WO2025132389A1 - Formulation - Google Patents

Abstract

The present invention relates to a composition comprising an insecticidal protein and maltodextrin having a DE of 5 to 50, wherein the composition has a pH of from 5.0 to 10. The present invention further relates to a process for preparing a composition according to the present invention and a method of controlling or preventing infestation of a plant, a part or a locus thereof by an insect pest, comprising applying to the plant, a part or a locus thereof a composition according to the present invention.

Description

FORMULATION

The present invention relates to a composition comprising an insecticidal protein and maltodextrin, a process for preparing a composition according to the present invention and a method of controlling or preventing infestation of a plant, a part or a locus thereof by an insect pest comprising applying to the plant, the part or the locus thereof a composition according to the present invention.

BACKGROUND

Insect pests are mainly controlled by chemical pesticides. Biological pest control agents for controlling insect pests are receiving increasing attention as alternative chemical control agents.

An example of a biological insect control agent is Bacillus thuringiensis expressing insecticidal proteins like delta (6)- endotoxins (also called Cry proteins) and vegetative insect proteins called Vip, which are secreted into the growth medium during vegetative growth. There are three subfamilies of Vip proteins, Vip1 , Vip2, and Vip3. Vip3 is the most extensively studied protein which possesses insecticidal activity against a wide spectrum of lepidopteran pests, Syed et al, Toxins 2020, 12, 522.

WO2013/122720 discloses an engineered pesticidal polypeptide which has activity against Ostrinia nubilalis (European corn borer).

When insecticidal proteins are used on plants, they need to be formulated such that they remain stable long enough to be ingested by the insect. Chang, L. and Pickal, M.J. (2009) describe various mechanisms of protein stabilization in solid state. It is concluded that protein stabilization in solid state is still a controversial topic. There is no teaching or suggestion how to stabilize proteins such as Vip. US2013/0296165 discloses a dry stabilizing composition for a bioactive material comprising a carbohydrate component and a protein component comprising a hydrolyzed protein. Disclosed is a pesticide formulated with pectin, sucrose, calcium phosphate dibasic, calcium chloride and gluconolactone. Also disclosed in US2013/0296165 is a stable dry powder containing an enzyme based on a hydrogel. There is no teaching or suggestion in US2013/0296165 of a Vip protein that is stable for a period long enough to be ingested by an insect.

W02020182994 discloses a pesticidal composition comprising maltodextrin, which comprises at least 75% oligosaccharides having a chain length of 3 to 7 based on the overall oligosaccharide mixture having chains of length 3 to 20. The maltodextrin composition is active against mites.

W02010081815 discloses an aqueous gelatin-free, egg portion-free two-phase coacervate composition comprising a water soluble biologically active agent, a polysaccharide-free tension-active system and a water-soluble carrier comprising maltodextrin having a Mw below 1 ,800, erythritol, xylitol, sorbitol, mannitol, maltitol, isomalt, lactitol. There is no teaching or suggestion in W02010081815 how to stabilize a Vip protein.

Behle et al. (1997), J. of Economic Entomology, Vol. 90, No. 6, p. 1561-1566, discloses Bacillus thuringiensis formulations with flour/gluten (2% wt:vol). Behle et al. does not disclose or teach a composition of insecticidal proteins.

There is a need for a composition comprising an insecticidal protein that is stable and/or persistent on plants long enough to be ingested by an insect. SUMMARY

The present invention relates to a composition comprising an insecticidal protein and maltodextrin having a DE of 5 to 50 wherein the composition has a pH from 5.0 to 10.

Surprisingly, it was found that a composition comprising an insecticidal protein and maltodextrin according to the present invention was able to control an insect pest for at least three days, preferably at least four, five, six or at least seven days.

In a second aspect the present invention relates to a process for preparing a composition according to the present invention, comprising a. cultivating microbial cells expressing an insecticidal protein; b. adding a salt solution having a pH of from 5.0 to 10 to the microbial cells comprising the insecticidal protein; c. optionally, disrupting the microbial cells; and d. adding maltodextrin having a DE of 5 to 50.

In a third aspect the present invention relates to a method for controlling or preventing infestation of a plant, a part or a locus thereof by an insect pest, comprising applying to the plant, a part or a locus thereof a composition according to the present invention.

In a fourth aspect, the present invention relates to a plant, a part or a locus thereof comprising a composition according to the present invention.

In a fifth aspect the present invention relates to the use of maltodextrin having a DE of 5 to 50 to stabilize an insecticidal protein for at least three days.

DETAILED DESCRIPTION

The present invention relates to a composition comprising an insecticidal protein and maltodextrin having a dextrose equivalent (DE) of 5 to 50 wherein the composition has a pH of from 5 to 10. Surprisingly, it was found that a composition comprising an insecticidal protein of the present invention was able to control an insect pest, such as a Spodoptera insect pest, for instance Spodoptera littoralis (Egyptian cotton leafworm), for at least three days after the composition was applied on a plant. Accordingly, it was surprisingly found that the composition was persistent on a plant, for at least three days preferably at least four, five, six, or at least seven days after application of the composition to the plant.

