Nematicidal composition comprising bacillus subtilis

Abstract

The present invention relates generally to compositions comprising Bacillus subtilis having nematicidal effects on plants and / or plant nematodes on their habitats, uses of the compositions, methods of preparing the compositions, uses of Bacillus subtilis, prevention, control, combat and / or induction of resistance to plant nematodes, and kits. In particular, the present invention relates to Bacillus subtilis (DSM 32324).

Description

Technical Field

[0001] The present invention generally relates to a composition comprising Bacillus subtilis having a nematicidal effect on plant nematodes on plants and / or their habitats, the use of the composition, a method for preparing the composition, the use of Bacillus subtilis, the prevention, control, combating and / or inducing resistance to plant nematodes, and a kit. In particular, the present invention relates to Bacillus subtilis (DSM 32324). Background Art

[0002] The ecological imbalance caused by the intensification of agricultural activities has made it necessary to use selective products that do not affect the balance between pests and their predators, parasitoids and pathogens (these balances are the main content of natural biological control) because they can keep the pest population at an acceptable level (DENT, D., Insect pest management. Cambridge: Cabi Bioscience 2000).

[0003] To reverse this situation, it is recommended to develop an integrated pest management program, which is defined as a decision-making system for the use of control strategies, either alone or in harmonious combination with cost / benefit-based management strategies, taking into account the benefits and / or impacts on farmers, society and the environment. Among the control measures that can be used in these management systems are entomopathogenic nematodes and insect parasitoids, covering different areas of biological pest control.

[0004] In the context of the current modern ecological society concerned with protecting the environment, biological control is considered an attractive alternative and / or complement to conventional control methods. Biological control is the use of one organism (predator, parasite or pathogen) to attack another organism that causes economic damage to crops. It is a very common strategy in agro-ecosystems as well as in conventional agriculture that relies on integrated pest management (IPM).

[0005] Although biological control has brought positive responses in terms of reducing or stopping the use of pesticides and improving farmers' income, a series of experimental analyses around the world have shown that the effects are still concentrated in a few crops. There is still much room for development in the field of pest and disease control.

[0006] Diseases caused by nematodes are difficult to control, and various approaches have been tested over the past few decades, such as resistant varieties, crop rotation with non-host crops, and application of nematicidal chemicals. However, available management tools have low efficacy or are unable to control the different nematode species that are often present in a mixture in the soil. Chemical nematicides are highly toxic compounds and overuse can cause serious harm to the environment. Restrictions on the use of chemical pesticides have increased interest in studying alternative methods of nematode control.

[0007] There is a great deal of emphasis on the use of bacteria that colonize plant roots, known as rhizobacteria, for biocontrol. Beneficial rhizobacteria that play a role in growth promotion and / or in the biocontrol of plant pathogenic bacteria are known as plant growth-promoting rhizobacteria or PGPRs. PGPRs increase the availability of plant nutrients and can produce combinations and concentrations of substances that promote growth.

[0008] Social pressure to replace nematicides with environmentally acceptable products or eco-friendly practices has encouraged the search for alternative methods of controlling nematodes. In this context, biological control is considered as one of the alternatives in an integrated approach aimed at ensuring sustainable agricultural development. The use of natural enemies has become an area of ​​research to reduce nematode populations below the threshold level of economic losses.

[0009] The risks to humans and the environment from the use of synthetic pesticides highlight the need for tools such as biological control in optimizing sustainable agricultural systems.

[0010] European patent application EP 0705807A1 relates to a bacterial preparation for soil conditioning, which contains bacteria belonging to the genus Bacillus, such as Bacillus subtilis and Bacillus licheniformis. The preparation can prevent nematode damage to crop roots.

[0011] Brazilian patent application BR PI 0604602-9A relates to the use of Bacillus subtilis in combination with Bacillus licheniformis for controlling plant nematodes.

[0012] Siddiqui and Mahmood (1999) described the role of bacteria in the management of plant parasitic nematodes, indicating that Bacillus subtilis and Bacillus licheniformis can be used against plant nematodes such as Meloidogyne spp., Heterodera spp., and Rotylenchulus.

[0013] WO 2012 / 020014 relates to a composition comprising Bacillus subtilis and Bacillus licheniformis having a nematicidal effect on plant nematodes.

[0014] Sikora, R.A. (Interrelationship between plant health promoting rhizobacteria, plant parasitic nematodes and soil microorganisms. Medicine Faculty Landbouww Rijksuniv Gent, Landbouww, Vol. 53, No. 2b, pp. 867-878, 1988) observed that treatment of seeds of various crops with Bacillus strains reduced infection with peanut root-knot nematode (Meloidogynearenaria), southern root-knot nematode (M. incognita) and kidney-shaped kidney-shaped nematode (Rotylenchulus reniformis) by about 60%-65%.

[0015] There is still a need for new nematicides with improved safety and efficiency to protect crops from nematode infestation.

[0016] The inventors of the present invention have conducted extensive screening and research to address the objective of providing a biological method for controlling nematodes based on the identification of novel Bacillus subtilis strains which exhibit different nematicidal modes of action and have been shown to be effective in reducing nematode diseases. Summary of the invention

[0017] The present invention provides a composition for use as a nematicide, comprising Bacillus subtilis or a variant thereof as an active ingredient and an agrochemically acceptable excipient and / or carrier, wherein the Bacillus subtilis has the characteristics of a strain deposited in the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 32324, wherein the variant is obtained using the deposited strain as a starting material, and wherein the variant retains or further improves the nematicidal properties of DSM 32324.

[0018] The composition comprising the Bacillus subtilis of the present invention may be in the form of a wettable powder or in the form of a liquid formulation, and has a nematicidal effect on plant nematodes on plants and / or their habitats, thereby preventing, controlling, combating plant nematodes and / or inducing resistance to plant nematodes.

[0019] The aim of the studies described in the examples was to investigate the effect of DSM 32324 on the penetration of nematode larvae into plant roots, based on the measurement of biofilm forming capacity, and on the number of galls and eggs on plant roots after inoculation with nematodes and application of the composition according to the invention.

[0020] The present invention describes and demonstrates that the Bacillus subtilis strain DSM 32324 has benefits in improving the health of plants to which it has been applied (by demonstrating nematicidal effects).

[0021] definition

[0022] Generally, the terms and phrases used herein have their art-recognized meanings, which can be found by reference to standard textbooks, journal literature and contexts known to those skilled in the art. The following definitions are provided to clarify their particular use in the context of the present disclosure.

