BRPI0911418A2 - selective herbicidal composition for dicotiledÈnea, and method of applying said composition - Google Patents

Abstract

SELECTIVE HERBICIDE COMPOSITION FOR DICOTHILEDEAN, AND APPLICATION METHOD OF THE REFERRED COMPOSITION. Herbicides are products used in agriculture to control weeds classified as harmful. The most used by the agricultural industry are chemical herbicides, however, several problems arising from the use of this type of herbicide need to be avoided or minimized, among them environmental contamination and toxicity to animals. Thus, chemical herbicides have been replaced by natural herbicides, which comprise biologically active substances in their formulation. The present invention relates to the herbicidal composition that comprises proteins with synergistic action Cerato-Platanins (CP), obtained from the fungus Moniliophthora perniciosa, pathogen that causes the Witches' Broom disease in cocoa (Theobroma cacau) and necrosis inducing and ethylene (NEP) and said use for selective action for dicot plants.

Description

"SELECTIVE HERBICIDE COMPOSITION FOR DICOTILEDONE, AND APPLICATION METHOD

COMPOSITION"

FIELD OF INVENTION

The present invention relates to a herbicidal composition comprising one or more proteins of the Cerato-Platanins (CP) family, obtained from the fungus MoniHophthora perniciosa, a pathogen that causes Cacao witches' broom disease (Theobroma cacau) and necrosis and ethylene inducers (NEP) and said use for selective action for dicotyledonous plants.

BACKGROUND OF THE INVENTION

GLOSSARY

• Cloning and Gene Expression: Refers to the technique whereby a gene is inserted into an expression vector to obtain recombinant DNA, allowing the transcription and translation of that gene into protein.

• Cotyledon: These are the first leaves that emerge from the embryos of Angiosperms, erupting during seed germination. They are reserve structures, nourishing developing plants until they can get nutrients through photosynthesis.

• Elicitor: Pathogen molecules that are recognized by the host triggering defense responses such as necrotic hypersensitivity, which may lead to acquired systemic resistance. They are of different chemical nature: proteins, lipids and oligosaccharides. Phytotoxicity: In the case of the present invention refers to the ability to induce cell death caused in plant tissues after contact with the protein (s) of the invention.

• Gene: Refers to a DNA fragment that is transcribed into an RNA molecule and encodes a protein.

• Herbicide: A chemical used in agriculture to control herbs classified as weeds, shrubs or other unwanted plants.

• Selective herbicide: is the herbicide that has specificity in its action, that is, when applied in the planting area, does not harm the main crop, although efficiently controls undesirable plants.

• Protein Inoculation in Plants: Procedure whereby protein (s) are placed in contact with plant surfaces and cells.

• Programmed cell death: It is a type of "cellular self-destruction" that occurs in an orderly manner and demands energy for its execution. It is related to the maintenance of homeostasis and physiological regulation of tissue size, but may also be caused by pathological stimulation.

• Necrosis: Refers to the development of lesions in plant cells or tissues following contact with the inducing protein (s) according to the invention. Necrosis becomes visible when a sufficient number of cells die after contact with the protein (s). • PCD: Programmed Cell Death

• Pfam: It is a protein family database that includes annotations and their multiple alignment sequences based on the hidden Markov model (HMM). Each Pfam HMM represents a protein family or domain.

• Dicotyledonous plants: Plants belonging to the Angiosperm group that have two cotyledons. They usually have 4 or 5 (or multiple) petals, reticularly arranged ribs and variable growth, and may reach a herbaceous, shrub or arboreal size.

• Monocotyledonous plants: Plants belonging to the Angiosperm group (flowering plants) that have a cotyledon. They usually have a number of three or multiple petals (6, 9 ...), they also have the leaf veins arranged in parallel and are usually herbaceous in size.

• Recombinant protein: Protein obtained by expressing a cloned gene in an expression vector.

• Acquired systemic resistance: Plant resistance response that occurs after exposure to molecules produced by a pathogen, usually those that cause necrosis. Plants sensitized by these molecules become more resistant to pathogen attacks (Djonovic et al., 2006).