As used herein a composition comprising an insecticidal protein that is persistent on a plant is a composition comprising an insecticidal protein that is stable on a plant. An insecticidal protein that is persistent for at least three days is used to indicate that the insecticidal protein shows activity against an insect pest on a plant for at least three days.

In one embodiment, the insecticidal protein is a protein that is soluble in an aqueous solution at a pH of 5 to 10, preferably at a pH of 6 to 9. An aqueous solution comprises water. A soluble protein as used herein means that the protein dissolves in an aqueous solution by forming a homogenous mixture at the molecular level. Soluble proteins comprise hydrophilic residues, both charged and non-charged, which can form dipole-dipole interactions or hydrogen bonding interactions with the surrounding aqueous solution.

A protein that is “insecticidal” or an insecticidal protein as used herein means a protein that is toxic to an insect or insect pest. The insecticidal protein may be any insecticidal protein, for instance a wildtype or naturally occurring insecticidal protein or a variant or engineered insecticidal protein. An insecticidal protein may be a vegetative insect protein (Vip), a Cry protein, and I or or Txp40 protein.

As used herein, the term “Cry protein” means an insecticidal protein of a Bacillus thuringiensis crystal delta-endotoxin type. The term “Cry protein” can refer to the protoxin form or any bio-active fragment or toxin thereof including partially processed and the mature toxin form, e.g., without the N- terminal peptidyl fragment and/or the C-terminal protoxin tail.

As used herein “Txp40 protein” is a toxin produced by two genera of bacteria, Xenorhabdus and Photorhabdus, which symbiotically associate with the nematode genera, Steinernema and Heterrhabditis, respectively. The nematode-bacteria pair are capable of invading and killing certain insects. A Txp40 protein was first isolated from P. luminescens and shown to have injectable toxicity to several insect pests. A Txp40 protein is for instance disclosed in WO2020/247465.

Preferably, the insecticidal protein as disclosed herein is a vegetative insecticidal protein, wherein the vegetative insecticidal protein comprises Vip1 , Vip2, Vip3 and I or Vip4, preferably Vip3A and I or Vip3D. A vegetative insecticidal protein preferably comprises a Vip3A protein which comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence which has at least 80%, preferably at least 85%, 90%, 95%, preferably at least 96%, 97%, 98%, or at least 99% identity to an amino acid sequence according to SEQ ID NO: 1. A vegetative insecticidal protein may also comprise a Vip3D protein comprising an amino acid sequence according to SEQ ID NO: 2, or a protein which comprises an amino acid sequence which has at least 80%, preferably at least 85%, 90%, 95%, preferably at least 96%, 97%, 98%, or at least 99% identity to an amino acid sequence according to SEQ ID NO: 2. A vegetative insecticidal protein may also comprise a variant of a Vip3D protein comprising an amino acid sequence according to SEQ ID NO: 3, or a protein which comprises an amino acid sequence which has at least 80%, preferably at least 85%, 90%, 95%, preferably at least 96%, 97%, 98%, or at least 99% identity to an amino acid sequence according to SEQ ID NO: 3. The amino acid sequence according to SEQ ID NO: 3 corresponds to amino acid sequence according to SEQ ID NO: 2, wherein the amino acid sequence comprises the amino acid substitution K455A as compared to SEQ ID NO: 2. Engineered vegetative insecticidal proteins are disclosed in for instance WO2013/12272. The amino acid sequence SEQ ID NO: 3 in WO2013/12272, which is annotated at Vip3E in WO2013/12272 corresponds to SEQ ID NO: 3 as disclosed herein.

As used herein, the terms "percent identity," and "percent identical" refer to the relatedness of two or more nucleotide or amino acid sequences, which may be calculated by (i) comparing two optimally aligned sequences over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100 percent to yield the percent identity. If the "percent identity" is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present invention, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the "percent identity" for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100 percent.

In one embodiment, the composition according to the present invention comprises maltodextrin having a dextrose equivalent (DE) of 5 to 50, preferably a DE of 6 to 40, preferably a DE of 7 to 35, preferably a DE of 8 to 32, preferably a DE of 9 to 30. Surprisingly, it was found that maltodextrin having a DE of 5 to 50 stabilizes the insecticidal protein on a plant, resulting in an active protein against insects for at least three days. It was surprisingly found that maltodextrin as disclosed herein stabilizes the insecticidal protein during storage and improved persistence on the plant surface, such as on a plant leaf.

We found that sugars like soluble potato starch, methyl cellulose and disaccharides did not result in a stable insecticidal protein on a plant for at least three days. It was found that glucose as monosaccharide also increased the persistence of the insecticidal protein on plants. However, a disadvantage of glucose as stabilizing agent is that glucose is a carbon substrate that promotes microbial, such as bacterial or fungal, growth and therefore not a suitable stabilizing agent.

