Nematode-killing composition containing Bacillus subtilis

Abstract

The present invention generally relates to a composition comprising Bacillus subtilis having a nematicidal effect against plants and / or plant nematodes on their habitats, its use, a process for its preparation, the use of Bacillus subtilis, the prevention, control, extermination and / or imparting of resistance against plant nematodes, and a kit. In particular, the present invention relates to Bacillus subtilis (DSM 32324).

Description

Technical Field

[0001] The present invention generally relates to a composition containing Bacillus subtilis having a nematicidal effect against plants and / or plant nematodes on their habitats, its use, the process for its preparation, the use of Bacillus subtilis, the prevention, control, extermination of plant nematodes and / or the imparting of resistance thereto, and a kit. In particular, the present invention relates to Bacillus subtilis (DSM 32324).

Background Art

[0002] The intensification of agricultural activities has caused ecological imbalances, and due to the reason that its products can maintain pest populations at an acceptable level, it has become necessary to use selective products that do not affect the balance of pests, their predators, parasitoids, and pathogens, which are responsible for much of the natural biological control (DENT, D., Insect pest management. Cambridge: Cabi Bioscience 2000).

[0003] To reverse this situation, it is recommended to develop a program for integrated pest management, defined as a decision-making system for the use of control tactics, alone or combined in a coordinated manner, in a cost / benefit-based management strategy that takes into account the interests and / or impacts on farmers, society, and the environment. Control measures available for these management systems include entomopathogenic nematodes and insect parasitoids that include biological pest control in different regions.

[0004] In the current situation of modern environmentally conscious societies, which are interested in protecting the environment, biological control is considered an attractive alternative and / or supplement to conventional methods of control. Biological control is the use of one organism (predator, parasite or pathogen) that attacks another organism that is causing economic damage to a crop. This is a very common strategy in agriculture and in systems related to the environment and integrated pest management (IPM).

[0005] Biological control has a positive impact in reducing or eliminating the use of pesticides and improving farmers' incomes, but an analysis of a series of experiments around the world shows that the results are still concentrated on only a few crops. There is still much to be developed in the area of pest and disease control.

[0006] Diseases caused by nematodes are very difficult to control, and over the past few decades, various methods such as resistant varieties, non-crop rotations and the application of nematicidal chemicals have been tested. However, the available management means are either of low effectiveness or cannot completely control the various nematode species commonly found in mixtures in the soil. Chemical nematicides are very toxic compounds that can cause serious harm to the environment after overuse. The restrictions on the use of chemical pesticides are increasing the interest in research on alternative methods of nematode control.

[0007] Considerable emphasis has been placed on research into biological control involving the use of bacteria that colonize the roots of plants, called rhizosphere bacteria. Beneficial rhizosphere bacteria that promote growth and / or act in the biological control of phytopathogenic bacteria are called plant growth-promoting rhizobacteria or PGPR. PGPR can bring about a combination and concentration of substances that increase the availability of nutrients to plants and promote growth.

[0008] Social pressure to replace nematicides with environmentally acceptable products or environmentally friendly practices is driving the search for alternative methods to control nematodes. In this context, biological control is considered to be one of the alternatives within an integrated approach aimed at ensuring the sustainable development of agriculture. The use of natural enemies that can play a role in reducing nematode populations below the economic injury threshold is an area of research.

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

[0010] European Patent Application Publication No. 0705807 A1 relates to a bacterial preparation for soil conditioning, including bacteria belonging to the genus Bacillus such as Bacillus subtilis and Bacillus licheniformis. This preparation can prevent root damage to crops caused by nematodes.

[0011] Brazilian Patent Application PI 0604602-9 A relates to the use of Bacillus subtilis in combination with Bacillus licheniformes for the control of plant nematodes.

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

[0013] International Publication No. WO 2012 / 020014 pamphlet relates to a composition comprising Bacillus subtilis and Bacillus licheniformis, which has a nematicidal effect against plant nematodes.

[0014] Sikora, R.A. (Interrelationship between plant health promoting rhizobacteria, plant parasitic nematodes and soil microorganisms. Medicine Faculty Landbouww Rijksuniv Gent, Landbouww, v.53, n.2b, p.867 - 878, 1988) used the treatment of seeds of various crops with Bacillus strains and observed a reduction in the infection of approximately 60% - 65% of Meloidogyne arenaria, M. incognita and Rotylenchulus reniformis.

[0015] There is still a need for a new nematicide with higher safety and effectiveness to protect crops from nematode infection.

[0016] The inventors have continued extensive screening and research to solve the problem of providing a biological method for controlling nematodes based on the identification of a new Bacillus subtilis strain that exhibits various nematicidal modes of action and has proven effectiveness in reducing nematode diseases.

Summary of the Invention

Means for Solving the Problems

[0017] The present invention provides a composition for use as a nematicide, comprising, as an active ingredient, Bacillus subtilis or a variant thereof having the characteristics of the strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 32324, which is obtained by using this deposited strain as a starting material and retains or further improves the nematicidal properties of DSM 32324, and an agriculturally acceptable excipient and / or carrier.

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

[0019] The purpose of the study described in the examples was to investigate the effect of DSM 32324 on the invasion of nematode larvae in plant roots based on the measurement of the biofilm-forming ability and the effect on the number of galls and eggs in plant roots after nematode planting and administration of the composition of the present invention.

[0020] The present invention explains and demonstrates the benefits of the Bacillus subtilis strain DSM 32324 in improving the health of plants to which it is administered by showing a nematicidal effect.

[0021] Definitions In general, the terms and expressions used herein have their meanings recognized in the art, which can be found by referring to standard textbooks, academic journal references and the context known to those skilled in the art. The following definitions are provided to facilitate understanding of their specific use in relation to the present disclosure.