[0023] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] As used herein, the term "and / or" is intended to connote both combinatorial ("and") and exclusive ("or") usage, ie, "A and / or B" is intended to mean "A alone, or B alone, or A and B together."

[0025] Composition: As used herein, the term "composition" refers to a composition comprising a vector and at least one bacterial strain as described herein.

[0026] Controlling plant nematode infestation: As used herein, the term "controlling plant nematode infestation" means methods and / or compositions that partially or completely inhibit plant nematode infestation in plants. Thus, the term "controlling plant nematode infestation" means that plant nematode infestation is reduced or completely eliminated, and the overall health of the plant is improved.

[0027] Effective amount / effective concentration / effective dose: As used herein, the term "effective amount", "effective concentration", or "effective dose" is defined as an amount, concentration or dose of one or more bacterial strains sufficient to improve the overall health of the plant and impart benefits similar to those demonstrated in the Examples.

[0028] The absolute number of the actual effective dose depends on factors including: the health of the plant in question, the presence of other ingredients. An "effective amount," "effective concentration," or "effective dose" of a bacterial strain can be determined by conventional assays known to those skilled in the art. Examples 3 and 4 give examples of effective doses.

[0029] Isolated: As used herein, the term "isolated" means that the bacterial strain described herein is in a form or environment that does not exist in nature, that is, the strain is at least partially separated from one or more or all of the naturally occurring components with which it is associated in nature.

[0030] Plant parasitic nematodes: As used herein, the term "plant parasitic nematodes" means nematodes that live on plants as parasites and cause serious damage to a variety of crops, resulting in annual yield declines and significant economic losses. The term plant nematodes is intended to mean the same, and plant nematodes and plant parasitic nematodes are used interchangeably in this application. Examples of plant parasitic nematodes include, but are not limited to, root knot nematodes (Meloidogyne species), cyst nematodes (cyst nematodes and Globodera species (Globodera spp.)) and root spot nematodes (Pratylenchus species (Pratylenchus spp.)). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1

[0032] Figure 1 DSM 33113 and DSM 32324 disclose biofilm (pellicle) formation and quantification. Spores were isolated at a final OD 600 0.05 was added to MSgg and incubated at 30°C for 3 days under static conditions. A) Pellicle formation after 3 days of incubation. B) Biofilm (pellicle) matrix quantification was performed using crystal violet and measuring absorbance at 570 nm.

[0033] Figure 2

[0034] Figure 2 Micrographs of A. thaliana roots colonized by DSM 33113 and DSM 32324 24 h after inoculation are disclosed. Vegetative cells were grown at a final OD 600 0.05 was inoculated into 1 / 2 Murashigue and Skoog containing 7-day-old Arabidopsis seedlings and incubated for 24 h at 22° C. The plant roots were then fixed on an agarose pad and a cover glass was placed on top before microscopic imaging.

[0035] Figure 3

[0036] Figure 3 Disclosed are the number of galls produced by the southern root-knot nematode in Arabidopsis thaliana seven days after treatment of the plants with vegetative cells of the strain DSM 32324. The plants were challenged with second-stage larvae of the nematode. A: Bars represent the mean of ten replicates. (p=0.07)

[0037] Figure 4

[0038] Figure 4 Disclosed are A) the number of eggs of Meloidogyne incognita extracted from tomato roots five weeks after DSM 32324 treatment and nematode inoculation. B) the weight of fresh roots of tomato plants.

[0039] Figure 5

[0040] Figure 5 The number of eggs of the southern root-knot nematode extracted from tomato roots five weeks after treatment with B. paralicheniformis DSM 33113 and Bacillus subtilis DSM 32324 strains (applied alone or in combination) and nematode inoculation is disclosed.

[0041] Brief description of sequence

[0042] SEQ ID NO:1

[0043] SEQ ID NO: 1 is the sequence of the purL gene from Bacillus subtilis DSM32324. DETAILED DESCRIPTION

[0044] Rhizobacteria

[0045] Soil is home to a complex community of organisms, in which microorganisms, prokaryotes, and eukaryotes dominate in both number and diversity. Some prokaryotes have ecological niches, such as the rhizosphere and / or root surfaces of plants, where they reproduce, survive, and protect themselves from antagonistic effects of the soil microbiota. These organisms are often referred to as rhizobacteria.

[0046] In relation to plants, rhizobacteria may have deleterious, ineffective or beneficial effects. Those rhizobacteria that exert beneficial effects (growth promotion and biological control of diseases) are known as PGPR (plant growth promoting rhizobacteria). It is estimated that only 0.6% of rhizobacteria have some beneficial effects on the plants they are associated with.

[0047] PGPRs have been used for biological control of plant diseases, thereby increasing crop productivity. How and why this biological control is performed remains a topic of research that requires additional study.

[0048] In some cases, biological control may occur through direct antagonistic effects exerted by PGPRs on pathogens, involving known antibiosis mechanisms: production of antimicrobial substances, direct parasitism, nutrient competition and ecological niche. Studies have shown that some PGPRs appear to act as elicitors of ISR (induced systemic resistance), i.e., plants can defend themselves systemically against more than one pathogen, which is different from classical biological control, which aims at more targeted control.

[0049] Some rhizobacteria produce antagonistic secondary metabolites that affect nematode motility in vitro, whereas others inhibit larval hatching and larval penetration into the roots.

[0050] An important parameter affecting the ability of PGPR to infect and colonize plant surfaces is the ability of PGPR to form biofilms.

[0051] The present invention provides a superior biofilm-forming Bacillus strain which produces a set of secondary metabolites having nematicidal effects, which have been described in the prior art.

[0052] Rhizobacteria can inhibit plant-parasitic nematodes through different direct and indirect methods. Direct antagonism is based on the synthesis of lytic enzymes, toxic proteins, volatile compounds or parasitism. Indirect antagonism manifests itself as nutrient competition, induction of systemic resistance (ISR) or release of molecules that modulate nematode behavior including recognition, feeding and sex ratio.

[0053] Therefore, rhizobacteria can exert control over nematodes in various ways and can affect different stages of the nematode life cycle:

[0054] - Eggs: Antibiotics and toxins produced by rhizosphere bacteria diffuse into the soil and can be absorbed by nematode eggs, killing the cells and preventing their embryonic development.

