ES2736016A1 - Methods for the biological control of phytopathogenic infections in plants and crops - Google Patents

Abstract

Methods for the biological control of infections produced by phytopathogens in plants and crops. The present invention refers to the use of a protein of fungal origin called AfpA and/or compositions comprising said protein, in the control of phytopathogenic agents, preferably phytopathogenic fungi and oomycetes of plants and/or crops.

Description

D E S C R I P C I Ó N

Methods for the biological control of phytopathogenic infections in plants and crops

The present invention is encompassed in the agri-food sector, preferably in the protection of plants and crops against phytopathogens. Specifically it refers to the use of a protein of fungal origin called AfpA and / or of compositions comprising said protein, in biological control of phytopathogens in plants and / or crops.

BACKGROUND OF THE INVENTION

Phytopathogens represent a major threat to agriculture, food security and public health. The most common pathogens in plants are fungi, which also encompass the group of oomycetes, although bacteria, and nematodes are also important. Diseases caused by mycoplasmas and viruses are not often recorded, as they are difficult to detect. Thus, for example, fungal infections destroy more than 125 million tons of the main crops every year, including rice, wheat, corn, potatoes and soybeans, amounts that would feed more than 600 million people. In addition, many of these phytopathogenic fungi are mycotoxin producers, contaminating many food products with these compounds harmful to animal and human health (Bebber, D.P. and Gurr, S.J. Fungal Genet. Biol., 2015; 74: 62-64). The situation is increasingly alarming in the face of climate change and globalization that promote the emergence of new, more aggressive fungal strains and the invasion of new territories. The control of phytopathogenic fungi depends exclusively on a few fungicides, which are not 100% effective due to the development of resistance due to the great plasticity of fungal genomes, and because the legislation is increasingly restrictive for phytosanitary products due to unwanted effects on the food chain (Montesinos, E., FEMS Microbiol. Lett., 2007; 270: 1-11; Perfect, JR, Expert Opin. Emerg. Drugs., 2016; 21: 129-131).

At present, the use of chemical biocides is the main method used to protect crops against infections caused by phytopathogens. Nevertheless, The application of these chemical compounds in agriculture presents serious disadvantages, such as their non-specific activity against other non-harmful microorganisms, the emergence of resistant strains or their negative effects on the environment or human health, which can range from simple skin irritations to damage to the reproductive system and possible carcinogenicity. These problems have prompted the study and the search for new alternatives for the control of diseases caused by phytopathogens, which have a greater specificity and few or no effects on the health of consumers, farmers and the environment without affecting the final quality of the products.

Antifungal proteins ( Anti-Fungal Proteins, AFPs) secreted by filamentous fungi are considered as promising candidates for the development of new molecules and antifungal therapies. AFPs are small, cationic, very stable proteins, rich in cysteines ( Cysteine-Rich Proteins, CRPs) and with a compact p-sheet structure stabilized by the formation of up to four disulfide bridges. AFPs are produced and secreted in large quantities to the culture medium by ascomycete filamentous fungi, and exhibit high antifungal activity in the micromolar range, and are harmless against bacterial, animal or plant cells. The first AFP identified was that produced by Aspergillus giganteus (AgAFP) (Nakaya, K., et al. Eur. J. Biochem., 1990; 193: 31-38). Another well-characterized protein is the PAF protein of Penicillium chrysogenum, which is secreted and produced in large quantities in the culture medium (up to 80 mg / L) (Marx, F., et al., Gene, 1995; 167: 167- 171).

Both proteins, AgAFP and PAF, inhibit the growth of filamentous fungi, including human and plant pathogens, without affecting yeasts, bacteria and plant or mammalian cells, both in vitro and in vivo (Moreno, AB, et al., Appl. Microbiol Biotech. 2006, 72: 883-895; Hegedüs, N. and Marx, F. Fungal Biol. Rev.

2013, 26: 132-145). The AfpB protein from the fungus Penicillium digitatum, whose antifungal potency is even greater, compared to different phytopathogenic fungi, than the antifungal activity of the PAF protein (Garrigues, S., et al. , Sci Rep., 2017; 7: 14663).

In the genome of filamentous fungi are between 1 and 3 genes that encode proteins with sequence homology with AFPs, and which have been noted as "antifungal proteins" (Garrigues, S., et al., Appl. Microbiol. Biotechnol. 2016; 100: 2243). However, in the vast majority of cases have not been able to detect, identify, or produce the corresponding antifungal proteins.There is still, in the state of the art, the need to effectively produce new and more potent AFPs to fight phytopathogenic infections, in plants and / or crops

DESCRIPTION OF THE INVENTION

The present invention provides a solution to the existing demand for new natural compounds useful for the biological control of phytopathogens in plants and / or crops.