[0022] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0023] As used herein, the term "and / or" is intended to mean both conjunctive ("and") and disjunctive ("or") usage, i.e., "A and / or B" is intended to mean "A alone or B alone or A and B together".

[0024] Composition: As used herein, the term "composition" means a composition comprising a carrier and at least one bacterial strain as described herein.

[0025] Controlling plant nematode infection: As used herein, the term "controlling plant nematode infection" means a method and / or composition that inhibits plant nematode infection in a plant to some extent or completely. Thus, the term "controlling plant nematode infection" means that plant nematode infection is reduced or completely eliminated, improving the overall health of the plant.

[0026] Effective amount / concentration / dosage: As used herein, the terms "effective amount", "effective concentration" or "effective dosage" are defined as the amount, concentration or dosage of a bacterial strain sufficient to improve the overall health of a plant and confer benefits similar to those demonstrated in the examples.

[0027] The actual effective dosage in absolute numbers depends on factors including the health status of the target plant and other components present. The "effective amount", "effective concentration" or "effective dosage" of a bacterial strain can be determined by conventional assays known to those of skill in the art. Examples of effective amounts are given in Examples 3 and 4.

[0028] Isolated: As used herein, the term "isolated" means that the bacterial strain described herein is in a form or environment that does not occur in nature, i.e., the strain is at least partially removed from one or more or all of the naturally occurring components it is associated with in nature.

[0029] Plant-parasitic nematodes: As used herein, the term "plant-parasitic nematodes" means nematodes that live as parasites on plants and cause significant damage to a very wide range of crops, which results in poor productivity and significant annual economic losses. The term plant nematodes is intended to mean that 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 (Heterodera and Globodera species), and lesion nematodes (Pratylenchus species). Brief Description of the Drawings

[0030]

Figure 1

[0031]

Figure 2

[0032]

Figure 3

[0033]

Figure 4

[0034]

Figure 5

Mode for Carrying Out the Invention

[0035] Brief Description of the Sequences SEQ ID NO: 1 SEQ ID NO: 1 is the purL gene sequence of Bacillus subtilis DSM32324.

[0036] Rhizosphere bacteria Soil is a base for complex biotic communities, and soil microorganisms, prokaryotes and eukaryotes account for the majority in both number and diversity. Some prokaryotes have an ecological niche as the rhizosphere and / or the root surface of plants, and these prokaryotes reproduce, survive and protect themselves from the antagonism of the soil microflora. These organisms are collectively called rhizosphere bacteria.

[0037] In relation to plants, rhizobacteria can have harmful, neutral or beneficial effects. Those that exert beneficial effects - growth promotion and biological control of diseases - are called PGPR (Plant Growth-Promoting Rhizobacteria). It is estimated that only 0.6% of rhizobacteria have beneficial effects on the plants associated with them.

[0038] PGPR are used for the biological control of plant diseases, thereby increasing crop productivity. The question of why and how this biological control is exerted remains a topic requiring further research.

[0039] In some situations, biological control can occur through direct antagonism exerted by PGPR against pathogens, which involves known mechanisms of antibiosis: production of antimicrobial substances, direct parasitism, and competition for nutrients and ecological niche. Research has shown that certain PGPR seem to act as inducers of ISR (Induced Systemic Resistance) in the sense that plants become protected systemically from two or more pathogens, unlike classical biological control which aims to perform control more specifically.

[0040] Some rhizobacteria produce antagonistic secondary metabolites that affect the movement of nematodes in vitro, while other rhizobacteria inhibit larval hatching and the process by which the larvae invade the roots.

[0041] An important parameter affecting the ability of PGPR to infect and colonize the plant surface is the ability of PGPR to form biofilms.

[0042] The present invention provides excellent biofilm-forming Bacillus strains that produce a series of secondary metabolites having nematicidal effects as described in the prior art.

[0043] Rhizosphere bacteria can inhibit plant parasitic nematodes through both direct and indirect different ways. Direct antagonism is based on the synthesis or parasitism of lytic enzymes, toxic proteins, and volatile compounds. Indirect antagonism is expressed through competition for nutrients, induction of systemic resistance (ISR), or the release of molecules that regulate nematode behavior including recognition, feeding, and sex ratio.

[0044] Therefore, the control of nematodes by rhizosphere bacteria can be carried out in various ways and may affect different stages of the nematode life cycle.

[0045] - Eggs: Antibiotics and toxins produced by bacteria in the rhizosphere can diffuse into the soil and may be absorbed by nematode eggs, killing the cells and preventing their embryogenesis.

[0046] - Hatching: Rhizosphere bacteria decompose root exudates that act as hatching factors for many species of nematodes. Then, the compounds absorbed by nematode eggs may be inactivated or cause deformities during development, which prevents hatching.

[0047] - Target orientation and motility: The conversion of root exudates into metabolic by-products of rhizosphere bacteria can prevent nematodes from simply recognizing chemotaxis, so that nematodes may continue to move randomly, and ultimately, their energy reserves are depleted and they die without invading the roots. When nematodes recognize root exudates and move towards the roots, some bacterial compounds exhibit nematostatic properties and can reduce nematode motility, preventing them from reaching the roots.

[0048] - Host recognition: Substances produced by rhizosphere bacteria can be absorbed by the roots and change their chemical composition, preventing nematodes from recognizing their host. It is also thought that rhizosphere bacteria bind to lectins on the root surface, which are the binding sites between nematodes and their plant hosts, thereby interfering with recognition.

[0049] - Root invasion: Toxins or repellents produced by rhizosphere bacteria at high concentrations in the root surface area or intracellular content of root epidermis cells can prevent nematodes from invading the host plant.

