Formulation comprising a protein with insecticidal and / or nematicidal activity and related methods

Abstract

Disclosed are a formulation comprising a protein with pesticidal activity, useful for a pesticidal formulation, and a method of controlling pests comprising the use of said formulation, wherein the pesticidal protein is a Txp40 protein or variant thereof.

Description

[0001] FORMULATION COMPRISING A PROTEIN WITH INSECTICIDAL AND / OR NEMATICIDAL ACTIVITY AND RELATED METHODS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a formulation comprising a protein with pesticidal activity, useful as a pesticidal formulation, and a method of controlling pests comprising the use of said formulation, wherein the pesticidal protein is a Txp40 protein or a variant thereof.

[0004] BACKGROUND

[0005] Within the agricultural field, there has been a growing concern in recent years over the continued use of traditional chemistry-based pesticides, particularly in relation to their environmental impact. Correspondingly, there has been a recent trend for developing biological pesticides, that is, pesticides derived from natural sources instead of synthetic ones, since they are usually more environmentally-friendly.

[0006] It is known that chemistry-based pesticides have the following disadvantages:

[0007] 1) Their prolonged use can trigger resistance;

[0008] 2) They can have undesired environmental effects;

[0009] 3) They are generally non-specific, harming non-target animals and plants;

[0010] 4) They have residual effects in the plants that can persist over time.

[0011] In this sense, the use of biological insecticides, based on the metabolic products of microorganisms, have the following advantages when compared to chemistry-based pesticides:

[0012] 1) They can be targeted towards a particular species or genus, presenting no harm to other organisms;

[0013] 2) They are less aggressive towards the environment, usually being non-toxic to higher organisms and plants.

[0014] Biological insecticides are known in the art, with commercial products already being available on the market. These commercial products are typically based on Bacillus thuringiensis, which exerts an insecticidal effect due to the production of a family of protein toxins referred to as Cry proteins, and they are widely used around the world.

[0015] However, some insect pests have been shown to be resistant to Cry proteins, which is why there is a constant need for further developing biological insecticides to control a wider range of insect pests, as well proper ways to formulate them so as to obtain an effective product.

[0016] The protein comprised within a formulation of the present invention belongs to the group of Txp40 proteins. It is known in the art that Txp40 proteins exhibit insecticidal activity, and that different microorganisms are capable of producing different types of Txp40 proteins. The following are some examples of these Txp40 proteins and their use as insecticides:

[0017] Brown S.E. et al., disclose the presence of the gene encoding Txp40 proteins, with a high degree of conservation, in several species of the genera Xenorhabdus and Photorhabdus. They further describe the insecticidal activity of the Tx40 protein of Xenorhabdus nematophila, mainly by injection into the hemocoel of insects, in particular in Spododoptera sp.

[0018] Mathur C. et al. disclose the toxic effects of a Txp40 protein obtained from Photorhabdus luminescens on Galleria mellonella.

[0019] Shankhu P.Y. et al. disclose the insecticidal activity of a Txp40 protein obtained from Photorhabdus akhurstii against larvae from Helicoperva armigera, Spodoptera litura and Spodoptera exigua.

[0020] Kinkar O.U. et al. disclose a characterization of a Txp40 protein obtained from Xenorhapdus nematophila, and show its toxic effect on larvae from Galleria mellonella.

[0021] Park J.M. et al. disclose a Txp40 protein obtained from Xenorhabdus nematophila and its insecticidal activity against larvae of Plutella xylostella.

[0022] Further, patent applications that also disclose Txp40 proteins and their use in pest control are available as follows:

[0023] US Patent application published under No. 2004 / 0055036 A1 discloses genes that encode toxic proteins from X. nematophila and P. luminisescens, among which Txp40 is included, and further describes a method for transforming microorganisms and plants with nucleic acids that encode these proteins.

[0024] US Patent application published under No. 2022 / 0324920 A1 discloses a general method for controlling Spodoptera pests by using a protein or variants thereof obtained from microorganisms.

[0025] In particular, US 2022 / 0324920 A1 discloses a Txp40 protein from Photorhabdus luminescens and variants thereof, and their use as an insecticide against Spodoptera frugiperda.

[0026] Despite the known insecticidal effects of Txp40 proteins and their variants, there is still a need for an effective pesticidal formulation that comprises a Txp40 protein.

[0027] SUMMARY OF THE INVENTION

[0028] In view of the above-mentioned need, a first aspect of the present invention is a pesticide formulation comprising: a) a polypeptide comprising a sequence as set forth in SEQ ID NO: 1 or a polypeptide variant thereof with at least 90% identity to the polypeptide sequence as set forth in SEQ ID NO: 1 , b) a pH buffer of pH 7-9, selected from HEPES, MOPS, PIPES, Tricine, Bicine, TES, Tris-HCI or Phosphate buffer; c) a chelating agent selected from citric acid, ethylenediaminetetracetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylenediaminetriacetic acid (HEEDTA), ethylenediaminedi-[o-hydroxyphenylacetic acid] (EDDHA), ethylene glyco I- bis- (2- aminoethyl ether)- tetracetic acid (EGTA), diethylenetriaminepentacetic acid (DTPA), or 1 ,2-diaminocyclohexanotetracetic acid (DCTA). d) an emulsifier or a non-ionic surfactant selected from polysorbate, octoxynol, glycerol, Sorbitan esters, polyethylene glycol (PEG), or polyvinyl alcohol, or any combination thereof; e) a protease inhibitor selected from Phenylmethylsulfonyl fluoride (PMSF), 4-(2- aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), E-64 or Pepstatin; and f) a preservative with biocidal activity selected from sodium azide, benzisothiazolinone (BIT), or bronopol.

[0029] In an embodiment, the pesticide formulation comprises a polypeptide variant having i) a deletion of at least one amino acid between residues 216 and 237 of the sequences as set forth in SEQ ID NO: 1 ; or ii) a substitution of at least one amino acid selected from the residues 35, 70, 77, 87, 100, 111 , 131 , 173, 180, 195, 264, 267, 275, or 308 of the sequence as set forth in SEQ ID NO: 1 , or iii) a sequence as set forth in any one of SEQ ID NO: 7-21 .