In this regard, the present invention relates to the use of a protein of fungal origin that exhibits antifungal activity and against oomycetes against infections in plants and / or crops. Said antifungal protein belongs to the family of proteins called AFPs, specifically called AfpA, hereinafter protein of the invention, and comprises an amino acid sequence that has a sequence identity of at least 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 1. More preferably, the AfpA protein comprises SEQ ID NO: 1. More preferably still, the AfpA protein consists of SEQ ID NO: 1. The protein-producing fungus of the invention belongs to the genus Penicillium, preferably to the species P. expansum, and more preferably still, it is strain P. expansum CECT 20906 .

Another aspect of the invention relates to the use of a composition comprising said AfpA protein of the invention (hereinafter composition of the invention), where optionally, said composition may further comprise excipients, additives and / or other additional active ingredients, for the biological control of phytopathogens in plants and / or crops.

Another aspect of the present invention relates to a method for the biological control of phytopathogens in plants and / or crops comprising administering an effective amount of the protein of the invention, or of a composition comprising the protein of the invention, to said plants, crops and / or the environment around them.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Production and purification of AfpA protein comprising SEQ ID NO: 1 from the culture supernatant of P. expansum CECT 20906 grown in Minimum Medium (MM). Photographs of the protein content analysis are shown by polyacrylamide gel electrophoresis (PAGE) and Coomasie blue staining. (A) Protein content of the P. expansum CECT 20906 culture supernatant grown in nutrient-rich medium (PDB) or in minimal medium (MM) for 5, 7, and 10 days of culture (5d, 7d and 10d, respectively ). (B) Protein content of pure preparations of the P. chrysogenum PAF protein (SEQ ID NO: 2) and the P. expansum AfpA protein (SEQ ID NO: 1) together with a protein marker of size known as control. AfpA protein is identified in gels with a black triangle and PAF protein with a white triangle.

FIG. 2. The AfpA protein comprising SEQ ID NO: 1 of the invention protects tomato plants from Botrytis cinerea infection . The figure shows images of tomato plant leaves inoculated with a suspension of conidia from B. cinerea (0.5x106 conidia / ml) together with different concentrations (1, 5 and 10 pM) of AfpA, AfpB and PAF proteins . The photos were taken 4 days after inoculation.

FIG. 3. The AfpA protein comprising SEQ ID NO: 1 significantly reduces the size of the lesion caused by B. cinerea in tomato plant leaves. Quantification of the injured area at 4 days of inoculation by image analysis with the Fiji Imagej program (https://imagej.net/ImageJ). The data correspond to the average value of the percentage of injured leaf of at least 3 leaves per treatment. Asterisks show statistically significant differences (Tukey test * p <0.005; ** p <0.001).

FIG. 4. The AfpA protein comprising SEQ ID NO: 1 maintains the protection of tomato plants against long-term B. cinerea infection. Images of tomato leaves inoculated with a suspension of conidia B. cinerea (0.5x106 conidia / ml) together with different concentrations of the indicated proteins. The photos were taken 7 days after inoculation.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to the use of the AfpA protein of the invention or the composition comprising it for the biological control of phytopathogens in plants and / or crops, where preferably the phytopathogens are fungi and / or oomycetes.

The AfpA protein used in the present invention belongs to the family of AFPs proteins, is secreted by the fungus Penicillium expansum, preferably by the strain P. expansum deposited in the Spanish Type Culture Collection (CECT) with the accession number CECT 20906.

In the context of the present invention, the term "AfpA protein" is defined by an amino acid sequence comprising SEQ ID NO: 1, more preferably it consists of SEQ ID NO: 1. The term "AfpA coding sequence" is defined as a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the AfpA protein of SEQ ID NO: 1, and which may comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of Ia SEQ ID NO: 1, b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d ) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% with SEQ ID NO: 1; in which the polypeptide encoded by said nucleic acids has the activity and structural characteristics of the AfpA protein. It includes, therefore, various variants of the AfpA protein, that is, proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation.

In the sense used in this description, the term "variant" refers to a protein substantially homologous to the AfpA protein. In general, a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation. These variants are also functionally equivalent to the AfpA protein of the invention.

