Compositions and methods for improving soil microbial populations

Abstract

A method of preserving or stimulating the growth of beneficial soil microorganisms in soil for crop or plant growth is disclosed, the method comprising applying to a plant, locus or propagation material thereof an effective amount of a composition comprising cyfluflurram.

Description

Background Art

[0001] The present invention relates to the use of nematicidal compositions comprising four-membered ring carboxamide compounds, particularly cyclobutylcarboxamide compounds, and their use in improving plant soil microbiome compatibility, thereby promoting crop growth in soil, enhancing pest control, and / or facilitating soil bioremediation. The present invention also relates to methods for using these compositions in agriculture. These compositions modify the soil microbiome in a manner beneficial to plants by promoting favorable microbial levels, microbial activity, microbial metabolism, and / or microbial diversity. The compositions described herein can be used to increase the usefulness of the soil microbiome to plants.

[0002] Nematodes (also called round worms) make up the phylum Nematoda, a diverse phylum of animals that inhabit a wide range of environments, particularly moist surfaces and tissues.

[0003] Many nematodes have evolved into successful plant and animal parasites and cause significant economic losses, particularly in agriculture. Nematode parasites of plants can infest all parts of a plant, including roots, developing flower buds, leaves, and stems. Therefore, there is a need for safe and effective nematode control in plants and / or soil.

[0004] Nematicides have been widely used in agricultural systems as part of pest and / or weed control strategies. However, due to the inherent properties of these products, their use can cause adverse effects on soil microflora (including bacteria, fungi, algae and protozoa), and thus also on their related functions and key roles in the cycling of organic and inorganic nutrients in the soil. A reduction in soil biodiversity, which is associated with a reduction in the available interactions between all soil organisms, is believed to reduce the soil's resistance to environmental changes and, therefore, its ability to recover.

[0005] Therefore, there remains an urgent need to develop environmentally safe, effective methods for controlling plant parasitic nematodes without negatively impacting soil microflora. Furthermore, it would be beneficial if the growth of beneficial microflora, such as bacterial and fungal species, could be supported. In particular, there remains a need for nematicides with reduced secondary effects.

[0006] For example, WO 2013 / 143811 and WO 2015 / 003951 disclose cyclobutylcarboxamide compounds and methods for their preparation. Particularly effective cyclobutylcarboxamide compounds (known as cyclobutrifluram in the ISO name) are used as potent nematicides. The chemical structure of cyclobutrifluram is a compound having formula (I):

[0007]

[0008] Cyclobutaflutol, its salts or N-oxides, and its close analogs have been reported to have some activity against root-knot nematodes (such as the Meloidogyne genus) and cyst-forming nematodes (such as the Heterodera genus), as well as some fungicidal activity. However, it has now been unexpectedly discovered that cyclobutaflutol is very effective in controlling and improving the diversity and health of the soil microbiome. Summary of the Invention

[0009] Thus, in a first aspect, the present invention relates to a method of stimulating the growth of beneficial soil microorganisms.

[0010] In a second aspect, the invention relates to the method, wherein stimulating the growth of beneficial soil microorganisms comprises stimulating the growth of bacterial and fungal species that exhibit biocontrol and / or soil remediation activity.

[0011] In a third aspect, the present invention relates to a method according to the invention, wherein the beneficial microorganism is selected from the group consisting of bacteria of the genera Sphingomonas, Bacillus and Pseudomonas, and fungi of the genus Trichoderma.

[0012] In a fourth aspect, the present invention relates to a method according to the invention, wherein cyclopentaflutamine is applied to seeds or propagation material in an amount of between 1 gram and up to and including 1000 grams of cyclopentaflutamine per 100 kg of seeds or propagation material, preferably wherein cyclopentaflutamine is applied to seeds or propagation material in an amount of between 10 grams and 400 grams of cyclopentaflutamine per 100 kg of seeds or propagation material; and still more preferably wherein cyclopentaflutamine is applied to seeds or propagation material in an amount of between 20 grams and 100 grams of cyclopentaflutamine per 100 kg of seeds or propagation material.

[0013] In a fifth aspect, the present invention relates to a method according to the invention, the composition being applied via drip irrigation, sprinkler irrigation, soil drench or flood irrigation; in furrow application, or as a seed treatment.

[0014] In a sixth aspect, the present invention relates to a method according to the invention, application of the composition is performed before or after planting.

[0015] In a seventh aspect, the present invention relates to a method according to the invention, the composition further comprising one or more additional biologically active agents, preferably one or more acaricides, bactericides, fungicides, insecticides, nematicides and / or plant activators.

[0016] In an eighth aspect, the present invention relates to the use of a composition comprising cyclohexane for maintaining or improving the integrity of the soil microbiome in soil in need of treatment.

[0017] In a ninth aspect, the present invention relates to a method according to the invention, wherein the soil in need of stimulation is selected by identifying a portion of the soil containing plant pests present in an amount sufficient to damage or reduce the growth of plants growing in the soil, preferably wherein the plant pests include plant parasitic nematodes, in particular endoparasitic nematodes, semi-endoparasitic nematodes and ectoparasitic nematodes.

[0018] In a tenth aspect, the present invention relates to the use of a composition comprising cyclohexane for improving the plant health of plants grown in soil in need of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The extent of the development of fungal microbial community modifications in the soil microbiome associated with cyclohexene treatment is shown. The differential abundance of fungal taxa in the treated soil after 97 days was detected by PCR amplification and sequential high-throughput sequencing of fungal taxonomic marker genes (small subunit ribosomal RNA gene (16S) and ribosomal internal transcribed spacer (ITS), respectively) in plant-associated soil samples.

[0020] Figure 2 Depicted in Figure 2 Variation of the Shannon-Wiener biodiversity index of bacterial (16S) and fungal (ITS) communities at the sites over time (T0, T1, T2 and T3).

