ES2900384B2 - STREPTOMYCES GENUS STRAIN AND USES IN AGRICULTURE - Google Patents

Description

DESCRIPTION

Strain of the genus Strepomyces and uses in agriculture

The present specification refers, as its title indicates, to a strain of the Streptomyces genus and its uses in agriculture. Its use is proposed as a manure/fertilizer and/or phytosanitary and/or biostimulant against biotic and abiotic stress.

The invention that is presented provides as one of the main advantages, that the use of this strain has a high efficiency in agriculture compared to the use of other fertilizers/fertilizers and/or biostimulants and/or chemical pesticides whose manufacture and use damage the environment. environment and human health.

field of invention

The invention falls within the field of agriculture, microbiology and fertilizer and/or phytosanitary and/or biostimulant products.

Current State of the Technique

We will previously establish a series of definitions for a better understanding of this specification:

Manure or fertilizer: product used in agriculture or gardening that, due to its supply of nutrients, facilitates the growth of plants, increases their yield and improves the quality of crops or that, due to its specific action, modifies, as appropriate, the fertility of the soil. soil or its physical, chemical or biological characteristics.

Nutrient: chemical element essential for plant life and plant growth. In addition to carbon (C), oxygen (O) and hydrogen (H), especially from air and water, the nutrient elements are classified as: main nutrients, secondary nutrients and micronutrients.

Main nutrients: exclusively, the elements nitrogen, phosphorus and potassium.

Secondary nutrients: calcium, magnesium, sodium and sulfur.

Micronutrients: the elements boron, cobalt, copper, iron, manganese, molybdenum and zinc, essential for plant growth, although in small amounts compared to the main and secondary nutrients.

Phytosanitary: substance or mixture of substances intended to prevent the action of, or directly destroy, insects, mites, molluscs, rodents, fungi, weeds, bacteria, nematodes and other forms of animal or plant life harmful to public health and also to the Agriculture. Included in this group are pesticides, defoliants, desiccants and substances that regulate plant growth or phytoregulators.

Biostimulant: is any substance that, when applied to plants, is capable of improving their efficiency in the absorption and assimilation of nutrients, tolerance to biotic or abiotic stress or improve any of their agronomic characteristics, regardless of the nutrient content of the plant. the substance

Biotic stress: stress caused in plants by living beings such as viruses, bacteria, fungi, nematodes, as well as insects and other plants.

Abiotic stress: stress caused in plants by non-biotic causes and which are mainly due to drought, high and low temperatures, salinity, heavy metals, ultraviolet radiation, soil acidity, and fundamentally applications of phytosanitary products.

Genus: category of the biological classification (taxonomic category) that is located between the family and the species, and that includes one or more morphologically similar species related phylogenetically. By "taxonomic category" is meant the level of hierarchy used for the classification of organisms.

Nematode - multicellular worms belonging to the group of helminths. They can be found both in water and on land. In reference to terrestrial nematodes, there are bacterivorous nematodes, fungivores, predators of other nematodes, insect parasites, other animals and plant parasites (phytoparasites). For the present memory when we talk about nematodes we will refer to those that live in the terrestrial environment and have a parasitic activity of plants and are called in the agricultural field as "phytoparasitic nematodes".

Today's modern intensive agriculture depends on the constant use of chemical products that seek to improve the productivity and quality of agricultural products. However, the continuous use of all these chemical substances is generating serious environmental problems. Every day there are increasing restrictions on the use of chemical fertilizers that cause contamination in rivers and land due to excess nitrogen or phosphorous, the presence of heavy metals or other polluting substances. Similarly, the use of phytosanitary products with hazardous waste encounters increasing problems both from the regulatory level and from the user level. All this has given rise to the fact that many of the existing products on the market have had or will have to be banned and withdrawn from the market due to their high toxicity to humans or the environment or due to the presence of toxic residues or due to their high manufacturing costs that do not make them competitive. This has caused farmers to have a limitation in terms of the products to be used with the consequent economic losses.

For all these reasons, any professional in the agricultural sector knows that the development of new substances and, specifically, the discovery and development of new strains of microorganisms or agricultural compositions that not only have a single use as fertilizers/fertilizers or phytosanitary products or biostimulants, but also have several uses in a single product and that also leave no residue, are safe and competitive from an economic point of view is of great interest to the sector.

Background of the invention

Actinobacteria are a ubiquitous group of microorganisms widely distributed in natural ecosystems, they can be found in terrestrial, freshwater and marine aquatic environments. Specifically, bacteria of the genus Streptomyces are found freely in the soil. There, among other actions, they act in the decomposition of organic waste. About 500 species have been represented and they produce more than 80% of known antibiotics as well as other antitumor and antiparasitic substances. For a few years these bacteria have been used and marketed in the agricultural sector.