As used herein, a protein is "substantially homologous to the AfpA protein" when its amino acid sequence has a good alignment with the amino acid sequence SEQ ID NO: 1, that is, when its amino acid sequence has a degree of identity regarding the amino acid sequence SEQ ID NO: 1, of at least 80%, advantageously of at least 85%, preferably of at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, and more preferably at least 99%. The term "identity", as used herein, refers to the proportion of identical amino acids between two amino acid sequences that are compared. The percentage of identity existing between two sequences can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences, which includes, but is not limited to, the BLASTP or BLASTN program , and FASTA (Altschul et al., J. Mol. Biol., 1999; 215: 403-410).

The term "fragment", as used in the present description refers to a portion of the AfpA protein or one of its variants.

The term "functionally equivalent", as used herein, means that the protein or the fragment of the protein in question essentially maintains the properties related to the biological control of phytopathogens described herein. Such properties can be determined by conventional methods such as those described in the examples that accompany this description.

The AfpA protein of the invention can be obtained by culturing P. expansum fungus under particular conditions. Thus, a preferred method for obtaining the protein of the invention comprises the following steps:

(a) Cultivate a fungus of the Penicillium expansum species in a minimum culture medium under conditions that allow the fungus to grow,

(b) Collect the culture supernatant from step (a),

(c) Purify the protein of the invention from the supernatant of step (b).

P. expansum, preferably strain CECT 20906 is grown in minimal culture medium under conditions that allow the growth of said fungus. Said minimum culture medium comprises the nutrients and requirements necessary for the development and growth of the fungus. Preferably, the minimum culture medium comprises 2% sucrose, 0.3% NaNO ³ , 0.05% MgSO ⁴ x 7H ² O, 0.005% FeSO ⁴ x 7H ² O, 0.1% trace elements solution A, 25 mM potassium phosphate buffer, pH 5.8. The trace elements of solution A are preferably: 0.1% FeSO ⁴ x 7H ² O, 0.9% ZnSO ⁴ x 7H ² O, 0.4% CuSO ⁴ x 5H ² O, 0.01% MnSO ⁴ x H ² O, 0.01% H ³ BO ³ , 0.01% Na ² MoO ⁴ x 2H ² O.

A suspension of P. expansum CECT 20906 conidia is inoculated in said culture medium, preferably equal to or greater than 5x10 ⁴ conidia / mL of culture, for example, 5x10 ⁶ conidia / ml of culture.

The conditions for the cultivation and growth of P. expansum CECT 20906 are the conventional conditions, known to those skilled in the art, for the development and growth of said fungi, and comprise, for example, the incubation of the culture medium, previously inoculated , in an orbital shaker with stirring, at a temperature between 26 and 28 ° C for a period of time between 24 and 96 hours. Example 1 describes conditions and culture media appropriate for the development and growth of P. expansum CECT 20906.

To obtain the protein of the invention, the P. expansum fungus is grown in a minimum medium for at least 5 days, preferably for at least 7 days, more preferably for at least 10 days. After this time, the mycelium is removed from the culture medium and the recovery of the supernatant (step (b)). The mycelium can be removed by any appropriate solid-liquid separation method, for example, by decantation, filtration, centrifugation, etc .; however, in a particular embodiment, the removal of the mycelium is performed by centrifugation. The collected supernatant is subjected to a dialysis process in 20 mM sodium phosphate buffer pH 6.6.

The purification of step (c) is performed by chromatographic or ultrafiltration techniques.

Alternatively, the AfpA protein of the present invention can also be synthesized by means of any technique known in the state of the art. The synthesis technique can be, but not limited to, a macromolecular synthesis technique, such as, but not limited to, solid phase peptide synthesis. Any of the peptides of the invention can also be obtained by recombinant DNA techniques, that is, by means of adapted techniques of molecular and biotechnological biology, in microorganisms or genetically modified plants, or by transient expression.

The AfpA protein of the invention can be combined or mixed with other products that facilitate its vehicle (if any) and / or stabilize and / or enhance its effect, such as other active ingredients, preferably other compounds for the biological control of phytopathogens. Such combinations give rise to the composition of the invention, which comprises the AfpA protein and which is used in the biological control of plant and / or crop phytopathogens.

As used in this description, the term "biological control" refers to the control of a pathogenic agent, organism or microorganism of plants and / or crops, that is to say a phytopathogen, through the use of products of biological origin, preferably AFPs, more preferably the AFP protein of the invention, or of a composition comprising said protein of the invention, being able to control said pathogenic agents, organisms or microorganisms of plants and / or crops and which can be applied directly or indirectly to the plant, crop, and / or the environment around them.