[0021] Figure 3 Depicted are the differentially abundant beneficial bacterial taxa (DA) found in four different treatments of potato plants in a field trial, when compared to control conditions at different time points. Different letters indicate locations (A) and (B). Different shapes indicate different treatments: squares for the comparative example fluopyram; triangles (A'200) and diamonds (A"250) for cytofluanid according to the invention at two different concentrations. The size of the corresponding symbol indicates the degree of significance (p-value), where larger sizes are more significant and the smallest sizes indicate no significant fold change in the observed taxa. The gradient scale ranges from dark grey (negative values) to light grey (positive values).

[0022] Figure 4Depicted are the differential beneficial fungi found in four different treatments of potato plants in a field trial. Again, different letters indicate locations (A) and (B). Different shapes indicate different treatments: squares for the comparative example fluopyram; triangles (A'200) and diamonds (A"250) for cytofluanid according to the invention at two different concentrations. The size of the corresponding symbol indicates the degree of significance (p-value), where larger sizes are more significant and the smallest sizes indicate no significant fold change in the observed taxon. The gradient scale ranges from dark grey (negative values) to light grey (positive values). DETAILED DESCRIPTION

[0023] The compositions according to the invention can advantageously be used for controlling or destroying pests (such as insects and / or fungi) which occur, in particular, on plants, especially useful and ornamental plants in agriculture, horticulture and forestry, or on organs of such plants, such as fruits, flowers, leaves, stalks, rhizomes, seeds or roots, and in some cases even on plant organs formed at a later point in time, for protection against these pests.

[0024] It is known that soil with a healthy microbial population is best suited for effective pest control, robust plant growth, and efficient biodegradation of unwanted soil pollutants. Soil microorganisms decompose dead plant and animal material and mediate the biodegradation of most man-made pesticides. To thrive, these microorganisms require a readily available carbon source as food. Furthermore, stimulating the proliferation of native soil microorganisms is more desirable than adding microorganisms from external sources.

[0025] The compounds of formula (I) according to the invention are active ingredients of preventive and / or therapeutic value in the field of pest control. Even when applied at low application rates, they can be used to combat pests (such as insects) and fungi that are resistant to pesticides. These compounds of formula (I) have a very favorable biocidal spectrum and are well tolerated by warm-blooded species, fish and plants.

[0026] It has now been found that the compounds of formula I according to the invention have (for practical purposes) a very advantageous spectrum of activity for protecting the health of plant and soil microbiota and are therefore particularly useful for protecting plants from attack and damage by nematodes and for promoting plant and soil health.

[0027] The present invention therefore also provides for compositions comprising compounds of the invention having formula (I) to be used for improving soil microbiome.

[0028] It has now also been found that the compounds of formula I according to the invention have (for practical purposes) a very advantageous spectrum of activity for protecting useful plants against attack and damage by fungi. The present invention therefore also makes available fungicidal compositions comprising the compounds of the invention, such as formula (I).

[0029] Cyclobutaflutol as disclosed above represents a cis racemate: the phenyl ring on the left and the pyridyl-C(=O)-NH group on the right are cis to each other on the cyclobutyl ring, as illustrated for compounds of formula (Ia) and formula (Ib):

[0030]

[0031] The racemic compound of cyclohexane is a 1:1 mixture of compounds of formula (Ia) and (Ib). The wedge-shaped bonds shown in the compounds of formula (Ia) and (Ib) represent the absolute stereochemistry, while the thick straight bonds, as shown for the compound of formula (I), represent the relative stereochemistry in the racemic compound. It has also been unexpectedly discovered that one enantiomer of cyclohexane is particularly useful in methods for controlling or preventing infection of plants by phytopathogenic microorganisms of the genus Aspergillus.

[0032] Therefore, preferably, there is provided a process according to the invention, wherein cyclohexane is in the form of the (1S,2S) stereoisomer (Ia):

[0033]

[0034] As is known to those skilled in the art, cyclohexane is typically applied as part of a pesticidal composition according to the methods of the present invention, and compounds of formula (I) are therefore particularly useful for nematode control. Thus, a method for controlling or preventing infestation of plants by nematode plant pests is provided, comprising applying a pesticidal composition comprising cyclohexane and one or more formulation adjuvants to a plant crop, its locus, or its propagation material, and, according to the present invention, simultaneously improving the soil microbiome. The preparation of cyclohexane is disclosed in WO 2013 / 143811 and WO 2015 / 003951, which are incorporated herein by reference.

[0035] Crop or plant health is closely linked to the balance of beneficial microbial species in the soil, whereby soil type, soil fertility, water, competing microorganisms, and plants interact in complex ways. The interactions between microbial species and plants are further influenced by agricultural practices, which can either improve or degrade the soil microbiome. Thus, fertile or highly productive soils may contain a different natural microbial composition than soils that are nutrient-depleted and associated with low crop productivity.

[0036] Different microbial species are closely associated with plants, either on above-ground plant surfaces in the phyllosphere, at root surfaces in the soil rhizosphere, or intimately as endophytes.

[0037] Large-scale DNA analyses of these microbial associations have revealed unexpected phylogenetic complexity, and it has been hypothesized that complex microbiomes may be associated with plant health, stress tolerance, secondary metabolite accumulation, and disease tolerance.

[0038] Furthermore, plants specifically select microbial communities from their local environment and fine-tune the microbiome at the crop variety level. Root-associated microbes can enhance plant and root growth by promoting nutrient cycling and acquisition, providing growth advantages through direct plant stimulation, by mediating biofertilization, or through biocontrol of pathogens.

[0039] Agriculturally useful beneficial microbial groups include plant growth-promoting rhizobacteria (PGPR), pathogen-suppressing bacteria, mycorrhizae, nitrogen-fixing bacteria, stress-tolerant endophytes, and other microorganisms with a range of biodegradation capabilities.