Surfactants: molecules used as adjuvants in herbicide compositions that decrease the surface tension of aqueous environments, facilitating the penetration of the formulation into plant tissues via both stomata and cuticle. This effect ensures greater effectiveness to the herbicide.

• Expression vector: DNA molecule consisting of regulatory regions, in particular an inducible promoter, allowing expression of a downstream inserted gene by recombinant DNA techniques.

Herbicides are products used in agriculture to control weeds classified as weeds. The most used by the agricultural industry are chemical herbicides, however, several problems arising from the use of this type of herbicide need to be avoided or minimized, including environmental contamination and animal toxicity.

Thus, chemical herbicides have been replaced by natural herbicides, which include biologically active substances in their formulation. However, the emergence of herbs resistant to such formulations has been a problem to be overcome.

Today, the choice of a selective herbicide that is suitable for a particular crop depends largely on the differences between the crop and weeds. The use of an herbicide in adequate concentration to eliminate weeds should not be detrimental to the crop grown. An important factor to consider when choosing a herbicide is its mode of action and the way it is metabolized by plants. Herbicides have different mechanisms of action, for example:

a) Mitotic root inhibitors: These compounds are capable of blocking cell division. Examples: Trifluralin and Pendimethalin.

Cause inhibition of root development leading to dwarfism of the plant.

b) Pigment inhibitors: Contributes to the destruction of chlorophyll. Examples: Clomazone and Norflurazon. Cause leaf chlorosis.

c) Sprout inhibitors: Affect cell growth and division. Examples: Alachlor and Metachlor. Cause growth retardation of plants and shoots.

d) Photosynthesis inhibitors: They interfere with photosynthesis by blocking electron transport, causing cell membrane damage and death. Examples: Atrazine and Cyanazine.

e) Auxin-like herbicides: Affect the growth of new stems and leaves by altering protein synthesis and normal cell division. Examples: 2,4-D and 2,4-DB. They produce rickets and malformed seedlings.

f) Acetolactate Iyase Enzyme (ALS) inhibitors: Block the normal activity of ALS by inhibiting metabolism and mitosis.

Examples: Imazethapyr and Nicosulfuron. They inhibit normal plant growth by causing dwarfism.

g) Meristematic inhibitors: Block the formation of lipids in meristemas and grass roots. Examples: Sethoxydim and Quizalofop. They cause slow growth leading to dwarfism. h) Membrane Disruptors: Cause rupture of the cell's internal membranes. Examples: Acifluorfen and Lactofen.

Non-selective herbicides acting on the amino acid biosynthetic pathway include glyphosate (N- (phosphonomethyl) glycine, C3H8N05P). Glyphosate is one of the most effective systemic herbicides to inhibit the growth of both weeds such as perennials and cultivated plants.

An effective herbicide must be absorbed by the target plant and translocated to the molecular site of action. In this scope, the related studies vary according to the plant species studied, but also, these processes are influenced by certain environmental conditions, such as temperature, humidity and light.

The advantages sought in the use of herbicides are: speed of action, specificity to unwanted plants, reduced cost, low residual effect and no soil tillage.

Herbicides with specificity to different types of plants exist, but are less common than generics. There are herbicides specific to dicotyledonous plants that do not harm a monocotyledonous crop, such as those made with synthetic auxin, being the 2.4D product the most common and most used. The 2,4D product acts on plants as an auxin mimic. Although pharmacological studies show that it is not accumulated in the human body, the World Health Organization (WHO) considered 2,4-D to be moderately toxic (class II) and recommended the use of a maximum dissolved concentration in 30μg of drinking water. / L.

In order to overcome all drawbacks of the art, the present invention aims to overcome the deficiency of a selective and non-toxic weed herbicide by the use of biologically active substances from the fungus Moniliophthora perniciosa to replace chemical herbicides.