For the purposes of the present invention, the term "acceptable vehicle" refers to a substance or combination of substances that can be used in the agricultural or food sector, and includes, for example, adjuvants, solids or liquids, diluents , solvents, surfactants, etc. Said compounds allow a better application of the protein of the invention and / or alternatively of the active ingredients that accompany it in the compositions of the invention. Compositions comprising said vehicles can be formulated by conventional procedures known in the state of the art.

Additionally, the compositions of the invention may further comprise a acceptable excipient, diluent, adjuvant and / or stabilizer. As the person skilled in the art understands, the protein of the invention will be present in the composition of the invention in an agriculturally effective amount, that is, in an amount such that it is sufficient to alleviate, improve, stabilize, reverse, prevent, retard or delay the progression of stages of diseases caused by phytopathogens in plants and / or crops. Said compounds allow a better application of the protein of the invention and / or alternatively of the active ingredients that accompany it in the compositions of the invention, especially to plants and / or crops treated according to the invention.

The composition of the invention may also contain, if desired, one or more products and / or active ingredients that stabilize and / or enhance their effect. Alternatively, said products may be one or more proteins with different enzymatic activities required for the modification or degradation of cell walls of the pathogen, such as, one or more lytic enzymes capable of modifying or degrading cell walls, for example, enzymes with cellulolytic activity, Mannanolytic, chitinolytic, proteolytic, etc. In another particular embodiment, the composition of the invention further comprises one or more chemical compounds for the biological control of phytopathogens. As a chemical compound, any of the commonly used chemical compounds can be used, preferably a chemical compound selected from the group consisting of chemical compounds for the control of phytopathogens that affect the membrane, which affect the synthesis of the cell wall and mixtures thereof. Alternatively, said active ingredients are selected from the list consisting of insecticides, baits, sterilizing agents, bactericides, acaricides, nematicides, growth regulators, herbicides, protectants, fertilizers, etc.

Both the protein of the invention and the composition of the invention can be presented in any form suitable for transport, administration or application, for example, in liquid or solid form, such as in the form of a granulate or powder. Liquid forms of presentation are suitable, for example, for spraying on plants and / or crops, as well as on the surrounding environment. These compositions include not only compositions that are ready to be applied by a suitable device, such as a spraying or dusting device, but also concentrated commercial compositions, They must be diluted before application to plants, crops, as well as the environment around them.

In another aspect, the invention relates to a method for the biological control of plant and / or crop phytopathogens comprising administering an effective amount of the protein of the invention and / or the composition of the invention, to said plants and / or crops In a preferred embodiment of the method of the invention, this also comprises administering an effective amount of the protein of the invention and / or the composition of the invention to the environment surrounding said plants and / or crops.

For the purposes of the present invention, the term "effective amount" or "effective amount" refers to the amount necessary to alleviate, improve, stabilize, prevent, retard or delay the progression of developmental stages of the phytopathogenic agent in question in the plant. or cultivation and thus obtain beneficial or desired results. An effective amount can be administered at one time or in several administrations. In terms of treatment and protection, an effective amount is the amount sufficient to alleviate, improve, stabilize, reverse, prevent, delay or delay the progression of disease stages caused by plant and / or crop pathogens.

The effective amount of the AfpA protein of the invention, or of a composition comprising it, may vary within a range of concentrations, preferably in the range of 1 to 10 pM. In yet another more preferred embodiment, the concentration will depend on each specific species or crop, and a person skilled in the art is able to determine the concentration of said compound to be administered. The amounts or doses indicated in the present invention are given as illustrative examples of the process according to the invention. One skilled in the art will know how to adapt the application doses, especially according to the nature of the plant and / or crop, as well as the method of administration.

In the method of the invention, the AfpA protein of the invention, and / or the composition comprising it, can be applied preventively to plants and / or crops, to prevent the occurrence of infections and diseases caused by phytopathogens that preferably they are selected among fungi and / or oomycetes. Preventive treatment is based on the fact that the peptides of the invention inhibit the growth of phytopathogens, preferably fungi and / or pathogenic oomycetes of plants and / or crops.

In the method of the invention, the AfpA protein of the invention, and / or the composition comprising it, can be used to treat said infections and diseases once their presence has been detected in plants and / or crops, since the peptide of the invention and the composition comprising it, inhibit the growth of phytopathogens, preferably fungi and / or phytopathogenic oomycetes causing said infections and diseases.

In this description the term infection refers to the invasion and destruction of an organ of the plant, for example, leaves, roots, stems, flowers, seeds, etc., by the pathogenic microorganisms. Therefore, it is a localized process. On the other hand, the term disease refers to a process that affects the plant, giving rise to symptoms.

