ES2684858B1 - New strain of Paecilomyces variotii, compositions and applications thereof - Google Patents

Description

DESCRIPTION

New strain of paecilomyces variotii, compositions and applications thereof.

Technical sector

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

State of the art

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

There are numerous beneficial microorganisms, and their use in agricultural systems can improve nutrition, resistance to abiotic and biotic stresses, and provides 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 plant growth promoting bacteria (PGPR, Plant Growth Promoting Rhizobacteria), mycorrhizal fungi, and plant growth promoting fungi (PGPF, Plant Growth Promoting Fungí). 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 various crops, especially in the sowing and transplanting 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 live 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 they trigger different metabolic events such as stimulating the growth, development and yield of crops ”(Dibut, 2006).

The term biofertilizer can be defined as "preparation containing live or latent cells of efficient microbial strains, which are used to apply them in seeds or to the soil with the aim of increasing the number of these microorganisms in the medium and accelerating microbial processes in such a way. that the amounts of nutrients that can be assimilated by plants be increased or physiological processes that influence the development and yield of crops become faster ”(Dibut, 2006).

Unlike fertilizers, biostimulators are not directly associated with the substitution of doses of chemical fertilizers in crops, but are used independently of the application or not of these inputs. Its activity producing physiologically active substances and its effect on the plant, reaches its maximum expression when the plant is properly nourished. Thus, even when fertilizers are not applied, a marked stimulating effect on yield is obtained, but in this case it must be fertilized with organic amendments to avoid soil impoverishment over several harvest cycles (Sánchez, 2011).

It is in this aspect that the registration of products containing microorganisms for use in agriculture is supported, since by using the word "fertilizer" in some countries, it allows easier registration. Based on these facts, it is common to find biostimulants and biofertilizers on the market but rather oriented towards a use as a means of phytosanitary defense.

The genus Paecilomyces is found in a wide range of habitats within which are cultivated soils, forests, grasslands, deserts, sediments and sewage sludge. It has also been detected inside nematode eggs and even inside nematode females. Likewise, a great diversity of crops has been detected frequently in the rhizosphere. This species of fungus can develop at different temperatures from 5 ° C to 50 ° C. It also has a wide pH tolerance and can be developed in a wide variety of substrates (Linares, I.2009). There are several species such as Paecilomyces lilacinus, which is considered a natural enemy of insects such as whiteflies and salivate bedbugs, and is also considered to be effective for the control of Meloidogyne, Pratylenchus and Radopholus nematodes producing enzymes capable of acting on eggs , and larvae causing deformations, and loss of movement.

Paecilomyces variotii is the asexual state of Byssochlamys spectabilis, a member of the Phylum Ascomycota (Family Trichocomaceae). However, the status of Byssochlamys is rarely observed in the crop due to the heterotolic nature of it. Paecilomyces variotii grows rapidly, producing a yellow-brown powdery colony. This species has also been described as a nematicide for nematodes of the genus Meloidogyne, Pratylenchus and Radopholus.

Object of the invention

The object of the invention is a new strain of Paecilomyces variotii with deposit number CECT No. 20957.

Likewise, the use of the strain, and the composition comprising it, for biofertilization, to enhance the growth and / or production of plants, for example horticultural crops, ornamental plants and even forestry, is an object of the invention.

The object of the present invention is all the embodiments mentioned in the dependent claims herein.

"Biofertilizer" and "biostimulant" have the same meaning, as substances that stimulate plant growth.

Detailed description of the invention

The present invention relates to a new plant growth promoting strain, called Pae10773, of the species Paecilomyces variotii, which was deposited on June 26, 2016, in the Spanish Type Culture Collection) with CECT No. 20957, (University of Valencia, 56100 Burjassot, Valencia, Spain) according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure.

Said strain constitutes an effective biostimulant microorganism and plant growth promoter, such as crops. This promoter fungus, grown in a biologically pure form, is called Pae10773 and has been identified by sequencing the ITS (Internal Transcriber Spacer) region of the rDNA (listed as SEQ ID No. 1).

Pae10omy3 strain of Paecilomyces variotii was isolated from natural soils from the coast of Almeria. The growth rate of Paecilomyces variotii in agar culture medium (agar-malt) and in an oven at 25 ° C has been:

- Day 0: 0 mm

- Day 2: 2.5 mm

- Day 4: spore dispersion through the plate (multiple isolated colonies)

- Day 6: general growth.

The Pae10773 strain of Paecilomyces variotii Pae10773 has been tested in seed 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 it has been tested has a disinfection or sterilization of the substrates or floors in which it has been carried out inoculation, a practice that, in general, is recommended for many products of this type, which is an advantage over existing biofertilizer products.