[0030] In an embodiment, the pesticide formulation of the first aspect of the invention comprises the polypeptide as set forth in SEQ ID NO: 3 or a variant thereof. Preferably, wherein said variant of the polypeptide as set forth in SEQ ID NO: 3 comprises at least one of the following:

[0031] • a deletion of at least one amino acid between residues 239 and 260; or

[0032] • a substitution of at least one amino acid selected from the residues 58, 93, 100, 110, 123, 134, 154, 196, 203, 218, 287, 290, 298, 331.

[0033] More preferably, the pesticide formulation comprises a polypeptide having a sequence as set forth in any one of SEQ ID NO: 7-21 .

[0034] In an embodiment, the pesticide formulation comprises a homogenized bacterial extract in an amount such that the optic density of the formulation is between 0.5-5 at 600 nm, for example, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5. Preferably the optic density is of 1 at 600 nm, and wherein the extract comprises a polypeptide comprising the sequence as set forth in SEQ ID NO: 1 or a variant thereof.

[0035] In an embodiment, the pesticide formulation comprises a pH buffer at a concentration of 0.1-50 mM, at the pH of the formulation is between 6-9, preferably the pH is between 7.2-8.2.

[0036] In an embodiment, the pesticide formulation comprises a chelating agent at a concentration of 0.05-5 mM.

[0037] In an embodiment, the pesticide formulation comprises an emulsifier or a non-ionic surfactant at a concentration of 0.01-20% w / v.

[0038] In an embodiment, the pesticide formulation comprises a protease inhibitor at a concentration of 0-2 mM.

[0039] In an embodiment, the pesticide formulation comprises a preservative with biocidal activity at a concentration of 0.00001-0.1% w / v.

[0040] In a particularly preferred embodiment, the pesticide formulation comprises: a) a bacterial homogenate with an optic density of 1 , and further comprising the pesticidal polypeptide, b) Tris-HCI or Phosphate buffer at a concentration of 2-50 mM, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 mM, more preferably 2-25 mM, and pH 7.2-8.2, for example pH 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, 8.1 or 8.2; c) EDTA at a concentration of 0.2-2.0 mM, for example 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1 .7, 1.8, 1.9 or 2.0 mM; d) PEG400, PEG600 at a concentration between 5%-20% v / v, for example 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% v / v; or glycerol at a concentration between 5-12% w / v, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12% w / v; or polysorbate 65 (Tween65) at a concentration of 0.05-0.20% w / v, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11 %, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20% w / v; e) PVA at a concentration of 0.1-1 % w / v, for example, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1 % w / v; f) PMSF at a concentration of 0.1-2 mM, for example 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,

[0041] 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1 .7, 1.8, 1.9 or 2.0 mM; g) BIT or sodium azide at a concentration of 0.1-0.005% w / v, for example 0.1 %, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01 %, 0.009%, 0.008%, 0.007%, 0.006% or 0.005% w / v.

[0042] In an embodiment, the pesticide formulation comprises a) a bacterial homogenate with an optic density of 1 , and further comprising the pesticidal polypeptide of SEQ ID NO: 1 or a variant thereof with at least 90% identity to the polypeptide sequence as set forth in SEQ ID NO: 1 , b) Tris-HCI or Phosphate buffer at a concentration of 2-25 mM, c) EDTA at a concentration of 0.2-2 mM, d) PEG400 at a concentration of 5-20% v / v, or polysorbate65 (tween65) at a concentration of 0.05-0.2% v / v, orglycerol at a concentration of 5-12% v / v, or PEG600 at a concentration of 5-20% v / v, e) PVA at a concentration of 0.1 -0.5% w / v, f) PMSF at a concentration of 0.1-2 mM, g) BIT or sodium azide at a concentration of 0.0005-0.05% w / v.

[0043] In an embodiment, the pesticidal formulation of the first aspect of the present invention in any of its embodiments comprises an insecticide and / or nematicide formulation. A second aspect of the present invention refers to a method for controlling pests comprising applying a pesticide formulation of the first aspect of the present invention to a plant or plant part.

[0044] In an embodiment the method for controlling pests comprises applying a pesticide formulation to a plant or plant part, wherein the plant is selected from main row crops, fruits and vegetables, preferably selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp. In particular, the plant is selected from Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Allium sativum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum. More preferably, the plant is selected from Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum.

[0045] In an embodiment, the method for controlling pests of the present invention comprises controlling a pest, wherein said pest comprises a pathogenic fungi, bacteria, nematode or insect, preferably, controlling an insect or nematode pest.

[0046] In an embodiment, the method for controlling a pest comprises controlling an insect pest by applying an insecticide formulation to a plant or plant part.

[0047] In an embodiment, the method for controlling an insect pest, comprises controlling an insect pest selected from the genera Spodoptera spp., Tuta spp., Trialeurodes spp., Anticarsia spp., Helicoperva spp., Rachiplusia spp., Dalbulus spp., Tetranychus spp., Bemisia spp., Aphis spp., Myzus spp., Rachiplusia spp., Alphitobus spp. Anthonomus spp., Thri ps spp., or Frankliniella spp. In a particular embodiment the method for controlling an insect pests comprises controlling a pest selected from Tuta absoluta, Trialeurodes vaporariorum, Anticarsia gemmatalis, Helicoverpa armigera, Spodoptera frugiperda, Spodoptera exigua, Spodoptera cosmioides, Tetranychus urticae; Bemisia tabaci; Aphis gossypii, Myzus persicae, Rachiplusia nu, Alphitobius diaperinus, Anthonomus grandis, Helicoverpa zea, Helicoverpa gelotopoeon, Thrips simplex, Frankliniella schultzei Trybom, Frankliniella occidentalis, Frankliniella insularis Franklin, Frankliniella gemina.

[0048] In an embodiment, the method for controlling a pest comprises controlling a nematode pest by applying a nematicide formulation to a plant or plant part.

[0049] In an embodiment, the method for controlling a nematode pest, comprises controlling a nematode pest selected from the genera Meloidogyne spp, Nacobbus spp, Heterodera spp, Giobodera spp, Pratyienchus spp, Paratylenchus spp, Rotylenchus spp, Xiphinema spp, Trichodorus spp.

[0050] In an embodiment, the method for controlling pests comprises applying a pesticide formulation of the present invention in a dose between 500-10000 mL / ha, for example, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 mL / ha.