In this sense, and although Streptomyces are a group of actinobacteria considered to be soil dwellers and have been scarcely studied in the field of plant biofertilization, some effects have been described as fertilizers/fertilizers or effects as a biostimulant against biotic or abiotic stress. Thus, some strains of the bacterium Streptomyces spp. they are capable of colonizing and growing in association with plant roots, significantly increasing their growth and development (Condori-Pacsi, et al. IDESIA (Chile). 37: 109-116, 2019). In other cases, they have been described as producers of PGP (vegetative growth promoters) (Minuto et al. Crop Protection.

25: 468-475, 2006).

In the phytosanitary aspect, and as a fungicide, there are currently a total of 2 species of the Streptomyces spp. that are registered as phytosanitary products acting against pathogenic fungi or bacteria in the EU. In this sense, application WO2017/055939A1 describes the use of the Streptomyces Jofer strain as a fungicide. Patent US53968503A describes the use of Streptomyces YCED9 strains in the control of plant pathogens susceptible to fungal infections, particularly in grass seeds and roots. Similarly, patent US5527526A describes the use of Streptomyces WYEC108 strains for the protection of plants such as chickpea seeds also against fungal infections. Other patent applications such as CN102206593A with the use of Streptomyces aureoverticillatus HN6 or CN102329755A with the use of Streptomyces ahygroscopicus BWL58 describe the use of these bacteria against fungal problems in bananas.

In the phytosanitary aspect and as a nematicide, we can indicate that numerous microorganisms have been described for the control of nematodes such as Sphingobacterium spiritivorum strain C-926 and Corynobacterium paurometabolum C-924 (MX250042), Pasteruria spp (MX193344), fungi of the genus Arthrobotrys (US4 666,714), Bacillus thuringiensis (US5,270,448), Pochonia chlamydosporia (US2009169518), Verticillium chlamydosporium CC334168 (WO1991/001642), and Paecilomyces carneus (WO2014/092529) and others. Similarly, within the Streptomyces genus, some strains of the species Streptomyces dicklowii ( WO1993018135A1) and Streptomyces albogriseolus have shown nematicidal activity. Finally, scientific works on bacteria of the Streptomyces genus with a nematicidal effect have been described in the literature. However, at this time there is no species of bacteria of the Streptomyces genus that has a phytosanitary registration as a nematicide in the EU.

The Streptomyces strain presented in this report has numerous advantages, such as that it sporulates, which favors the production of commercial products for use in agriculture with a longer duration compared to other microorganisms that do not have this characteristic. Furthermore, the described strain does not produce streptomycin, which is an antibiotic. In many areas of the world, such as Europe, the use of antibiotics in agriculture is not authorized due to the possible contamination and creation of resistance in microorganisms to these substances.

In the same way, the Streptomyces strain has other clear advantages, such as the fact that it does not produce toxic chemical residues that are problematic from a health point of view, nor do they pollute the soil or watercourses. In addition, the present strain has several joint uses, it has a use as a manure/fertilizer and as a phytosanitary product and as a biostimulant against biotic and abiotic stress. All this means that, by using a single strain, we can replace the use of numerous products with the economic and practical advantages that this entails for the farmer.

Description of the invention

To solve the problem that currently exists in agriculture with the use of chemical products such as fertilizers, phytosanitary products and biostimulants with the problem of using several products (each one with a function) and the production of environmental contamination or toxic waste in its manufacture or use, the use in agriculture of a new strain of the genus Streptomyces not described until now in this field of application has been devised.

The strain of the Streptomyces genus is deposited in the Spanish Collection of Type Cultures (CECT) with the deposit number CECT30147, from now on "strain of the invention". It is a strain isolated from a natural environment from samples of I usually.

The strain of the Streptomyces genus of this invention does not have the ability to synthesize the antibiotic streptomycin. After the analysis of the biosynthesis of streptomycin and of the genes involved and the study of the genome of the strain of the invention, no homology has been found in the genome. The conclusion indicates that the strain of the invention does not have all the genes required for the production of this antibiotic from the biosynthetic pathways.

In a preferred embodiment, the strain of the invention is in a sporulated state. By "sporulated state" is meant the stage of the bacterium in which it is in the form of a spore.

Another aspect of the invention refers to a composition, hereinafter "composition of the invention", which comprises, among other elements, an element selected from the list:

(a) Strain of the invention, preferably in the sporulated state.

(b) Products of metabolism (metabolites) and/or of the strain of the invention.