[0050] - Food: Rhizosphere bacteria or their metabolites can be absorbed by plants, which can recognize the presence of nematodes and induce hypersensitive reactions in giant cells, which is the main mechanism of host resistance to nematodes of the genus Meloidogyne. This resistance, called systemic resistance, is not specific to plants, i.e., it is a reaction induced therein by the presence of PGPR.

[0051] - Reproduction: Some rhizosphere bacteria have a greater effect on reducing the number of galls than on reducing the number of eggs, which can be one of the mechanisms of action.

[0052] The present invention discloses Bacillus strains identified as having this series of characteristics, which are powerful agents for the management of plant parasitic nematodes. The identified main characteristics are, in addition to the ability to form strong biofilms, the ability to synthesize surfactin, dimethyldisulfide and extracellular protease (all secondary metabolites), and enzyme activities with demonstrated inhibitory effects on plant parasitic nematodes. As shown in Examples 3 and 4, this special combination of these modes of action in a single microorganism has a great potential to interfere with nematode behavior around the rhizosphere and reduce nematode invasion and its reproduction in the plant host.

[0053] Bacteria of the genus Bacillus Spore-forming rhizosphere bacteria have several advantages over chemical pesticides or other biological control agents. They are easy to mass-produce, they are easy to store, they can be adapted to formulation technology, and they do not require genetic manipulation.

[0054] Spore-forming rhizobacteria can be applied by treating the culture medium, immersing the roots of seedlings in the bacterial suspension, wetting the plants with the bacterial suspension by soaking the seeds in the suspension of rhizobacteria, or applying PGPR using seed pelleting.

[0055] An example of spore-forming rhizobacteria is the Bacillus species, which has received considerable attention in recent years for its safety to the environment and its ability to deliver different modes of action in suppressing nematode populations in the soil.

[0056] Bacillus species are Gram-positive bacteria, characterized by having a thick cell wall and no outer membrane, different from Gram-negative bacteria. Most of the wall of Gram-positive bacteria is composed of peptidoglycan.

[0057] Gram-positive species are grouped according to their morphological and biochemical characteristics. The genus Bacillus belongs to the group of spore-forming bacteria. Species that form spore structures resistant to environmental changes can withstand dry heat and certain chemical disinfectants for a moderate period. They can survive in dry soil for several years.

[0058] For example, the beneficial effects of Bacillus species such as B. subtilis, when applied near the seeds or soil, are not only due to the antagonistic effect on pathogens. PGPR has a positive impact on germination, growth, and crop yield due to the production of substances that promote plant growth (e.g., volatile organic compounds, plant hormones) and improve plant nutrition (e.g., solubilization of phosphorus).

[0059] Use of Bacillus species in nematode control Plant-parasitic nematodes cause significant damage to a wide range of crops worldwide, resulting in poor productivity and significant economic losses in agricultural production. The estimated losses are approximately $100 billion per year globally for economically important crops. Root-knot nematodes (Meloidogyne species), cyst nematodes (Heterodera and Globodera species) and lesion nematodes (Pratylenchus species) are among the most economically and chemically important species due to their complex relationships with their host plants, wide host ranges and levels of damage caused by infection.

[0060] Due to the lack of resistance sources for breeding, the lack of adaptability of resistant cultivars to certain regions and planting seasons or the breakdown of resistance under outdoor conditions, the use of cultivars resistant to nematodes is not always possible. Chemical control of nematodes is generally not recommended. This is because it leaves residues in food, causes environmental pollution, is less effective and expensive. These drawbacks have led to increased social pressure to limit the use of chemicals, which in turn creates a demand for the agriculturalization of products that are non-toxic to humans and animals, inexpensive and very effective in controlling nematodes.

[0061] Many soil microorganisms are known as nematode parasites or predators. The actions of these microorganisms can result in direct or indirect effects through interference with steps in the pathogen life cycle.

[0062] Genus Meloidogyne Nematodes of the genus Meloidogyne (root-knot nematodes) exhibit remarkable diversity on plant hosts, occur in various regions of the earth, and cause losses to various crops. The main symptom is the presence of galls on the roots of plants. These galls are abnormal or hypertrophied root system formations. Affected plants grow poorly, have low yields, early leaf drop, and pre-mature senescence, and death of the plant can sometimes occur when symptoms are exacerbated under conditions of nutritional stress and drought.

[0063] First, second-stage juveniles (J2) of the genus Meloidogyne invade the roots and establish feeding sites in the region of the root stele. Throughout nematode development, J2s differentiate into adult males or females. Adult males leave the root system, while females remain within the roots. During the development of female Meloidogyne, approximately 500 eggs are laid. The eggs are deposited in a gelatinous matrix outside the roots, where J2s hatch, thereby reinfecting the root system. The life cycle of Meloidogyne is approximately four weeks and can be extended under less favorable temperature conditions. Temperatures below 20°C or above 35°C and conditions of soil drying or waterlogging reduce nematode occurrence and survival.

[0064] Control of Meloidogyne incognita and Meloidogyne javanica can be carried out, depending on the crop in question, using chemicals containing certain active ingredients, such as carbofuran, ethoprophos, aldicarb, metam-sodium, or fenamiphos, among others. The implementation of crop rotation is also important for control for the placement of non-host crops such as peanuts, pineapples, rice, or the use of plant species as ground cover that are also non-hosts, such as Sudan grass.

[0065] The nematode Meloidogyne exigua is very active and spreads over a very wide area in cultivation fields. Another method of chemical control in areas infected by M. exigua includes the use of chemicals containing either the active ingredient terbufos or carbofuran, depending on the type of crop. Among non-host crops, outstanding ones are cotton, peanuts, rice and oats.

[0066] Similar to M. exigua, M. paranaensis also spreads over a wide area in coffee plantations, but is less of a problem in other crops. Regarding its control, positive results have been obtained with the fungus Paecilomyces lilacinus, which reduced the nematode population in the roots of tomato "Santa Clara" in the greenhouse.

