Microbial mixtures comprising carbon sequestering fungi and methods of the use thereof

Abstract

The present disclosure provides a mixture comprising: a first composition comprising a fungal strain belonging to a genus selected from the group consisting of: Acrocalymma, Leptodontidium, Phaeosphaeria, Thozetella, Trichoderma, Periconia , and Clonostachys; and a second composition comprising rhizobial bacteria, an arbuscular mycorrhizal fungus (AMF), or free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria. Also disclosed are methods of plant enhancement, increasing organic carbon in a soil, and sequestering atmospheric carbon for storage as organic carbon in a soil with the mixtures.

Description

[0001] MICROBIAL MIXTURES COMPRISING CARBON SEQUESTERING FUNGI AND METHODS OF USE THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 386,195, filed on December 6, 2022, the contents of which are incorporated herein by reference in their entirety.

[0004] STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0005] A Sequence Listing in XML format, submitted under 31 C,F.R. § 1.821, entitled LOAM-M002-01WO.xml, 28, 649 bytes in size, generated on December 5, 2923, and filed via EFS-Web, is provided to lieu of a paper copy. This Sequence Listing is incorporated by reference into the specification for its disclosures.

[0006] TECHNICAL FIELD

[0007] The present invention generally relates to mixtures and methods for increasing carbon commit to the soil, mitigating atmospheric carbon dioxide, and improving plant growth and yield. The present invention particularly relates to mixtures comprising carbon: sequestering fungal cells and biological control agents,

[0008] BACKGROUND

[0009] Carbon dioxide and methane absorb and retain heat in the atmosphere; therefore, both gases play a pivotal role in the greenhouse effect. However, as methane is much more short- lived than carbon dioxide, the effect of carbon dioxide is often considered more important than that of methane to the greenhouse effect.

[0010] The life cycle of carbon includes the removal of carbon dioxide from the atmosphere by plants through photosynthesis. During the process of photosynthesis, the carbon dioxide gets absorbed through the stroma of leaves, and the carbon dioxide is further converted into sugars. Such sugars become nutrients for plants and microbes present in the soil. Carbon enters hack into the atmosphere in the form of carbon dioxide by respiration and combustion. Hence, a balanced amount of release and absorption of carbon dioxide is essential for balancing the ecosystem. Human activities such as the combustion of fuels, overpopulation, forest degradation, soil erosion, etc. have led to an increase in atmospheric carbon dioxide. Approaches for sequestering carbon dioxide from the atmosphere, therefore present an important component of a strategy for reducing or controlling atmospheric carbon dioxide. However, for this to be successful, there must also be a reduction in the release of carbon dioxide from the soil back into the atmosphere.

[0011] Decay of plants, animals, and microbes into the soil can lead to the build-up of soil organic carbon (SOC), an essential nutrient that promotes physical stability of the structure of the soil, soil aeration, water drainage and retention, thus reducing soil erosion and nutrient leaching. However intensive cultivation has also led to a decline in SOC, eventually making the land unsuitable for commercial crop production. As such, the benefits associated with SOC can be seen as twofold, namely, the sequestration of atmospheric carbon, provided the carbon is retained by the soil and the overall improvement of the soil quality.

[0012] It would be advantageous to develop compositions, treatments, and methods for increasing soil carbon in a manner that will produce more stable carbon in the soil by sequestering atmospheric carbon, as well as provide benefits to commercial crop plants. The present invention provides active compound combinations, compositions, and treatments which in some aspects at least achieve the stated objective.

[0013] SUMMARY

[0014] The present disclosure provides amixture comprising: a) a first composition comprising a fungal strain belonging to a genus selected from the group consisting of; Acrocalymma, Leprtadontudium, Phaeosphaeria, Thozetella, Trichoderma , Periconia and Clonastachys; and b) a second composition comprising rhizobial bacteria selected from the group consisting of; Brtidyrhizobium japonicum, Mesorhizobium cicera , Mesorhizobium loti, Rhizobium leguninosrum, Sinorhizobium meliloti and combinations thereof; Wherein a combination of the first composition and the second composition exhibits synergy. In one aspect, the rhizobial bacteria comprise Bradyrhizobium japonicum. In another aspect, the rhizobial bacteria comprise Bradyrhizobium japonicum US-B-1237 (NRRL Accession No. B-68330), In one aspect, the rhizobial bacteria comprise Sinorhizobium meliloti (also known as Ensifer meliloti In another aspect, the rhizobial bacteria comprise Ensifer meliloti US-B-12.97 (NRRL Accession No. B-68331). In other aspects, the present disclosure provides a mixture comprising; a) a first composition comprising a fungal strain belonging to a genus selected from the group consisting of: Aerocalymma, Leptodonridium, Phaeosphaeria, Thozetella, Trichoderma, Periconia, and Clonostachys; and b) a second composition comprising an arbuscular mycorrhizal fungus (AMF) selected from the group consisting of: Acaulaspora mellea, Claroideoglomus etunicatum, Dominikia aurea, Funnelformis masseae, Rhizophagus clarus , Rhizaphagus custos, Rhizophagus diaphanous, Rhizophagus irregularis, Rhizophagus intraradices, Paraglomus brasilianum, and combinations thereof; wherein a combination of the first composition and the second composition exhibits synergy;