[0055] - Hatching: Rhizobacteria degrade root exudates (hatching factors for many nematode species), and the compounds absorbed by the nematode eggs may then be inactivated during development or cause deformation, thus preventing hatching.

[0056] - Targeting and Mobility: Root exudates are converted into metabolic byproducts of rhizobacteria, which can cause the nematode to not recognize the tropism stimulus at all, so it will continue to move randomly, eventually exhausting its energy reserves and dying without being able to enter the roots. If the nematode recognizes the root exudates and moves towards the roots, some bacterial compounds can present tropism characteristics and reduce the nematode's mobility, preventing it from reaching the roots.

[0057] - Host recognition: Rhizobacteria produce substances that are absorbed by the roots and can change their chemical composition, resulting in the nematode being unable to recognize its host. It is also believed that rhizobacteria bind to lectins on the root surface, which feature as binding sites between the nematode and its plant host, thus preventing recognition.

[0058] - Penetration into roots: Toxins or repellents produced by rhizosphere bacteria are present in higher concentrations in the root surface area or in the contents of root epidermal cells, which can prevent nematodes from penetrating the host plant.

[0059] - Food: Rhizobacteria or their metabolites can be absorbed by the plant, and the plant senses the presence of the nematodes, triggering a hypersensitive response in the giant cells, which is the main mechanism of host resistance to nematodes of the genus Meloidogyne. This resistance (called systemic resistance) is not intrinsic to the plant, but a response induced by the presence of PGPR.

[0060] - Reproduction: Some rhizobacteria may have a greater effect on reducing eggs than gall numbers, which may be one of the mechanisms of action.

[0061] The present invention discloses strains of the genus Bacillus that have been identified as having a range of these qualities making them potent agents for managing plant parasitic nematodes. The key characteristics identified include: the ability to form strong biofilms, the ability to synthesize surfactins, dimethyl disulfide, and extracellular proteases, all secondary metabolites and enzyme activities that have been shown to have inhibitory effects on plant parasitic nematodes. The unique combination of these modes of action in a single microorganism has great potential to interfere with nematode behavior around the rhizosphere, thereby reducing nematode penetration and its reproduction within the plant host, as shown in Examples 3 and 4.

[0062] Bacillus bacteria

[0063] Spore-forming rhizobacteria have many advantages over chemical pesticides or even other biological control agents: they are easy to produce on a large scale, they are easy to store, they are amenable to formulation techniques, and they do not require genetic manipulation.

[0064] Spore-forming rhizobacteria can be applied by treating the substrate, dipping the roots of seedlings into the bacterial suspension, watering the plants with the bacterial suspension (by dipping the seeds into the rhizobacteria suspension) or pelleting the PGPR with the seeds.

[0065] An example of spore-producing rhizobacteria are Bacillus species, which have attracted much attention in recent years due to their environmental safety and ability to offer different modes of action to suppress nematode populations in soil.

[0066] Bacillus species are Gram-positive bacteria characterized by a thick cell wall and the absence of an outer membrane, unlike Gram-negative bacteria. Most of the wall of Gram-positive bacteria is composed of peptidoglycan.

[0067] Gram-positive species are divided into several groups based on their morphological characteristics and biochemical features. The genus Bacillus belongs to the group of spore-forming bacteria. Species that form spore structures are able to resist environmental changes and withstand dry heat and certain chemical disinfectants for moderate periods of time. They can survive for many years in dry land.

[0068] When Bacilli (such as Bacillus subtilis) are applied near seeds or soil, their beneficial effects are not only due to their antagonistic effects against pathogens. PGPR can produce substances that promote plant growth (such as volatile organic compounds, phytohormones) and improve plant nutrition (such as phosphorus solubilization), thus having a positive impact on germination, development and crop yield.

[0069] Use of Bacillus species in controlling nematodes

[0070] Plant parasitic nematodes cause severe damage to a wide range of crops worldwide, resulting in low agricultural production yields and significant economic losses. It is estimated that losses in economically important crops worldwide amount to approximately US$ 100 billion per year. Root-knot nematodes (Meloidogyne species), cyst nematodes (Sporadenoma and Coccidioides species), and root spot nematodes (Pratylenchus species) rank among the most economically and scientifically important species due to their complex relationships with host plants, their wide host range, and the level of damage caused by infection.

[0071] Cultivars resistant to nematodes are not always available due to lack of sources of resistance for breeding, insufficient adaptation of resistant cultivars to certain regions and growing seasons, or breakdown of resistance under field conditions. Chemical control of nematodes is generally not recommended because it is not very effective; it is expensive because it can remain in food and cause environmental pollution. Due to these shortcomings, there is increasing social pressure to limit the use of chemicals, resulting in farmers demanding products that are non-toxic to both humans and animals, inexpensive, and very effective in controlling nematodes.

[0072] Many soil microorganisms are parasites or predators of nematodes. The actions of these microorganisms can have direct or indirect effects by interfering with steps in the pathogen's life cycle.

[0073] Meloidogyne

[0074] Nematodes of the genus Meloidogyne (root-knot nematodes) show great diversity on plant hosts and occur in different regions of the world, causing losses to different crops. The main symptom is the presence of galls on the roots of plants. These galls are deformed or thickened root systems. Affected plants suffer from stunted growth, low yields, early defoliation and premature decline, and sometimes even plant death, with symptoms exacerbated under conditions of nutritional stress and drought.

[0075] Initially, the second stage larvae (J2) of the root-knot nematode species penetrate the roots and establish a feeding site in the central cylindrical area of ​​the roots. Through the development of the nematodes, the J2 differentiates into adult males or females. The adult males leave the root system, while the females remain in the roots. During the development of female root-knot nematodes, about 500 eggs are laid. They are deposited in a gelatinous matrix outside the roots (where the J2 hatches), thereby infecting the root system again. The life cycle of root-knot nematodes is about four weeks and may be prolonged under less favorable temperature conditions. Temperatures below 20°C or above 35°C and drought or waterlogged conditions of the soil reduce the development and survival of the nematodes.

[0076] Chemicals containing certain active ingredients such as furadan, ethoprophos, aldicarb, metamphos or fenamiphos (depending on the culture in question) can be used to control southern root-knot nematodes and Javan root-knot nematodes (M. javanica). Crop rotation practices are also important in control, as is the deployment of non-host crops (such as peanuts, pineapples, rice) or the use of plant species as ground covers, such as non-host plants such as oats.