[0067] The use of rhizosphere bacteria related to biological control is already known. However, the applicant is currently developing a composition containing Bacillus subtilis DSM 32324 or its variants, which have a nematicidal effect against plant nematodes.

[0068] Accordingly, a first aspect of the present invention relates to the novel strain or its variants described herein for use as a nematicide.

[0069] It is obvious to those skilled in the art that by using the deposited strain as a starting material, readers skilled in the art can routinely obtain further variants or derivatives thereof that retain the appropriate characteristics and advantages described herein by methods of natural strain improvement or reisolation techniques. Therefore, the term "its variants" in the first aspect relates to variant strains obtained by methods of natural strain improvement using the deposited strain as a starting material.

[0070] Plant nematodes and / or plant-parasitic nematodes are widely regarded as any nematodes that have a negative impact on commercial crops. Nematodes that can be controlled using the compositions of the present invention include nematodes of the following genera: Meloidogyne, Pratylenchus, Heterodera, Globodera, Ditylenchus, Tylenchulus, Xiphinema, Radopholus, Rotylenchulus, Helicotylenchus, and Belonolaimus. Species of the genus Meloidogyne are considered particularly suitable because they are responsible for approximately 95% of all parasitism on crops, causing approximately 5% of all crop losses worldwide. The compositions of the present invention can contain, in addition to the active ingredients, agriculturally acceptable excipients and / or vehicles. The compositions of the present invention further comprise an agriculturally acceptable carrier, vehicle, and / or adjuvant.

[0071] Agriculturally acceptable carriers, vehicles, and / or adjuvants are considered known to the reader skilled in the art and can be selected from the group consisting of, but not limited to, maltodextrin, silicon dioxide, modified zeolite, kaolinite, lignin, starch, chitosan, and calcium carbonate. In a preferred embodiment, the agriculturally acceptable carrier, vehicle, and / or adjuvant are maltodextrin and silicon dioxide.

[0072] The compositions of the present invention play a particular role in controlling nematodes in crops.

[0073] In one embodiment, the composition of the present invention may be mixed with additional components relevant to the agrochemical field, including but not limited to microorganisms, biological and / or chemical insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators, and / or fertilizers (present in an amount suitable to benefit plant growth and / or confer protection against pathogen infection in susceptible plants), carriers, surfactants, dispersants, enzymes, and / or yeast extracts.

[0074] The main crops of plants include sugarcane, coffee, soybeans, cotton, corn, potatoes, tomatoes, tobacco, bananas, rice, wheat, avocados, pineapples, pumpkins, cacao, coconuts, oats, onions, lettuce, sugar beets, carrots, cassava, legumes, sunflowers, peppers, turnips, apples, strawberries, okra, radishes, and onions.

[0075] Regarding fruit cultivation, citrus fruits, grapes, guavas, papayas, figs, peaches, plums, and loquats are particularly suitable, and regarding horticulture, eggplants and cruciferous plants are particularly suitable.

[0076] Regarding flower gardening, roses, chrysanthemums, lisianthus, gerberas, amaryllises, begonias, and carnations.

[0077] The present invention relates to a composition comprising Bacillus subtilis DSM 32324 or a variant thereof, a kit prepared by a process comprising or preparing this composition, and an instruction manual and a suitable container.

[0078] A process for preparing a composition comprising Bacillus subtilis DSM 32324 or a variant thereof, together with an agriculturally acceptable carrier, vehicle, and / or adjuvant, and the use of said composition for controlling, repelling, and / or conferring specific resistance against plant nematodes are also provided.

[0079] Furthermore, the present invention relates to the use of an effective amount of Bacillus subtilis DSM 32324 or a variant thereof in the manufacture of a pesticidal composition having a nematicidal effect against plant nematodes in plant cultivation, and to a process for controlling plant nematodes, eradicating plant nematodes, and / or conferring specific resistance to plant nematodes.

[0080] Suitable Bacillus strains of the present invention are provided in commercially suitable forms known to those skilled in the art. Thus, in certain embodiments, the Bacillus strains of the present invention are present in a dried (e.g., spray-dried) or frozen form.

[0081] The compositions of the present invention can be coated onto plant seeds and can contain from about 1.0×10 2 CFU / seed to about 1.0×10 9 CFU / seed of Bacillus, such as Bacillus subtilis DSM 32324 spores. In another embodiment of the present invention, the composition can be coated onto plant seeds in an amount of from about 1.0×10 6 CFU / g (seed) to about 1.0×10 11 CFU / g (seed) of Bacillus, such as Bacillus subtilis DSM 32324 spores.

[0082] Plant seeds can include, but are not limited to, seeds of monocotyledonous and dicotyledonous plants, such as grains, corn, sweet corn, popcorn, seed corn, silage corn, feed corn, rice, wheat, barley, sorghum, cruciferous vegetables, broccoli, cabbage, cauliflower, Chinese cabbage, collard, kale, mustard green, kohlrabi, bulb vegetables, onion, garlic, shallot, fruiting vegetables, pepper, tomato, eggplant, ground cherry, tomatillo, okra, grape, herb / spice, cucurbit vegetables, cucumber, cantaloupe, melon, honeydew, squash, watermelon, pumpkin, eggplant, leafy vegetables, lettuce, celery, spinach, parsley, chicory, leguminous plants / vegetables (moist and dried beans and peas), bean, green bean, snap bean, shell bean, soybean, dried bean, chickpea, lima bean, pea, split pea, lentil, oilseed crops, canola, sunflower, cotton, flax, peanut, rapeseed, safflower, sesame, sunflower, soybean, root / tuber and bulb vegetables, carrot, potato, sweet potato, sugar beet, ginger, horseradish, radish, Chinese carrot, turnip, sugarcane, sugar beet, grass or turfgrass seeds.