Given the properties of this new strain, the present invention contemplates its use to enhance the growth of plants, for example, horticultural crops, ornamental or forest plants.

The present invention also relates to a biofertilizing composition comprising a biologically pure culture of strain Pae10773.

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

- a biologically pure culture of the strain Pae10773.

- A vehicle.

From the concept "biologically pure culture of strain Pae10773" it should be understood that the culture may contain metabolites derived from the strain itself.

In this report "biologically pure culture of strain Pae10773", "strain Pae10773", "spores of strain Pae10773" or "Pae10773" 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 Paecilomyces variotii, for example, Trichoderma.

The vehicle has the function of being a suitable and necessary agent 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 cultivation of strain Pae10773 as a means to obtain a composition that can be manipulated, 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 vehicle can 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 fungus spores 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 plant growth. Said substrate may be, for example, any of those commonly used 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 substrate chosen, the pot size, 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 marc, mushroom, horticultural waste, urban solid waste, garden waste, vermicompost and mixtures thereof. In preferred embodiments of the composition of the invention, substrates formulated with peat or compost of the TCP type (Peat + compost + perlite), compost-peat-vermiculite, compost-peat-coconut fiber, or any mixture of 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 Pae10773 Paecilomyces variotii 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, that is, the strain Pae10773, can be mixed by way of example, with saline solution, or it can be encapsulated, or a substrate, such as peat, can be mixed with the spores of Pae10773.

According to particular embodiments, the composition of the invention comprises a culture of Pae10773 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, one can also optionally 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 the strain Pae10773.

- an agent selected from:

- a plant

- a seed.

In the case of the combination of the invention, the agent - the plant itself that is intended to be biostimulated or a seed of a plant - is the object on which the Pae10773 strain is intended to produce its effects.

The combination according to particular embodiments comprises:

- a biologically pure culture of the strain Pae10773.

- a plant.

The combination according to particular embodiments comprises:

- a biologically pure culture of the strain Pae10773.

- a seed.

- A vehicle.

A vehicle, which has the same functions mentioned above for the compositions, in combinations comprising seeds, according to the present invention is selected from:

to. 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 may 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 using a substance such as alginate, so that the seeds are impregnated with the strain.

That is, the strain can be incorporated into the seed, to the substrate where it is going to be sown or simply added in irrigation water.

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

In the combinations defined above, seeds can be impregnated with the spore suspension, or a substrate, such as peat, can be mixed with the spores of Pae10773 -. In both the biostimulant compositions and the defined combinations, the strain Pae10773 is present in the form of conidia. The concentration of conidia that is added to the composition or combination may be between 1108 and 11012 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 other or other growth stimulating and biological control agents.

Obtaining the biofertilizing composition of the invention or the defined combinations, can be carried out by inoculating strain Pae10773 in the corresponding vehicle, either incorporated into a commercial substrate such as peat, or by 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. Plants that may be susceptible to growth promotion include horticultural, ornamental and even forest plants.

Without establishing any limitation, the effects caused by Paecilomyces variotii Pae10773 affect plant growth, which can be in the form of increasing root mass, root depth, stem size or plant length.

Paecilomyces variotii Pae10773 can be applied in any environment and environment without limitation (to the soil, to a substrate, to a hydroponic crop simply by adding it to the water) as long as it is in the right conditions (i.e. the normal conditions of a crop) .

In another main aspect the invention relates to the use of Paecilomyces variotii Pae10773, the use of the described composition, or the use of the described combinations, to enhance the growth and / or production of fruit and vegetable crops or other plants in soils with phytosanitary problems or stress.

In another main aspect the invention relates to the use of Paecilomyces variotii Pae10773 with compatible fungicides. To facilitate the tasks for the farmer, in the same irrigation can be used other compatible microorganisms or fungicides, in a single application.

The methods of application of Paecilomyces variotii Pae10773 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 may be between 1108 and 11012 conidia / mL of water. Said microorganism has been produced in a solid base culture medium supplemented with the nutritional elements required for each case.

Both the biostimulant compositions and the combinations defined above may include adjuvants, such as emulsifiers, suspending agents, tackifiers, etc., as well as other or other growth stimulating and biological control agents.

Obtaining the biofertilizing composition of the invention or the defined combinations, can be carried out by inoculating strain Pae10773 in the corresponding vehicle, either incorporated into a commercial substrate such as peat, or by 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. Plants that may be susceptible to growth promotion include horticultural, ornamental and even forest plants.

Without establishing any limitation, the effects caused by Paecilomyces variotii Pae10773 affect plant growth, which can be in the form of increasing root mass, root depth, stem size or plant length.

Paecilomyces variotii Pae10773 can be applied in any environment and environment without limitation (to the soil, to a substrate, to a hydroponic crop simply by adding it to the water) as long as it is in the right conditions (i.e. the normal conditions of a crop) .