[0051] BRIEF DESCRIPTION OF THE FIGURES

[0052] Figure 1 shows the performance results of an embodiment of the formulation of the present invention in an experiment of biocontrol of Tuta absoluta (tomato moth) by foliar application.

[0053] Figure 2 shows the performance results of an embodiment of the formulation of the present invention in an experiment of biocontrol of Trialeurodes vaporariorum (whitefly) by foliar application.

[0054] Figure 3 shows the performance results of an embodiment of the formulation of the present invention in an experiment of biocontrol of Nacobbus aberrans, measured as the average nodules found in each treatment and the quantification of the biocontrol of (C= nodule without treatment).

[0055] DETAILED DESCRIPTION OF THE INVENTION Examples given herein shall be interpreted only as exemplary embodiments of the different aspects, objects, and / or methods of the present invention and are not intended to limit the scope of the present invention in any way.

[0056] Any technical terminology used herein shall be understood by the common definition utilized in the art and / or by those skilled in the art, unless otherwise explicitly stated or inferred by context.

[0057] Each and every embodiment of any aspect, object, and / or method of the present invention resulting from the combination of particular embodiments of said aspect, object and / or method described herein are to be considered as falling within the scope of the present invention.

[0058] The terms “formulation” or “composition” will be used throughout the present specification interchangeably, and both refer to a mixture of substances suitable for agricultural use.

[0059] The terms “a”, “an”, and “the” refer to “one or more” or “at least one” when used in this application, including the claims. Thus, for example, reference to “a polypeptide” includes a plurality of polypeptides.

[0060] As used herein, the terms “comprise(s)”, “comprising”, “have”, “has”, “having” is intended to be inclusive and open-ended. It means ’’including, but not limited to,“ and does not exclude additional, non-recited elements or steps. Thus, a system, device, method, or process that comprises one or more components or steps does not exclude the possibility of additional components or steps being present, even though they are not expressly listed.

[0061] As used herein, the term “embodiment”, and similar expressions like “an embodiment”, or “one embodiment”, or “a preferred embodiment”, among others, indicates the description of a particular embodiment of a product or method of an aspect of the present invention that may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include that particular feature, structure, or characteristic. Furthermore, the expression may not necessarily refer to the same embodiment. When a particular feature, structure, or characteristic is associated or connected to a particular embodiment of a product or method, it is submitted that is within the knowledge of the person skilled in the art to implement such feature, structure, or characteristic in connection with other embodiments, whether or not explicitly described.

[0062] For the purpose of providing sufficient disclosure and / or description, each and every patent, patent application, established publication and cited document are expressly incorporated herein by reference.

[0063] The terms “from X to Y”, or “between X and Y” or “from / between X-Y” should be interpreted to include X and Y, unless the context indicates otherwise.

[0064] Throughout the following specification, when referring to a concentration as weight / volume (w / v), this should be understood as g / 100 mL.

[0065] Throughout the following specification, when referring to a concentration as volume / volume (v / v), this should be understood as mL / 100 mL.

[0066] As used herein, the term “pesticide”, “pesticidal” or variants thereof, when referring to a substance (e.g. a protein), or combination of substances (e.g. a formulation) refers more specifically to the insecticidal and / or nematicidal properties of said substance, or combination of substances.

[0067] As used herein, the term “insecticide”, “insecticidal” and variants thereof, when referring to a substance (e.g. a protein), or combination of substances (e.g. a formulation) refers to the ability of said substance or combination of substances to inhibit the ability of insect pests to survive, grow, feed, and / or reproduce, or to limit insect-related damage or loss in plants or plant parts. Preferably, these effects are the result of a toxic effect. The term “insecticide” may or may not mean killing insects, although it preferably means killing the insects. Said insects may be in any stage of their life cycle, for example neonate, larva, pupa (if corresponding) or adult.

[0068] As used herein, the term “nematicide”, “nematicidal” and variants thereof, when referring to a substance (e.g. a protein), or combination of substances (e.g. a formulation) refers to the ability of said substance or combination of substances to inhibit the ability of nematode pests to survive, grow, feed, and / or reproduce, or to limit nematode-related damage or loss in plants or plant parts. Preferably, these effects are the result of a toxic effect. The term “nematicide” may or may not mean killing nematodes, although it preferably means killing the nematodes. Said nematodes may be in any stage of their life cycle, for example neonate, larva, or adult. As used herein, “pesticidal protein”, “insecticidal protein”, “nematicidal protein” or“Txp40 protein” when referring to the protein contained in a formulation of the first aspect of the present invention, refers to a protein (i.e., a polypeptide) comprising the sequence as set forth in SEQ ID NO: 1 or a variant thereof. The polypeptide of SEQ ID NO: 1 corresponds to a Txp40 protein of the microorganism Photorhabdus hindustanensis.

[0069] By a “variant” of the aforementioned pesticidal, insecticidal, nematicidal or Txp40 proteins, throughout the present description it is made reference to proteins comprising an amino acid sequence with a similar amino acid sequence compared to SEQ ID NO: 1 , while maintaining the pesticidal function of a pesticidal protein comprising the amino acid sequence as set forth in SEQ ID NO: 1 .

[0070] According to the invention, variants of polypeptide of SEQ ID NO: 1 comprise polypeptide sequences with at least 90% identity to SEQ ID NO: 1. Said variants may include, for example, homologue variants of the polypeptide of SEQ ID NO: 1 that can be naturally found in other microorganisms from the genus Xenorhabdus or Photorhabdus, for example, Xenorhabdus nematophila, Photorhabdus luminescens, or Photorhabdus akhurstii. Photorhabdus laumondii, Photorhabdus bodei, Photorhabdus caribbeanensis, as well as allelic variants, and natural or synthetic mutations of a Txp40 from Photorhabdus hindustanensis. In an embodiment, these variants of the polypeptide of SEQ ID NO: 1 comprise the polypeptides of SEQ ID NO: 7-21 , more preferably variants of SEQ ID NO 7-16, or variants of SEQ ID NO17-21 .

[0071] In another embodiment, said variants comprise amino acid sequences with at least 97.5% identity to SEQ ID NO: 1 , or polypeptide sequences with up to 8 mutations when compared to SEQ ID NO: 1 .