[0083] In one or more embodiments, plant seeds can include dried beans, corn, wheat, soybeans, canola, rice, cotton, grass and turfgrass seeds.

[0084] In an alternative embodiment, the compositions of the invention comprising Bacillus or Bacillus subtilis DSM 32324 can be added to: soil or growth medium surrounding a plant; soil or growth medium before sowing plant seeds in the soil or growth medium; or soil or growth medium before planting a plant, a plant cutting, a plant transplant or a plant callus tissue in the soil or growth medium.

[0085] In a preferred embodiment, the composition of the present invention comprising Bacillus or Bacillus subtilis DSM 32324 is added to the soil or growth medium surrounding the plant.

[0086] When the composition of the present invention is added to the soil or growth medium surrounding the plant; the soil or growth medium before sowing the plant seeds in the soil or growth medium; or the soil or growth medium before planting the plant, plant cutting, plant transplant or plant callus tissue in the soil or growth medium, the composition contains an amount of Bacillus, for example, about 1.0×10 6 CFU / ml to about 1.0×10 9 CFU / ml of Bacillus subtilis DSM 32324 spores.

[0087] In one or more embodiments, the plant, plant cutting, plant transplant or plant callus tissue may include soybean, bean, green bean, wheat, cotton, corn, pepper, tomato, potato, cassava, grape, strawberry, banana, peanut, pumpkin, squash, eggplant, sugarcane and cucumber.

[0088] The composition containing the Bacillus strain as described herein may be in the form of a liquid formulation, an oily suspension, a powder, a dry wettable powder, a sprayable granule or a dry granule wettable powder. More specifically, the composition may be, for example, an emulsion concentrate (EC), a suspension concentrate (SC), a suspoemulsion (SE), a capsule suspension (CS), a granule wettable powder (WG), an emulsion granule (EG), an oil-in-water emulsion (EO), a water-in-oil emulsion (EW), a microemulsion (ME), an oily suspension (OD), an oil miscible flowable (OF), an oil miscible liquid (OL), a soluble concentrate (SL), a microspray suspension (SU), a microspray liquid (UL), a dispersion concentrate (DC), a wettable powder (WP) or any technically possible formulation in combination with an agriculturally acceptable adjuvant.

[0089] The present invention relates to a composition comprising Bacillus subtilis DSM 32324 or a variant thereof, a kit prepared by a process comprising or preparing this composition, and an instruction manual and a suitable container.

[0090] Also provided is a process for preparing a composition comprising Bacillus subtilis DSM 32324 or a variant thereof together with a carrier, vehicle and / or adjuvant acceptable as a pesticide, and the use of said composition for controlling, repelling 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 in the manufacture of a pesticidal composition having a nematicidal effect in plant cultivation and a process for promoting the health of plants.

[0092] In the context of the present invention, "a variant thereof" should be understood as a Bacillus subtilis having a change in the wild-type nucleotides of the genome of the organism (e.g., Bacillus subtilis DSM 32324) that results in a phenotypic change in the organism, and the change can 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 should be understood as a genetic mutation that results in the removal of one or two nucleotides from the wild-type nucleotide sequence of the genome of the organism; an insertion should be understood as the addition of one or more nucleotides to the wild-type nucleotide sequence; a substitution (or point mutation) should be understood as a genetic mutation in which a nucleotide of the wild-type nucleotide sequence is changed by another nucleotide; a frameshift should be understood as a genetic mutation caused by the insertion or deletion of several nucleotides within the wild-type nucleotide sequence that is not divisible by 3, thus changing the reading frame and resulting in a translation that is completely different from the original reading frame; the introduction of a stop codon should be understood as a point mutation in the DNA sequence that results in a premature stop codon; and the inhibition of substrate binding of the encoded protein should be understood as any mutation in the nucleotide sequence that results in a change in the protein sequence responsible for preventing the binding of the substrate to its catalytic site of the protein. Furthermore, a knockout mutant should be understood as a genetic mutation that results in the removal or deletion of a gene, such as an entire gene or an entire open reading frame, from the genome of the organism.

[0093] Algorithms for aligning arrays and determining the degree of array identity between arrays are well known in the art. For the purposes of the present invention and by way of example, one of these algorithms is to apply standard parameter settings (matrix: BLOSUM62, gap cost: existence: 11 extension: 1, conditional combination score matrix correction), using blastp provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov to align both arrays, and subsequently, based on the quantification of identical amino acid pairs at the same positions across the aligned amino acid sequences. A similar process can be carried out using blastn provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov, which in this case applies standard parameters, to align nucleotide sequences.

[0094] In one embodiment, the present invention relates to a variant of Bacillus subtilis DSM 32324, and the average nucleotide identity (ANI) of this Bacillus subtilis variant is at least 99%, for example at least 99.5%, for example at least 99.8%, for example at least 99.9% identical to the genome of the strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 32324.

[0095] The following listed aspects are further included in the present invention.

[0096] Aspect 1. A Bacillus subtilis that expresses the purL gene, wherein the purL gene is encoded by a sequence having at least 95%, for example at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 1.

[0097] Aspect 2. The Bacillus subtilis according to Aspect 1, wherein the genome of the strain is at least 99%, for example at least 99.5%, for example at least 99.8%, for example at least 99.9% identical to the genome of the strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 32324.

[0098] Aspect 3. The Bacillus subtilis according to any one of Aspects 1 to 2, which produces secondary metabolites surfactin, fengycin, dimethyldisulfide and exhibits extracellular protease activity.