The composition according to the present invention has a pH of 5 to 10, preferably a pH 5.5 to 9.5, preferably a pH of 6 to 9, a pH of 6.5 to 8.8, preferably a pH of 6.8 to 8.5, preferably a pH of from 7 to 8.3, preferably a pH of from 7.2 to 8. It was found that the pH of the composition influences the activity of the insecticidal protein. It was found that a vegetative insecticidal protein such as Vip3A or Vip3D as disclosed herein was active at a pH of from 6 to 10 preferably a pH from 6.5 to 9.5, preferably a pH from 7 to 9, preferably a pH of from 7.2 to 8.8, preferably a pH of from 7.5 to 8.5. The vegetative insecticidal protein is soluble at a pH of between 5 and 10.

In one embodiment, the composition according to the present invention comprises an insecticidal protein and maltodextrin having a DE of 5 to 50, at a ratio of insecticidal protein to maltodextrin having a DE of 5 to 50 of 1 :200 to 1 :1 wt/wt, preferably a ratio of 1 :150 to 1 :2 wt/wt, preferably a ratio of 1 :100 to 1 :5 wt/wt, preferably a ratio of 1 :80 to 1 :8 wt/wt, preferably a ratio of 1 :70:1 :10 wt/wt, preferably a ratio of 1 :60 to 1 :15 wt/wt, preferably a ratio of 1 :50 to 1 :20 wt/wt, preferably a ratio of 1 :40 to 1 :25 wt/wt.

In one further embodiment, the composition as disclosed herein comprises an agricultural acceptable carrier. As used herein an “agricultural acceptable carrier” can include natural or synthetic, organic or inorganic material which is combined with the active protein to facilitate its application to or on the plant, or a part or locus thereof. Examples of agricultural acceptable carriers include, without limitation, powders, dusts, pellets, granules, sprays, emulsions, colloids, and solutions. Agricultural acceptable carriers further include, but are not limited to, inert components, dispersants, surfactants, adjuvants, tackifiers, stickers, binders, or combinations thereof, that can be used in agricultural formulations. A suitable surfactant may for instance comprise non-ionic surfactant such as butyl-based block polyalkylene oxide copolymer Toximul®8320 or a polysorbate 20, such asTween® 20, or an alcohol ethoxylate such as Synperonic® 10/6. Such compositions can be applied in any manner that bring pesticidal proteins or other pest control agents in contact with the insect pests. Accordingly, the compositions can be applied to the surfaces of plants or plant parts, including seeds, leaves, flowers, stems, tubers, roots, and the like.

In one embodiment, the composition according to the present invention comprises a salt. Any suitable salt can be used, preferably a buffer salt. Preferably the buffer salt results in a composition having a pH of 5 to 10. Preferably the salt comprises a phosphate salt, for instance a phosphate salt plus sodium chloride, ammonium bicarbonate, or N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), or tris(hydroxymethyl)aminomethane (TRIS).

A composition as disclosed herein may be used as such, as a tank mix or it may be conveniently formulated in a known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions or suspensions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations e.g. in polymeric substances. As with the type of the compositions, the methods of application, such as spraying, atomising, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances. The compositions may also contain further adjuvants such as stabilizers, antifoams, viscosity regulators, binders or tackifiers as well as fertilizers, micronutrient donors or other formulations for obtaining special effects.

In one embodiment the composition according to the present invention may be a soluble powder or a tank mix, known to a person skilled in the art. A soluble powder is a formulation of an insecticide where an active ingredient, such as an insecticidal protein as disclosed herein is in powder form (much like wettable powders) and is able to blend into a complete solution. A soluble powder formulation is also known as water-soluble powder or water-soluble packets (WSP). A tank mix as used herein is a composition that is obtained by mixing all constituents making-up the tank mix into a single tank to make a single spray application. A tank mix may also be called soluble concentrate.

In a further embodiment, the insecticidal composition comprises a microbial cell, preferably wherein microbial cell produces an insecticidal protein in the composition as disclosed herein. Preferably, the microbial cell has been inactivated or disrupted. Preferably, the microbial cell is a non-living or inactivated or disrupted microbial cell. Such an insecticidal composition can be prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture comprising a microorganism expressing the insecticidal protein as disclosed herein. A composition as disclosed herein may comprise at least about 1 weight percentage (wt %), preferably at least 5, at least 10, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least about 60, at least 70, at least 80, at least 90, at least 95, at least 97, or at least 99 by weight percentage (wt %) of a protein of the invention. In additional embodiments, the composition comprises from about 1 to about 99 by weight of the insecticidal protein of the invention.

In one aspect the present invention relates to a process for preparing a composition according to the present invention, comprising a. cultivating microbial cells expressing an insecticidal protein; b. adding a salt solution having a pH of from 5.0 to 10 to the microbial cells comprising the insecticidal protein; c. optionally disrupting the microbial cells; and d. adding maltodextrin having a DE of 5 to 50.

The wording microbial cell or microorganism are used interchangeably herein.