[0040] Microorganisms involved in the nitrogen cycle include nitrogen-fixing bacteria (Azotobacter zo tobacter and Bradyrhizobium zo bium), nitrogen-fixing cyanobacteria, ammonia-oxidizing bacteria (such as Nitrosomonas and Nitrospira), nitrite-oxidizing genera (such as Nitrospira and Nitrobacter), and heterotrophic denitrifying bacteria, particularly Pseudomonas and Azotobacter.

[0041] Bacterial genera thought to be active in solubilizing and increasing phosphorus availability to plants include Pseudomonas, Bacillus, Micrococcus, and Flavobacterium, as well as various fungal genera, particularly Trichoderma. Bacillus and Clostridium species help solubilize and mobilize potassium.

[0042] Phytostimulation of plant growth, and alleviation of biotic and abiotic stresses are delivered by many bacterial and fungal associates, either directly through the production of stimulatory secondary metabolites or indirectly by triggering low-level plant defense responses.

[0043] Applicants have discovered that cyclohexane can, among other things, enhance the growth of beneficial microorganisms, including bacteria of the genera Sphingomonas, Bacillus, and / or Pseudomonas, and fungi of the genus Trichoderma. Sphingomonas species (such as S. sediminicola, S. japsi, and S. daechungensis) are widely associated with bioremediation and fungicide properties. Bacillus species (such as Bacillus halmapalus and B. cereus) are associated with several beneficial functions in the soil and for plant health (such as plant hormone production, pathogen protection, and abiotic stress protection).

[0044] Advantageously, this growth enhancement can be carried out simultaneously or subsequently in combination with a method for controlling damage to plants and parts thereof by phytoparasitic nematodes, in particular endoparasitic nematodes, semi-endoparasitic nematodes and ectoparasitic nematodes, in particular the following phytoparasitic nematodes, such as the root knot nematode, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne arenaria and other root knot nematode species; the cyst-forming nematode, Globodera rostochiensis and other Globodera species; the cereal cyst nematode, Heterodera glycines, Heterodera schachtii, Heterodera rufipogon trifolii, and other Heterodera species; Seed gall nematodes, Anguina species; Stem and foliar nematodes, Aphelenchoides species; Stingnematode, Eelonolaimus longicaudatus, and other Belonolaimus species; Pine nematodes, Bursaphelenchus xylophilus, and other Bursaphelenchus species; Ring nematodes, Criconema species, Criconemella species, Criconemoides species, Mesocriconema species; Stem and bulb nematodes, nematodes), Ditylenchus destructor, Ditylenchus dipsaci, and other Ditylenchus species; Awl nematodes, Dolichodorus species;Spiral nematodes, Heliocotylenchus multicinctus, and other species of the genus Helicotylenchus; Sheath and sheathoid nematodes, species of the genus Hemicycliophora, and species of the genus Hemicriconemoides; Hirshmanniella species; Lance nematodes, species of the genus Hoploaimus; false rootknot nematodes, species of the genus Nacobbus; Needlenematodes, Longidorus elongatus, and other species of the genus Longidorus; Pin nematodes nematodes), Pratylenchus species; Lesionnematodes, Pratylenchus neglectus, Pratylenchuspenetrans, Pratylenchus curvitatus, Pratylenchusgoodeyi, and other Pratylenchus species; Burrowingnematodes, Radopholus similis, and other Radopholus species; Reniform nematodes, Rotylenchus robustus, Rotylenchus reniformis, and other Rotylenchus species; Scutellonema species; Stubby root nematodes nematodes), Trichodorus primitivus, and other species of the genera Trichodorus and Paratrichodorus;Stunt nematodes, Tylenchorhynchus claytoni, Tylenchorhynchus dubius, and other Tylenchorhynchus species; Citrus nematodes, Tylenchulus species; Dagger nematodes, Xiphinema species; and other plant-parasitic nematode species, such as Subanguina spp., Hypsoperine spp., Macroposthonia spp., Melinius spp., Punctoderas spp., and Quinisulcius spp. It should be noted that where the term "species" is used above, it may include one or more species; as often abbreviated by the term "species (spp.)".

[0045] The term "microorganism" herein refers to microorganisms including, but not limited to, bacteria, archaea, fungi, and algae such as microalgae. In some instances, the microorganism is a unicellular organism, such as bacteria, cyanobacteria, some fungi, or some algae. In other instances, the term microorganism includes multicellular organisms, such as certain fungi or algae, for example, multicellular filamentous fungi or multicellular algae.

[0046] The term "preserving or improving the soil microbiome" refers to the composition of the soil microbiome in which the presence of useful microorganisms is maintained or improved, while plant pathogenic microorganisms are preserved or reduced. This means that soil biodiversity is preserved or improved to preserve a range of vibrant life forms in the soil and enable plants to develop larger root systems in infested fields.

[0047] In the method according to the invention, for the beneficial composition comprising both the (1S,2S) and (1R,2R) stereoisomers of cyclohexane, the ratio of the (1S,2S) stereoisomer to its enantiomer (1R,2R) is preferably greater than 1: 1. More preferably, the ratio of (1S,2S) to (1S,2S) is greater than 1.5:1, more preferably greater than 2.5:1, especially greater than 4:1, advantageously greater than 9:1, desirably greater than 20:1, and especially greater than 35:1.

[0048] Also understood as part of the present invention are mixtures containing up to 50%, preferably up to 40%, more preferably up to 30%, especially up to 20%, advantageously up to 10%, desirably up to 5%, in particular up to 3% of the trans stereoisomer of the compound of formula (I) (i.e., wherein the phenyl and pyridyl-C(=O)-NH groups are trans to each other). Preferably, the ratio of cis isomers to their trans isomers is greater than 1.5:1, more preferably greater than 2.5:1, especially greater than 4:1, advantageously greater than 9:1, desirably greater than 20:1, in particular greater than 35:1.

[0049] According to another embodiment of the present invention, a method is provided, wherein the composition is a suspension concentrate composition. According to another embodiment of the present invention, a method for using the composition is provided, the method comprising the steps of: providing cyclopentaflutol or a composition comprising cyclopentaflutol as defined in any one of the above embodiments; and applying the composition to a propagation material; and planting the propagation material in a medium, preferably a soil portion.