[0099] Aspect 4. The Bacillus subtilis according to any one of Aspects 1 to 3, which shows better or equivalent suppression of nematode diseases in plants when compared with the effect of the Bacillus paralicheniformis strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 33113, either alone or in combination with the said Bacillus subtilis.

[0100] Aspect 5. A composition comprising the Bacillus subtilis according to any one of Aspects 1 to 4.

[0101] Aspect 6. A composition comprising the Bacillus subtilis according to any one of the preceding Aspects 1 to 5 and its excipient and / or carrier acceptable as a pesticide.

[0102] Aspect 7. A composition for use as a nematicide, comprising Bacillus subtilis or a variant thereof having the characteristics of the strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under deposit number DSM 32324 as an active ingredient, which is obtained by using the deposited strain as a starting material and retains or further improves the nematicidal properties of DSM 32324, including said variant, and a pesticidally acceptable excipient and / or carrier, the Bacillus subtilis as described in any of the preceding aspects 1 to 6.

[0103] Aspect 8. The composition according to any of the preceding aspects 6 to 7, wherein the pesticidally acceptable excipient and / or carrier is selected from the group consisting of maltodextrin, silicon dioxide, modified zeolite, kaolinite, lignin, starch, chitosan, and calcium carbonate.

[0104] Aspect 9. The composition according to any of the preceding aspects 5 to 8, which is in the form of a wettable powder.

[0105] Aspect 10. The composition according to any of the preceding aspects 5 to 8, which is in the form of a liquid formulation.

[0106] Aspect 11. The composition according to any of the preceding aspects 5 to 10, which contains at least 1×10 9 CFU / gram of said Bacillus subtilis.

[0107] Aspect 12. The composition according to any of the preceding aspects 5 to 11, which further contains one or a combination of a microorganism, a biological and / or chemical insecticide, fungicide, nematicide, bactericide, herbicide, plant extract, plant growth regulator and / or fertilizer, a carrier, a surfactant, a dispersant, an enzyme and / or a yeast extract, in an amount suitable for benefiting plant growth and / or preventing pathogen infection in plants susceptible to infection, and preferably, the microorganism is a Bacillus strain.

[0108] Aspect 13. Use of the composition according to any one of Aspects 5 to 12 or Bacillus according to any one of Aspects 1 to 4 as a biocidal nematicide, and / or plant growth promoter, and / or plant health promoter, and / or plant disease or pest control agent.

[0109] Aspect 14. Use of the composition according to Aspect 13 or Bacillus according to any one of Aspects 1 to 4 for controlling plant nematodes, eradicating plant nematodes, and / or conferring specific resistance to plant nematodes.

[0110] Aspect 15. The plant nematodes are selected from the group consisting of Meloidogyne, Pratylenchus, Heterodera, Globodera, Ditylenchus, Tylenchulus, Xiphinema, Radopholus, Rotylenchulus, Helicotylenchus and Belonolaimus, and the use according to any one of Aspects 13 or 14.

[0111] Aspect 16. The plant nematodes are selected from the group consisting of Meloidogyne incognita, Meloidogyne javanica, Meloidogyne exigua, Meloidogyne paranaensis, Heterodera glycines and Pratylenchus zeae, and the use according to any one of Aspects 13 to 15.

[0112] Aspect 17. The composition according to any one of Aspects 5 to 12 or the Bacillus according to any one of Aspects 1 to 4 is used according to any one of Aspects 13 to 16, which is applied to a plant, on a seed, or within the habitat of the plant.

[0113] Aspect 18. The use according to Aspect 17, wherein the plant is selected from the group consisting of corn, rice, sugarcane, soybean, potato, sugar beet, carrot, coffee, tomato, and banana.

[0114] Aspect 19. A process for preventing, controlling, and / or eradicating plant nematodes on a plant and / or its habitat, the process comprising applying 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 to the plant and / or its habitat.

[0115] Aspect 20. A process for preventing, controlling, and / or eradicating plant nematodes on a plant and / or its habitat, the process comprising applying 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 to the plant nematodes and / or their habitat.

[0116] Aspect 21. The process according to Aspect 19 or 20, wherein the composition acts by reducing the infection rate of plant nematodes.

[0117] Aspect 22. The process according to Aspect 19 or 20, wherein the composition acts by reducing the invasion of plant nematodes into the roots of plants.

[0118] Aspect 23. A kit comprising a composition as defined in any one of Aspects 5 to 12, instructions for use, and a suitable container.

[0119] Aspect 24. Plant seeds coated with a composition according to any one of Aspects 5 to 12, present in an amount suitable to be beneficial for plant growth and / or to provide protection against pathogen infection in plants susceptible to infection.

[0120] Aspect 25. The composition is about 1.0×10 2 CFU / seeds to about 1.0×10 9 CFU / seeds of the amount of spores of Bacillus subtilis as described in any of Aspects 1 to 4, and the plant seeds of Aspect 24.

[0121] Aspect 26. The composition is about 1.0×10 6 CFU / g (seeds) to about 1.0×10 11 CFU / g (seeds) of the amount of spores of Bacillus subtilis as described in any of Aspects 1 to 4, and the plant seeds of Aspect 24.

[0122] Aspect 27. The composition further comprises one or a combination of a microorganism, a biological or chemical insecticide, a fungicide, a nematicide, a bactericide or a plant growth regulator present in an amount suitable to be beneficial for plant growth and / or to prevent pathogen infection in plants susceptible to infection, preferably, the microorganism is a Bacillus strain, and the plant seeds of Aspect 24.

[0123] Aspect 28. purL is encoded by SEQ ID NO: 1 or its homolog, and Bacillus as described in any of Aspects 1 to 27.

