WO2019063865A1 - Novel strain of trichoderma aggresivum fsp europaeum, compositions and applications thereof - Google Patents

Abstract

The present invention relates to a new strain of Trichoderma aggressivum fsp europaeum, having the CECT deposit number 20974 and which has been named Tae52481. Due to the properties of this new strain, the present invention contemplates its use for potentiating the growth of plants, such as than horticultural or ornamental crops. Thus, the invention relates to a composition which comprises said strain, which can promote the growth of plants in saline soils, in the presence of pathogenic stress conditions. The invention therefore describes its use as a biofertilizer for plants.

Description

 D E S C R I P C I O N

NEW CEPA DE Tríchoderma aggressivum fsp europaeum, COMPOSITIONS AND APPLICATIONS OF THE SAME.

SECTOR OF THE TECHNIQUE

This invention is framed in the technical sector of agricultural microbiology, more specifically in that relating to the application of microorganisms in agricultural crops for biofertilization and promotion of plant growth.

BACKGROUND OF THE INVENTION

The use of microorganisms in agriculture has experienced a remarkable development in recent years. Its use is oriented mainly for two purposes: to act as an agent of biological control of pests or diseases of crops and / or to exert a plant growth promoting effect.

 There are numerous beneficial microorganisms, and their use in agricultural systems can improve nutrition, resistance to abiotic and biotic stresses, and provide an effective transition from production systems based on the use of chemical products to more sustainable ones. The search for new tools to reduce the use of pesticides and chemical fertilizers is an objective that must be achieved in the medium term.

The most effective microorganisms as plant growth promoters are the plant growth promoting bacteria (PGPR, Plant Growth Promoting Rhizobacteria), mycorrhizal fungi, and the plant growth promoting fungi (PGPF, Plant Growth Promoting Fungi). Microorganisms with beneficial effects on plants have considerable potential as biofertilizers and as biological control agents. Currently, in agriculture, treatments with biopreparations are applied to different crops, especially in the sowing and transplant phase, since the need arises to protect the seedling at this time of stress, making it strong and vigorous. Microorganisms can also be used as bioactivators or biostimulators, which are defined as "products containing living or latent cells of previously selected microbial strains, which are characterized by producing physiologically active substances (auxins, gibberellins, cytokinins, amino acids, peptides and vitamins) that when interacting with the plant trigger different metabolic events such as stimulating the growth, development and yield of crops "(Dibut, 2006). Plant biostimulants are formulated with various microorganisms and / or substances that are applied to different crops with the aim of improving growth, development and adaptation to abiotic stress, products formulated based on the genus Trichoderma have been very successful due to their great capacity to control phytopathogenic fungi, due to their mycopathogenic nature and also , to promote the development of plants Some strains of Trichoderma have a predominant biostimulating action that makes them unique for different crops, they are safe for humans, livestock and crop plants and in their natural state.

They colonize the roots of plants without apparent adverse reactions. Both solid and liquid formulations containing conidia can be used to produce adequate amounts of active and viable inoculants from the time the formulation is produced until it is used in the field. The mechanism of phytostimulation by Trichoderma involves multiple levels: communication with the root and shoot systems, since it releases auxins, small peptides, volatile and other active metabolites in the rhizosphere, which promote the branching of the roots and the absorption of nutrients , thus increasing the growth and performance of the plants.

The species belonging to the genus Trichoderma are characterized by being saprophytic fungi, which survive in soils with different amounts of organic matter, which are capable of decomposing it and under certain conditions can be facultative anaerobes, which allows them to show a greater ecological plasticity. Trichoderma species are present in all latitudes, from the polar to the equatorial zones. This wide distribution and its ecological plasticity are closely related to the high enzymatic capacity they possess to degrade substrates, a versatile metabolism and resistance to microbial inhibitors. However, the most serious diseases in the cultivation of mushrooms are the so-called green molds caused by fungi of the genus Trichoderma, giving rise to enormous yield losses in mushroom plantations, due to their mycopathogenic nature. In Europe, the most aggressive form is Trichoderma aggressivum f. europaeum This variety is a non-aggressive biotype of T. harzianum but differs considerably from it, mainly because of the speed of mycelial growth. However, this species does not affect other species than fungi, so it can be used as a plant promoting agent, as well as controlling phytopathogenic fungi. The need to find mechanisms that increase the productivity of the field has driven the search for strategies of control of agricultural diseases that are efficient alternatives to chemical control and that also involves lowering the environmental and health risk without risking human health, today constitutes a great challenge for agriculture and its development.