[0077] Meloidogyne exigua is very aggressive and widespread in plantations. Different chemical control methods used in areas infested with Meloidogyne exigua include the use of chemicals containing either terbufos or furadan (depending on the species of the culture) as the active ingredient. Among non-host crops, cotton, peanuts, rice and oats are the most prominent.

[0078] Like the short root-knot nematode, the Parana root-knot nematode (M. paranaensis) is widespread in coffee plantations but is less of a problem for other crops. In terms of control, positive results were obtained with the fungus Paecilomyces lilacinus, which reduced the nematode population on the roots of Santa Clara tomatoes in a greenhouse.

[0079] The use of rhizobacteria in biocontrol is known. However, the applicant has now developed a composition comprising Bacillus subtilis DSM 32324 or a variant thereof having nematicidal action against plant nematodes.

[0080] Thus, a first aspect of the present invention relates to a novel strain or variant thereof as described herein for use as a nematicide.

[0081] It is clear to the skilled person that by using the deposited strain as the starting material, the skilled person can routinely obtain additional variants or derivatives thereof retaining the relevant features and advantages described herein by natural strain improvement methods or re-isolation techniques. Therefore, the term "variants thereof" of the first aspect relates to variant strains obtained by natural strain improvement methods using the deposited strain as the starting material.

[0082] Plant nematodes and / or plant parasitic nematodes are widely considered to be any nematodes that have a negative impact on economic crops. The nematodes that can be antagonized using the composition of the present invention include nematodes of the following genera: root-knot nematodes, Pratylenchus, cyst nematodes, spherospore cyst nematodes, Ditylenchus, Tylenchulus, Xiphinema, Radopholus, Renal-shaped nematodes, Helicotylenchus and Belonolaimus. Species of the genus genus Root-knot nematodes are considered to be of particular significance because they account for about 95% of all crop infestations, resulting in approximately 5% crop losses worldwide. In addition to the active ingredient, the composition of the present invention may also contain an agrochemically acceptable excipient and / or vehicle. The composition of the present invention further comprises an agrochemically acceptable carrier, vehicle and / or adjuvant.

[0083] Agrochemically acceptable carriers, vehicles and / or adjuvants are considered to be known to the skilled person and may be selected from, but not limited to, the group consisting of maltodextrin, silicon dioxide, modified zeolites, kaolinite, lignin, starch, chitosan and calcium carbonate. In a preferred embodiment, the agrochemically acceptable carrier, vehicle and / or adjuvant is maltodextrin and silicon dioxide.

[0084] The compositions of the invention are particularly useful for combating nematodes in crop plants.

[0085] In one embodiment, the composition of the present invention can be mixed with additional ingredients, carriers, surfactants, dispersants, one or more enzymes and / or yeast extracts relevant to the field of agrochemicals, including but not limited to: microbial insecticides, biological insecticides and / or chemical insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators and / or fertilizers, in an amount suitable for benefiting plant growth and / or protecting susceptible plants from pathogenic infection.

[0086] The main plant crops are sugar cane, coffee, soybeans, cotton, corn, potatoes, tomatoes, tobacco, bananas, rice, wheat, avocados, pineapples, squash, cocoa, coconuts, oats, onions, lettuce, beets, carrots, cassava, beans, sunflowers, peppers, turnips, apples, strawberries, okra, radishes, and onions.

[0087] In fruit cultivation: citrus, grapes, guava, papaya, fig, peach, plum and loquat (nespeira) are particularly relevant; and in horticulture: eggplant and the cruciferous family are particularly relevant.

[0088] In floriculture: roses, chrysanthemums, lisianthus, gerberas, amaryllis, begonias and gentiana are particularly relevant.

[0089] The present invention relates to a composition comprising Bacillus subtilis DSM 32324 or a variant thereof, and to a kit comprising the composition (or prepared by a method for preparing the composition), as well as instructions and a suitable receptor.

[0090] Also provided is a method for preparing a composition comprising Bacillus subtilis DSM 32324 or a variant thereof and an agrochemically acceptable carrier, vehicle and / or adjuvant, and use of the composition for controlling, combating and / or conferring specific resistance to plant nematodes.

[0091] Furthermore, the present invention relates to the use of an effective amount of Bacillus subtilis DSM 32324 or a variant thereof for the preparation of an agrochemical composition having a nematicidal effect on plant nematodes in plant culture, and a method for controlling, combating and / or conferring specific resistance to plant nematodes.

[0092] The relevant Bacillus strains of the present invention are provided in commercially relevant forms known to the skilled person. Thus, in an embodiment, the Bacillus strain of the composition is present in a dried (eg spray-dried) or frozen form.

[0093] The composition of the present invention can be coated on plant seeds and can include about 1.0x10 2 CFU / seed about 1.0x 10 9 CFU / seed of Bacillus spores, such as, for example, Bacillus subtilis DSM 32324 spores. In another embodiment of the present invention, the composition can be coated on plant seeds at about 1.0 x 10 6 CFU / g seeds about 1.0x 10 11 CFU / g seed of Bacillus sp., such as, for example, Bacillus subtilis DSM 32324 spores.

[0094] Plant seeds may include, but are not limited to, seeds of monocots and dicots, such as cereals, corn, sweet corn, popcorn, seed corn, silage corn, forage corn, rice, wheat, barley, sorghum, Brassica vegetables, broccoli, cabbage, cauliflower, Brussels sprouts, collard greens, kale, mustard greens, kohlrabi, bulb vegetables, onions, garlic, shallots, fruiting vegetables, peppers, tomatoes, eggplant, ground cherries, tomatillos, okra, grapes, herbs / spices, cucurbits, cucumbers, cantaloupe, melons, cantaloupe, winter squash , watermelon, pumpkin, eggplant, leafy vegetables, lettuce, celery, spinach, parsley, endive, legumes / vegetables (juicy, dry and peas), beans, green beans, snap beans, shell beans, soybeans, dry beans, chickpeas, lima beans, peas, chickpeas, split peas, lentils, oilseed crops, canola, castor, cotton, flax, peanuts, rapeseed, safflower, sesame, sunflower, soybeans, root / tuber and bulb vegetables, carrots, potatoes, sweet potatoes, beets, ginger, horseradish, radish, ginseng, turnips, sugarcane, sugar beets, grass or turf grass seeds.