[0124] Aspect 29. A method of treating a plant to enhance plant growth and / or promote plant health and / or control plant diseases, comprising 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, said step comprising a) the step of applying a Bacillus strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under deposit number DSM 33113, or b) a method that results in similar biofilm formation or similar pellicle biofilm formation when compared to the method comprising the step of applying a composition having a Bacillus strain deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under deposit number DSM 33113.

[0125] Aspect 30. The method according to the preceding Aspect 29, further comprising the step of applying a Bacillus strain according to any one of the preceding Aspects 1 to 4 or a composition according to any one of the preceding Aspects 5 to 12 to the soil.

[0126] Aspect 31. The method according to any one of the preceding Aspects 29 to 30, wherein the step of applying a Bacillus strain according to any one of the preceding Aspects 1 to 4 or a composition according to any one of the preceding Aspects 5 to 12 is applied before, during or after the plant or plant part comes into contact with the soil.

[0127] 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, bulbs, tubers, corms and rhizomes.

[0128] Aspect 33. A process for preparing a composition as defined in any one of claims 1 to 4, comprising mixing an amount of Bacillus subtilis effective for application in a desired ratio with a carrier, vehicle and / or adjuvant acceptable as a pesticide.

[0129] The examples shown below serve to explain the present invention in more detail. However, the formulations described merely refer to some means of some embodiments of the present invention and should not be regarded as limiting its scope.

Example

[0130] Tests were conducted to evaluate the nematicidal effect of the Bacillus strains of the present invention both in vitro and under greenhouse conditions.

[0131] Example 1 Root colonization and biofilm formation: Bacillus subtilis DSM 32324 has the ability to form a strong biofilm on the surface of plant roots. This biofilm formation was analyzed both in vitro on multi-well plates and in vivo on plant roots. The ability of the Bacillus subtilis DSM 32324 strain to form a biofilm was compared with that of the Bacillus paralicheniformis DSM 33113 strain, another rhizosphere bacterium with the ability to colonize plant roots. The ability of Bacillus subtilis DSM 32324 to form a biofilm under in vitro conditions was stronger compared to Bacillus paralicheniformis DSM 33113 (Figure 1). In Figure 2, how the colonization of Bacillus subtilis DSM 32324 on plant roots in vivo was observed under the microscope and how it was compared with the colonization of Bacillus paralicheniformis DSM 33113 can be seen.

[0132] Example 2 Secondary metabolite production: It is reasonable to investigate the production of a series of secondary metabolites whose nematicidal effects have already been described in various scientific studies, along with the biofilm-forming ability of Bacillus subtilis DSM 32324 of the present invention. The main effects of these secondary metabolites on nematodes are described herein and summarized in Table 1.

[0133] Surfactin is a cyclic lipopeptide compound with the properties of inhibiting the hatching of eggs of the genus Meloidogyne species (Kavitha et al., 2012) and increasing the mortality rate of larvae. When produced by bacteria living in association with plants, surfactin and fengycin trigger plant immune responses mediated by the induction of hydroperoxides and lipoxygenases (Ongena et al., 2007).

[0134] Dimethyldisulfide can reduce the motility, gall formation rate, and nematode reproduction of Meloidogyne incognita larvae (Bui & Desaeger, 2021). Dimethyldisulfide is considered one of the main volatile organic compounds (VOCs) with nematicidal properties, and its use as a fumigant is being explored (Yin et al., 2021).

[0135] Bacillus subtilis DSM 32324 also exhibits extracellular protease activity widely distributed in Bacillus strains with nematicidal activity, suggesting that these enzymes may play important roles in the bacteria-nematode-plant-environment interactions and that these enzymes can play a role as important nematicidal factors in balancing nematode populations in the soil (Lian et al., 2007). This main role is associated with the degradation of the nematode cuticle.

[0136] The Bacillus subtilis DSM 32324 strain further has the gene purL (99% identity), which has been identified as being responsible for mediating nematicidal activity against various plant parasitic nematodes such as Ditylenchus destructor, Bursaphelenchus xylophilus, and Meloidogyne javanica (Xia et al., 2011).

[0137]

Table 1

[0138] Example 3 Nematode disease suppression: The suppression of nematodes in plants was evaluated under two different conditions. In the first experiment, the roots of Arabidopsis thaliana were treated with vegetative cells of DSM32324, and then larvae of Meloidogyne incognita were inoculated onto them. The purpose of this experiment was to measure the ability of the bacteria to reduce the invasion of nematodes into the roots compared to untreated roots. Bacillus subtilis DSM 32324 reduced the invasion of nematodes into the plants, suggesting that the ability of this strain to form strong biofilms and produce metabolites promotes the protection of the roots from nematode invasion and thus nematode infection (Figure 3).

[0139] The second experiment was conducted under greenhouse conditions. One tomato seedling (second week) was transplanted into each pot, and the area around the stem was perfused with 50 ml of a DSM 32324 spore suspension at a concentration of 10 6 spores / ml. 3000 eggs of Meloidogyne incognita were inoculated into each plant through two holes around the stem. The plants were maintained at 26°C under greenhouse conditions for 5 weeks. For the evaluation of the experiment, the roots were lifted, cleaned, weighed, and used for nematode extraction. The plants treated with DSM 32324 had lower infection and better root development than the untreated roots (Figure 4).

[0140] Example 4 A greenhouse experiment was set up to evaluate the effectiveness of B. paralicheniformis DSM 33113 and B. subtilis DSM 32324 strains against nematode control when applied alone or in combination. Tomato seedlings (Solanum lycopersicum "Tiny Tim" variant) (second week) were transplanted into pots filled with 2 liters of non-sterile soil (one seedling per pot). After transplanting the seedlings, the area around the plant stems was irrigated with 50 ml of each treatment solution. Each treatment solution consisted of spores suspended in water at a concentration of 10 6 spores / ml. After treatment with the bacteria, 3000 eggs of M. incognita were planted into each plant through two holes around the stem. The plants were maintained for 5 weeks under greenhouse conditions at 26 °C. For the evaluation of the experiment, the roots were lifted, cleaned, weighed, and used for the extraction of nematode eggs. The number of eggs was used as a parameter for nematode development inside the tomato roots. This experiment was set up in a completely randomized design using 9 biological replicates per treatment. The data were subjected to ANOVA followed by Tukey's test grouping.