The prevention and / or treatment of plants and / or cultures with the protein and / or composition of the invention is carried out by conventional procedures, such as direct administration on the aerial parts of plants and / or crops, on the roots, or about the environment around them. In a particular embodiment, the administration of the protein and / or the composition of the invention is carried out by, for example, spraying, immersion, vaporization, atomization, dissemination, sprinkling, foaming, irrigation, etc.

For the purposes of the present invention, agents, organisms or microorganisms pathogenic to plants and / or crops, also called phytopathogens, include any agent, organism or microorganism capable of causing damage or infection in said plants and / or crops, for example, fungi, oomycetes, bacteria, viruses, nematodes and insects. In a more preferred embodiment, the pathogenic pathogens are fungal fungi and / or oomycetes, in another more preferred embodiment, the phytopathogens are fungi.

For the purposes of the present invention, the phytopathogens are selected from the list consisting of: Botrytis spp., For example Botrytis cinerea; Magnaporthe spp., For example Magnaporthe oryzae; Fusarium spp., For example Fusarium oxysporum, Fusarium graminearum; Puccinia spp .; for example Puccinia graminis f. sp. tritici, Puccinia stríiformis f. sp. triticium , Puccinia triticin ; Blumería spp., For example Blumería graminis ; Mycosphaerella spp., For example Mycosphaerella graminicola; Erysiphe spp., For example Erysiphe cichoracearum, Erysiphe betae, Erysiphe necator, Erysiphe orontii; Colletotrichum spp., For example Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum graminicola, boninense Colletotrichum, Colletotrichum trifolii Colletotrichum capsici, Colletotrichum destructivum, Colletotrichum caudatum, Colletotrichum crassipes, Colletotrichum dematium, Colletotrichum kahawae, Colletotrichum orbiculare Colletotrichum sublineolum, Colletotrichum truncatum , Colletotrichum musae, Colletotrichum fragariae, Colletotrichum spinaceae, Colletotrichum lindemuthianum; Ustilago spp., For example Ustilago maydis; Melampsora spp., For example Melampsora lini; Phakopsora spp., For example Phakopsora pachyrhizi; Rhizoctonia spp., For example Rhizoctonia solani, Phytophthora spp, for example, Phytophthora capsici, Phytophthora infestans, Phytophthora nicotinae.

Plants and crops likely to be treated with the protein and / or with the compositions of the invention include plants used for the production of renewable energies, for human or animal nutrition, for wood production and ornamental plants.

Examples of plants and crops used in the production of fuels or renewable energy include, but are not limited to (i) plants used in the production of electrical energy: mainly obtained from fast-growing wood energy crops, such as poplar, willow , eucalyptus, conifers, acacias, bananas, etc., and herbaceous plants, such as thistles, reeds, euphorbias, nopales, etc .; and (ii) plants used in the production of biofuels: production of bioalcohols obtained from beets, corn, sweet sorghum, sugarcane, topinambur, etc., and bio-oils obtained from rapeseed, sunflower, soybeans, etc. Examples of wood plants include, but are not limited to, pines, eucalyptus, cork oaks, cedar, oak, oak, etc. Illustrative non-limiting examples of ornamental plants of interest include plants belonging to the genus Aeschynantus; Canna; Column; Anemone; Azalea; Begonia; Calceolaria; Camellia; Dianthus; Freessia; Gerbera; Hibiscus; Hypoestes; Kalanchoe; Nicotiana; Pelargonium; Petunia; Príkmula; Rannunculus; Rhipsalidopsis; Pink; Saintpaulia; Sinningia-gloxinia; Streptocarpus; Tigridia; Verbena; and Zinnia. Other ornamental plants include orchids ( Orchidaceae) and ornamental shrubs, which include bay laurel ( Laurus nobilis), honeysuckle ( Lonicera fragrantissima), star magnolia ( Magnolia stellata), hydrangea ( Hydrangea macrophylla), Laburnum ( Laburnum watereri), Japanese rose or kerria ( Kerria japonica), etc.