In another main aspect the invention relates to the use of Paecilomyces variotii Pae10773, the use of the described composition, or the use of the described combinations, to enhance the growth and / or production of fruit and vegetable crops or other plants in soils with phytosanitary problems or stress.

In another main aspect the invention relates to the use of Paecilomyces variotii Pae10773 with compatible fungicides. To facilitate the tasks for the farmer, in the same irrigation can be used other compatible microorganisms or fungicides, in a single application.

The methods of application of Paecilomyces variotii Pae10773 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 may be between 1108 and 11012 conidia / mL of water. Said 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 plant growth 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 on the essential aspects of the object thereof, as set forth in the previous sections of this description.

Brief description of the figures

Figure 1. Effect of the application of P. variotii Pae10773 on broccoli seedlings. In each parameter the unit is indicated, so that the "y" axis are only numbers that correspond to the x axis.

Figure 2. Effect of the application of P. variotii Pae10773 on the leaf area in broccoli plants.

Figure 3. Effect of the application of P. variotii Pae10773 on a broccoli crop in soil.

Figure 4. Development of the pepper root expressed in Dry Weight (g) after the application of two doses of Paecilomyces variotii Pae10773 (PD1SN and PD2SN). Ss N: Witness plants sensitive to nematodes; RSN: Nematode resistant plants witness.

Figure 5. Effect of the application P. variotti Pae10773 at three different doses (104, 105, 106 spores / mL) in the development of tomato seedlings. CA: witness without P. variotti Pae10773. PAE D1, PAED2 AND PAED3: 104, 105, 106 spores / mL, respectively.

Figure 6. Effect of the application P. vañotti Pae10773 at three different doses (104, 105, 106 spores / mL) in the development of tomato seedlings (Stem diameter and number of leaves). CA: witness without P. variotti Pae10773. PAE D1, PAED2 AND PAED3: 104, 105, 106 spores / MI, respectively.

Figure 7. Dry weight of the leaves (g) of tomato plants transplanted in the field containing P. variotti Pae10773 at three different doses (104, 105, 106 spores / mL).

Figure 8. Dry root weight (g) of transplanted tomato plants containing P. variotti Pae10773 at three different doses (104, 105, 106 spores / mL).

Figure 9. Total dry weight (g) of transplanted tomato plants containing P. variotti Pae10773 at three different doses (104, 105, 106 spores / mL).

Figure 10. Ql quality index of transplanted tomato plants containing P. variotti Pae10773 at three different doses (104, 105, 106 spores / mL).

Figure 11. Stem-root index of transplanted tomato plants containing P. variotti Pae10773 at three different doses (104, 105, 106 spores / mL).

Examples

Example 1

A strain of the genus Paecilomyces variotii was isolated from a natural soil of the Natural Park of Cabo de Gata (Nijar) in the province of Almeria as follows:

A suspension was made with 1 g of soil in sterile water. Subsequently, successive dilutions were made, and for each of the suspensions Petri dish extension, incubation and subsequent identification of the colonies formed were performed. The resulting pure strain was named Pae10773 and was identified by PCR (Polymerase Chain Reaction) and subsequent sequencing of the ITS (Internal Transcriber Spacer) region of the rDNA, belonging to the Paecilomyces variotii species.

Example 2

The strain was grown in PDA (Potato Dextrose Agar) agarose culture medium in a laboratory environment and the amount of conidia produced by Petri dish was evaluated. Then, the conidia were extracted and hematocimeter quantification of their concentration. The final product in sterile water reached a concentration between 1107 - 5108 conidia / mL.

Feasibility tests in saline media showed conidia viability in 100% of cases, after 12-month storage periods. As well as adequate conservation in these formulations, which manifested themselves in the absence of germination in them during the storage period.

Example 3

Figures 1 and 2 show the results obtained after the application of Paecilomyces variotii Pae10773 in the development of broccoli seedlings in pots of a volume of 300 mL and the substrate used was peat. Paecilomyces variotii Pae10773 was applied at the dose of 106 spores / mL of water. The application was made by irrigation (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, the morphological parameters were collected. The results reveal that there is an increase in the morphological parameters evaluated after the application of Paecilomyces variotii Pae10773. An increase of 20.26 is observed; 10.02; 9.18; 14.73 and 9.12% in height, fresh weight, number of leaves, dry weight and leaf area, respectively. The letters a, and b, indicate statistically significant differences between treatments.

Example 4

Inoculation assays of Paecilomyces variotii Pae10773 were performed in a commercial broccoli greenhouse crop. The inoculation was performed by applying a dose of 4106 spores / mL of water in irrigation. The application was performed only once at the time of transplantation, adding 10 mL of the conidia suspension per plant. The duration of the trial was 60 days.