[0095] In one or more embodiments, the plant seeds may include seeds of dry beans, corn, wheat, soybeans, canola, rice, cotton, grasses, and turf grasses.

[0096] In alternative embodiments, the Bacillus or the composition of the invention comprising Bacillus subtilis DSM 32324 can be added to the soil or growing medium surrounding the plant, added to the soil or growing medium before sowing the seeds of the plant in the soil or growing medium, or added to the soil or growing medium before planting the plant, plant cuttings, plant transplants or plant callus in the soil or growing medium.

[0097] In a preferred embodiment, the Bacillus sp. or the composition of the invention comprising Bacillus subtilis DSM 32324 is added to the soil or growing medium surrounding the plant.

[0098] When the composition of the present invention is added to the soil or growing medium surrounding a plant, added to the soil or growing medium before sowing plant seeds therein, or added to the soil or growing medium before planting a plant, plant cutting, plant transplant, or plant callus therein, the composition may include about 1.0 x 10 6 CFU / ml to about 1.0x 10 9 CFU / ml of Bacillus sp., such as, for example, Bacillus subtilis DSM 32324 spores.

[0099] In one or more embodiments, the plants, plant cuttings, plant grafts, or plant callus may include soybeans, beans, snap beans, wheat, cotton, corn, peppers, tomatoes, potatoes, cassava, grapes, strawberries, bananas, peanuts, squash, pumpkin, eggplant, sugarcane, and cucumber.

[0100] Compositions comprising Bacillus strains as described herein can be in the form of liquids, oil dispersants, dusts, dry wettable powders, spreadable granules or dry wettable granules. More particularly, the composition can be, for example, an emulsion concentrate (EC), a suspension concentrate (SC), a suspoemulsion (SE), a capsule suspension (CS), a water-dispersible granule (WG), an emulsifiable granule (EG), an oil-in-water emulsion (EO), an oil-in-water emulsion (EW), a microemulsion (ME), an oil dispersant (OD), an oil suspension (OF), an oil-soluble liquid (OL), a soluble concentrate (SL), an ultra-low volume suspension (SU), an ultra-low volume liquid (UL), a dispersible concentrate (DC), a wettable powder (WP) or any technically feasible formulation in combination with an agriculturally acceptable adjuvant.

[0101] The present invention relates to a composition comprising Bacillus subtilis DSM 32324 or a variant thereof, and to a kit comprising the composition (or prepared by a method for preparing the composition), as well as instructions and a suitable receptor.

[0102] Also provided is a method for preparing a composition comprising Bacillus subtilis DSM 32324 or a variant thereof and an agrochemically acceptable carrier, vehicle and / or adjuvant, and use of the composition for controlling, combating and / or conferring specific resistance to plant nematodes.

[0103] Furthermore, the present invention relates to the use of an effective amount of Bacillus subtilis DSM 32324 or a variant thereof for the preparation of an agrochemical composition having a nematicidal effect in plant cultures, and a method for promoting plant health.

[0104] In the context of the present invention, "variants thereof" are understood as Bacillus subtilis having a change in the wild-type nucleotides of the genome of an organism (e.g. Bacillus subtilis DSM 32324), resulting in a change in the phenotype of the organism, wherein the change may be a deletion of one or more nucleotides, a substitution of one or more nucleotides, an insertion of one or more nucleotides and / or a modification of one or more nucleotides. In the context of the present invention, a deletion is understood as a genetic mutation resulting in the removal of one or two nucleotides of the wild-type nucleotide sequence of the genome of an organism; an insertion is understood as the addition of one or more nucleotides to a wild-type nucleotide sequence; a substitution (or point mutation) is understood as a genetic mutation in which a nucleotide of a wild-type nucleotide sequence is changed by another nucleotide; a frameshift is understood as a genetic mutation resulting from the insertion or deletion of a certain number of nucleotides in the wild-type nucleotide sequence that is not divisible by three, thereby changing the reading frame and resulting in a translation that is completely different from the original reading frame; the introduction of a stop codon is understood as a point mutation in the DNA sequence resulting in a premature stop codon; an inhibition of substrate binding of the encoded protein is understood as any mutation in the nucleotide sequence resulting in a change in the protein sequence that prevents the substrate from binding to its protein catalytic site. Furthermore, a knockout mutant is to be understood as a genetic mutation which results in the removal or deletion of a gene (such as an entire gene or an entire open reading frame) from the genome of an organism.

[0105] Algorithms for aligning sequences and determining the degree of sequence identity between them are well known in the art. For the purposes of the present invention and as an example, one of these algorithms is based on comparing two sequences with the blastp provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov, applying standard parameter settings (matrix: BLOSUM62, gap existence penalty: 11, gap extension penalty: 1, conditional composition score matrix adjustment); then the same amino acid pairs in the same position are quantified in the amino acid sequence of the comparison. A similar process can be performed to align nucleotide sequences, in this case using the blastn provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov, applying standard parameters.

[0106] In one embodiment the present invention relates to a variant of Bacillus subtilis DSM 32324, wherein the average nucleotide identity (ANI) of the Bacillus subtilis variant is at least 99%, such as at least 99.5%, such as at least 99.8%, such as at least 99.9% identical to the genome of the strain deposited with the German Collection of Microorganisms and Cell Cultures under the accession number DSM 32324.

[0107] The present invention further includes the aspects listed below:

[0108] Aspect 1. A Bacillus subtilis expressing a purL gene, wherein the purL gene is encoded by a sequence having at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 1.

[0109] Aspect 2. The Bacillus subtilis according to any of the preceding aspects, wherein the genome of the strain is at least 99%, such as at least 99.5%, such as at least 99.8%, such as at least 99.9% identical to the genome of the strain deposited with the German Collection of Microorganisms and Cell Cultures under the accession number DSM 32324.

[0110] Aspect 3. The Bacillus subtilis according to any one of the preceding aspects, which produces secondary metabolites surfactin, fengin, and dimethyl disulfide and exhibits extracellular protease activity.

[0111] Aspect 4. The Bacillus subtilis according to any one of the preceding aspects, which shows better or equal inhibition of nematode diseases in plants compared to the effect of the Bacillus paralicheniformis strain deposited with the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 33113, alone or in combination with the Bacillus subtilis.

[0112] Aspect 5. A composition comprising the Bacillus subtilis according to any one of the preceding aspects.