[0050] According to another embodiment of the present invention, a method for using a composition is provided, comprising the steps of: providing cyclohexane or a composition comprising cyclohexane as defined in any one of the above embodiments; and applying the composition to a plant, its propagation material or a locus.

[0051] According to another embodiment of the present invention, there is provided a method for growing plants, the method comprising applying or treating their propagation material with cyclohexane or a composition comprising cyclohexane as defined above, for simultaneously or subsequently enhancing the activity of beneficial microorganisms in the surrounding soil.

[0052] According to another embodiment of the present invention, there is provided a method for selectively reducing the population of Fusarium in soil, for example by at least 50%, preferably by at least 80%, for example by no more than 20%, preferably no more than 10%, while leaving the level of arbuscular mycorrhizalfungi substantially unchanged, the method comprising applying or treating the soil with cyclohexane as defined herein or a composition comprising cyclohexane.

[0053] It has also been found that cybutaflutamine can be combined with arbuscular mycorrhizal fungi to have a synergistic effect in promoting plant growth and / or plant health. Such fungi can be fungi already naturally present in the soil, or can be additional arbuscular mycorrhizal fungi that are added to the soil separately or simultaneously with cybutaflutamine.

[0054] Preferably, the propagation material is a seed. More preferably, cyclobutaflutol is applied to the seed in an amount between 1 gram and 1000 grams of cyclobutaflutol / 100kg seeds. Still more preferably, cyclobutaflutol is applied to the seed in an amount between 10 grams and 400 grams of cyclobutaflutol / 100kg seeds, and again more preferably, wherein cyclobutaflutol is applied to the seed in an amount between 20 grams and 100 grams of cyclobutaflutol / 100kg seeds. Preferably, the method provided herein can be applied to useful plants. Applying the compound of the present invention to seed is a preferred method of application. The term "seed" includes all types of seeds, plant propagation materials and plant propagators, including but not limited to real seeds, seed pieces, suckers, grains, bulbs, fruits, tubers, cereals, rhizomes, cuttings, cut branches and the like and in a preferred embodiment means real seeds.

[0055] The application according to the method or use according to the invention is preferably carried out on crops or plants, their loci or their propagation material. Preferably the application is to plants or their propagation material, more preferably to the propagation material.

[0056] Application of cyclohexane or a pesticidal composition comprising cyclohexane can advantageously be carried out according to any conventional application route, such as foliar application, drench application, soil application, in-furrow application, etc. The applicant has found that plant health can be significantly improved due to the beneficial effect of the composition on the soil microbiome composition over time.

[0057] The methods as defined above are suitable for use on any plant, including those that have been genetically modified to be resistant to active ingredients such as herbicides, or those that have been genetically modified to produce biologically active compounds that control infestation by plant pests.

[0058] Typically, cyclohexane is used in the form of a composition (eg, a formulation) containing a carrier. The cyclohexane or a composition comprising cyclohexane as defined in any of the above embodiments can be used in different forms, such as aerosol sprays, capsule suspensions, concentrated cold mists, dustable powders, emulsifiable concentrates, oil-in-water emulsions, water-in-oil emulsions, encapsulated granules, fine granules, flowable concentrates for seed treatment, gases (under pressure), gas-producing products, granules, concentrated hot mists, large granules, microgranules, oil-dispersible powders, oil suspensions, oil-soluble liquids, pastes, plant sticks, powders for dry seed treatment, seeds coated with pesticides, soluble concentrates, soluble powders, solutions for seed treatment, suspension concentrates (flowable concentrates), ultra-low volume (ulv) liquids, ultra-low volume (ulv) suspensions, water-dispersible granules or tablets, water-dispersible powders for slurry treatment, water-soluble granules or tablets, water-soluble powders for seed treatment and wettable powders.

[0059] The formulations typically comprise a liquid or solid carrier and optionally one or more customary formulation auxiliaries which may be solid or liquid, for example, non-epoxidized or epoxidized vegetable oils (e.g., epoxidized coconut oil, rapeseed oil or soybean oil), defoamers (e.g., silicone oils), preservatives, clays, inorganic compounds, viscosity modifiers, surfactants, binders and / or tackifiers.

[0060] The compositions may further comprise fertilizers, micronutrient donors or other agents that influence plant growth, and include combinations containing a compound of the invention and one or more other biologically active agents, such as bactericides, fungicides, nematicides, plant activators, acaricides and insecticides.

[0061] The compositions are prepared in a manner known per se, in the absence of auxiliaries, for example by grinding, screening and / or compressing the solid compound according to the invention, and in the presence of at least one auxiliary agent, for example by intimately mixing and / or grinding the compound according to the invention with one or more auxiliary agents. In the case of the solid compound according to the invention, the grinding / milling of the compound is to ensure a specific particle size.

[0062] Examples of compositions for use in agriculture are emulsifiable concentrates, suspension concentrates, microemulsions, oil dispersibles, directly sprayable or dilutable solutions, spreadable pastes, dilute emulsions, soluble powders, dispersible powders, wettable powders, dusts, granules or capsules in a polymeric substance, these compositions comprising at least cyclohexane and the type of composition being chosen to be appropriate for the intended purpose and the prevailing circumstances.

[0063] Typically, these compositions comprise from 0.1% to 99% (especially from 0.1% to 95%) of cyclohexane and from 1% to 99.9% (especially from 5% to 99.9%) of at least one solid or liquid carrier, it being generally possible that from 0 to 25% (especially from 0.1% to 20%) of the composition are surfactants, the % in each case being percentages by weight.