[0141] Conclusion Strain DSM 32324 has the ability to prevent the invasion of nematode larvae into the roots of the host and thus reduce nematode development inside the plant. Due to the various nematicidal modes of action demonstrated for DSM 32324 and its proven effectiveness in reducing nematode diseases, this strain has the potential to fill the lack of an excellent means for controlling various plant-parasitic nematode species.

[0142] Deposit and solutions for experts The Applicant requests that samples of the deposited microorganisms specified below be made available only to experts, subject to the samples being maintained in accordance with the available regulations administered by the Industrial Property Offices of the Contracting States of the Budapest Treaty until the date of grant of this patent.

[0143] [Table 2]

[0144] Sequence Part of this description consists of the Sequence Listing attached hereto. As specified herein, Sequence Listing SEQ ID NO:1 is the purL gene sequence of Bacillus subtilis DSM32324. [Chemical Formula] [Chemical Formula]

[0145] References Dent, D. (2000) Insect pest management. Cambridge: Cabi Bioscience Sikora, R. A. (1988). Interrelationship between plant health promoting rhizobacteria, plant parasitic nematodes and soil microorganisms. Medicine Faculty Landbouww Rijksuniv Gent, Landbouww, v. 53, n. 2b, p. 867 - 878 Gu, Y.Q., Mo, M.H., Zhou, J.P., Zou, C.S., & Zhang, K.Q. (2007). Evaluation and identification of potential organic nematicidal volatiles from soil bacteria. Soil Biology and Biochemistry, 39(10), 2567 - 2575. https: / / doi.org / 10.1016 / J.SOILBIO.2007.05.011 Ongena, M., Jourdan, E., Adam, A., Paquot, M., Brans, A., Joris, B., Arpigny, J.L., & Thonart, P. (2007). Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environmental Microbiology, 9(4), 1084 - 1090. https: / / doi.org / 10.1111 / j.1462 - 2920.2006.01202.x 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 for rhizobacterial suppression of nematode populations. Letters in Applied Microbiology, 45(3), 262 - 269. https: / / doi.org / 10.1111 / j.1472 - 765X.2007.02184.x Xia, Y., Xie, S., Ma, X., Wu, H., Wang, X., & Gao, X. (2011). The purL gene of Bacillus subtilis is associated with nematicidal activity. FEMS Microbiology Letters, 322(2), 99 - 107. https: / / doi.org / 10.1111 / j.1574-6968.2011.02336.x 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 cereus Strain Bc-cm103 Exhibit Fumigation Activity against Meloidogyne incognita. Plant Disease, 105(4), 904 - 911. https: / / doi.org / 10.1094 / PDIS-04-20-0783-RE Bui, H.X., & Desaeger, J.A. (2021). Volatile compounds as potential bio-fumigants against plant-parasitic nematodes - a mini review. Journal of Nematology, 53, 1 - 12. https: / / doi.org / 10.21307 / jofnem-2021-014 Kavitha, P.G., Jonathan, E.I., & Nakkeeran, S. (2012). Effects of crude antibiotic of Bacillus subtilis on hatching of eggs and mortality of juveniles of Meloidogyne incognita. Nematologia Mediterranea, 40, 203 - 206. Z.A. Siddiqui, I. Mahmood. (1999) Role of bacteria in the management of plant 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 a Bacillus subtilis or a variant thereof having the characteristics of the strain deposited under deposit number DSM 32324 at Deutsche Sammlung von Mikroorganismen und Zellkulturen as an active ingredient, obtained by using the deposited strain as a starting material and retaining or further improving the nematicidal properties of DSM 32324, and an excipient and / or carrier acceptable as a pesticide.

2. The composition according to claim 1, wherein the Bacillus subtilis is the strain deposited under deposit number DSM 32324 at Deutsche Sammlung von Mikroorganismen und Zellkulturen.

3. The composition according to claim 1 or 2, comprising the Bacillus subtilis, wherein the excipient and / or carrier acceptable as a pesticide is selected from the group consisting of maltodextrin and silicon dioxide.

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

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

6. 1.0×10 ６ CFU / gram to 1.0×10 ９ The composition according to any one of claims 1 to 5, comprising 1.0×10 ６ CFU / gram to 1.0×10 ９ CFU / gram.

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

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

9. The use according to claim 8, wherein the plant nematode is selected from the group consisting of the genus Meloidogyne, Pratylenchus, Heterodera, Globodera, Ditylenchus, Tylenchulus, Xiphinema, Bursaphelenchus, Radopholus, Rotylenchulus, Helicotylenchus, Nacobbus, Aphelenchoides and Belonolaimus.

10. The use according to claim 8 or 9, wherein the plant cultivation is selected from the group consisting of sugarcane, soybean, corn, wheat, potato, tomato, carrot, coffee and banana.

11. A process for preventing, controlling and / or exterminating plant nematodes on plants and / or their habitats, wherein the composition according to any one of claims 1 to 6 is applied to the plant nematodes and / or their habitats.

12. The process according to claim 11, wherein the composition acts by reducing the infection rate of the plant nematodes.

13. The process according to claim 11, wherein the composition acts by reducing the invasion of the plant nematodes into the roots of the plants.

14. A process for imparting induction of resistance to plant nematodes, the process comprising applying an effective amount of the composition according to any one of claims 1 to 6 to plants and / or their habitats.

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

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