[0072] As used herein, the term “% identity” when referring to a sequence, refers to the degree of similarity between two polynucleotide or polypeptide sequences. More specifically, it refers to the number of nucleotides (in case of a polynucleotide) or amino acids (in case of a polypeptide) that share positions in an alignment where the nucleotides or amino acids are identical when comparing a given sequence (e.g., a query sequence) to a reference sequence over a defined region. This value may be determined using specific algorithms (e.g., BLAST, ClustalW, or a similar sequence alignment tool) under defined parameters (e.g., gap penalties, scoring matrices, etc.). The identity is determined by dividing the number of identical residues in the aligned sequences by the total number of residues in the alignment (excluding gaps introduced for alignment) and multiplying by 100. For example, if two sequences of 100 residues align with 90 identical residues and 10 mismatches, the % identity would be 90%. In an embodiment, the pesticidal protein of the formulation comprises a polypeptide of SEQ ID NO: 1 , or a variant of the polypeptide sequence as set forth in SEQ ID NO: 1 . Preferably said variant comprises a deletion or substitution of at least one amino acid of the polypeptide sequence as set forth in SEQ ID NO: 1 , wherein said variant preserves the protein conformation, the pesticidal activity, or both, of the polypeptide of SEQ ID NO: 1 .

[0073] In an embodiment, said variant of the polypeptide of SEQ ID NO: 1 comprises at least one of the following:

[0074] • a deletion of at least one amino acid between residues 216 and 237; or

[0075] • a substitution of at least one amino acid selected from the residues 35, 70, 77, 87, 100, 111 , 131 , 173, 180, 195, 264, 267, 275, 308.

[0076] In an embodiment, the variant of the polypeptide of SEQ ID NO: 1 comprises at least one of the following substitutions, or combination thereof: i) Substitution of residue 35 by Asp, ii) Substitution of residue 70 by Glu or Asn, iii) Substitution of residue 77 by Ser or Ala, iv) Substitution of residue 87 by Thr, v) Substitution of residue 100 by Asn, vi) Substitution of residue 11 1 by Arg or His, vii) Substitution of residue 131 by Asp or Gin, viii) Substitution of residue 173 by Glu or Ala, ix) Substitution of residue 180 by Vai or Gly, x) Substitution of residue 195 by Gin or Glu, xi) Substitution of residue 264 by Glu, xii) Substitution of residue 267 by Thr, xiii) Substitution of residue 275 by Arg, xiv) Substitution of residue 308 by Asn or Glu.

[0077] In a preferred embodiment, the variant of the polypeptide of SEQ ID NO: 1 comprises the polypeptides of SEQ ID NO: 7-21 . Preferably, the variant comprises a polypeptide of SEQ ID NO: 7-16 or a variant of SEQ ID NO 17-21.

[0078] The pesticidal protein of the formulation may further comprise additional polypeptide sequences extending from the sequence as set forth in SEQ ID NO: 1 or the corresponding variant thereof. For instance, such an additional polypeptide sequence may extend from the N-terminal of the sequence as set forth in SEQ ID NO: 1 or the corresponding variant thereof. For example, said additional polypeptide sequence can result from the translation of a polynucleotide sequence that is added to the polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1 or a variant thereof, for example, for its insertion into an expression cassette or vector.

[0079] As an example, the amino acid sequence of SEQ ID NO: 3 is the result of adding the polypeptide sequence as set forth in SEQ ID NO: 2 to the polypeptide sequence as set forth in SEQ ID NO: 1 at its N-terminus. The amino acid sequence of SEQ ID NO: 3, in turn, is the result of the translation of a polynucleotide sequence as set forth in SEQ ID NO: 6. The sequence as set forth in SEQ ID NO: 6 is the result of adding the polynucleotide sequence as set forth in SEQ ID NO: 5 to the polynucleotide sequence as set forth in SEQ ID NO: 4 at its 5’ end, for its proper expression in E. coli.

[0080] In a particular embodiment, the pesticidal protein of the formulation comprises a polypeptide of SEQ ID NO: 3, or a variant of the polypeptide sequence as set forth in SEQ ID NO: 3 that comprises 90% of sequence identity to the sequence as set forth in SEQ ID NO: 3. Preferably said variant comprises a deletion or substitution of at least one amino acid of the polypeptide sequence as set forth in SEQ ID NO: 3, wherein said variant preserves the protein conformation, the pesticidal activity, or both, of the polypeptide of SEQ ID NO: 3.

[0081] In an embodiment, said variant of the polypeptide of SEQ ID NO: 3 comprises at least one of the following:

[0082] • a deletion of at least one amino acid between residues 239 and 260; or

[0083] • a substitution of at least one amino acid selected from the residues 58, 93, 100, 110, 123, 134, 154, 196, 203, 218, 287, 290, 298, 331.

[0084] In an embodiment, the variant of the polypeptide of SEQ ID NO: 3 comprises at least one of the following substitutions, or combination thereof: i) Substitution of residue 58 by Asp, ii) Substitution of residue 93 by Glu or Asn, iii) Substitution of residue 100 by Ser or Ala, iv) Substitution of residue 110 by Thr, v) Substitution of residue 123 by Asn, vi) Substitution of residue 134 by Arg or His, vii) Substitution of residue 154 by Asp or Gin, viii) Substitution of residue 196 by Glu or Ala, ix) Substitution of residue 203 by Vai or Gly, x) Substitution of residue 218 by Gin or Glu, xi) Substitution of residue 287 by Glu, xii) Substitution of residue 290 by Thr, xiii) Substitution of residue 298 by Arg, xiv) Substitution of residue 331 by Asn or Glu.

[0085] In an embodiment, the variant of the polypeptide of SEQ ID NO: 3 comprises the polypeptides of SEQ ID NO: 7-21 . Preferably, the variant comprises a polypeptide of SEQ ID NO: 7-16 or a variant of SEQ ID NO 17-21.

[0086] A person skilled in the art would be able to determine the optimal polynucleotide sequence that would result in the proper expression of a protein comprising the polypeptide sequence of SEQ ID NO: 1 or a variant thereof according to the invention. The exact polynucleotide sequence will depend on the expression cassette or vector used and the organism selected for expression. Preferably, the optimal polynucleotide sequence when expressed results in an additional polypeptide sequence that is added to the N-terminus of the polypeptide sequence of SEQ ID NO: 1 or the corresponding variant thereof.

[0087] The pesticide protein in any of its embodiments, can be used for the production of a pesticide formulation of the first aspect of the present invention.