[0113] Aspect 6. A composition comprising the Bacillus subtilis according to any one of Aspects 1 to 5 and an agrochemically acceptable excipient and / or carrier thereof.

[0114] Aspect 7. A composition comprising the Bacillus subtilis or a variant thereof according to any one of Aspects 1 to 6, for use as a nematicide, the composition comprising Bacillus subtilis or a variant thereof as an active ingredient and an agrochemically acceptable excipient and / or carrier, the Bacillus subtilis having the characteristics of a strain deposited in the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 32324, wherein the variant is obtained using the deposited strain as a starting material, and wherein the variant retains or further improves the nematicidal properties of DSM 32324.

[0115] Aspect 8. The composition according to any one of the preceding aspects 6 to 7, wherein the agrochemically acceptable excipient and / or carrier is selected from the group consisting of maltodextrin, silicon dioxide, modified zeolite, kaolinite, lignin, starch, chitosan and calcium carbonate.

[0116] Aspect 9. The composition according to any one of the preceding aspects 5 to 8, wherein the composition is in the form of a wettable powder.

[0117] Aspect 10. The composition according to any one of the preceding aspects 5 to 8, wherein the composition is in the form of a liquid formulation.

[0118] Aspect 11. A composition according to any one of aspects 5 to 10, wherein the composition comprises at least 1x10 9 CFU / gram of the Bacillus subtilis.

[0119] Aspect 12. A composition according to any one of the preceding aspects 5 to 11, further comprising one or a combination of microbial insecticides, biological insecticides and / or chemical insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators and / or fertilizers present in the following amounts, which are suitable for benefiting plant growth and / or protecting susceptible plants from pathogenic infection, and further comprising a carrier, a surfactant, a dispersant, one or more enzymes and / or a yeast extract, preferably wherein the microorganism is a strain of the genus Bacillus.

[0120] Aspect 13. Use of the composition according to any one of aspects 5 to 12 or the Bacillus according to any one of aspects 1 to 4 as a bionematicide and / or plant growth enhancer and / or plant health promoter and / or plant disease or pest control agent.

[0121] Aspect 14. Use of the composition according to aspect 13 or the Bacillus according to any one of aspects 1 to 4 for controlling, combating and / or conferring specific resistance to plant nematodes.

[0122] Aspect 15. The use according to any one of aspects 13 or 14, wherein the plant nematodes are selected from the group consisting of: root-knot nematode, Pratylenchus, cyst nematode, spherocytosis, Ditylenchus, Xiphinema, Radopholus, Renniformes, Heliconematode and Lancetella.

[0123] Aspect 16. The use according to any one of aspects 13 to 15, wherein the plant nematode is selected from the group consisting of: southern root-knot nematode, javanic root-knot nematode, short root-knot nematode, parana root-knot nematode, soybean cyst nematode (Heterodera glycines) and corn nematode (Pratylenchus zeae).

[0124] Aspect 17. The use according to any one of aspects 13 to 16, wherein the composition according to any one of aspects 5 to 12 or the Bacillus according to any one of aspects 1 to 4 is applied to a plant, a seed or a habitat of a plant.

[0125] Aspect 18. The use according to aspect 17, wherein the plant is selected from the group consisting of corn, rice, sugar cane, soybean, potato, beet, carrot, coffee, tomato and banana.

[0126] Aspect 19. A method for preventing, controlling and / or combating plant nematodes on a plant and / or its habitat, the method comprising applying to the plant and / or its habitat an effective amount of the Bacillus according to any one of Aspects 1 to 4 or the composition according to any one of Aspects 5 to 12.

[0127] Aspect 20. A method for preventing, controlling and / or combating plant nematodes on plants and / or their habitats, the method comprising applying to the plant nematodes and / or their habitats an effective amount of the Bacillus according to any one of Aspects 1 to 4 or the composition according to any one of Aspects 5 to 12.

[0128] Aspect 21. The method according to aspect 19 or 20, wherein the composition acts by reducing the infestation rate of the plant nematodes.

[0129] Aspect 22. The method according to aspect 19 or 20, wherein the composition acts by reducing the penetration of the plant nematodes into the roots of the plant.

[0130] Aspect 23. A kit comprising the composition according to any one of aspects 5 to 12, instructions for use and a suitable container.

[0131] Aspect 24. A plant seed coated with the composition of any one of aspects 5 to 12, the composition being present in an amount suitable for benefiting plant growth and / or protecting susceptible plants from pathogenic infection.

[0132] Aspect 25. The plant seed according to aspect 24, wherein the composition comprises about 1.0×10 2 CFU / seed is about 1.0×10 9 The Bacillus subtilis spores according to any one of aspects 1 to 4 in an amount of CFU / seed.

[0133] Aspect 26. The plant seed according to aspect 24, wherein the composition comprises about 1.0×10 6 CFU / g seeds is about 1.0×10 11 The Bacillus subtilis spores according to any one of aspects 1 to 4 in an amount of CFU / g seeds.

[0134] Aspect 27. The plant seed according to aspect 24, wherein the composition further comprises one or a combination of a microbial insecticide, a biological insecticide or a chemical insecticide, a fungicide, a nematicide, a bactericide or a plant growth regulator, present in an amount suitable for benefiting plant growth and / or protecting susceptible plants from pathogenic infection, preferably wherein the microorganism is a strain of Bacillus.

[0135] Aspect 28. The Bacillus according to any of the preceding aspects, wherein purL is encoded by SEQ ID NO: 1 or a homolog thereof.

[0136] Aspect 29. A method for treating plants to enhance plant growth and / or promote plant health and / or control plant diseases, wherein the method comprises the step of applying a Bacillus strain according to any one of the preceding aspects 1 to 4 or the step of applying a composition according to any one of the preceding aspects 5 to 12, wherein the step results in similar biofilm formation or similar pellicle biofilm formation compared to the following method a) comprising the step of applying a Bacillus strain deposited with the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 33113 or b) comprising the step of applying a composition having a Bacillus strain deposited with the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 33113.

[0137] Aspect 30. The method according to aspect 29, further comprising the step of applying the Bacillus strain according to any one of aspects 1 to 4 or the composition according to any one of aspects 5 to 12 to the soil.

[0138] Aspect 31. The method according to any one of aspects 29 to 30, wherein the step of applying the Bacillus strain according to any one of aspects 1 to 4 or the composition according to any one of aspects 5 to 12 is performed before, during or after the plant or plant part contacts the soil.