MoniHophthora perniciosa is a hemibiotrophic basidiomycete fungus [Purdy & Schmidt1 1996] that causes cacao witches' broom disease. There is considerable variation in the symptoms of witches' broom depending on the cultivar, the type of infected tissue and the stage of tissue development [Pereira, 2000]. M. pernicious invades meristematic tissues of the host causing swelling of the affected part. This symptom is the product of rapid cell division (hypertrophy) and increased cell size (hyperplasia) that appears accompanied by proliferation of overgrowth due to loss of apical dominance. Large, curved, twisted broom-like leaf structures form the disease's name [Griffith et al., 2003]. When young, the brooms are bright green (green broom) but die in 1 -2 months giving a brown color to the canopy of infected trees (dry broom).

Due to the complexity of the witch's broom disease, the Laboratory of Genomics and Expression of the Unicamp Institute of Biology started in 2001 the genome project of M. perniciosa (http://www.lge.ibi.unicamp.br/vassoura). ), to identify genes that encode proteins potentially related to the development of the disease. The genome survey was published by Mondego et al. (2008) in the journal BMC Genomics [Mondego et al., 2008].

Genomic analysis of M. perniciosa showed the existence of protein coding genes capable of inducing necrosis in plant tissues. Specifically, sequences similar to genes encoding proteins belonging to two families of elicitors were identified:

Kerato platanin [Zaparoli et al, 2008] and Necrosis and Ethylene Inducing Proteins (NEPs) [Garcia et al, 2007]. Representative genes from each elicitor family produced by M. perniciosa, both NEPs (MpNEPI and MpNEP2) and Keratoplanin (MpCP1, MpCP2 and MpCP3), were cloned and expressed using E. coli as an expression system. Proteins produced from both families were able to induce necrosis in leaves of cocoa and tobacco plants [Garcia et al., 2007; Zaparoli et al., 2008].

NEP1 protein, a precursor of the NEPs family, was discovered in 1995 from culture filtrate of a pathogenic isolate of the fungus Fusarium oxysporum [Bailey, 1995]. Similar proteins have been described with the development of genomic and proteomic analysis methods. Nepl in a wide variety of organisms, rapidly expanding this family of proteins in both size and distribution - bacteria, oomycetes and fungi [Gijzen & Nurnberger, 2006; Garcia et al., 2007],

All described NEPs are characterized by having a NPP1 domain (Necrosis-inducing Phytophthora Protein) [Fellbrich et al., 2002] with two or four conserved cysteine residues, and a conserved seven amino acid sequence (GHRHDWE) located in the central region of the region. protein.

An important feature of NEPs is the ability to activate plant defense responses such as programmed cell death and pathogenicity-related gene induction [Jennings et al., 2001; Veit et al., 2001; Fellbrich et al., 2002; Keates et al., 2003; Verica et al., 2004; Bailey et al., 2005], NEP family proteins are capable of inducing necrosis only in dicotyledonous plants [Qutob et al., 2006].

NEP family proteins can be used as an active compound of herbicides specific to dicotyledonous plants (Bailey et al., 2000). In document W02006068481, the inventors claimed the use of necrosis and ethylene inducing proteins (NEP) from species of the genus Botrytis, among other purposes, as herbicide components.

Cerato-platanin is a family of proteins first described in 1999 in total protein extract from the fungus Ascomyceto Ceratocystis fimbriata f.sp. platani, which causes machete malaria in cacao [Pazzagli et al., 1999]. 07/2009) the Cerato-plataninas family has 70 entries in InterPro (IPR010829). This family is characterized by low molecular weight secreted proteins with high amino acid sequence similarity and the presence of four conserved cysteines that can form two disulfide bridges. All described proteins of the family are secreted by fungi and in some cases, such as Ceratocystis fimbriata's Cerato-platanin, it has been experimentally shown that the protein is capable of inducing phytoalexins production and cell death in host and non-host plants of the pathogen. , acting as a likely elicitor of plant defense responses [Pazzagli et al, 1999].