[0088] The formulation of the present invention comprises a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1 or a variant thereof, a pH buffer, a chelating agent, an emulsifier and / or non-ionic surfactant, a protease inhibitor, and a preservative with biocidal activity. Possible pH buffers, chelating agents, emulsifiers and / or nonionic surfactants, protease inhibitors, and preservatives with biocidal activity will be further described herein.

[0089] The formulation comprises an active ingredient with pesticide activity, wherein said active ingredient with pesticide activity is a pesticide protein. Furthermore, said pesticide protein is obtained from a homogenized bacterial extract. In an embodiment, the formulation comprises a homogenized bacterial extract comprising a pesticidal polypeptide, preferably a diluted homogenized bacterial extract. Preferably, the formulation comprises the homogenized bacterial extract in an amount such that the optic density (OD) of the formulation at 600 nm is between 0.5-5, more preferably the OD at 600 nm is 1. The formulation can comprise a diluted homogenized bacterial extract, wherein said homogenized bacterial extract is diluted between 2-fold to 100-fold. Preferably the diluted bacterial homogenate is diluted 5-fold.

[0090] The homogenized bacterial extract comprises a pesticidal polypeptide comprising a polypeptide sequence as set forth in SEQ ID NO: 1 or a variant thereof with at least 90% identity to the polypeptide sequence as set forth in SEQ ID NO: 1. Preferably, wherein the pesticidal polypeptide variant comprises

[0091] • a deletion of at least one amino acid between residues 216 and 237 of the sequences of SEQ ID NO: 1 ; or

[0092] • a substitution of at least one amino acid selected from the residues 35, 70, 77, 87, 100, 111 , 131 , 173, 180, 195, 264, 267, 275, or 308 of the sequence as set forth in SEQ ID NO: 1 , or

[0093] • a sequence as set forth in any one of SEQ ID NO: 7-21 .

[0094] In another embodiment, the homogenized bacterial extract comprises a pesticidal polypeptide comprises a polypeptide as set forth in SEQ ID NO: 3 or a variant thereof. Preferably, wherein said variant of the polypeptide as set forth in SEQ ID NO: 3 comprises at least one of the following:

[0095] • a deletion of at least one amino acid between residues 239 and 260; or

[0096] • a substitution of at least one amino acid selected from the residues 58, 93, 100, 110, 123, 134, 154, 196, 203, 218, 287, 290, 298, 331 ; or

[0097] • a sequence as set forth in any one of SEQ ID NO: 7-21 .

[0098] More preferably, the homogenized bacterial extract comprises a polypeptide having a sequence as set forth in any one of SEQ ID NO: 7-21 .

[0099] In an embodiment, the formulation comprises a pH buffer selected from HEPES, MOPS, PIPES, Tricine, Bicine, TES, Tris-HCI or Phosphate buffer. In a particularly preferred embodiment, the formulation of the invention comprises a buffer in a concentration from 0.1-50 mM, preferably between 5-20mM. Alternatively, the formulation comprises a concentration of the pH buffer in a suitable concentration such that the final pH of the formulation ranges between 6-9, preferably 7.2-8.2. Preferably, the formulation comprises Tris-HCI or Phosphate buffer.

[0100] The formulation further comprises a chelating agent selected from citric acid, ethylenetriaminepentaace acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylenediaminetriacetic acid (HEEDTA), ethylenediaminedi-[o- hydroxyphenylacetic acid] (EDDHA), ethylene glycol-bis-(2-aminoethyl ether)- tetracetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), or 1 ,2- diaminocyclohexanotetracetic acid (DCTA). In a particularly preferred embodiment, the formulation of the first aspect of the invention comprises a chelating agent in a concentration from 0.05-5 mM, preferably, between 0.2-3 mM. Preferably the formulation comprises EDTA.

[0101] In an embodiment, the formulation comprises an emulsifier and / or non-ionic surfactant selected from octoxinol (e.g. Triton-X100), polysorbate (e.g. tween 60, tween 65, tween 80), glycerol, Sorbitan esters, polyethylene glycol (PEG; e.g. PEG400 or PEG600), or polyvinyl alcohol (PVA), or any combination thereof. Preferably, the formulation comprises PEG400 and PVA. In a particularly preferred embodiment, the formulation of the invention comprises PEG400 and PVA, in a concentration of 0.1-20% v / v for PEG400, or 0.1-20% w / v for PVA. In a particular preferred embodiment, the formulation comprises 1-15% v / v of PEG400, 0.1-5% w / v of PVA, or both.

[0102] The formulation further comprises a protease inhibitor selected from Phenylmethylsulfonyl fluoride (PMSF), 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), E-64 or Pepstatin. In a particularly preferred embodiment, the formulation of the invention comprises PMSF in a concentration from 0-10 mM preferably between 0 and 2 mM.

[0103] In an embodiment, the formulation comprises a preservative with biocidal activity selected from sodium azide, benzisothiazolinone (BIT), or bronopol. Preferably, the formulation comprises BIT or sodium azide. In a particularly preferred embodiment, the formulation of the invention comprises BIT in a concentration from 0.00001 -0. 1 % w / v, preferably between 0.0005 and 0.05% w / v.

[0104] In a particularly preferred embodiment, the preservative with biocidal activity maintains the pesticidal properties of the formulation between 12-28 months, preferably 12-24 months, more preferably for 24 months. The formulation of the present invention can comprise at least one further active ingredient, preferably with pesticidal activity. This may be selected from a pesticidal active ingredient compatible with the pesticidal protein of the formulation. For instance, the at least one additional pesticidal active ingredient may be a pyrethroid. Alternatively, it may be a pesticide belonging to the family of the neonicotinoids, or the organophosphates.

[0105] The formulation of the invention can comprise water. The water present in the formulation forms the aqueous phase of the formulation. Unless specified otherwise, the amount of water present in the formulation is the one required to reach a volume such that the concentration of the other ingredients of the formulation is as specified.

[0106] The formulation of the present invention may be subjected to a freeze-drying process, thereby dehydrating partially or completely said formulation, and producing a solid-state product. A freeze-dried formulation may be re-hydrated afterward for its application.

[0107] The formulation of the present application may be in the form of dusts, powders, granules, sprays, emulsions, pellets, colloids or solutions. Thus, the formulation of the present invention may further comprise excipients suitable for agricultural use that are compatible with these particular forms, including but not limited to, inert components, dispersants, surfactants, adjuvants, tackifiers, stickers, binders, or combinations thereof.