OBJECT OF THE INVENTION

The subject of the invention is a new strain of Trichoderma aggressivum fsp europaeum with deposit number CECT No. 20974.

Likewise, the object of the invention is the use of the strain, and of the composition comprising it, for biofertilization, to enhance the growth and / or production of plants, for example, horticultural crops, ornamental plants and even forestry. All the embodiments mentioned in the dependent claims are herein object of the present invention.

"Biofertilizer" and "biostimulant" have the same meaning, as substances that stimulate the growth of plants. EXPLANATION OF THE INVENTION

The present invention relates to a new plant growth promoter strain, named Tae52481, of the species Trichoderma aggressivum fsp europaeum, which was deposited on May 23, 2017, in the Spanish Type Culture Collection, with CECT No. 20974, ( University of Valencia, 56100 Burjassot, Valencia, Spain) according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of the Patent Procedure.

Said strain constitutes an effective biostimulating microorganism and promoter of the growth of plants, such as crops. This promoter fungus, cultivated in a Biologically pure, it is called Tae52481 and has been identified by sequencing the ITS (Infernal Transcriber Spacer) region of the rDNA using the ITS-1 and ITS-4 primers. Strain Tae52481 of Trichoderma aggressivum fsp europaeum was isolated from substrates with Agaricus bisporus (mushroom) with symptoms of the disease known as green mold caused by T. aggressivum fsp europaeum from the Manchuela region (Cuenca). The growth rate of T. aggressivum fsp europaeum in agar culture medium (agar-malta) and in an oven at 25 ° C has been:

- Day 1: 1 cm

 - Day 2: 2.7 cm

 - Day 3: 4.4 cm

 - Day 4: 6, 1 cm

-Day 5: 7,7 cm

-Day 6: the whole plate (petri dish of 9 cm in diameter).

The strain Tae52481 of Trichoderma aggressivum fsp europaeum has been tested under nursery and field conditions. The tests have been carried out under conditions of commercial use in which, by way of example, it can be indicated that in no case in which has been tested has a disinfection or sterilization of the substrates or soils in which it has been carried out inoculation, a practice that is generally recommended for many products of this type, which is an advantage over existing biofertilizers. Given the properties of this new strain, its use is contemplated in the present invention to enhance the growth of plants, for example, horticultural crops, ornamental or forest plants.

The present invention also relates to a biofertilizer composition comprising a biologically pure culture of the strain Tae52481.

 According to particular embodiments, the present invention also relates to a biofertilizing composition comprising at least:

 - a biologically pure culture of strain Tae52481 and

- A vehicle. From the concept "biologically pure culture of the strain Tae52481" it should be understood that the culture may contain metabolites derived from the strain itself.

In this specification "biologically pure culture of the strain Tae52481", "the strain Tae52481", "spores of the strain Tae52481" or "Tae52481" are used interchangeably. The composition of the invention may be in solid, liquid, gelled or colloidal form.

The composition of the invention may further comprise one or more microorganisms other than Trichoderma aggressivum fsp europaeum, for example, another species of Trichoderma.

The vehicle has the function of being a suitable agent and necessary for the dispersion of the strain, multiplication, or both, or also for the conservation of the spores without germinating, for example, when the vehicle is a saline solution.

The vehicle can be one or more products that are mixed with the culture of the strain Tae52481 as a means to obtain a composition that can be handled, and which at the same time favors the dispersion and / or multiplication of the strain. A vehicle in the compositions of the present invention is selected from: a) - substrate

b) - irrigation water,

c) - saline solution

d) - mixtures of at least two of a) b) and c).

The carrier may be solid, liquid, colloidal or gelled, for example, it is gelled in the case that alginate is added. Thus, in a particular embodiment of the composition of the invention, the vehicle is selected from a solid, liquid, colloidal or gelled medium, where the spores of the fungus are to be incorporated.