Non-limiting illustrative examples of plants used in human or animal nutrition include fruit trees, which include, but are not limited to, cherry, plum, peach, apricot, olive, mango, pear, apple, medlar, quince, orange, lemon, fig, papaya, chestnut, oak, oak, coscoja, hazelnut, almond, walnut, etc .; forage plants, which include but are not limited to legumes (for example, clovers, alfalfa, clitories, arachis, leucaena, bluebells, beans, lentils, chickpeas and peas, etc.), grasses (for example, rye grass, fescue, orchard grass, blue grass, rhodes grass, buffalo grass, andropogones, brachiarias, Bermuda grass and elephant grass, merkeron, sugarcane, Taiwan grass and corn grass, etc.), grains (for example, sorghum, wheat , rye, barley, corn, oats, etc.); plants for human consumption (lettuce, cabbage, spinach, chard, green beans, tomato plants, cabbage, watermelon, melon, cucumber, zucchini, eggplant, pepper, strawberry, broccoli, cauliflower, garlic, onion and carrot, apple tree, pear tree, apricot tree, cherry, peach, plum, banana, avocado, raspberry, kiwi, olive vine, etc.), etc. This list is not limiting but illustrative of plants and crops potentially susceptible to being treated with the protein and composition of the invention.

Therefore, the AfpA protein of the invention constitutes a real alternative to the use of pesticides of chemical origin, which have high contaminating capacity and harmful effects on health, since the use of biopesticides based on the natural capacity of living organisms leads to lower risks to the environment and the health of consumers. In addition, as observed in the examples included herein, the AfpA protein of the invention is an extremely active protein against plant and / or crop phytopathogens, compared to other previously known AFP-like proteins, substantially improving the known results to date regarding the use of other proteins for the biological control of known phytopathogens.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or Steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.

Materials and methods

Microorganisms

The microorganisms used in the antimicrobial assays include: Botrytis cinerea CECT 2100. To obtain the conidia, the fungus was grown in solid PDA medium ( potato dextrose agar) for 7-10 days at 25 ° C to subsequently obtain the necessary conidia suspensions for the essays.

Production and purification of the AfpA protein of the invention.

For the production of the AfpA protein of the present invention, the parental strain of P. expansum CECT 20906 was grown in 200 mL of Minimum Medium for P. chrysogenum (2% sucrose, 0.3% NaNO3, 0.05% MgSO4 x 7 H2O, 0.005% FeSO4 x 7 H2O, 0.1% trace elements solution A, 25 mM potassium phosphate buffer, pH 5.8; trace elements solution A: 0.1% FeSO4 x 7 H2O, 0.9% ZnSO4 x 7 H2O , 0.4% CuSO4 x 5 H2O, 0.01% MnSO4 x H2O, 0.01% H3BO3, 0.01% Na2MoO4 x 2 H2O) for 10 days, using an initial inoculum of 5.5 x 105 conidia / mL .

After removing the mycelium residues by centrifugation (10,000 rpm, 30 min), the supernatant was dialyzed (Snake Skin Dialysis Tubing 3.5 k MWCO) against 20 mM sodium phosphate buffer pH 6.6. Subsequently, a liquid chromatography was carried out in an AKTA Purifier (GE Healthcare) system using a cation exchange column (ReSourceS 6 mL; GE Healthcare) balanced in the dialysis buffer. The non-adsorbed proteins were eluted in the same phosphate buffer, and the proteins adsorbed to the chromatographic resin were eluted by a linear gradient of NaCl (0-0.5 M) in 20 mM phosphate buffer pH 6.6. Chromatographic fractions (6 mL) were evaluated by 16% SDS-PAGE and the fractions containing the AfpA protein of the invention were mixed, and then dialyzed against double-distilled water and lyophilized and subsequently used in antimicrobial assays.

The concentration of the AfpA protein of the invention was determined using the extinction coefficient S280 = 0.639 determined experimentally.

The molecular mass and peptide fingerprint of the AfpA protein of the invention was determined in the Proteomics Service of the University of Valencia.

Example 1. Production and purification of AfpA protein (SEQ ID NO: 1) from the culture supernatant of P. expansum CECT 20906 grown in Minimum Medium.

P. expansum CECT 20906 secretes the AfpA protein (SEQ ID NO: 1) to the culture supernatant after 5 days of growth in Minimum Medium ( FIG. 1 ). It is noteworthy that when P. expansum CECT 20906 is grown in PDB rich medium, AfpA protein (SEQ ID NO: 1) is not detected in the culture supernatant ( FIG. 1A ). These data demonstrate that the production and obtaining of the AfpA protein in the culture supernatant is only possible when the P. expansum CECT 20906 fungus is grown in Minimum Medium.

The optimal production of AfpA protein is reached between 7-10 days of growth. As described in materials and methods, the purification of the protein is carried out in a single chromatographic cation exchange step, obtaining yields of between 100 to 150 mg / L.

The identity and amino acid sequence of the AfpA protein was confirmed by peptide fingerprint analysis, confirming that the amino acid sequence of the AfpA protein of the present invention (SEQ ID NO: 1) is different from the amino acid sequence of other AFPs proteins previously known, for example, the AFP antifungal protein from Aspergillus giganteus, the P. chrysogenum PAF protein and the P. digitatum AfpB protein .