[0108] The inclusion of other excipients in the formulation of the present invention may also vary depending on the intended method of application. For example, a dispersing agent may be incorporated into the formulation when the form of application is by spraying. Likewise, the formulation can be formulated for a foliar or seed application.

[0109] In an embodiment, the formulation of the present invention can be applied to the surfaces of a plant or to a plant part, including seeds, leaves, flowers, stems, tubers, roots, and the like.

[0110] The production of the pesticidal protein of the formulation, in any of its embodiments can be carried out by following a procedure as described as follows. An exemplary method of production is further described in Example 2 below.

[0111] The production of the pesticidal protein of the formulation, in any of its embodiments, can be performed by recombinant technology in a non-pathogenic Escherichia coli bacterial strain, for example, strain BL21 (DE3), alternatively it can be produced in another organism such as yeast, Saccharomyces cerevisiae and Pichia pastoris.

[0112] The corresponding coding sequence of the pesticidal protein of the formulation, i.e., a polynucleotide comprising the sequence as set forth in SEQ ID NO: 4, in any of its embodiments, can be cloned into a vector or plasmid in which protein expression is inducible, for example, a bacterial expression vector or plasmid for a recombinant protein. Preferably a bacterial expression vector or plasmid selected from pET, pDEST, pRSET, pBAD, pGEX . Preferably, the vector or plasmid is a pET28 plasmid.

[0113] Induction may be carried out by IPTG, arabinose, lactose or methanol, in a concentration from 0.1-100 mM. The selected induction method depends on the chosen expression system, which can be determined by the person skilled in the art. In a preferred embodiment, the induction is performed by IPTG.

[0114] The vector can further comprise an epitope tag. Non-limiting examples of epitope tags are HIS, GST or MBP tags. These epitope tags may be included in either the N-terminal or C-terminal of an insecticidal protein of a formulation of the present invention.

[0115] Expression can be carried out in the laboratory in an Erlenmeyer flask, or in a 15-liter vessel or reactor, a pilot plant reactor, or a larger reactor supplemented with the appropriate culture medium, for example, a Luria-Bertani (LB) medium, and at a suitable temperature, for example, 37 °C.

[0116] The culture medium may be a rich medium, for example, Luria-Bertani (LB) medium, or any synthetic medium with the minimum necessary concentrations of essential compounds for bacterial growth. The exact formulation of said synthetic medium depends on the species and strain of bacteria grown. This knowledge falls within the expected technical knowledge of a person skilled in the art.

[0117] An antibiotic may be added to maintain a selection pressure. Examples of antibiotics include, but are not limited to kanamycin, ampicillin or chloramphenicol. The antibiotic may be at a concentration between 25-150 pg / mL.

[0118] After induction with IPTG, bacteria can be incubated for between 4-16 hours under constant agitation, in a temperature range varying between 18-37 °C. Afterwards, the crude extract obtained is subjected to a lysis process to release the cellular content, which can be a chemical or mechanical process, for example with glass beads, treatment with lysozyme, ultrasound, or by homogenization at high pressures. The remaining cellular and membrane components can be separated by cold centrifugation at 5000x g.

[0119] The homogenized bacterial extract may be included in a formulation of the first aspect of the present invention directly without any further separation. Optionally, a cell system can be used that allows the secretion of proteins directly into the culture medium, thus optimizing isolation and purification.

[0120] In a preferred embodiment, a formulation of the first aspect of the present invention comprises unpurified homogenized bacterial extract.

[0121] A second aspect of the present invention is a method for controlling an insect and / or nematode pest comprising applying a pesticide formulation of the present invention to a plant or plant part, preferably, applying an insecticide and / or nematicide formulation.

[0122] The term “controlling insect pests”, as used herein, means the action of inhibiting the ability of insect pests to survive, grow, feed, and / or reproduce, orthat limits insect-related damage or loss in plants or plant parts. The term “controlling insect pests” may or may not mean the action of killing insects, although it preferably means the action of killing insects. Said insects may be in any stage of their life cycle, for example neonate, larva, pupa (if corresponding) or adult.

[0123] The term “controlling nematode pests”, as used herein, means the action of inhibiting the ability of nematode pests to survive, grow, feed, and / or reproduce, or that limits nematode-related damage or loss in plants or plant parts. The term “controlling nematode pests” may or may not mean the action of killing nematodes, although it preferably means the action of killing nematodes. Said nematodes may be in any stage of their life cycle, for example neonate, larva, or adult.

[0124] In an embodiment, the method of the present invention comprises applying a formulation of the first aspect of the present invention to a plant or a plant part of agronomical value or interest, wherein the plant is selected from main row crops, fruits and vegetables, preferably selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp. In particular, the plant is selected from Solanum lycopersicum, Solanum tuberosum, Solarium melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Allium sativum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum. More preferably, the plant is selected from Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum.

[0125] In an embodiment, the method of the present invention comprises applying the formulation of the present invention to the surfaces of a plant or plant part, including seeds, leaves, flowers, stems, tubers, roots, or any combination thereof.

[0126] In an embodiment, the method of the present invention comprises applying a pesticide formulation of the present invention in a dose between 500-10000 ml / ha, preferably between 1000-5000 mL / ha, more preferably, a dose between 2000-5000 mL / ha, or2000, 2500, 3000, 3500, 4000, 4500, or 5000 mL / ha, more preferably in a dose of 5000 mL / ha.

[0127] In an embodiment, the method of the present invention further comprises contacting an insect or a nematode with a formulation of the present invention. Said contact may be by direct, for example, by directly applying the formulation of the present invention over an insect or nematode, or may be indirect, for example, by applying a formulation of the present invention over a plant or plant part, and the insect or the nematode contacting the formulation of the present invention after a certain amount of time.

[0128] Said contact or contacting comprises a physical interaction between an insect or a nematode and the formulation of the present invention. For example, contacting an insect or nematode or body part of said insect or nematode with a formulation of the present invention, wherein said body part may be external or internal. Thus, contact between the insect or nematode and the formulation of the present invention includes the ingestion of the formulation.

[0129] In an embodiment, the method for controlling pests of the present invention comprises controlling a pathogenic fungi, bacteria, nematode or insect, preferably, controlling an insect or nematode pest.