In the context of the present invention, a substrate is a substance or a mixture of substances that can be used for the growth of plants. Said substrate can be, for example, any of those commonly employed in horticulture. A substrate generally comprises one or more types of neutral light aggregates (perlite, vermiculite among others) and an organic constituent (Sphagnum peat, compost, among others), optionally with soil. In the formulation of substrates the proportions used of the aforementioned ingredients may vary, depending on the chosen substrate, the size of the pot, type of plant to be produced, irrigation system, among other factors, and therefore, the quantity must be adapted for each case.

The substrate can be for example peat, coconut fiber, compost and mixtures thereof. In particular embodiments the substrates are compost, preferably those obtained from grape pomace, mushroom, horticultural waste, urban solid waste, garden waste, vermicompost and mixtures thereof. In preferred embodiments of the composition of the invention, substrates formulated based on peat or compost of the TCP type (peat + compost + perlite), compost-peat-vermiculite, compost-peat-coconut fiber, or any mixture of the same. The solid compositions may be in the form of powder, granules or wettable powders, while the liquid compositions may be in the form of aqueous or non-aqueous media, in suspensions, dispersions or in concentrated form. Likewise, the strain Tae52481 of Trichoderma aggressivum fsp europaeum can be included in matrices of biopolymers (without excluding other possible formulations) such as calcium alginate, agar, and the possible additives that are considered for each situation to which the product is destined.

The microorganism, ie the strain Tae52481, can be mixed, for example, with saline, or it can be encapsulated, or a substrate, such as peat, can be mixed with the spores of Tae52481.

According to particular embodiments the composition of the invention comprises a culture of Tae52481 mixed with a substrate, such as peat, which acts as a vehicle, for its application.

In the case of mixing the culture of the strain with the substrate, it is also optionally possible to obtain a composition in which the culture of the strain is mixed with saline, and this mixture in liquid form, is added to a substrate that also acts - in addition to the saline solution - as a vehicle. The present invention also relates to a combination comprising at least:

- a biologically pure culture of strain Tae52481 and

 - an agent selected from:

 - a plant.

 - a seed.

In the case of the combination of the invention, the agent, - the plant itself to be biostimulated or a seed of a plant - is the object on which the strain Tae52481 is intended to produce its effects.

The combination according to particular embodiments comprises:

 - a biologically pure culture of the strain Tae52481

 - a plant.

The combination according to particular embodiments comprises:

 - a biologically pure culture of the strain Tae52481

 - a seed and

 - A vehicle.

A vehicle, having the same functions mentioned above for the compositions, in the combinations comprising seeds, according to the present invention is selected from: a) - a substrate

b) - irrigation water,

c) - saline solution

d) - mixtures of at least two of a), b) and c).

The substrate in the defined combinations can be any of those defined above for the compositions of the invention.

In the case that the agent is a seed, the strain can be mixed with the seed by the use of a substance such as alginate, so that the seeds are impregnated with the strain. That is, the strain can be incorporated into the seed, the substrate where it is going to be planted or simply adding it to irrigation water.

The composition of the invention can therefore be part of a seed, which results in a combination as defined above. In this case, the seeds themselves also act as a vehicle for dispersion and / or multiplication of the strain. Optionally, a combination containing the microorganism impregnated on seeds can be prepared, and in turn forming part of a substrate.

In the combinations defined above, seeds may be impregnated with the spore suspension, or a substrate, such as peat, mixed with the spores of Tae52481. Both in the biostimulant compositions and in the defined combinations, the strain Tae52481 is present in the form of conidia. The concentration of conidia that is added to the composition or combination can be comprised between 1-108 and 1-1012 conidia / mL of water. Both the biostimulant compositions and the combinations defined above may include adjuvants, such as emulsifiers, suspending agents, tackifiers, etc., as well as one or more other growth-stimulating and biological control agents. The obtaining of the biofertilizing composition of the invention or the defined combinations can be carried out by inoculating the strain Tae52481 in the corresponding vehicle, either incorporated into a commercial substrate as peat, or resuspending the spores at the commercial dose in the vehicle that is desired either liquid or solid and that allows its survival.