A microbial cell or microorganism expressing an insecticidal protein may be a naturally occurring microorganism or genetically modified or recombinant microorganism. The microorganism may be a Bacillus sp. for instance Bacillus thuringiensis, Escherichia coll, or a yeast such as Pichia pastoris or a filamentous fungus such Aspergillus sp. such as Aspergillus niger or Aspergillus oryzae. Preferably, the microorganism is a microorganism expressing a gene encoding a Vip protein, preferably the microorganism is an E. coll genetically modified with a nucleic acid sequence encoding a Vip protein, wherein the Vip protein is Vip1 , Vip2, Vip3 and I or Vip4, preferably Vip3A and I or Vip3D. The Vip protein may be a naturally occurring or wild type protein or a variant or engineered protein. Preferably, the microorganism expresses at least a Vip3A protein which comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence which has at least 80%, preferably at least 85%, 90%, 95%, preferably at least 96%, 97%, 98%, or at least 99% identity to an amino acid sequence according to SEQ ID NO: 1 . The microorganism may also express a Vip3D protein which comprises an amino acid sequence according to SEQ ID NO: 2, or a protein which comprises an amino acid sequence which has at least 80%, preferably at least 85%, 90%, 95%, preferably at least 96%, 97%, 98%, or at least 99% identity to an amino acid sequence according to SEQ ID NO: 2. The microorganism may also express a variant of a Vip3D protein comprising an amino acid sequence according to SEQ ID NO: 3, or a protein which comprises an amino acid sequence which has at least 80%, preferably at least 85%, 90%, 95%, preferably at least 96%, 97%, 98%, or at least 99% identity to an amino acid sequence according to SEQ ID NO: 3. The amino acid sequence according to SEQ ID NO: 3 corresponds to amino acid sequence according to SEQ ID NO: 2, wherein the amino acid sequence comprises the amino acid substitution K455A as compared to SEQ ID NO: 2.

As used herein, the term "recombinant" refers to a form of nucleic acid (e.g., DNA or RNA) or protein or an organism that would not normally be found in nature and as such was created by human intervention. As used herein, a "recombinant microorganism" is a microorganism that would not normally exist in nature, is the result of human intervention, and contains a transgene or heterologous nucleic acid molecule incorporated into its genome.

"Transformed I transgenic I recombinant" refers to a host organism such as a microorganism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of a host or the nucleic acid molecule can also be present as an extrachromosomal molecule. A "non-transformed", "non-transgenic", or "non- recombinant" host refers to a wild-type organism, e.g., a microorganism, which does not contain the heterologous nucleic acid molecule.

Cultivating a microbial cell may be performed by known methods in the art. Usually cultivating a microbial cell is performed in batch culture, a fed-batch culture or a continuous culture. Cultivating a microbial cell, comprises cultivating the microbial cell in a fermentation medium which comprises a suitable carbon and nitrogen source. A suitable carbon source may be molasses, such as beet or cane molasses, polysaccharides, flour, starch, sugar, or glucose. A suitable nitrogen source may be casein hydrolysate, tryptone, ammonium sulphate, ammonia, yeast extract, peptone or urea. Cultivating the microorganism may be performed under aerobic and I or anaerobic conditions.

Cultivating microbial cells expressing an insecticidal protein typically comprises producing a fermentation broth comprising microbial cells comprising the insecticidal protein.

Preferably, the process according to the present invention comprises a step of harvesting the microbial cells comprising the insecticidal protein. Harvesting the microbial cells, preferably microbial cells comprising the insecticidal proteins, may be performed by known methods in the art such a centrifugation or filtration. The insecticidal protein may be intracellular and/or extracellular of the microbial cells. In one embodiment the process according to the present invention comprises harvesting microbial cells, preferably microbial cells and the insecticidal protein, from the fermentation broth prior to adding a salt solution to the microbial cells.

The process according to the present invention further comprises adding a salt solution having a pH of 5.0 to 10 to the microbial cells comprising the insecticidal protein. Preferably the salt solution is a salt buffer solution having a pH 5.5 to 9.5, preferably a pH of 6 to 9, a pH of 6.5 to 8.5, preferably a pH of 7 to 8. Preferably, the salt solution is a solution of a phosphate salt, for instance a phosphate salt plus sodium chloride, ammonium carbonate, or N-cyclohexyl-3-aminopropanesulfonic acid (CAPS). Preferably, the salt solution is added at a concentration from 1 to 100 mM, for instance from 5 to 80 mM, for instance from 10 to 60 mM.

The process according to the present invention preferably comprises disrupting the microbial cells. Disrupting microbial cells or lysing microbial cells may be performed by any suitable known technology, for instance mechanical disruption, for instance using microfluidics using a French press, or a hydraulic pump, or sonification using ball mills. Disrupting the microbial cells may further comprise a step of removing the disrupted cells, such as cell debris or cell material, for instance by centrifugation or filtration, to obtain a clarified solution comprising the insecticidal protein.

The process according to the present invention further comprises adding maltodextrin having a DE of 5 to 50. Preferably the maltodextrin has a DE of 6 to 40, preferably a DE of 7 to 35, preferably a DE of 8 to 32 preferably a DE of 9 to 30. The maltodrextrin that is added in a process as disclosed herein may an aqueous solution comprising maltodextrin or a powder of maltodextrin.

In one embodiment, the process according to the present invention further comprises a step of removing water after step a), b), c) and I or d).

Water can be removed during or after step a), b), c) and I or d) using technologies known in the art. Known technologies of removing water are for instance lyophilization, spray drying, spray agglomeration or spray freeze drying.