Example 2. Antifungal activity of the AfpA protein of the invention against infection caused by B. cinerea in tomato plant leaves.

The tests were carried out on Mediterranean tomato leaves ( Solanum lycopersici, Marmande cultivar) of 21-day plants grown at 22 ° C with 16 h day. The leaves were inoculated by placing two drops (20 pl each) at two different points in the leaf beam with a suspension of 0.5x106 conidia / ml, together with the AfpA protein at different concentrations (1, 5, 10 pM). In addition to a control with sterile water (positive infection control) and a fungus-free control (negative infection control), PAF antifungal proteins (SEQ ID NO: 2) from the fungus P. chrysogenum and AfpB protein were included (SEQ ID NO: 3) from the fungus P. digitatum.

The development of the lesions was followed visually for 7 days. At 4 days of inoculation, and before the adjacent lesions join the control without protein, the area of the injured leaf was quantified by image processing.

In FIG. 2 the appearance of the leaves is shown at 4 days after inoculation, in FIG. 3 quantification of the injured area, and in FIG. 4 the appearance of the leaves at 7 days after inoculation. These results show a clear ability to inhibit the infection dependent on the concentration of the AfpA protein of the invention, clearly superior to that observed with the AfpB protein and with the PAF protein. The effectiveness of the protection of the leaves of the tomato plant infected with B. cinerea and in the presence of the AfpA protein of the invention, is maintained over time until 7 days after inoculation, while the case of the protein AfpB protection only lasts over time when the concentrations used are very high.

It is known that B. cinerea is a pathogen with a broad spectrum of action that infects more than 200 plant species causing gray rot and causing significant economic losses globally (over € 1 billion per year). It is considered a typical necrotroph that causes the death of the tissue from which it feeds. Its control is mainly carried out with chemical fungicides assuming a cost of more than € 40 / ha per year, and more than € 540 million in the fungicide market (2002 estimates). The almost total control of B. cinerea infection in plants Tomato mediated by the AfpA protein of the invention at working concentrations as low as 1 pM demonstrates its use in plant protection and represents a strategy applicable to many host plant species of B. cinerea, as well as other fungal pathogens of plants and crops.

Claims (11)