The composition of the invention is effective to enhance the growth and / or production of plants. The plants that may be susceptible to growth promotion include horticultural, ornamental and even forestry plants. Without establishing any limitations, the effects caused by Trichoderma aggressivum fsp europaeum Tae52481 affect the growth of the plant, which can be in the form of increasing the root mass, root depth, stem size or plant length. Trichoderma aggressivum fsp europaeum Tae52481 can be applied in any medium and environment without limitation (to the soil, to a substratum, to a hydroponic culture simply adding it to water) as long as it is in the right conditions (that is, the normal conditions of a culture). In another main aspect the invention relates to the use of Trichoderma aggressivum fsp europaeum Tae52481, to the use of the composition described, or to the use of the combinations described, to enhance the growth and / or production of horticultural crops or other plants in soils with problems phytosanitary or stress. In another principal aspect the invention relates to the use of Trichoderma aggressivum fsp europaeum Tae52481 with compatible fungicides. To facilitate the tasks for the farmer, in the same irrigation you can use other compatible microorganisms or fungicides, in a single application. The application methods of Trichoderma aggressivum fsp europaeum Tae52481 are mainly carried out by means of conidia. Without establishing limitations, the composition usually comprises one or more adjuvants and the microorganism in question. The concentration of conidia that is added to the composition or combination can be comprised between 1-108 and 1-1012 conidia / mL of water. This microorganism has been produced in a solid base culture medium supplemented with the nutritional elements required for each case.

The present invention also relates to a method for stimulating the growth of plants comprising obtaining a composition as defined above and contacting the plants or seeds of said plants with the composition.

The following examples illustrate the present invention, but should not be considered as limitations to the essential aspects of the object of the same, as they have been exposed in the previous sections of this description. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 on different morphological parameters of tomato seedlings in the big-plant culture system. In each parameter the unit is indicated, so the "y" axis are only numbers that correspond to the treatments performed (x axis).

Figure 2. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 on different morphological parameters of melon seedlings in the big-plant culture system.

Figure 3. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 on a broccoli culture in soil.

Figure 4. Effect of the application of Trichoderma aggressivum fsp europaeum plantlets broccoli in pot.

Figure 5. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 at three different doses (TAE D1, TAED2 and TAED3: 104, 105, 106 spores / mL, respectively) on the total fresh weight of tomato seedlings. T0: witness.

Figure 6. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 at three different doses (TAE D1, TAED2 and TAED3: 104, 105, 106 spores / mL, respectively) on the dry weight of the root of tomato seedlings. T0: witness.

Figure 7. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 at three different doses (TAE D1, TAED2 and TAED3: 104, 105, 106 spores / mL, respectively) on the number of leaves of tomato seedlings. T0: witness.

Figure 8. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 by irrigation on the incidence of gray rot in peppers.

Figure 9. Effect of the application of Trichoderma aggressivum fsp europaeum Tae52481 on the development of papaya seedlings PREFERRED EMBODIMENT OF THE INVENTION

Example 1

A strain of Trichoderma genus, Trichoderma aggressivum fsp europaeum Tae52481, was isolated from compost, used as a substrate in culture of Agaricus bisporus, with symptoms of green mold disease, in the La Manchuela region, Cuenca province, as follows: a sowing of the compost in Petri dish and a subsequent identification of the colonies formed. The resulting pure strain was named Tae52481 and was identified by PCR (Polymerase Chain Reaction) and subsequent sequencing of the ITS region (Internal! Transcriber Spacer) of the rDNA, belonging to the species Trichoderma aggressivum fsp europaeum

Example 2

 The strain was grown in a PDA agar medium (Potato Dextrose Agar) in a laboratory environment and the amount of conidia produced by the Petri dish was evaluated. Then, conidial extraction and hematocimeter quantification of its concentration were carried out. The final product in sterile water reached a concentration between 1 - 106-107 conidia / mL.

Feasibility tests in saline medium showed viability of conidia in 100% of the cases, after storage periods of 24 months. As well as an adequate conservation in said formulations, which were manifested in the absence of germination in them during the storage period.