Figure 3 shows the results obtained after the application of Paecilomyces variotii Pae10773 in the development of broccoli. As you can see there has been an increase of 34.18; 2.70; 1.68 and 0.92% in the dry weight of aerial part, dry root weight, number of leaves and plant diameter, respectively. The letters a, and b, indicate statistically significant differences between treatments.

Example 5

Two doses of conidia of Paecilomyces variotii Pae10773 (PD1SN: 105 spores / mL of water and PD2SN: 106 spores / mL) of water were applied to the substrate (peat) 5 ml of spore suspension to each pot of 300 mL of the suspension with spores, prior to the development of pepper seedlings sensitive to nematodes, in total absence of nematodes in the substrate. In Figure 4, the development of the root expressed in Dry Weight (g) is observed after the application of said doses of Paecilomyces variotii Pae10773. The results show that the application of both doses increases the dry weight of the pepper root, with PD2SN showing significant differences with the control (SSN). The dry weight of the root corresponding to the application of the PD2SN dose: 106 spores / mL, does not show significant differences with the use of a commercial variety of pepper (RSN) with greater vigor and resistance to nematodes. The letters a, b and c indicate statistically significant differences between treatments.

Example 6

Three doses of conidia of Paecilomyces variotii Pae10773 (PaeD1: 104 spores / mL, PaeD2: 105 spores / mL of water and PaeD3: 106 spores / mL of water) were applied to the development of tomato seedlings. The application was made by irrigation (R) to the substrate, adding 1 mL of spore suspension to 300 mL pots per plant in a single application. The results reveal that there is an increase in stem length for the three doses applied, there being significant differences with respect to the control (Figure 5). The greatest increase is observed for D1, assuming more than 30% of the stem length with respect to the control (CA). Also, this same effect is observed for the parameters stem diameter and number of leaves. (Figure 6). The letters a, b and c indicate statistically significant differences between treatments.

Example 7

Three doses of conidia of Paecilomyces variotii Pae10773 (PaeD1: 104 spores / mL, PaeD2: 105 spores / mL of water and PaeD3: 106 spores / mL of water) were applied to the development of tomato seedlings. The application was made by irrigation (R) to the substrate. The application was made by irrigation (R) to the substrate, adding 1 mL of spore suspension to 300 mL pots, per plant in a single application. Said seedlings were sown in a sanded crop in a parral greenhouse and the same doses were applied again Paecilomyces variotii Pae10773, by irrigation to the seedling 15 days after transplantation, with the dose indicated above. The results reveal that there is a greater development of the plant as evidenced by the values reached for the dry weight of the root, the dry weight of the leaves and the total dry weight, (figures 7, 8 and 9). Likewise, the plant quality index indicates that the best doses correspond to D1 and D3, (figure 10), although the root stem index, which corresponds to low values, better plant quality, the three doses were satisfactory ( Figure 11). The radical volume was higher for all three doses, which means less stress in the transplant. The letters a, and b, indicate statistically significant differences between treatments.

Claims (19)

1. The Pae10773 strain of Paecilomyces variotii that was deposited in the Spanish Type Culture Collection with CECT No. 20957. 2. Partial non-coding sequence of the ribosomal gene of strain Pae10773 defined in claim 1, SEQ ID NO: 1. 3. Biofertilizing composition comprising a biologically pure culture of strain Pae10773. 4 Biofertilizer composition according to claim 3, comprising at least: - a biologically pure culture of the strain Pae10773. - A vehicle. 5. Composition according to one of claims 3 or 4, which is in solid, liquid, gelled or colloidal form. 6. A combination comprising at least: - a biologically pure culture of strain Pae10773 defined in claim 1. - an agent selected from: - a plant - a seed. 7. A combination according to claim 6, comprising at least: - a biologically pure culture of strain Pae10773 defined in claim 1. - a plant. 8. A combination according to claim 6, comprising at least: - a biologically pure culture of strain Pae10773 defined in claim 1. - a seed. - A vehicle. 9. Composition according to claim 4, or a combination according to claim 8, 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). 10. 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 Paecilomyces variotii. 11. 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 Pae10773 is present in the form of conidia. 13. Composition or combination according to claim 12 to which conidia have been added at a concentration between 1108 and 11012 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 stimulating agent - a biological control agent - combinations of two or more of the above. 15. Composition or combination according to claim 14, wherein the adjuvant is selected from an emulsifier, a suspending agent and a tackifying agent. 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, to enhance growth, production, or Plant growth and production. 17. Use according to claim 16 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 wherein the strain Pae10773 is used simultaneously with compatible fungicides.