[0130] In an embodiment, the method of the present invention comprises applying a pesticide formulation of the present invention at least twice, preferably between 2 and 5 times. In an embodiment the method of the present invention comprises applying a pesticide formulation of the present invention at least when the pest is discovered and at least one additional time, after a certain amount of days, for example, after 7 or 14 days.

[0131] It should be understood that when the method for controlling pests comprises controlling an insect pest, said method in any of its embodiments comprises the use of an insecticidal formulation. Likewise, when the method for controlling pests comprises a nematode pest, said method in any of its embodiments comprises the use of a nematicidal formulation.

[0132] EXAMPLES

[0133] EXAMPLE 1 : Preferred embodiments of the formulation of the present invention

[0134] EXAMPLE 1.1 : Formulation A

[0135] Table 1 . Exemplary embodiment of the formulation of the present invention.

[0136] EXAMPLE 1.2: Formulation B

[0137] Table 2. Exemplary embodiment of the formulation of the present invention.

[0138] EXAMPLE 1.3: Formulation C

[0139] Table 3. Exemplary embodiment of the formulation of the present invention.

[0140] EXAMPLE 2: PESTICIDAL PROTEIN PRODUCTION PROTOCOL

[0141] For the production of pesticidal protein, an E. coli strain BL21 (DE3) was used, which allows high levels of recombinant protein production. This strain was transformed with a plasmid pET28 containing the coding sequence for an insecticidal and / or nematicidal protein of SEQ ID NO 3 (i.e., the polynucleotide sequence as set forth in SEQ ID NO: 6).

[0142] The clone to be expressed was inoculated in liquid LB medium supplemented with kanamycin (final concentration 50 pg / mL) and left to grow overnight at 37°C with constant agitation at 200 rpm. The next day, a 1 / 50 dilution of the saturated culture was prepared, also in LB kanamycin medium, and allowed to grow for approximately 2 hours until an optical density (OD) at 600 nm of 1 was reached. At this time, expression was induced with the addition of IPTG (final concentration 0.1 mM) and the same growth conditions were maintained for 4 additional hours. Afterwards, the OD was determined at 600 nm.

[0143] After expression, cell disruption was followed by the application of high pressures. A homogenizer was used for this purpose, at pressures of about 1000 bar. The process ended with the synthesis of the formulation, as indicated in any of the tables as shown in Example 1 , and the final volume was completed with purified sterile water. The effectiveness of the whole process was verified by SDS-PAGE.

[0144] EXAMPLE 3: PREPARATION OF THE FORMULATION OF THE PRESENT INVENTION

[0145] The formulation according to any one of the tables provided in Example 1 was made from the homogenized crude extract, starting with a bacterial culture with a final optical density of 1 . After cell disruption, first the buffer, the protease inhibitor and the necessary amount of water were added. Then, the volume was completed with the additives, and finally, the preservative was added to the final product.

[0146] EXAMPLE 4: INSECTICIDAL FORMULATION FIELD TRIALS

[0147] The formulation A was also tested as an insecticide in field trials. It showed surprisingly effective results in trials for the control of the pest Tuta absoluta, a Lepidopteran insect of the Gelechiidae family. Mainly Solanum lycopersicum (tomato) crops are susceptible to this pest, while as secondary hosts the inventors found potato, eggplant and other Solanaceae crops. T. absoluta causes crop losses for two reasons: yield reduction due to the destruction of leaves and flowers, and fruit damage that reduces their commercial value.

[0148] Treatments of the pest are usually late, so the pest causes serious crop damage. Biocontrol of tomato moth by the insecticidal formulation was tested by foliar application (Figure 1). The efficacy on tomato crops with doses up to 3000 mL / ha was comparable to that of both the chemical control (emamectin benzoate) and the biological control (Bacillus thuringiensis), while treatments with higher doses, such as 5000 mL / ha, showed a greater effect, progressively increasing with the number of applications over time. In some conditions, a 5% greater control was achieved than the chemical control used and a 150% greater effect than the biological control used.

[0149] Additionally, the formulation A showed a biocontrol capacity when challenging Trialeurodes vaporariorum, a whitefly that affects tomato, potato and other species (Figure 2). These insects produce chlorosis, deformation and dehydration of the leaves, causing the weakening of the plant. Fumagine then develops on the liquids excreted by the fly, causing the fruit to appear covered with spots. The treatment with extracts containing the insecticidal protein showed a similar effect to the biological control (Beauveria kopper) during the first applications and greatly exceeded it in the following applications, between 20% and 30% more. These observations were repeated in both adults and nymphs.

[0150] EXAMPLE 5: IN VITRO RESULTS FOR THE INSECTICIDAL PROTEIN

[0151] Also, in vitro laboratory assays were performed. The insecticidal protein (formulation A) showed efficacy in the control of lepidoptera, a diverse order of insects that are important pests of crops, such as soybeans and corn. Basically, two techniques were used:

[0152] 1) Incorporated diet. It consisted of incorporating the extract into the diet at a 1 :10 ratio (that is, it represents 10% of the diet).

[0153] 2) Tissue immersion. It consisted of immersing a disc of tissue in the protein solution.

[0154] In both cases, different concentrations are used in order to generate a dose-response curve.

[0155] The most important results were observed in Anticarsia gemmatalis and Spodoptera frugiperda, especially in the tests with the lyophilized samples that have a higher protein concentration. An increase in mortality was observed in them, consistent with the increase in doses, which at the highest concentrations was comparable with the commercial control, reaching 100%. In addition, there is a notable reduction in the larva size. The latter causes a lower defoliation capacity of the insect and triggers death without the larva reaching adulthood.

[0156] Table 2. Insecticidal effect of a particular embodiment of the formulation of the present invention on Anticarsia gemmatalis, induced by tissue submersion.

[0157] Table 3. Insecticidal effect of a particular embodiment of the formulation of the present invention on Anticarsia gemmatalis, induced by dietary incorporation

[0158] Table 4. Insecticidal effect of a particular embodiment of the formulation of the present invention on Spodoptera frugiperda , induced by tissue submersion.

[0159] Table 5. Insecticidal effect of a particular embodiment of the formulation of the present invention on Spodoptera frugiperda , induced by dietary incorporation.

