending on the phytopathogenic fungus.

This allows to classify the strain Trichoderma longibrachiatum THICA-4 as highly inhibitory of the growth of the phytopathogenic fungi evaluated.

Table 2. Mycopasitic capacity of the Trichoderma longibrachiatum THICA-4 strain against phytopathogenic fungi at three days of contact under in vitro conditions.

Example 3. Experiment selection of the substrate for the production of inoculum by fermentation in solid medium of the strain Trichoderma longibrachiatum ThlCA-4.

The selection of optimal substrate to obtain biomass of the strain Trichoderma longibrachiatum THICA-4, to be used in the product formulation process was carried out in the following way: petri dishes were prepared with Potato Dextrose Agar, (PDA) where I place a 5 mm diameter disc of a culture of the strain Trichoderma longibrachiatum THICA-4. Subsequently, it was allowed to grow for three days and after this time, sterile water was added and a spore suspension was obtained at a concentration of approximately 1 x 103 spores / ml of water. Subsequently, 30 grams of the following substrates were placed in jars of 100 ml: rice (1); wheat straw (2) and forest land (3) independently and in triplicate. To the jars with the substrates were added sterile water until obtaining a humidity of 30% and later they were sterilized by moist heat at 120 ° C and 15 pounds of pressure for 2 hours. Once the substrate was sterilized, it was inoculated with 5 ml of the spore suspension of the strain Trichoderma longibrachiatum THICA-4, previously obtained at a concentration of 1 x 103 spores / ml. The bottles with the respective substrates were incubated in a humid chamber with a temperature range between 30 to 35 ° C and 60% relative humidity. Daily observations were made and the results showed that the jars with rice as substrate, was the only one that showed a 100% growth at seven days after the inoculation with respect to the straw of wheat and earth of mount that presented 0% of growth, respectively, during the period evaluated (Table 3).

Table 3. Growth of the strain Trichoderma longibrachiatum THICA-4, in different substrates The application form of the strain Trichoderma longibrachiatum THICA-4, and / or its spores, is carried out by means of the formulation of a product obtained from the solid fermentation having rice as substrate. Once obtained these spores, they are extracted with water to have a suspension that is mixed with diatomaceous earth.

The excess moisture of said mixture is eliminated by drying. Subsequently, a formulation was made that includes, in addition to a diatomaceous earth base with the spores of the THICA-4 strain, a carbon source, preferably disaccharides, and an adherent, generating at the end an agronomic formulation with a 1.5 ratio X 107 spores per gram of the above-described mixture. A preferred embodiment having the formulation consists of a mixture with a 50% proportion of the diatomaceous earth base with the spores of the ThlCA-4 strain, 25% of adherent preferably gelatin and 25% of disaccharides preferably sucrose. Said formulation can be prepared in granules or powders.

Example 4. Experiment for the determination of the effect of the strain Trichoderma longibrachiatum THICA-4 on the growth of plants.

In order to evaluate the effect of the inoculation with Trichoderma longibrachiatum ThlCA-4 on the physiological development of agricultural seedlings, experiments were carried out with seeds of cucumber and transgenic cotton. For this purpose transgenic Bollgard® cotton seeds, and cucumber seeds previously disinfected with a 0.5% NaOCI solution for 3 min followed by four washes with sterile deionized water were put to germinate at 30 ° C and 60% relative humidity. When the seeds presented 15 days after germination, they were inoculated, with a formulation containing the THICA-4 strain, at an approximate concentration of 1.5 X 107 spores per gram of product. With direct application to the root system at the time of transplanting into 0.3 Liter pots containing a commercial mix of soil for horticulture (peat moss 20% and agrolita 30%).

The seedlings were kept for 30 days in a growth chamber with a temperature range between 30 and 35 ° C during the day and between 24 and 26 ° C during the night, with a photoperiod of 12-h light: dark and a 60% relative humidity. Where at the end of the experiment the variables were determined: relative growth rate, doubling time, height and number of leaves. As well as the number of roots of 1, 2, 3 and 4 order in the plants inoculated and not inoculated. The results on the growth and development of the number of roots in the inoculated plants are shown in Tables 4 and 5, respectively.