[0139] Aspect 32. The method according to any one of the preceding aspects 29 to 31, wherein the plant part is selected from the group consisting of seeds, roots, corms, tubers, bulbs and rhizomes.

[0140] Aspect 33. A method for preparing the composition according to any one of aspects 1 to 4, comprising mixing an effective amount of the Bacillus subtilis with an agrochemically acceptable carrier, vehicle and / or adjuvant in a desired ratio for application.

[0141] The illustrative examples presented below serve to better describe the present invention. However, the described formulations relate only to some means of some embodiments of the present invention and should not be considered to limit the scope thereof.

[0142] Examples

[0143] Tests have been conducted to evaluate the nematicidal effect of the Bacillus strains of the present invention in vitro and under greenhouse conditions.

[0144] Example 1

[0145] Root Colonization and Biofilm Formation:

[0146] Bacillus subtilis DSM 32324 is able to form strong biofilms on plant root surfaces. Biofilm formation was analyzed in vitro on multiwell plates and in vivo on plant roots. The ability of the Bacillus subtilis DSM 32324 strain to form biofilms was compared with the Bacillus paralicheniformis DSM 33113 strain, another rhizobacterium with the ability to colonize plant roots. Compared to Bacillus paralicheniformis DSM 33113, Bacillus subtilis DSM 32324 was more capable of forming biofilms under in vitro conditions ( Figure 1 ).exist Figure 2 In the figure, the colonization of plant roots by Bacillus subtilis DSM 32324 can be seen under a microscope and compared with the colonization by Bacillus paralicheniformis DSM 33113.

[0147] Example 2

[0148] Secondary metabolite production

[0149] In addition to the biofilm forming ability of the present Bacillus subtilis DSM 32324, it is also of interest to study the production of a group of secondary metabolites whose nematicidal effects have been described in different scientific studies. The main effects of these secondary metabolites on nematodes are described herein and summarized in Table 1.

[0150] Surfactin is a cyclic lipopeptide compound that has the property of inhibiting egg hatching and increasing larval mortality of root-knot nematode species (Kavitha et al., 2012). When surfactin and fengyogenin are produced by bacteria that live in symbiosis with plants, they trigger plant immune responses mediated by the induction of hydroperoxides and lipoxygenases (Ongena et al., 2007).

[0151] Dimethyl disulfide can reduce the activity of southern root-knot nematode larvae, gall incidence, and nematode reproduction (Bui and Desaeger, 2021). Dimethyl disulfide is considered to be one of the main volatile organic compounds (VOCs) with nematicidal properties, and its use as a fumigant has been explored (Yin et al., 2021).

[0152] Bacillus subtilis DSM 32324 also showed extracellular protease activity, which is widely distributed among Bacillus strains with nematicidal activity, indicating that these enzymes may play an important role in bacteria-nematode-plant-environment interactions and that they can serve as important nematicidal factors to balance nematode populations in soil (Lian et al., 2007). The main effect is related to the degradation of the nematode cuticle.

[0153] In addition, Bacillus subtilis DSM 32324 strain also possesses the gene purL (99% identity), which has been identified as the gene responsible for mediating nematicidal activity against different plant parasitic nematodes such as Ditylenchus destructor, Bursaphelenchus xylophilus and javanica (Xia et al., 2011).

[0154]

[0155]

[0156] Table 1. List of metabolites and enzyme activities as well as bionematicidal activities and the associated described mechanisms of Bacillus subtilis DSM 32324. + means that the enzyme activity or metabolite is produced at low concentrations, ++ means that the enzyme activity or metabolite is produced at medium concentrations, and +++ means that the enzyme activity or metabolite is produced at high concentrations (compared to well described Bacillus subtilis strains).

[0157] Example 3

[0158] Nematode Disease Suppression:

[0159] The suppression of nematodes in plants was evaluated under two different conditions. In the first experiment, Arabidopsis roots were treated with vegetative cells of DSM 32324 and then inoculated with larvae of the southern root-knot nematode. The goal of the experiment was to measure the ability of the bacteria to reduce the penetration of the roots by the nematodes (compared to untreated roots). Bacillus subtilis DSM 32324 reduced the penetration of the nematodes in the plants, indicating that the strain's ability to form a strong biofilm and its ability to produce metabolites help protect the roots from penetration by the nematodes, thereby preventing nematode infection ( Figure 3 ).

[0160] The second experiment was conducted under greenhouse conditions. One two-week-old tomato seedling was transferred to each pot and 50 ml of 10 6 The area around the stem was drenched with a spore suspension of DSM 32324 at spores / ml. Each plant was inoculated with 3000 eggs of the southern root-knot nematode through two holes around the stem. The plants were kept in a greenhouse at 26°C for five weeks. To evaluate the experiment, the roots were lifted, cleaned, weighed and subjected to nematode extraction. Plants treated with DSM 32324 had a lower degree of infection and better root development ( Figure 4 ).

[0161] Example 4

[0162] A greenhouse experiment was conducted to evaluate the efficacy of Bacillus paralicheniformis DSM 33113 and Bacillus subtilis DSM 32324 strains for nematode control when applied alone or in combination. Two-week-old tomato seedlings (Solanum lycopersicum var. 'Tiny Tim') were transferred to pots (one seedling per pot) containing two liters of non-sterile soil. After transplanting the seedlings, the area around the plant stem was drenched with 50 ml of each treatment. Treatments consisted of spores suspended in water at a concentration of 10 6 spores / ml. After treatment with bacteria, each plant was inoculated with 3000 southern root-knot nematode eggs through two holes around the stem. The plants were kept under 26°C greenhouse conditions for five weeks. To evaluate the experiment, the roots were lifted, cleaned, weighed and nematode eggs were extracted. The number of eggs was used as a parameter for nematode development in tomato roots. The experiment was set up as a completely randomized design with nine biological replicates per treatment. The data were subjected to ANOVA followed by Tukey's test for grouping.

[0163] in conclusion

[0164] Strain DSM 32324 is able to prevent nematode larvae from penetrating the host roots, thereby reducing nematode development within the plant. Due to the different nematicidal modes of action exhibited by DSM 32324 and its proven efficacy in reducing nematode disease, this strain has the potential to be a good tool for controlling different plant-parasitic nematode species.