[0160] EXAMPLE 6: IN VITRO RESULTS FOR THE NEMATICIDAL FORMULATED PROTEIN

[0161] Additionally, the nematicidal activity of the formulated protein (formulation A) was tested in vitro. The formulation demonstrated efficacy against the nematode Nacobbus aberrans in tomato plants.

[0162] N. aberrans caused crop damage including patches of plants with low growth, reduced apical growth, chlorotic and rolled leaves and small fruits. In the root, spherical galls with secondary roots are observed.

[0163] In this assay, tomato seedlings were infested with N. aberrans. 10 plants were used for each treatment. Each plant was inoculated with 1 mL of the treatment and with 1 mL of suspension of N. aberrans eggs and larvae (200 larvae or eggs). The inoculation of the treatments were carried out four times, every 7 days. The tomato plants were grown for 60 days. Subsequently, the roots were extracted, washed with sterile water and the nodules found were counted. The presence of mature females, larvae and eggs of A / , aberrans in the nodules was confirmed by microscope observation.

[0164] Table 6. Nematicidal effect of a particular embodiment of the formulation of the present invention on Nacobbus aberrans. Number of nodules / tomato plant evaluated at 60 days after transplant and control percentage (%). Different letters indicate significant differences between the means.

[0165] LIST OF SEQUENCES

Claims

CLAIMS1. A pesticide formulation comprising: i) a polypeptide comprising a sequence as set forth in SEQ ID NO: 1 or a polypeptide variant thereof having at least 90% of identity to the sequence as set forth in SEQ ID NO: 1 , ii) a pH buffer selected from HEPES, MOPS, PIPES, Tricine, Bicine, TES, Tris- HCI and Phosphate buffer, wherein the buffer has a pH value of between 7 to 9; iii) a chelating agent selected from citric acid, ethylenediaminetetracetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylenediaminetriacetic acid (HEEDTA), ethylenediaminedi-[o-hydroxyphenylacetic acid] (EDDHA), ethylene glycol-bis-(2-aminoethyl ether)- tetraacetic acid (EGTA), diethylenetriaminepentacetic acid (DTPA), and 1 ,2- diaminocyclohexanotetracetic acid (DCTA), iv) an emulsifier or a non-ionic surfactant selected from polysorbate, octoxynol, glycerol, Sorbitan esters, polyethylene glycol (PEG), or polyvinyl alcohol, and combinations thereof, v) a protease inhibitor selected from Phenylmethylsulfonyl fluoride (PMSF), 4-(2- aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), E-64 and Pepstatin; and vi) a preservative with biocidal activity selected from sodium azide, benzisothiazolinone (BIT), and bronopol.

2. The pesticide formulation according to claim 1 , wherein the polypeptide variant comprises: i) a deletion of at least one amino acid between residues 216 and 237 of the sequences as set forth in SEQ ID NO: 1 ; or ii) a substitution of at least one amino acid selected from the residues 35, 70, 77, 87, 100, 111 , 131 , 173, 180, 195, 264, 267, 275, or 308 of the sequence as set forth in SEQ ID NO: 1 ; or iii) a sequence as set forth in any one of SEQ ID NO: 7-21 .

3. The pesticide formulation according to claim 1 , wherein the polypeptide variant comprises the polypeptide as set forth in SEQ ID NO: 3 or a variant thereof comprising i) a deletion of at least one amino acid between residues 239 and 260; or ii) a substitution of at least one amino acid selected from the residues 58, 93, 100,110, 123, 134, 154, 196, 203, 218, 287, 290, 298, 331 , or iii) a sequence as set forth in any one of SEQ ID NO: 7-21 .

4. The pesticide formulation of any one of the preceding claims, wherein the polypeptide variant comprises a polypeptide having a sequence as set forth in any one of SEQ ID NO: 7-21.

5. The pesticide formulation according to any one of the preceding claims, further comprising a homogenized bacterial extract in an amount such that the optic density of the formulation is between 0.5-5 at 600 nm.

6. The pesticide formulation according to any one of the preceding claims, wherein the pH buffer is at a concentration of 0.1 to 50 mM and the pH of the formulation is between 7.2-8.2.

7. The pesticide formulation according to any one of the preceding claims, wherein the chelating agent is at a concentration of 0.05-5 mM.

8. The pesticide formulation according to any one of the preceding claims, wherein the emulsifier or the non-ionic surfactant is at a concentration of 0.01 to 20% v / v.

9. The pesticide formulation according to any one of the preceding claims, wherein the protease inhibitor is at a concentration of 0.1-2 mM.

10. The pesticide formulation according to any one of the preceding claims, wherein the preservative with biocidal activity is at a concentration of 0.00001 to 0.1% w / v.

11. The pesticide formulation according to claim 1 , comprising: i) a bacterial homogenate with an optic density of 1 , comprising a polypeptide having a sequence as set forth in SEQ ID NO: 3; ii) Tris-HCI at a concentration of 20 mM; iii) EDTA at a concentration of 0.2 mM, iv) PEG400 at a concentration of 5% v / v; v) PVA at a concentration of 0.1 w / v, vi) PMSF at a concentration of 0.5 mM; vii) BIT at a concentration of 0.001% w / v.

12. A method for controlling a pest comprising applying the formulation of any one of the preceding claims to a plant or a plant part.

13. The method of claim 12, wherein the pest is a pathogenic insect pest.

14. The method of claim 12, wherein the pest is a pathogenic nematode pest.

15. The method of any one of the preceding claims, wherein the plant or plant part is selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicerspp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., and Allium spp.

16. The method of any one of the preceding claims, wherein the method comprises applying the pesticide formulation of claim 1 in a dose between 500 and 10000 mL / ha.

17. The method of claim 13, wherein the insect pest is selected from the genera Spodoptera spp., Rachiplusia spp., Tuta spp., Trialeurodes spp., Anticarsia spp., Helicoperva spp., Dalbulus spp., Tetranychus spp., Bemisia spp., Aphis spp., Myzus spp., Rachiplusia spp., Alphitobus spp. Anthonomus spp., Thrips spp., or Frankliniella spp.

18. The method of claim 14, wherein the nematode pest is selected from the genera Meloidogyne spp, Nacobbus spp, Heterodera spp, Globodera spp, Pratyienchus spp, Paratylenchus spp, Rotylenchus spp, Xiphinema spp, and Trichodorus spp.

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