Example 3

Figure 1 shows the results obtained after the application of Trichoderma aggressivum fsp europaeum Tae52481 in the development of tomato seedlings in the "big-plant" culture system and the substrate used was peat. Trichoderma aggressivum fsp europaeum Tae52481 was applied at a dose of 106 spores / mL of water. The application was made by watering (R) to the substrate. 5 mL of the suspension was added with the spores per plant and / or pot in a single application. The duration of the trial was 55 days, and subsequently, the data of the parameters was taken morphological The results reveal that there is an increase in the morphological parameters evaluated after the application of Trichoderma aggressivum fsp europaeum Tae52481. An increase of 2.7% is observed; 18.4%; 19%; 22.31% in the diameter, dry weight of the leaves, dry weight of the stem and foliar area, respectively. The letters a, and b, indicate statistically significant differences between treatments.

Example 4

 Figure 2 shows the results obtained after the application of Trichoderma aggressivum fsp europaeum Tae52481 in the development of melon seedlings in the "big-plant" culture system and the substrate used was peat. Trichoderma aggressivum fsp europaeum Tae52481 was applied at a dose of 106 spores / mL of water. The application was made by watering (R) to the substrate. 5 mL of the suspension was added with the spores per plant and / or pot in a single application. The duration of the trial was 55 days, and subsequently, we proceeded to take data of the morphological parameters. The results reveal that there is an increase in the morphological parameters evaluated after the application of Trichoderma aggressivum fsp europaeum Tae52481. An increase of 26.26% is observed; 9.3%; 25.8%; 4.41% in the height of the plant, dry weight of the stem and dry weight of the root and foliar area, respectively. The letters a, and b, indicate statistically significant differences between treatments.

Example 5

 Inoculation trials of Trichoderma aggressivum fsp europaeum Tae52481 were conducted in a commercial broccoli crop in a greenhouse. The inoculation was done by applying a dose of 106 spores / mL of water in irrigation. The application was made only once at the time of the transplant, adding 10 mL of the suspension of conidia per plant. The duration of the trial was 60 days.

In Figure 3, the results obtained after the application of Trichoderma aggressivum fsp europaeum Tae52481 in the development of broccoli are shown. As can be seen, there has been an increase of 51, 24; 7.6; 6 and 18.87% in dry weight of aerial part, root dry weight, number of leaves and diameter of the plant, respectively. The letters a, and b, indicate statistically significant differences between treatments. Example 6 In figure 4, the results obtained after the application of Trichoderma aggressivum fsp europaeum Tae52481 in the development of broccoli seedlings in pots of a volume of 300 ml_ are shown and the substrate used was peat. T aggressivum fsp europaeum Tae52481 was applied at a dose of 106 spores / mL of water. The application was made by watering (R) to the substrate. 5 ml_ of the suspension was added with the spores per plant and / or pot in a single application. The duration of the trial was 30 days, and subsequently, we proceeded to take data of the morphological parameters. The results reveal that there is an increase in the morphological parameters evaluated after the application of Trichoderma aggressivum fsp europaeum Tae52481. An increase of 8.5 is observed; 25.20; 21, 46; and 14, 19% in the number of leaves, height, fresh weight and total dry, respectively. The letters a, and b, indicate statistically significant differences between treatments.

Example 7

Three doses of conidia of Trichoderma aggressivum fsp europaeum Tae52481 (TaeD1: 104 spores / mL, TaeD2: 105 spores / mL of water and TaeD3: 106 spores / mL of water) were applied to the development of tomato seedlings. The application was made by watering (R) to the substrate, adding 1 mL of spore suspension per plant in a single application. The results reveal that there is an increase in the fresh weight of the plant for the three doses applied, there being significant differences with respect to the control (Figure 5). The greatest increase was observed for dose 2 (105 spores / mL), assuming an increase of 20.96% in fresh weight with respect to the control (T0). Also, this same effect is observed for root dry weight parameters (Figure 6) and number of leaves (Figure 7). The letters a, b and c indicate statistically significant differences between treatments.