[0165] Preservation and Expert Solutions

[0166] The applicant requests that, prior to the date of grant of the patent, only samples of the deposited microorganisms described below be made available to the expert, subject to the regulations in force in the industrial property offices of the contracting states of the Budapest Treaty.

[0167] Table 2: Deposit made with a depository granted the status of an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0168] strain Accession number Date of deposit (year month day) Bacillus subtilis DSM 32324 08 June 2016

[0169] sequence

[0170] The sequence listing attached hereto forms part of this specification.

[0171] As specified herein, the sequence

[0172] SEQ ID NO: 1 is the purL gene sequence from Bacillus subtilis DSM32324

[0173]

[0174]

[0175] References

[0176] Dent,D.(2000)Insect pest management.Cambridge:Cabi Bioscience

[0177] Sikora,R.A.(1988).Interrelationship between plant health promotingrhizobacteria,plant parasitic nematodes and soil microorganisms.MedicineFaculty Landbouww Rijksuniv Gent,Landbouww,v.53,n.2b,p.867-878

[0178] Gu,Y.Q.,Mo,M.H.,Zhou,J.P.,Zou,C.S.,&Zhang,K.Q.(2007).Evaluation andidentification of potential organic nematicidal volatiles from soilbacteria.Soil Biology and Biochemistry,39(10),2567–2575.https: / / doi.org / 10.1016 / J.SOILBIO.2007.05.011

[0179] Ongena,M.,Jourdan,E.,Adam,A.,Paquot,M.,Brans,A.,Joris,B.,Arpigny,J.L.,&Thonart,P.(2007).Surfactin and fengycin lipopeptides of Bacillussubtilis as elicitors of induced systemic resistance in plants.EnvironmentalMicrobiology,9(4),1084–1090.https: / / doi.org / 10.1111 / j.1462-2920.2006.01202.x

[0180] Lian,L.H.,Tian,B.Y.,Xiong,R.,Zhu,M.Z.,Xu,J.,&Zhang,K.Q.(2007).Proteases from Bacillus:a new insight into the mechanism of action forrhizobacterial suppression of nematode populations.Letters in AppliedMicrobiology,45(3),262–269.https: / / doi.org / 10.1111 / j.1472-765X.2007.02184.x

[0181] Xia,Y.,Xie,S.,Ma,X.,Wu,H.,Wang,X.,&Gao,X.(2011).The purL gene ofBacillus subtilis is associated with nematicidal activity.FEMS MicrobiologyLetters,322(2),99–107.https: / / doi.org / 10.1111 / j.1574-6968.2011.02336.x

[0182] Yin,N.,Liu,R.,Zhao,J.-L.,Khan,R.A.A.,Li,Y.,Ling,J.,Liu,W.,Yang,Y.-H.,Xie,B.-Y.,&Mao,Z.-C.(2021).Volatile Organic Compounds of Bacillus cereusStrain Bc-cm103 Exhibit Fumigation Activity against Meloidogyneincognita.Plant Disease,105(4),904–911.https: / / doi.org / 10.1094 / PDIS-04-20-0783-RE

[0183] Bui,H.X.,&Desaeger,J.A.(2021).Volatile compounds as potential bio-fumigants against plant-parasitic nematodes–a mini review.Journal ofNematology,53,1–12.https: / / doi.org / 10.21307 / jofnem-2021-014

[0184] Kavitha,P.G.,Jonathan,E.I.,&Nakkeeran,S.(2012).Effects of crudeantibiotic of Bacillus subtilis on hatching of eggs and mortality ofjuveniles of Meloidogyne incognita.Nematologia Mediterranea,40,203–206.

[0185] Z.A.Siddiqui,I.Mahmood.(1999).Role of bacteria in the management ofplant parasitic nematodes:A review.Bioresource Technology,69(2),167-179.https: / / doi.org / 10.1016 / S0960-8524(98)00122-9.

Claims

1. A composition for use as a nematicide, comprising as an active ingredient Bacillus subtilis or a variant thereof and an agrochemically acceptable excipient and / or carrier, the Bacillus subtilis having the characteristics of a strain deposited in the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 32324, wherein the variant is obtained using the deposited strain as a starting material, and wherein the variant retains or further improves the nematicidal properties of DSM 32324.

2. The composition according to claim 1, wherein the Bacillus subtilis is a strain deposited in the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 32324.

3. The composition according to any one of claims 1 and 2, comprising the Bacillus subtilis, wherein the agrochemically acceptable excipient and / or carrier is selected from the group consisting of maltodextrin and silicon dioxide.

4. The composition according to any one of claims 1 to 3, wherein the composition is in the form of a wettable powder.

5. The composition according to any one of claims 1 to 3, wherein the composition is in the form of a liquid formulation.

6. The composition according to any one of claims 1 to 5, wherein the composition comprises 1.0 x 10 6 CFU / g to 1.0x 10 9 CFU / g.

7. A method for preparing the composition according to any one of claims 1 to 6, which comprises mixing an effective amount of the Bacillus subtilis with an agrochemically acceptable carrier, vehicle and / or adjuvant in a desired ratio for application.

8. Use of a composition according to any one of claims 1 to 6, or a composition obtainable from a process according to claim 7, for preventing, controlling, combating and / or inducing resistance to plant nematodes.

9. The use according to claim 8, wherein the plant nematodes are selected from the group consisting of: root-knot nematode, Pratylenchus, cyst nematode, spherosporean cyst nematode, Ditylenchus, Xiphinema, Bursaphelenchus, Radopholus, Reniform nematode, Heliconematode, Pearl nematode, Bursaphelenchus and Aphelenchus.

10. Use according to any one of claims 8 to 9, wherein the plant culture is selected from the group consisting of sugar cane, soybean, corn, wheat, potato, tomato, carrot, coffee and banana.

11. A method for preventing, controlling and / or combating plant nematodes on plants and / or their habitat, wherein the composition according to any one of claims 1 to 6 allows to act on these plant nematodes and / or their habitat.

12. The method of claim 11, wherein the composition acts by reducing the infestation rate of the plant nematodes.

13. The method of claim 11, wherein the composition acts by reducing the penetration of the plant nematodes into the roots of the plant.

14. A method for inducing resistance to plant nematodes, the method comprising applying to the plant and / or its habitat an effective amount of the composition according to any one of claims 1 to 6.

15. A kit comprising a composition according to any one of claims 1 to 5 or a composition obtainable from a method according to claim 6, instructions and a suitable receptor.

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