[0064] Although concentrated compositions tend to be preferred for commercial purposes due to their smaller size and inherent greater stability in the absence of water, end users typically use dilute compositions having significantly lower concentrations of active ingredients. Examples of foliar formulation types for premix compositions are: GR: granules; WP: wettable powder; WG: water-dispersible granules (dusts); SG: water-soluble granules; SL: soluble concentrate; EC: emulsifiable concentrate; EW: oil-in-water emulsion; ME: microemulsion; SC: aqueous suspension concentrate; CS: aqueous capsule suspension; OD: oil-based suspension concentrate, and SE: aqueous suspoemulsion.

[0065] Examples of seed treatment formulation types for premix compositions are: WS: wettable powder for seed treatment; LS: solution for seed treatment; ES: emulsion for seed treatment; FS: suspension concentrate for seed treatment; WG: water-dispersible granules; and CS: aqueous capsule suspension. Examples of formulation types suitable for tank-mix compositions are solutions, diluted emulsions, suspensions, or mixtures thereof, and dusts.

[0066] In view of the nature of the formulation, the application method (such as foliar application, drench application, spray application, atomization application, dusting application, broadcast application, coating application or pouring application) can be selected according to the intended purpose and the prevailing circumstances.

[0067] Tank-mix compositions are typically prepared by diluting one or more premix compositions containing different pesticides and, optionally, additional adjuvants, with a solvent (eg, water).

[0068] Suitable carriers and adjuvants can be solid or liquid and are the substances customary in formulation technology, such as natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or fertilizers.

[0069] Typically, tank mix formulations for foliar or soil application contain 0.1% to 20%, especially 0.1% to 15%, of the desired ingredients and 99.9% to 80%, especially 99.9% to 85%, of solid or liquid adjuvants (including, for example, solvents such as water), wherein these adjuvants may be surfactants, in amounts of 0 to 20%, especially 0.1% to 15%, based on the tank mix formulation.

[0070] Typically, premix formulations for foliar application contain 0.1% to 99.9%, especially 1% to 95%, of the desired ingredients and 99.9% to 0.1%, especially 99% to 5%, of solid or liquid adjuvants (including, for example, solvents such as water), where these adjuvants may be surfactants, in an amount of 0 to 50%, especially 0.5% to 40%, based on the premix formulation.

[0071] Typically, tank mix formulations for seed treatment applications contain 0.25% to 80%, especially 1% to 75%, of the desired ingredients and 99.75% to 20%, especially 99% to 25%, of solid or liquid adjuvants (including, for example, solvents such as water), wherein these adjuvants may be surfactants, in an amount of 0 to 40%, especially 0.5% to 30%, based on the tank mix formulation.

[0072] Typically, premix formulations for seed treatment applications contain 0.5% to 99.9%, in particular 1% to 95%, of the desired ingredients and 99.5% to 0.1%, in particular 99% to 5%, of solid or liquid adjuvants (including, for example, solvents such as water), wherein these adjuvants may be surfactants, in an amount of 0 to 50%, in particular 0.5% to 40%, based on the premix formulation.

[0073] While commercial products are preferably formulated as concentrates (ie pre-mix compositions or formulations), end users typically use diluted formulations (eg so-called tank-mix compositions).

[0074] Preferred seed treatment premix formulations are aqueous suspension concentrates. Conventional processing techniques and machines can be used, such as fluidized bed technology, drum milling method, static rotation (rotostatic) seed treatment machine and drum coating machine, to apply the formulation to seeds. Other methods (such as spouted bed) can also be useful. Seeds can be pre-sized before coating. After coating, seeds are typically dried and then transferred to a sizing machine to be sized. Such procedures are known in the art. Cyclobutaflutamide is particularly suitable for use in soil and seed treatment applications.

[0075] While commercial products will preferably be formulated as concentrates (eg, premix compositions (formulations)), the end user will typically employ a diluted formulation (eg, a tank-mix composition).

[0076] Preferred seed treatment premix formulations are aqueous suspension concentrates. Conventional processing techniques and machines can be used, such as fluidized bed technology, drum milling method, static rotation (rotostatic) seed treatment machine and drum coating machine, to apply the formulation to seeds. Other methods (such as spouted bed) can also be useful. Seeds can be pre-sized before coating. After coating, seeds are typically dried and then transferred to a sizing machine to be sized. Such procedures are known in the art. Compounds of the present invention are particularly suitable for use in soil and seed treatment applications.

[0077] In general, the premix composition of the present invention contains 0.5% to 99.9% by mass, in particular 1% to 95%, advantageously 1% to 50% by mass of the desired ingredients, and 99.5% to 0.1% by mass, in particular 99% to 5% by mass of solid or liquid adjuvants (including, for example, solvents such as water), wherein the adjuvant (or adjuvants) may be surfactants, the amount of which is 0 to 50% by mass, in particular 0.5% to 40% by mass, based on the premix formulation.

[0078] In a preferred embodiment, independent of any other embodiment, the compound of formula (I) is in the form of a composition for treating (or protecting) plant propagation material, wherein the composition for protecting plant propagation material may additionally comprise a colorant. The composition or mixture for protecting plant propagation material may also comprise at least one polymer from the group consisting of water-soluble and water-dispersible film-forming polymers that improve the attachment of the active ingredient to the treated plant propagation material, the polymer generally having an average molecular weight of at least 10,000 to about 100,000.

[0079] Examples of methods of application of the compounds and compositions of the present invention, i.e., methods for controlling pests in agriculture, are spraying, atomizing, dusting, brushing, seed dressing, broadcasting or pouring, which are selected to suit the intended purpose under the prevailing circumstances.

[0080] One method of application in agriculture is application to the leaves of the plant (foliar application), with the frequency and rate of application potentially being selected to overcome the risk of infestation by the pest / fungus in question. Alternatively, the active ingredient can reach the plant via the root system (systemic action), which is achieved by applying the compound to the site of the plant, for example, by applying a liquid composition of the compound to the soil (by drenching), or by applying the compound in solid form to the soil in the form of granules (soil application). In the case of rice plants, such granules can be metered into flooded paddies. Applying the compounds of the invention to the soil is a preferred method of application.