The process according to the present invention may further comprise adding an agricultural acceptable carrier. Any suitable agricultural acceptable carrier may be used and may be a carrier as defined herein above.

The composition according to the present invention is obtainable by a process according to the present invention.

In a third aspect the present invention relates to a method for controlling or preventing infestation of a plant, a part or a locus thereof by an insect pest, comprising applying to the plant, a part or a locus thereof a composition according to the present invention. Also disclosed herein is a method for controlling an insect pest, comprising contacting the insect pest with a composition according to the present invention.

The method according to the present invention does not include a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practiced on the human or animal body.

The method for controlling or preventing infestation of an insect pest as disclosed herein, comprises controlling or preventing the insect pest on a plant or a part or a locus thereof, which comprises applying to the plant, the part or the locus thereof a composition according to the present invention.

Applying a composition according to the present invention to a plant, a part or a locus thereof preferably comprises contacting the insect pest with the composition according to the present invention.

Contacting an insect pest with a composition as disclosed herein involves delivering the composition to the insect. To "deliver" the composition comprising an insecticidal protein means that the insecticidal protein comes into contact with an insect, resulting in a toxic effect and control of the insect. An insect pest as used herein includes larvae of the insect and adults of the insect, preferably the insect pest comprises larvae.

The composition comprising the insecticidal protein provided control of or prevented investation of an insect pest for at least three, preferably at least four, five, six or at least seven days. Surprisingly it was found that the composition comprising the insecticidal protein of the present invention is persistent on the plant, the part or the locus thereof, for at least three, preferably at least four, five, six or at least seven days, after applying the composition to the plant, the part or the locus thereof.

The composition comprising the insecticidal protein is applied at a suitable amount. A suitable amount is from 2 to 200 g protein per hectare (ha), such as from 5 to 150 g protein per ha, or from 10 to 100 g per ha, or from 15 to 75 g protein per ha.

Applying the insecticidal protein to the plant, a part or a locus thereof may be performed by any suitable methods in the art. Suitable methods of application to a plant surface, or contacting a plant or a part, or a locus thereof include spraying, atomising, dusting, scattering, misting, sprinkling, coating, dipping or pouring, in accordance with the intended objectives and the prevailing circumstances. The compositions may also contain further adjuvants such as stabilizers, antifoams, viscosity regulators, binders or tackifiers as well as fertilizers, micronutrient donors or other formulations for obtaining special effects.

In one embodiment, the method according to the present invention comprises controlling an insect pest, wherein the insect pest is a Coleopteran, Hemipteran or Lepidopteran insect pest. Preferably the insect pest is a Lepidopteran insect pest, such as Ostrinia nubilalis (European corn borer), Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Spodoptera littoralis (Egyptian cotton leafworm), Spodoptera eridania (Southern armyworm moth), Spodoptera exigua (beet armyworm), Spodoptera litura (tobacco cutworm), Agrotis ipsilon (black cutworm), Agrotis orthogonia (pale western cutworm), Striacosta albicosta (western bean cutworm), Helicoverpa zea (corn earworm), Helicoverpa armigera (cotton bollworm), Heliothis virescens (tobacco budworm), Helicoverpa punctigera (native budworm), , Manduca sexta (tobacco hornworm), Trichoplusia ni (cabbage looper), Mamestra brassicae (cabbage moth), Tuta absoluta (tomato leafminer), Lobesia botrana (European grapevine moth), Grapholita molesta (Oriental fruit moth), Eupoecilia ambiguella (vine moth), Cydia pomonella (codling moth), Chrysodeixis includens ( soybean looper moth), Chilo suppressalis (striped rice stem borer) Pectinophora gossypiella (pink bollworm), Diatraea grandiosella (southwestern corn borer), Diatraea saccharalis (sugarcane borer), Elasmopalpus lignosellus (lesser cornstalk borer), , Anticarsia gemmatalis (velvetbean caterpillar), Plathypena scabra (green cloverworm), and Cochylis hospes (banded sunflower moth).

Preferably the insect pest is a Ostrinia nubilalis (European corn borer), Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Spodoptera littoralis (Egyptian cotton leafworm), Spodoptera eridania (Southern armyworm moth), Spodoptera exigua (beet armyworm), Spodoptera litura (tobacco cutworm), Agrotis ipsilon (black cutworm), Helicoverpa zea (corn earworm), Helicoverpa armigera (cotton bollworm), Heliothis virescens (tobacco budworm), Trichoplusia ni (cabbage looper), Mamestra brassicae (cabbage moth), Tuta absoluta (tomato leafminer), Lobesia botrana (European grapevine moth), Grapholita molesta (Oriental fruit moth), Eupoecilia ambiguella (vine moth), Cydia pomonella (codling moth), Chrysodeixis includens ( soybean looper moth), Chilo suppressalis (striped rice stem borer)

Preferably the insect pest is a Spodoptera insect pest, preferably Spodoptera littoralis (Egyptian cotton leafworm).

The term “plant” refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits.

The term plants involve “useful plants” or “crops”. The wording “Useful plants” and “crops” are used interchangeably herein.