The results showed that the inoculation with the strain THICA-4 with the registry key NRRL 50762, but more specifically with an agronomic formulation containing spores of the Trichoderma longibrachiatum strain THICA-4, favors a greater absorption of nutrients and water. The above was demonstrated since plants were obtained with higher height, a greater time of duplication of biomass and relative growth rate, as well as a greater number of leaves and root development compared with plants that were not treated with our formulation (Table 4 and 5). The data obtained is shown in Tables 4 and 5, where the data of inoculated plants, ie, plants treated with the present invention, had a significant effect with a value of p < 0.05 Table 4. Effect of the inoculation of Trichoderma strain THICA-4 on the growth of the seedlings.

Different letters between inoculated and non-inoculated plants indicate a significant effect with a value of p < 0.05 Table 5. Effect of inoculation Trichoderma strain THICA-4 on the number of roots of 2, 3 and 4 order with respect to non-inoculated plants.

Different letters between inoculated and non-inoculated plants indicate a significant effect with a value of p < 0.05

Claims (17)

CLAIMS Having described the invention, as above, I consider as a novelty and claim of my property contained in the following claims:

1. A strain of Trichoderma longibrachiatum THICA-4 with accession number NRRL 50762.

2. The strain of Trichoderma longibrachiatum THICA-4 according to claim 1, further characterized in that it contains SEQ ID: 1.

3. The strain of Trichoderma longibrachiatum THICA-4 according to the preceding claims, further characterized because it has the ability to control the development of phytopathogenic fungi causing plant diseases.

4. The strain of Trichoderma longibrachiatum THICA-4 according to the preceding claim, further characterized by the phytopathogenic fungi are: Fusarium moniliforme, Aspergillus flavus, Phymatotrichum omnivorum, Colletotrichum spp., Botrytis cinerrea, Penicillium spp., And Macrophomina phaseolina.

5. The strain of Trichoderma longibrachiatum THICA-4 according to the previous claims, further characterized because it has the ability to promote the growth of plants.

6. A formulation of an agronomic product characterized in that it contains: a strain of Trichoderma longibrachiatum THICA-4 with accession number NRRL 50762, as claimed in the preceding claims; and an agronomically acceptable vehicle.

7. The formulation of the preceding claim, characterized in that it contains 1.5 X 107 spores of the strain Trichoderma longibrachiatum THICA-4 per gram.

8. The formulation of claim 6, characterized in that said agronomically acceptable carrier consists of a mixture containing diatomaceous earth, an adherent and a carbon source.

9. The formulation of the preceding claim, characterized in that said mixture has the following proportions: 50% diatomaceous earth, 25% adherent and 25% carbon source.

10. The formulation of claims 8 and 9, characterized in that said adherent is preferably gelatin.

11. The formulation of claims 8 and 9, characterized in that said carbon source is preferably a disaccharide.

12. The formulation of the preceding claim, characterized in that said disaccharide is preferably sucrose.

13. The formulation of claims 5, 6, 7, 8, 9, 10, 11 and 12, further characterized in that it is useful for promoting the growth of plants.

14. The formulation of claims 6, 7, 8, 9, 10, 11, and 12, characterized in that its presentation is allowed to be in the form of powder and granules.

15. A production method by fermentation in solid medium of the strain Tríchoderma longibrachiatum THICA-4, characterized in that it comprises the following steps: a) cultivate the strain Tríchoderma longibrachiatum THICA-4 with accession number NRRL 50762 in a moist substrate, preferably sterilized rice; b) ferment for seven days at 33 ± 2 ° C in a growth chamber with 60% relative humidity and; c) obtain the cultured spores of the said strain with sterile distilled water.

16. A method for promoting growth in plants, characterized in that it comprises the following steps: a) sowing the seeds, allowing a seedling for 15 days and, b) apply to the root system of said plants the agronomic formulation of claim 6 which contains the strain Trichoderma longibrachiatum THICA-4 with accession number NRRL 50762.

17. The method of the preceding claim, further characterized in that the plant is transgenic cotton and cucumber.