[0081] Typical application rates per hectare are generally 10 to 500 g of active ingredient per hectare, in particular 50 to 250 g / ha, preferably 100 to 250 g / ha, such as 150 to 200 g / ha.

[0082] Compound of the present invention and composition thereof are also suitable for the protection (such as seed, tuber or grain, or nursery plant) of plant propagation material, to resist the harmful organism of above-mentioned type.Can be used compound before planting, propagation material is processed, for example, can be processed before sowing seed.Alternately, compound can be applied to seed grain (coating), and this is by being immersed in liquid composition by grain or by using solid composition layer and realize.Also may apply composition when propagation material is planted in application site, for example, during drilling, composition is applied in the seed furrow.These treatment methods for plant propagation material and the plant propagation material of so processing are other themes of the present invention.

[0083] The method according to the invention can be used to control, i.e. to suppress or destroy harmful organisms of the above-mentioned type, which occur in particular on plants, in particular on useful and ornamental plants in agriculture, horticulture and forestry, or on organs of such plants, such as fruits, flowers, leaves, stems, tubers or roots, and in some cases, even plant organs formed at a later point in time still remain protected against these harmful organisms. In particular, suitable target crops are cereals, such as wheat, barley, rye, oats, rice, maize or sorghum; beets, such as sugar beets or fodder beets; fruits, such as pome fruits, stone fruits or soft fruits, such as apples, pears, plums, peaches, apricots, cherries or berries, such as strawberries, raspberries or blackberries; leguminous crops, such as beans, lentils, peas or soybeans; oilseed crops, such as rapeseed, mustard, olives, sunflowers, coconuts, castor beans, cocoa beans, beans or peanuts; cucurbit crops such as pumpkin, cucumber or melon; fiber plants such as cotton, flax or jute; citrus fruits such as oranges, lemons, grapefruits or tangerines; vegetables such as spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes or bell peppers; Lauraceae such as avocado, cinnamon or camphor; and also tobacco, nuts, coffee, eggplant, sugarcane, tea, pepper, grapevine, hops, plantaginaceae and latex plants. The methods of the present invention can also be used on any ornamental and / or vegetable crops (including flowers, shrubs, broadleaf trees and evergreen plants).

[0084] The invention will now be illustrated by the following non-limiting examples.All references cited are incorporated by reference.

[0085] Biological Examples

[0086] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and techniques are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0087] Plant-associated soil microbiome

[0088] To examine microbial community modifications in the soil microbiome associated with cyclohexene treatment against root-knot nematodes, plant-associated soil samples were subjected to PCR amplification and sequential high-throughput sequencing of bacterial and fungal taxonomic marker genes (small subunit ribosomal RNA gene (16S) and ribosomal internal transcribed spacer (ITS), respectively).

[0089] In general, differential abundance analysis of the microbiome data did not detect significant modifications of bacterial communities at the genus level associated with cyclohexetine treatment. At the ASV level, no significant ecologically relevant ASVs were found after cyclohexetine treatment (relative abundance >1%, Table 3a). The 14 ASVs that were found to be significantly altered were characterized by relative abundance <0.1% and therefore had little ecological relevance.

[0090] bacterial communities

[0091] No modifications in bacterial diversity were found. Bacterial ASVs were detected by 16S rRNA gene amplicon sequencing, with taxonomic assignments at the ASV level. The relative abundances in control and cyclohexane-treated samples, and the relative abundance variations when statistically significant (Deseq2, p=0.1), are reported in Table 1a, which reports bacterial ASVs detected by 16S rRNA gene amplicon sequencing, with taxonomic assignments at the ASV level. The relative abundances in control and cyclohexane-treated samples, and the relative abundance variations when statistically significant (Deseq2, p=0.1), are reported here. Bacteria with an abundance greater than 0.01% are shown.

[0092] Table 1a: Table of reported bacterial ASVs

[0093]

[0094]

[0095] fungal communities

[0096] Comparison of the fungal communities of control and RKN-cyclopentafen plant-associated soil samples highlighted a microbiome shift characterized primarily by a reduction in the plant pathogen Fusarium spp., a ubiquitous pathogenic fungus that constituted an abundant taxonomic unit in the control soil. Following cyclopentafen treatment, the relative abundance of Fusarium spp. decreased from 17.8% in the control sample to 7.5%. In addition, the variation in other ecologically relevant taxonomic units (as shown in Table 1b) was low. Table 1b reports the fungal genera detected by ITS gene amplicon sequencing, with taxonomic assignment at the genus level; statistically significant (Deseq2, p = 0.1) relative abundance, and relative abundance variation in control and cyclopentafen-treated samples.

[0097] Table 1b: Fungal communities

[0098]

[0099]

[0100] Figure 1 The magnitude of the effects on various fungal taxa in direct comparisons is shown, both for stimulatory and inhibitory conditions. Surprisingly, in addition to suppressing Fusarium, cyclohexene treatment preserved soil microbiome diversity, as the very negligible inhibitory or stimulatory effects on other species had very low or no impact from a microbial ecology perspective.

[0101] Effects of cyclohexane on soil microbial communities - field trials

[0102] In this experiment, assuming that biodiversity can be measured by the taxonomic richness and abundance of each taxon, differences between treatments were determined and their effects on the community and beneficial species over time were examined. This led to testing two hypotheses. First, that cyclofluamine application had an impact on microbial diversity. Second, that cyclofluamine application improved beneficial microbial diversity over time. These hypotheses were tested on two potato farms in the United States.

[0103] This experiment was conducted in two potato (Solanum tuberculosum L.) fields at two test sites in Louisiana, USA (described as Sites A and B in Table 2). 2 Three products (Table 3) were tested in an area of ​​​​1000 m2 (1000 m2) and three replicates were established for each product using complete block randomization. To evaluate the effect on the microbiome, a "check" plot (untreated) was established. In addition, fluopyram was used as a comparative example. Fluopyram is a widely used fungicide and nematicide and represents the industry standard here. The application method was to inoculate the slurry at the concentration given in Table 3.