In addition to necrosis induction and cell death, another function for proteins of the Cerato-platanins family is described. Trichoderma spp Sm 1 protein induces systemic resistance to pathogens after being applied to plants (host or not) by activating plant defense mechanisms [Djonovic et al, 2006; Djonovic et al, 2007; Vargas et al., 2008],

There is evidence of a possible relationship between the state of aggregation of kerato-platanin family proteins and biological activity (induction of necrosis and systemic resistance). Both Sm1 and EpH, both belonging to the family of Trichoderma-expressed and secreted kerato- platanines, when present as monomers are capable of inducing systemic resistance but not inducing necrosis. However, dimeric forms of these proteins cause necrosis and do not induce resistance responses in plants [Vargas et al, 2008], Zaparoli et al. (2008) showed that a protein from the M. perniciosa Cerato-platanin family (MpCPI) is present in dimer form and is capable of inducing necrosis in dicotyledonous plants. The necrosis produced, however, is not proportional to the increase of the inoculated protein concentration, and the induction of symptoms only occurs at concentrations higher than or equal to 0.5 mg / mL. At subnecrotic kerato-platanin concentrations, the plant is capable of expressing pathogen resistance-related genes (Djonovic et al., 2006, Djonovic et al., 2007).

Inoculation of MpCPI protein (Seq. 6) in cocoa and tobacco plants together with a NEP-like M. pernicious protein, MpNEP2 (Seq. 4), showed an increase in the intensity of necrosis symptoms observed, suggesting a synergy between these two proteins. Both MpCPI and MpNEP2 protein were resistant to high temperatures maintaining biological activity even after temperature stress (30 minutes at 100 ° C) [Garcia et a /., 2007; Zaparoli et al., 2008],

The use of proteins produced by living beings as active ingredients or adjuvants in industrial products, in order to increase their efficiency, has been increasingly used. Products such as soap powders use different enzymes or enzyme sets in their formulations in order to increase their cleaning effect. These enzymes are artificially produced, and added to the formulation. The main types of enzymes used in the detergent industry include: a) amylases, which degrade starch and other carbohydrates; b) proteases, which degrade peptide bonds; c) lipases, which degrade lipids; d) cellulases, which degrade cellulose (Bom, 1995). Most of the soaps are added with enzymes that will degrade organic matter, according to their affinity for the substrate, present in the stains of clothes. This is a clear example of the feasibility of using proteins as active ingredients of compounds on an industrial scale.

BRIEF DESCRIPTION OF THE INVENTION

Herbicides used today in agriculture to control weeds classified as weeds present several problems such as environmental contamination and toxicity. Faced with this problem, chemical herbicides are being replaced by natural herbicides, which include biologically active substances in their formulation.

The present invention relates to the herbicidal composition with biologically active components, specifically one or more necrosis inducing proteins of the Cerato-platanins group and NEPs, with synergistic action to cause necrosis specifically in dicotyledonous plants, more efficiently than applied separately. The composition may be used in addition to other agribusiness compositions such as bactericides, fungicides, herbicides, insecticides, nematicides, soil conditioners, fertilizers, or other additives already employed to increase the yield of the crop of interest.

By having a synergistic effect of proteins on CP and NEP proteins, the present invention enables efficient and specific pre- or post-emergent control of weeds and dicot plants. These characteristics allow the use of chemical herbicides in lower concentrations, reducing their toxic effect, environmental impact and damage to monocotyledonous crops. The composition may contain, in addition to said proteins and herbicides, a liquid carrier (such as water, alcohol or nonpolar compounds) or solid carrier (such as clay, talc or inorganic minerals), emulsifiers, pH buffers, stabilizers, antifreeze, surfactants , gelling agents, dispersing agents, adhesives or other additives employed depending on the conditions of the crop to be protected, the pest to be fought, climate, soil, the method of use and application, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses the proteins belonging to the family of kerato-platanins (CP) of the fungus Moniliophthora perniciosa MpCPI [EU250339], MpCP2, [EU250341], MpCP3 [EU250343] or any other homologous protein belonging to this family - (pfam). A herbicidal composition may comprise a single protein in the formulation or an association with other necrosis inducing proteins. Possible combinations include NLP class proteins (ΝΕΡ1 like proteins), among others. The CP: NEP association of M. perniciosa shows an increase in necrosis intensity compared to their separate application, accelerating leaf death [Zaparoli et al., 2008].