The plant includes, but is not limited to, corn (maize), soybean, rice, wheat, barley, rye, oat, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanut, vegetable (including, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melon, pepper, celery, squash, pumpkin, zucchini, and the like), fruit (including, apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, and the like), a specialty plant (such as Arabidopsis), or a woody plant (such as coniferous and/or deciduous trees). In embodiments, a plant in a method of the invention is a crop plant such as maize, sorghum, wheat, sunflower, tomato, a crucifer, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, oilseed rape plant and the like. Preferably the plant comprises soybean and I or cotton.

The term "useful plants" is to be understood as also including useful plants that have been rendered tolerant to herbicides like bromoxynil or classes of herbicides (such as, for example, HPPD inhibitors, ALS inhibitors, for example primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol- pyrovyl-shikimate-3-phosphate-synthase) inhibitors, GS (glutamine synthetase) inhibitors or PPO (protoporphyrinogen-oxidase) inhibitors) as a result of conventional methods of breeding or genetic engineering.

The term “part” of a plant comprises seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, leaves and fruits. The term “locus” as used herein means fields in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed into the soil.

In a fourth aspect the present invention relates to a plant, a part or a locus thereof comprising a composition according to the present invention. Surprisingly, it was found that a plant, a part or a locus thereof comprising a composition according to the present invention suffered less, or not, from insect pests for a least three days, preferably at least four, five, six, or at least seven days, as compared to a plant, a part or locus thereof, which did not comprise a composition according to the present invention.

In one further aspect the present invention relates to the use of maltodextrin having a DE of 5 to 50 to stabilize an insecticidal protein for at least three days, preferably at least four, five, six or at least seven days. Preferably the use comprises stabilizing the insect protein on a plant, a part or a locus thereof. The use further comprises a salt solution at pH 5.0 to 10. All embodiments described above are applicable to the use according to the present invention.

EXAMPLES

Methods

Fermentative production of Vip3A (SEQ ID NO: 1) and variant Vip3D (SEQ ID NO: 3) having a substitution at position K455A as compared to SEQ ID NO: 2.

To produce Vip3A (SEQ ID NO: 1) and the Vip3D variant protein (SEQ ID NO: 3), recombinant Escherichia coli strains were generated to enable expression of the Vip3 proteins, in line with the methods disclosed in Estruch et al¹ and Khan et. al². Nucleic acid sequences for the Vip3 proteins were codon optimised for expression in E. coli and were cloned into a standard protein expression vector. The vectors containing the Vip3 encoding sequences were introduced into an E. coli protein expression host via chemical transformation, to provide recombinant strains for Vip3 protein production. The recombinant E.coli strains were cultivated by batch fermentation in a complex liquid medium as disclosed in Estruch et aP and Khan et. at² using a stirred tank bioreactor. Expression of Vip3 protein was induced by the addition of IPTG and incubation was continued for a further period followed by the harvesting of biomass by centrifugation.

References:

1) Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5389-94.

2) M. H. Khan, G. Jander, Z. Mukhtar, M. Arshad, M. Sarwar, S. Asad, Comparison of in Vitro and in Planta Toxicity of Vip3A for Lepidopteran Herbivores, Journal of Economic Entomology, Volume 113, Issue 6, December 2020, Pages 2959-2971

Recovery of Vip3 protein

Cell mass was collected from the fermentation broth using a disk stack centrifuge. The cell mass was resuspended in an aqueous sodium phosphate or Tris buffer at variable strength and a 137mM sodium chloride or Tris-buffer unless indicated otherwise. The cells were disrupted using a microfluidics device. Cell debris was removed by centrifugation followed by chemical inactivation of the cells using a biocide (benzisothiazolinone, BIT).

The clarified material contained soluble protein at a concentration of 2 to 10 mg / ml, measured by SDS- PAGE and densitometry against a purified standard.

Preparation of Vip3/sugar Soluble concentrate (SL) or tank mix

Vip3 clarified lysate either as a solution or lyophilized powder, was directly added in a 10, 20 or 30% w/w sugar (see table 1 below) stock solution at a weight ratio Vip3 to sugar of 1 :2, 1 :4, 1 :10, 1 :20, 1 :30, 1 :50 or 1 :100.

Spray tank preparation: the above solution of solube concentrate (SL) was diluted with water to achieve the correct application rate (g Al/ha), to make up 90% of the spray volume, and lastly the adjuvants (retention aids, wetting agents, rain fastness agents, photoprotectants) were added as a tank mix to achieve the right concentration with the remaining 10% of the spray volume.

Table 1. Sugars used to stabilize Vip proteins on the plant

1- Preparation of Vip3/sugar Soluble Powder (SP) formulations

Sugar (See Table 1) was directly dissolved into Vip3 clarified solution at a weight ratio Vip3 to sugar of 1 :10, 1 :30, 1 :50 or 1 :100, and water was removed via lyophilization, thereby preparing a soluble powder formulation.

Spray tank preparation: the above soluble powder (SP) was added and dissolved in water to achieve the correct application rate (g Al/ha) to make up 90% of the spray volume, and lastly the adjuvants (eg. retention aids, wetting agents, rain fastness agents, or photoprotectants) were added as a tank mix where needed to achieve the right concentration with the remaining 10% of the spray volume.