[0104] Table 2: Trial locations

[0105]

[0106] Table 3: Processing List

[0107]

[0108] To analyze microbial biodiversity, three soil samples were taken per treatment, and each sample consisted of soil collected from three different points within the plot. In this way, the effects of soil variability were minimized, as there were nine samples per treatment.

[0109] Soil samples were collected four times (T) during the harvest season to understand the effects of treatments on the microbiome over time, where T0 represents the time before treatment application, T1 represents 30 days after treatment, T2 represents 60 days after treatment, and T3 represents 120 days after treatment.

[0110] As described above, in order to detect microbial community modifications associated with cyclohexene treatment in the soil microbiome, plant-associated soil samples were subjected to PCR amplification and continuous high-throughput sequencing of bacterial and fungal taxonomic marker genes (small subunit ribosomal RNA gene (16S) and ribosomal internal transcribed spacer (ITS), respectively). Exploratory data analysis was performed to describe the behavior of microbial biodiversity and species abundance percentages under the factors evaluated. 16S and internal transcribed spacer (ITS) ribosomal RNA (rRNA) sequencing are common amplicon sequencing methods used to identify and compare the bacteria or fungi present within a given sample taken at a given time and place. This is also expressed in the graphs depicting the Shannon-Wiener biodiversity index of bacterial (16S) and fungal (ITS) communities over time (T0, T1, T2 and T3), as shown Figure 2 Described in.

[0111] Beneficial bacteria

[0112] The site A plots were the plots that showed the greatest effect of the applied treatments. More specifically, the treatments with cyclohexane (Examples 1 and 2, A_200 and A_250) were shown to be the most effective by stimulating the growth of beneficial bacterial species with biocontrol activity. These treatments caused a significant increase in Pseudomonas putida, a plant growth promoting rhizobacterium (PGPR) with genes that facilitate nutrient mobilization, prevention of pathogen development, and effective niche colonization. In addition, for Site A in T3 (120 days after application) and Site B in T2 (60 days after application), Sphingomonas species (Sphingomonas sedimentum, Sphingomonas japsi, and Sphingomonas ohata) tended to increase significantly, which is consistent with the application of the different treatments ( Figure 3 This bacterial genus is widely associated with bioremediation and fungicide properties. The Bacillus genus is associated with several beneficial functions in soil and for plant health, such as phytohormone production, pathogen protection, and protection against abiotic stresses. Significant increases in the abundance of Bacillus haloperidol and Bacillus cereus were observed in T2.

[0113] Furthermore, the complete microbial species biodiversity was analyzed, whereby it was observed that, although there were variations in microbial richness and evenness over time and at each measurement, the treatment according to the invention did not induce negative changes at any location (cf. Figure 2 ).

[0114] Beneficial fungi

[0115] The treatments of Example 1 and Example 2 in Table 3 according to the present invention (A_200 and A_250) increased the relative abundance of Trichoderma brevis in Site B ( Figure 4 ). Trichoderma species are a well-known group of ascomycetes that are believed to provide several beneficial functions, including natural fungicide function, thereby protecting plants against several pathogenic fungi.

[0116] Effect of Fluopyram as Comparative Example: The findings of a beneficial or at least neutral effect with cytofluam contrast strongly with the findings of the comparative examples with fluopyram, which show an effect in promoting in particular phytopathogenic microorganisms.

[0117] Conclusion

[0118] Treatment with cyclohexane against nematode infestation demonstrated a positive and preservative effect on soil microbiome diversity, resulting in a vibrant microbiome. Furthermore, no significant negative effects of cyclohexane on the soil bacterial community of tomato plants were observed in the presence of RKN infestation over the medium to long term. Quite the contrary, cyclohexane treatment had a positive effect on the soil fungal community of tomato plants in the presence of RKN infestation over the medium to long term. In particular, treatment with cyclohexane significantly reduced the relative abundance of the plant pathogen Fusarium spp.

[0119] Experiments conducted in potatoes suggest that the use of cyclohexane does not cause negative changes in the soil microbiome community, but rather its application leads to a significant enhancement of beneficial bacterial and fungal species such as Sphingomonas sedimentaris, Sphingomonas japsi, Sphingomonas otago, Bacillus halosus, Bacillus cereus, Pseudomonas putida, and Trichoderma brevis, which play an important role in pathogen protection, bioremediation, and plant growth promoting rhizobacteria (PGPR). Comparative examples did not show any beneficial effects.

[0120] Thus, the compositions comprising cytofluanid were ultimately shown to benefit the soil microbiome by preserving the diversity of beneficial bacterial species and by inhibiting pathogenic fungi, particularly when compared to the compositions comprising fluopyram. It is therefore clear that cytofluanid treatment preserves soil biodiversity and, therefore, will preserve a range of vibrant life forms in the soil and enable plants to develop larger root systems in infested fields.

[0121] Effects on arbuscular mycorrhizal fungi and compatibility with them

[0122] Selection of active ingredients

[0123] A panel of cyclohexene analogs with high nematicidal activity and varying degrees of fungicidal activity against a broad spectrum of fungal and oomycete diseases was selected based on toxicity screening against: nematodes (Heterodera exigua, Meloidogyne incognita species); and fungal lineages including Ascomycetes (Dothideomycetes (Zymoseptoriatritici, Cercospora arachidicola, Pyrenophora teres, Alternaria solani, Phaeosphaeria nodorum species), Leotiomycetes (Erysiphe graminis, Blumeria graminis, Botryotinia fuckeliana, Sclerotinia sclerotiorum), Sordariomycetes (Glomerella anthracnose), and The compounds were tested against various fungicidal fungi including Fusarium ulmorum, Fusarium graminearum, Gaeumannomyces graeminis, Magnaporthe grisea, Monographella nivalis, Basidiomycota (Thanatephorus cucumeris, Puccinia recondita), and Oomycota (Plasmopara viticola, Pythium ultimum, Phytophthora infestans). The average activity across all tested groups within the phyla Nematode, Doctomycetes, Hammerworms, Sclerotiniae, Basidiomycota, and Oomycetes was calculated, indicating that all compounds exhibited nematicidal and fungicidal activity.