1. Use of a protein comprising SEQ ID NO: 1, or a composition comprising SEQ ID NO: 1, for the biological control of phytopathogens in plants and / or crops. 2. Use according to any of claims 1 to 2 wherein the phytopathogen is selected from the list consisting of: Botrytis spp., Magnaporthe spp., Fusarium spp., Puccinia spp .; Blumeria spp., Mycosphaerella spp., Erysiphe spp., Colletotrichum spp., Ustilago spp., Melampsora spp., Phakopsora spp., Rhizoctonia spp., Phytophthora spp., And / or any combination thereof. 3. Use according to claim 2 wherein the phytopathogen is selected from the list consisting of: Botrytis cinerea, Magnaporthe oryzae, Fusarium oxysporum, Fusarium graminearum, Puccinia graminis f. sp. tritici, Puccinia striiformis f. sp. tritici, Puccinia triticina, Blumeria graminis, Mycosphaerella graminicola; Erysiphe cichoracearum, Erysiphe betae, Erysiphe necator, Erysiphe orontii; Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum graminicola, boninense Colletotrichum Colletotrichum trifolii, Colletotrichum capsici, Colletotrichum destructivum, Colletotrichum caudatum, Colletotrichum crassipes, Colletotrichum dematium, Colletotrichum kahawae, Colletotrichum orbiculare, Colletotrichum sublineolum, Colletotrichum truncatum, Colletotrichum musae, Colletotrichum fragariae , Colletotrichum spinaceae, Colletotrichum lindemuthianum Ustilago maydis, Melampsora lini, Phakopsora pachyrhizi, Rhizoctonia solani Phytophthora capsici, Phytophthora infestans, Phytophthora nicotinae, and / or any combination thereof. 4. Use according to any of claims 1 to 3 wherein the plants and / or crops are selected from the list consisting of: fruit trees, selected from the list consisting of cherry, plum, peach, apricot tree, olive, mango, pear , apple, medlar, quince, orange, lemon, fig, papaya, chestnut, oak, oak, coscoja, hazelnut, almond, walnut; forage plants, selected from the list consisting of: clovers, alfalfa, clitories, arachis, leucaena, bells, beans, lentils, chickpeas, peas; pastures selected from the list consisting of: rye grass, fescue, orchard grass, blue grass, rhodes grass, buffalo grass, andropogones, brachiarias, Bermuda grass, elephant grass, merkeron, sugar cane, taiwan grass, corn grass; grain crops that are selected from the list consisting of: sorghum, wheat, rye, barley, corn, oats; plants for human consumption selected from the list consisting of: lettuce, cabbage, spinach, chard, green beans, tomato plants, cabbage, watermelon, melon, cucumber, zucchini, eggplant, pepper, strawberry, broccoli, cauliflower, garlic, onion , carrot, apple, pear, apricot, cherry, peach, plum, banana, avocado, raspberry, kiwi, vine, olive grove; ornamental plants that are selected from the genus Aeschynantus; Canna; Column; Anemone; Azalea; Begonia; Calceolaria; Camellia; Dianthus; Freessia; Gerbera; Hibiscus; Hypoestes; Kalanchoe; Nicotiana; Pelargonium; Petunia; Príkmula; Rannunculus; Rhipsalidopsis; Pink; Saintpaulia; Sinningia-gloxinia; Streptocarpus; Tigridia; Verbena; and Zinnia. 5. Method for the biological control of phytopathogens in plants and / or crops comprising administering an effective amount of the protein comprising SEQ ID NO: 1, or of a composition comprising SEQ ID NO: 1, to said plants and / or crops. 6. Method according to claim 5 wherein SEQ ID NO: 1, or the composition comprising it, is administered via root, foliar and / or in the irrigation water. 7. Method according to any of claims 5 to 6 wherein SEQ ID NO: 1, or the composition comprising it, is administered by injection, immersion, vaporization, spraying, dissemination, irrigation, dusting and / or foaming. Method according to any one of claims 5 to 7, wherein the effective amount of SEQ ID NO: 1, or of the composition comprising it, ranges from 1 to 10 qM. 9. Method according to any of claims 5 to 8 wherein the phytopathogen is selected from the list consisting of: Botrytis spp., Magnaporthe spp., Fusarium spp., Puccinia spp .; Blumeria spp., Mycosphaerella spp., Erysiphe spp., Colletotrichum spp., Ustilago spp., Melampsora spp., Phakopsora spp., Rhizoctonia spp. Phytophthora spp, and / or any combination thereof. 10. Method according to claim 9 wherein the phytopathogen is selected from the list consisting of: Botrytis cinerea, Magnaporthe oryzae, Fusarium oxysporum, Fusarium graminearum, Puccinia graminis f. sp. triticium, Puccinia stríiformis f. sp. triticium, Puccinia triticina, Blumeria graminis, Mycosphaerella graminicola; Erysiphe cichoracearum, Erysiphe betae, Erysiphe necator, Erysiphe orontii Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum graminícola, Ustilago maydis, Melampsora lini, Phakopiztoratoph Phytophthorphthorph, Phylophthorphthorphthorph, Phylophthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphthorphths the same. 11. Method according to any of claims 5 to 10 wherein the plants and / or crops are selected from the list consisting of: fruit trees, selected from the list consisting of cherry, plum, peach, apricot tree, olive, mango, pear , apple, medlar, quince, orange, lemon, fig, papaya, chestnut, oak, oak, coscoja, hazelnut, almond, walnut; forage plants, selected from the list consisting of: clovers, alfalfa, clitories, arachis, leucaena, bells, beans, lentils, chickpeas, peas; pastures selected from the list consisting of: rye grass, fescue, orchard grass, blue grass, rhodes grass, buffalo grass, andropogones, brachiarias, Bermuda grass, elephant grass, merkeron, sugarcane, Taiwan grass, grass of corn; grain crops that are selected from the list consisting of: sorghum, wheat, rye, barley, corn, oats; plants for human consumption selected from the list consisting of: lettuce, cabbage, spinach, chard, green beans, tomato plants, cabbage, watermelon, melon, cucumber, zucchini, eggplant, pepper, strawberry, broccoli, cauliflower, garlic, onion , carrot, apple, pear, apricot, cherry, peach, plum, banana, avocado, raspberry, kiwi, vine, olive grove; ornamental plants that are selected from the genus Aeschynantus; Canna; Column; Anemone; Azalea; Begonia; Calceolaria; Camellia; Dianthus; Freessia; Gerbera; Hibiscus; Hypoestes; Kalanchoe; Nicotiana; Pelargonium; Petunia; Príkmula; Rannunculus; Rhipsalidopsis; Pink; Saintpaulia; Sinningia-gloxinia; Streptocarpus; Tigridia; Verbena; Zinnia.