Persistence activity assay:

Soybean (Glycine max) or cotton plants (Gossypium hirsuturri) were sprayed with diluted test solutions in an application chamber. The plants were incubated in the greenhouse under controlled conditions (temperature at 22°C during day and 20°C during night and relative humidity of 65%).

The leaves were then cut and placed in petri dishes with wetted filter paper at different instances.

The petri dishes were infested (few hours after the application or 1 , 3, 4 or 5 days after the application day (DAA), with the Egyptian cotton leafworm, Spodoptera littoralis (8-10 L2 larvae per dish) and covered with a fabric filter and plastic lids. The larvae were assessed 5 days after infestation for mortality in each of the infestation instances.

All the assays were conducted in the laboratory under controlled environmental conditions (temperature 25°C and relative humidity 65% with 16 hours of light and 8 hours of dark conditions).

Abbott's formula (% test mortality - % control mortality/ 100 - control mortality x 100) was used to correct the larval mortality for the corresponding control response in the activity persistence assays (W. S. Abbott, A Method of Computing the Effectiveness of an Insecticide, Journal of Economic Entomology, Volume 18, Issue 2, 1 April 1925, Pages 265-267).

Example 1. Effect of maltodextrin and disaccharides on the stability of Vip3A

Soluble concentrate (SL) type formulations were prepared by adding a solution of Vip3 clarified lysate into a 10% maltodextrin or disaccharide solutions. A 1xPBS buffer solution (10mM sodium phosphate and 137mM sodium chloride) at pH 7.4 was used during the cell resuspension. Soybean plants were treated as described above with a solution of Vip3A and maltodextrin at 75 ppm (Table 2) and a solution of Vip3A and different disaccharides at 37.5 ppm (Table 3).

Cotton plants were treated with 200 ppm of Vip3A and polysaccharides (maltodextrin) and disaccharides (maltose and lactose) (Table 4). Karate (lambda cyhalothrin) was taken as a positive control.

The results in Table 2 show that the presence of maltodextrin having a DE of 5 to 50 protected the Vip3A protein and provided control against the Spodopetra litoralis L2 larvae up to three days after application on soybean plants. The value of Dextrose Equivalent of the maltodextrin types used in the experiment had a minor influence on the persistence of the Vip3A protein on soybean leaves (Table 2).

The results in Table 3 show that the presence of disaccharides protected the Vip3A protein and provided control of Spodopetra litoralis L2 larvae on soybean leaves for only 1 day. Control against S. litoralis was very weak when larvae were infested on leaves 3 days after the application of a composition of disaccharides and Vip3A. The results in Table 4 show that the addition of maltodextrin to Vip3A resulted in a longer persistence of Vip3A on cotton leaves (up to 4 DAA) as compared to maltose and lactose.

Table 2. Mortality of Spodopetra littoralis L2 larvae, exposed at 0, 1 and 3 DAA, assessed 5 days after infestation on Vip3A-treated soybean leaves with maltodextrins.

Table 3. Mortality of Spodopetra littoralis L2 larvae, exposed at 0, 1 and 3 DAA, assessed 5 days after infestation on Vip3A-treated soybean leaves with four disaccharides.

Table 4. Mortality of Spodopetra littoralis L2 larvae, infested at 0, 3 and 4 DAA, assessed 5 days after infestation on Vip3A-treated cotton leaves with the addition of maltodextrins and disaccharides at two ratios.

Example 2. Effect of maltodextrin on the stability of Vip3A and a variant Vip3D

Soluble concentrate (SL) type formulations, prepared by adding a solution of clarified Vip3 lysate into a 10% maltodextrin solution, were used in this example. A 1xPBS buffer solution (10mM sodium phosphate and 137mM sodium chloride) at pH 7.4 was used during the cell resuspension. Soybean plants were treated as described above with solutions of Vip3A or Vip3D containing maltodextrin Glucidex 29 at 75 ppm (Table 5). Karate (lambda cyhalothrin) was taken as a positive control.

The results in Table 5 show that Glucidex 29, at a 1 :100 weight ratio Vip3 to sugar, stabilized both Vip3A and a variant of Vip3D on plants and provided control against S. littoralis larvae up to 7 days after application on soybean plants.

Table 5. Mortality of Spodopetra littoralis L2 larvae, exposed at 0, 3, 5 and 7 DAA, assessed 5 days after infestation on Vip3A- and Vip3D variant K455A (SEQ ID NO: 3)-treated soybean leaves, with or without the addition of Glucidex 29

Example 3. Effect of different sugars on the stability of Vip3A

Soluble concentrate (SL) type formulations, prepared by adding a solution of Vip3 clarified lysate into a 10% sugar solution (potato starch, methyl cellulose, glucose and Glucidex 29), were used in this example. A 1xPBS buffer solution (10mM sodium phosphate and 137mM sodium chloride) of pH 7.4 was used during the cell resuspension.