Example 8

Figure 8 shows the results obtained after the application of Trichoderma aggressivum fsp europaeum Tae52481 in the control of the phytopathogenic fungus Botrytis cinerea in pepper seedlings in pots of a volume of 300 mL and the substrate used was peat. T aggressivum fsp europaeum Tae52481 was applied at a dose of 106 spores / mL of water. The application was made by watering (R) to the substrate. No control treatment against the disease was carried out by air. 5 mL of the suspension was added with the spores per plant and / or pot in a single application. The duration of the trial was 30 days, and subsequently, data was collected in terms of incidence of disease. The results reveal that there is a reduction of the disease after the application of Trichoderma aggressivum fsp europaeum Tae52481 in irrigation, and allows to control the disease, due to the good development and vigor of the plant. A reduction in the incidence of the disease of 51, 44% is observed.

Example 9

 In figure 9, the results obtained after the application of Trichoderma aggressivum fsp europaeum Tae52481 in the development of seedlings of papaya in nursery trays are shown and the substrate used was peat. T aggressivum fsp europaeum Tae52481 was applied at a dose of 106 spores / mL of water. The application was made by watering (R) to the substrate. 1 mL of the suspension was added with the spores per plant in a single application. The duration of the trial was 50 days, and later, we proceeded to take data of some morphological parameters before planting in soil. The results reveal that there is an increase of 35.76% in the height of the papaya seedlings after the application of Trichoderma aggressivum fsp europaeum Tae52481. The letters a, and b, indicate statistically significant differences between treatments.

Claims

The strain Tae52481 of Trichoderma aggressivum fsp europaeum that was deposited on May 23, 2017, in the Spanish Type Culture Collection) with CECT No. 20974. Method for obtaining the strain Tae52481 of Trichoderma aggressivum fsp europaeum defined in claim 1, comprising:  - isolating the strain from a soil comprising said strain, or from a culture medium of the strain e  - identify it by sequencing the ITS region of the rDNA using the ITS-1 and ITS-4 primers. Biofertilizing composition comprising a biologically pure culture of the strain Tae52481. Biofertilizer composition according to claim 3, comprising at least: - a biologically pure culture of strain Tae52481 and  - A vehicle. Composition according to one of claims 3 or 4, which is in solid, liquid, gelled or colloidal form. A combination comprising at least:  - a biologically pure culture of the strain Tae52481 defined in claim 1 and  - an agent selected from:  - a plant  - a seed. A combination according to claim 6, comprising at least:  - a biologically pure culture of the strain Tae52481 defined in claim 1 and  - a plant. A combination according to claim 6, comprising at least: - a biologically pure culture of the strain Tae52481 defined in claim 1  - a seed and  - A vehicle. Composition according to claim 4, or a combination according to claim 10, wherein the vehicle is selected from:  a) - substrate  b) - irrigation water,  c) - saline solution  d) - mixtures of at least two of a) b) and c). Composition according to one of claims 3 to 5, or a combination according to one of claims 6 to 8, further comprising one or more microorganisms other than Trichoderma aggressivum fsp europaeum. The composition according to claim 4, or a combination according to claim 9, wherein the substrate is selected from peat, coconut fiber, compost and combinations thereof. 12. Composition according to any of claims 3 to 5, or a combination according to one of claims 6 to 8, wherein the strain Tae52481 is present in the form of conidia. 13. The composition or combination according to claim 12, to which conidia have been added at a concentration comprised between 1-108 and 1-1012 conidia / ml_ of water. 14. Composition according to one of claims 3 to 5, or a combination according to one of claims 6 to 8, further comprising at least one of the following substances:  - an adjuvant  - a growth stimulation agent  - a biological control agent or - combinations of two or more of the above. 15. The composition or combination according to claim 14, wherein the adjuvant is selected from an emulsifier, a suspending agent and a tackifier. 16. Use of the strain defined in claim 1, or of a composition defined in one of claims 3-5, or of a combination defined in one of claims 6 to 8, for enhancing growth, production, or growth and the production of plants. 17. Use according to claim 17 wherein the plants are plants in soils with phytosanitary problems or stress. 18. Use according to claim 16 or 17 wherein the plants are horticultural crops, ornamental or forest plants. 19. Use according to one of claims 16 to 18 in which the strain Tae52481 is used simultaneously with compatible fungicides.