(c) A combination of (a) and (b).

As used in the present invention, the term "products of metabolism" or "metabolites" refers to any molecule excreted by the strain of the invention into the medium. Examples of these metabolites are, but are not limited to, amino acids, nucleotides, vitamins, organic acids, alcohols, such as polyols, sugars, such as polysaccharides, antibiotics, enzymes, such as, but not limited to, cell wall hydrolytic enzymes, etc.

The composition of the invention also comprises elements necessary for the growth and development of the strain of the invention. Thus, said composition includes, but is not limited to, carbon sources, mineral salts and/or organic and/or inorganic nutrients.

The composition of the invention can be presented in any presentation form suitable for its administration or application in agriculture, for example, but not limited to, in liquid or solid or water-soluble solid form. Liquid or solid water-soluble dosage forms are suitable for spraying onto soil, plant or plant material or for creating a solution in which plants or plant material are dipped. The solid presentation is suitable for dispersion on the ground in the form of powder, granular, pelletized, etc.

With respect to the composition of the invention where one of the elements is according to what is described in list (a), the strain of the invention can be administered alone both to the roots and to the aerial part of the plant, to the plant material including but not limited to seeds, leaves or fruit or soil.

With respect to the composition of the invention where one of the elements is according to what is described in list (b), once excreted, the products of the metabolism of the strain of the invention can be isolated and applied, alone or in combination with the strain (case (c) of the list), directly to the plant, both to the roots and to the aerial part, to the plant material, including but not limited to seeds, leaves or fruits, or to the soil.

When the metabolic products are administered in combination with the strain of the invention, the mode of administration can be simultaneous or sequential. Or, the strain of the invention can be administered alone both to the roots and to the aerial part of the plant, to the plant material including but not limited to seeds, leaves or fruits or to the soil, so that said metabolic products are produced. on-site

The term "use" or "treatment", as understood in the present invention, refers to the use of the strain of the invention as a fertilizer/fertilizer and/or biostimulant against biotic or abiotic stress and/or as a phytosanitary as established in the definitions of these terms in the present invention.

The strain of the invention, and/or the products of its metabolism, or the composition of the invention, can be used for the treatment of a plant, a soil, or plant material, including but not limited to seeds, leaves or fruits. , in combination with one or more fertilizer agents, and/or biostimulants and/or phytosanitary products simultaneously or sequentially.

The mode of use or treatment for the administration of the strain of the invention, and/or of the products of its metabolism, as well as of the composition of the invention, can be carried out, for example, although without limitation, through irrigation water. spraying, spraying, coating, fumigation, impregnation, mixing with the substrate, immersion of the root of the plant in a solution comprising the strain of the invention, or pre-germinated spores thereof, and/or the products of its metabolism, or the composition of the invention.

Advantages of the invention

This novel use of the composition based on a new strain of the Streptomyces genus for the treatment of problems in agriculture provides multiple advantages over currently available products, the most important being that it has a high agronomic efficacy and multifunctionality compared to chemical synthesis products that are currently known for very specific and limited uses.

It is important to note that the new strain of the Streptomyces genus can sporulate, which makes it possible to produce long-lasting commercial products, and also does not produce the antibiotic streptomycin, which is a clear drawback for the use of other strains of the Streptomyces genus in agriculture.

In addition, it is worth highlighting the advantage that the use of this composition presents a very positive toxicological profile according to international standards compared to other chemical synthesis products, thanks to the use of a strain of a microorganism of natural origin. This can lead to declaring this composition as free of phytosanitary residues with clear agricultural, health and economic advantages over synthetic products.

Likewise, it is necessary to highlight the advantage that originates from the use of this composition since, by not containing synthetic chemical products, it avoids the introduction of substances that can act as contaminants or increase the toxicity or phytotoxicity of the agricultural composition.

Another advantage of this new composition is that it is cheaper compared to traditional chemicals that are used as fertilizers/fertilizers, biostimulants or phytosanitary products.

examples

The invention will now be illustrated by means of tests carried out, which show the effectiveness of the strain and of the composition of the invention in the different uses described. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only and are not to be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention, without limiting its field of application.

Example 1: Study of the fertilizer/biostimulant capacity of the strain of the invention.

The objective of the protocol was to evaluate the biostimulant potential of the strain of the invention. Specifically, the nitrogen fixation capacity was studied. This ability does not exclude said microorganism possesses other biostimulant properties that have not been studied in this example.

Environmental nitrogen fixation capacity: This test evaluates the capacity of the strain of the invention to fix environmental nitrogen. For this, a triple streak seeding was carried out starting from a fresh plate culture of the isolate to be studied. At the same time, a positive control was carried out with reference strains with the capacity to fix nitrogen to verify the proper functioning of the method and the good condition of the culture medium.