Soybean plants were treated as described above with a solution of Vip3A and in the presence of various types of sugars (see Table 1 and Table 6) at 37.5 and 9.375 ppm (Table 6). Karate (lambda cyhalothrin) was used as a positive control. The results in Table 6 show that amongst all the sugars tested, maltodextrin Glucidex 29 D stabilized Vip3 both applied at 37.5 and 9.375 ppm, providing persistent control against S. littoralis up to 5 days after the application on soybean plants. Potato starch and methyl cellulose did not provide good persistent control against S. littoralis larvae at a concentration of 37.5 and 9.375 ppm and glucose was less effective against S. littoralis larvae at 9.375 ppm as compared to maltodextrin Glucidex 29.

Potato starch, methyl cellulose and glucose, applied as a standalone at 0.375% on soybean plants, did not show activity against S. littoralis larvae. Table 6. Mortality of Spodopetra littoralis L2 larvae, exposed at 0, 3 and 5 DAA, assessed 5 days after infestation on Vip3A-treated soybean leaves, with or without the addition of Glucidex 29 and other sugars.

* All sugars were added at a weight ratio of 1:100 (Al: sugar). Example 4. Effect of salt/ buffer solution on protein activity

Different buffer types and protein (Al) to sugar ratios were tested in this example.

As described above, the biomass collected from the fermenter was re-suspended in 50mM sodium phosphate or 50mM Tris-buffer solutions at pH 8 and 137mM sodium chloride. The cells were lysed, and the cell debris removed via centrifugation.

Soluble powder type formulations were prepared by mixing the Vip3 clarified lysate in a 20% Glucidex 29 solution followed by lyophilization to remove the water.

Soybean plants were treated by dissolving the above powder, at a 75 ppm Vip3A dose rate (Table 7 and 8). Karate (lambda cyhalothrin) was used as a positive control.

The results in Tables 7 and 8 show that a solution of sodium phosphate resulted in increased stability of Vip3A in comparison with Tris-buffer. Incorporating Glucidex 29 with Vip3A in a sodium phosphate buffer provided control against S. littoralis larvae up to 5 DAA (table 7).

Incorporating Glucidex 29 with Vip3A in Tris buffer provided control against S. littoralis larvae up to 3 DAA. The protein activity was significantly reduced when larvae were infested 5 DAA on soybean leaves (Table 8).

Table 7. Mortality of Spodopetra littoralis L2 larvae, infested at 0, 3 and 5 DAA, assessed 5 days after infestation on Vip3A-treated soybean leaves, with incorporated Glucidex 29 at different ratios in a sodium phosphate buffer.

Table 8. Mortality of Spodopetra littoralis L2 larvae, infested at 0, 3 and 5 DAA, assessed 5 days after infestation on Vip3A-treated soybean leaves, with incorporated Glucidex 29 D at different ratios in a Tris buffer.

Claims

1. A composition comprising an insecticidal protein and maltodextrin having a DE of 5 to 50, wherein the composition has a pH of from 5.0 to 10.

2. The composition according to claim 1 , wherein the insecticidal protein is soluble in aqueous solution at a pH of 5 to 10.

3. The composition according to claim 1 or 2, wherein the insecticidal protein comprises a vegetative insecticidal protein, wherein the vegetative insecticidal protein comprises Vip1 , Vip2, Vip3 and I or Vip4, preferably Vip3A and I or Vip3D.

4. The composition according to any one of the claims 1 to 3, wherein the insecticidal protein and maltodextrin are at a ratio of insecticidal protein to maltodextrin of 1 :200 to 1 :1 wt:wt.

5. The composition according to any one of the claims 1 to 4 wherein the composition further comprises a salt.

6. The composition according to any one of the claims 1 to 5, wherein the composition further comprises an agricultural acceptable carrier.

7. The composition according to any one of the previous claims, wherein the composition is a soluble powder or a tank mix.

8. A process for preparing a composition according to any one of the claims 1 to 7, comprising a. cultivating microbial cells expressing an insecticidal protein; b. adding a salt solution having a pH of from 5.0 to 10 to the microbial cells comprising the insecticidal protein; c. optionally disrupting the microbial cells; and d. adding maltodextrin having a DE of 5 to 50

9. The process according to claim 8, further comprising a step of removing water after step a), b), c) and I or d).

10. The composition according to any one of the claims 1 to 7, or the process according to any one of the claims 8 or 9, wherein the salt comprises a sodium phosphate salt ammonium bicarbonate, N- cyclohexyl-3-aminopropanesulfonic acid (CAPS), ortris(hydroxymethyl)aminomethane (Tris).

11. A method for controlling or preventing infestation of a plant, a part or a locus thereof by an insect pest, comprising applying to the plant, a part or a locus thereof a composition according to any one of the claims 1 to 7.

12. The method according to claim 11 , wherein the insect pest is a Spodoptera, preferably Spodoptera littoralis.

13. The method according to claim 11 or 12, wherein the plant is corn, rice, soybean, or cotton.

14. A plant, a part or a locus thereof comprising a composition according to any one of the claims 1 to 7.

15. Use of maltodextrin having a DE of 5 to 50 to stabilize an insecticidal protein for at least three days.