The present invention encompasses plant necrosis inducing proteins belonging to the family of Ceratoplatanins and NEPs in combination acting synergistically to specifically cause necrosis in dicotyledonous plants in a composition, thereby allowing pre- or post-emergent control. plants of this group, without prejudice to plants of the monocotyledonous group. This makes it possible to use less nonspecific chemical herbicides for a weed to be fought and / or toxic to humans and other animals and the environment, thanks to the characteristic of CP and NEP proteins that specifically cause necrosis. in dicotyledonous plants, without harming monocotyledonous plantations. Examples of monocotyledonous plants include wheat, rice, corn and sugarcane crops.

Said proteins may be added to any existing formulation used in the agro-industry, including bactericides, fungicides, herbicides, nematicides, soil conditioners or fertilizers, conferring or enhancing efficiency and specificity characteristics against dicotyledonous plants, preventing or retarding their growth. , reducing the need for the use of chemical herbicides. It is also necessary to ensure that once the composition in question has been applied, it remains in contact with the weed as long as possible, ensuring greater effectiveness. This can be achieved through additives such as adhesives, composition evaporation inhibiting substances (such as high vapor pressure nonpolar compounds), emulsifiers, composition preventing leaching substances [isocyanate-capped diols, triols or polyols ( BR951012) or other polymers, or wetting agents for solid carrier compositions.

The proteins selected for the combination of the present invention exhibit high temperature resistance to maintain induction and necrosis activity in dicotyledonous plants even when confronted with high temperatures - even after half an hour in boiling water, MpCPI (Cerato) proteins. -platinum) and MpNEP2 (NLPs) preserve their activity [Garcia et al., 2007; Zaparoli et al., 2008]. This characteristic indicates an excellent property for maintaining the stability of the proposed active herbicide in solution.

Still, stabilizers or antifreeze may be employed in herbicidal formulations, allowing them to be preserved longer, ensuring their effectiveness after some storage, or at higher concentrations, useful for economy during transport and space and effort during handling of the compositions in question. These may be pH buffers such as Tris-HCl, stabilizers, glycerol, ethylene glycol or propylene glycol (antifreeze).

Other optional additives include defoamers (e.g. based on silicone or magnesium stearate), humectants and colorants, etc.

The herbicidal composition can be converted into solution, suspension, emulsion, sprinkle, powder, paste or granule, which can be further diluted, resuspended, emulsified, always with a view to better application and efficiency of the final product, depending on the characteristics of the plantation. be protected, the pest to be fought, climatic, soil, etc.

Plant-protein contact (s) can be carried out either on leaf tissue by direct application to the leaf surface (leaf spraying, etc.) or root tissue (direct application to the soil, mixing with fertilizers or soil conditioners, etc.). etc). The compositions may, for example, be diluted or resuspended in the crop irrigation system.

Depending on the method used to favor protein solution contact with plant cells, the formulation (protein concentration and additives) to be used may vary, since the penetrability of the protein mixture into the tissues will directly influence the extent. of the injury.

The degree of penetration (absorption) will depend not only on the inoculation method used but also on the characteristics of the tissue (eg number of stomata on the leaf surface) and environmental factors such as moisture present at the time of inoculation. To facilitate the penetration of proteins into plant tissues, application may occur in humid weather, after a period of rain or irrigation, for example.

A formulation that is able to penetrate the surface of the target plants is desired in order to place the proteins inside the leaves and / or roots, thereby increasing the amount of necrosis causing proteins that reach their target - the plant cells -, decreasing the actual / actual concentration required in the final product.

The best way to accomplish this task is by adding organosulfactants to the herbicide formulation. The application of organosulfactants together with proteins shows an increase in the efficiency of the final product as it helps the penetration of proteins by the stomata and cuticle of the plant, thus reaching the target - the cells more easily. A group of surfactants widely used in commercial herbicides are organosilicones such as Silwet L-77 (1,1,1,3,5,5,5-heptamethyltrisiloxanyl propyl-methoxy-poly [ethylene oxide]), but others may be employed as long as they do not drastically reduce CP and NEP protein activity.