Results:

The results of the plate indicate that the culture of the invention gave the same results as the positive control and we can conclude that it is capable of fixing environmental nitrogen, which is a very important characteristic as a manure/fertilizer or biostimulant.

Example 2: Study of the in vitro chitinolytic activity of the strain of the invention.

The objective of the protocol was to evaluate the chitinolytic activity of the strain of the invention on certain substrates. Chitinolytic enzymes are produced by some microorganisms in conditions of absence of nutrients and are favored by the presence of complex chitin structures in the medium (as is the case of the nematode wall). With this, the nematicidal potential of the strain of the invention is measured. The method allows comparing microorganisms with each other not only from the qualitative point of view but also quantitatively.

The substrates analyzed were the following:

4-Nitrophenyl N,N’-diacetyl-p-D-chitobioside: A suitable substrate for exochitinase activity

4-Nitrophenyl N-acetyl-p-D-glucosaminide: A suitable substrate for exochitinase activity

4-Nitrophenyl PDN,N',N''-triacetylchitotriose: A suitable substrate for endochitinase activity

The activity of the strain of the invention was compared against a control microorganism that is described as having chitinolytic activity and with the name Serrata marcenses CECT846.

Results:

A summary table of chitinolytic activity is shown below. The indicated chitinolytic activity is the sum of the activities on the 3 indicated substrates.

The results indicate the high chitinolytic activity, which indicates a high potential as a nematicide product.

Example 3: Study of the nematicidal efficacy of the strain of the invention.

The objective of the protocol was to evaluate the mode of action and the efficacy of the activity against eggs and juveniles (J2) of the nematodes of the genus Meloidogyne incognita and the genus Pratylenchus penetrans under in vitro conditions. The compositions analyzed were two. Composition composed of the bacteria ( Streptomyces) plus the fermentation broth (metabolites) and the composition solely composed of the fermentation broth without the presence of the bacteria of the strain of the invention ( Streptomyces).

Tests with eggs of nematodes of the genus Meloidogyne incognita and the genus Pratylenchus penetrans

The tests were carried out under in vitro conditions in a Petri dish with disks containing the eggs. 100 eggs were applied in each Petri dish corresponding to each of the nematode genera studied. Subsequently, the indicated compositions were applied and kept at room temperature in the laboratory (22°C). for 7 days. Subsequently, the samples were studied under a microscope. Unhatched eggs and the presence of juveniles after the study time were evaluated.

In all cases, a sample without treatment and others that acted as controls were introduced to verify that the method and the assay worked properly.

Results:

Below is a summary table of the efficacies.

The results indicate high efficiency as a nematicide of the strain of the invention in the two compositions tested in the example.

Claims (1)

1 - Strain of the Streptomyces genus deposited in the Spanish Collection of Type Cultures (CECT) with deposit number CECT30147. 2 - Strain of the genus Streptomyces, according to claim 1, characterized in that it is in a sporulated state. 3 - Composition characterized in that it comprises a strain of the Streptomyces genus according to any of claims 1 or 2. 4 - Composition characterized in that it comprises the fermentation broth of the strain of the Streptomyces genus according to any of claims 1 or 2. 5 - Composition characterized in that it comprises a combination of a strain of the Streptomyces genus according to any of claims 1 or 2 and the fermentation broth of the strain of the Streptomyces genus according to any of claims 1 or 2. 6 - Composition according to any of claims 3, 4 and 5, characterized in that it is liquid. 7 - Composition according to any of claims 3, 4 and 5, characterized in that it is solid. 8 - Use of the strain of the Streptomyces genus or of the composition according to any of the preceding claims, characterized in that it is applied in agriculture. 9 - Use of the strain of the Streptomyces genus or of the composition, according to claim 8, characterized in that the use is as manure / fertilizer in agriculture. 10 - Use of the strain of the Streptomyces genus or of the composition, according to claim 8, characterized in that it is used as a biostimulant in agriculture. 11 - Use of the strain of the Streptomyces genus or of the composition, according to claim 8, characterized in that it is used as a phytosanitary product in agriculture. 12 - Use of the strain of the Streptomyces genus or of the composition, according to claim 8, characterized in that it is the prevention and/or treatment of conditions caused by nematodes in agriculture. 13 - Use of the strain of the Streptomyces genus or of the composition, according to claim 12, characterized in that the treatment against nematodes is carried out on crops chosen from the group consisting of bananas, plantains, table grapes, wine grapes, fruit trees, citrus, strawberry, vegetables and soybeans.