[0124] Testing of cyclohexane and comparative examples in soybeans

[0125] Soybeans of the Toliman variety were treated with 0.15 mg ai / seed of cytofluanid and the like and planted in 1.5 L pots with 0-3 mm sieved soil. The seeds were inoculated with Bradyrhizobium japonicum (Bradyrhizobium japonicum) using a commercially available product for soybeans. o bium japonicum).

[0126] In the first series, for each active ingredient tested, 15 replicate pots were sown. In each pot, 3 seeds were sown. After germination, 2 seedlings were removed from each pot, and the final number was 1 plant per pot. Each pot was inoculated with a small amount of mycorrhizal inoculant (blend A, commercially available from Symbiom, Czech Republic). Plants were grown in a greenhouse for 81 days under the following conditions: 45%-64% humidity, 22°C to 24°C temperature, with a 16 / 8 hour day / night cycle. In another test series, the soybean seeds processed as described above were then planted in a 1L pot with 0-3mm sieved soil. For each active ingredient, 8 replicate pots were sown, thus 3 seeds were sown in each pot. After germination, 2 seedlings were removed from each pot, and the final number was 1 plant per pot.

[0127] The plants were then grown in a growth chamber for 42 days under the following conditions: temperature of 20°C and 12 / 12 hour day / night.

[0128] After harvest, the percentage of mycorrhization was quantified using the method of McGonigle et al. (TPMcGONIGLE, MHMMILLER and DGEVANS, et al., "A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizalfungi". New Phytol. 115(3): 495-501).

[0129] Table 1 below shows the structures of compounds A1 to A7 and the effects of the different compounds on mycorrhization in soybean roots. It was unexpectedly found that cyclohexane not only showed higher colonization compared to the comparative compound, but also showed higher colonization compared to the control, while the tested analog comparative examples, despite their structures being very similar to cyclohexane, showed reduced colonization levels.

[0130] Table 1. Compounds tested for mycorrhizal compatibility; effect on the level of mycorrhization in soybean roots.

[0131]

[0132]

[0133]

[0134] Conclusion

[0135] Surprisingly, for soybeans, cypermethrin treatment showed a very positive effect on the growth of arbuscular mycorrhizal fungi, while also strongly suppressing nematodes and undesirable fungi.

[0136] Comparative examples using structurally and chemically very close analogs showed negative effects on the growth of arbuscular mycorrhizal fungi under the same conditions despite the high structural similarity.

[0137] Thus, it was conclusively shown that cyclohexene has no negative effects on arbuscular mycorrhizal fungi and can therefore support plant growth. This could also potentially reduce the need for fertilization. On the contrary, cyclohexene preserves soil biodiversity and, therefore, a range of vibrant life forms in the soil, allowing plants to develop larger root systems in infested fields.

Claims

1. A method for preserving or stimulating the growth of beneficial soil microorganisms and / or inhibiting plant pathogenic microorganisms in the soil for the purpose of crop or plant growth, the method comprising applying an effective amount of a composition comprising cyclohexane to the plant, its locus or propagation material.

2. The method of claim 1, wherein stimulating the growth of beneficial soil microorganisms comprises stimulating the growth of bacterial and fungal species that exhibit biocontrol and / or soil remediation activity.

3. The method according to any one of the preceding claims, wherein the beneficial microorganisms comprise bacteria of the genera Sphingomonas, Bacillus and / or Pseudomonas and fungi of the genus Trichoderma.

4. The method according to claim 1, wherein the beneficial microorganisms include the following bacteria: Sphingomonas sedimentum, Sphingomonas japsi, Sphingomonas ohta, Bacillus haloperidol, Bacillus cereus, Pseudomonas putida and Growth-Promoting Rhizobacteria (PGPR).

5. The method of claim 3, wherein the beneficial microorganism comprises the fungus Trichoderma brevis.

6. The method according to any one of the preceding claims, wherein the composition comprising cyclohexane further comprises one or more additional biologically active agents, preferably one or more acaricides, bactericides, fungicides, insecticides, nematicides and / or plant activators.

7. The method according to any one of the preceding claims, for preserving or improving the integrity of the soil microbiome in soil in need of treatment, in particular wherein the soil contains nematode plant pests and / or phytopathogenic Fusarium fungi.

8. The method according to any one of the preceding claims, wherein the composition comprising cyclohexane is applied via irrigation, soil drench, in-furrow application and / or as a seed treatment or inoculation, wherein soil treatment is carried out in an amount ranging between 10 and 500 grams of cyclohexane per hectare.

9. The method according to any one of the preceding claims, wherein the composition comprising cyclohexane is applied to, or inoculated into, the seeds or propagation material in an amount of between 1 gram and 1000 grams of cyclohexane per 100 kg of seeds or propagation material.

10. The method according to any one of the preceding claims, wherein the application of the composition comprising cyclohexane is carried out before, during or after planting.

11. A method according to any one of the preceding claims, wherein root development of plants grown in said soil is increased compared to roots of plants grown in untreated soil.

12. Use of a composition comprising cyclohexane for preserving or improving the integrity of the soil microbiome in soil in need of treatment.

13. Use of a composition comprising cyclohexane for improving plant health and / or plant root growth in plants grown in soil in need of treatment.

14. Use according to claim 13, wherein the soil in need of stimulation is selected by identifying a portion of the soil containing plant pests present in sufficient amounts to harm or reduce the growth of plants growing in the soil.

15. The use according to claim 14, wherein the plant pests include: Plant-parasitic nematodes, in particular endoparasitic, semiendoparasitic and / or ectoparasitic nematodes; and / or harmful bacteria or fungi, in particular fungi of the genus Fusarium.

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