The compatibility of NEPs proteins with chemical herbicides (glyphosate and 2,4D) has already been tested according to Bailey et al. (2000). This work showed that NEP1 does not interfere with the action of these chemical herbicides and vice versa, while protein utilization accelerated the necrosis process. These studies were the basis for the proposed use of proteins now selected in combination with known chemical herbicides.

The scope of this invention includes combinations of the herbicidal composition with other agrochemicals already employed in the agribusiness, such as bactericides, fungicides, herbicides, nematicides, fertilizers, soil conditioners, etc.

The herbicidal composition needs a vehicle to be applied to crops, which may be liquid (water, alcohol, ketones, esters, paraffins, hydrocarbons, aromatic compounds or mixtures thereof, eg) or solid (inorganic clays, talc or minerals such as silica, eg).

EXAMPLES

The examples described below are intended solely to exemplify some of the numerous ways of carrying out the invention, but without limiting the scope or scale thereof.

EXAMPLE 1

CP and NEP1 proteins present from 5 µg / ml to 500 µg / ml, preferably 100 to 200 µg / ml, are added to a soil fertilizer or soil conditioner which is applied prior to planting the desired monocotyledonous crop. . In this way, dicotyledonous weeds that come into contact, undergo the necrotic process, which causes growth retardation or hindering, however, without causing damage to monocotyledonous plantation.

EXAMPLE 2 CP and NEP1 proteins present from 5 µg / ml to 500 µg / ml, preferably 100 to 200 µg / ml, are added to a nematicide applied to the soil of a monocot plantation. Thus, in addition to the control of plant-damaging nematodes, there will be control of pre- or post-emergence dicotyledonous weeds.

EXAMPLE 3

CP and NEP1 proteins present from 5 µg / ml to 500 µg / ml, preferably 100 to 200 µg / ml, are added to a spray-applied foliar fungicide on monocotyledonous plantation. In this way a composition is obtained that, in addition to fighting fungi, helps in the control of unwanted dicotyledonous plants.

EXAMPLE 4

Herbicidal formulations (2,4D or glyphosate, e.g.) containing the CP and NEP proteins present from 5 µg / ml to 500 µg / ml, preferably 100 to 200 µg / ml. The characteristic of causing necrosis in CP and NEP plant tissues specifically in dicotyledonous plants allows smaller amounts or concentrations of such chemical herbicides to be used to combat these pests, as well as minimizing their toxicity and impacts on the environment and monocotyledonous plantation. of interest. Such formulations are dependent on the crop to be protected, the pest to be fought, the stage of pest development to be fought (pre- or post-emergent), climatic characteristics, soil, etc. REFERENCES

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Claims (7)

1. Herbicidal composition, characterized in that it comprises in effective quantities the components (A) and (B) with selective synergistic effect for dicotyledonous plants, where: (A) represents one or more selected proteins of the Cerato-Platanina family; (B) represents one or more proteins selected from the Necrosis and Ethylene-Inducing (NEP) family. Herbicidal composition according to Claim 1, characterized in that the protein of the Cerato Platanina family is preferably MpCPI. Herbicidal composition according to Claim 1, characterized in that the protein of the Necrosis and Ethylene-Inducing family is preferably MpNEP2. Herbicidal composition according to claim 1, characterized in that it further comprises one or more components selected from the group of agrochemically active compounds, auxiliary formulations and additives conventionally used in agricultural and gardening activities. Dicotyledonous weed control method, characterized in that the application of the composition as described in the preceding claims, is carried out jointly or separately, preemergent, postemergent, or preemergent and post-emergent, on plant parts, seeds or in the area in which the plant grows. Method according to claim 4, characterized by applying the composition described in claims 1 to 3 for controlling dicotyledonous weeds in a monocotyledonous crop. Method according to Claim 4, characterized in that the application of the composition takes place in previously moistened plants.