[0015] In yet other aspects, the disclosure relates to a mixture comprising: a) a first composition comprising a fungal strain belonging to a genus selected from the group consisting of; Acrocalymma, Leptodonridium, Phaeosphaeria, Thozetella, Trichodenna, Periconia, and Clonostachys; and b) a second composition comprising free-living nitrogen-fixing bacteria and / or phospbate-solubilizing bacteria belonging to a genus selected from the group consisting of; Klebsiella, Pseudomonas, Azolobacter, Gluconacetobacter , Azospirillum, Asarhizobium, Rhadopsendomonas , Rhodobacter, Burkholderia, Herbaspirillum, Cyanothece, Enterbacter , Bacillus, Paenibacillus, Achromobacter, Kosakonia, Arthrobacter, Rhodococcus, Nocardioiedes, Curtroacterium, and combinations thereof; wherein a combination of the first composition and the second composition exhibits synergy.

[0016] In some aspects, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria are selected from the group consisting of; Klebsiella variicola, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas argentihensis, Pseudomonas azotoformans, Pseudomonas lini Azotobacter vinelandii, Azotobacter chroococcum, Gluconacetobacter azotocaptans, Gluconacetobacter diazotrophicus, Azospirillum brasilense, Azaspirtllum lipoferum, Azorhizobium caulinodans, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Burkholderia unamae, Burkholderia tropica, Burkholderia cepacian, Burkholderia xenavorans, Herbaspirillum seropedicae, Herbaspirillum frisingense. Achromobacter xylosodidans, Kosakonia saccharl, Kosakonia radicincitans, Kosakonia (Enterabacter) oryzae, Arthrobacter agilis, Arthrobacter aurescens, Arthrobacter giacomelloi, Rhodococcus qingshengii, Rhodococcus erythropolis, Rhodococcus baikonurensis, Rhodococcus fascians, Rhodococcus opacus, Nocardioides hanchengensis, Nocardioides ginkgobilobae, Nocardioides aromaricivorans Nocardioides albus , Nocardioides yefusinii Curtobacterium herbarum, Curtobacterium flaccumfaciens, Curtobacterium pusillum. Curtobacterium cltreum, and combinations thereof In one aspect, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Pseudomonas argenlinensis. In another aspect, the free-living nitrogen- fixing bacteria and / or phosphate-solubilizing bacteria comprise Pseudomonas argentinensis AU-B-13 (Accession No. V23 / 0.18893).

[0017] In some aspects, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Pseudomonas lini. In one aspect, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Pseudomonas lini AU-EM4 (Accession No. V23Z018895).

[0018] In other aspects, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Pseudomonas azotoformans. In one aspect, die free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Pseudomonas asotoformans AU-B- 130 (Accession Np. V23 / 018897).

[0019] In yet other aspects, the free-living nitrogen-fixing bacteria and / or phosphate- solubilizing bacteria comprise Kosakonia (Enterobacter) oryzae. In one aspect, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Kosakania (Enterobacter) oryzae AU-B-27 (Accession No. V23 / 018894).

[0020] In certain aspects, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Arotobacter chroococcum. to one aspect, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Azotobacter chroococcum AU-B-122 (Accession No. V23 / 018896),

[0021] In yet other aspects, the free-living nitrogen-fixing bacteria and / or phosphate- solubilizing bacteria comprise Sphingomamas sp. In one aspect, the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise Sphingomonas sp. AU-B-172 (Accession No. V23 / 018898).

[0022] In certain aspects, the fungal strain has a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence dial is at least 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-22. In one aspect, the nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1- 22.

[0023] In one aspect, lhe fungal strain is selected from the group consisting of: Acracalymma vagum DMTR-CTR-11556 (NMI Accession No. V22 / 006357), Clonaslachys rosea DMTR- CTR-US-173 (ATCC Accession No. PTA-I27299). Clonostachys rosea DMTR-CTR-1081 (NMI Accession No. V22'003495), Leptodontidium orchidicola DMTR-CTR-4873 (NMI Accession No. Y22 / 003497), Periconia sp; DMTR-CTR-6649 (NMl Accession No. V22 / 006356), Periconia macrospinosa DMTR-CTR-US-125 (ATCC Accession No. PTA- 127300), Periconia macraspiaosa DMTR-CTK-1852. (NMI Accession No. V22 / 006358) Phaeosphderia luctuasa / vagans DMTR-CTR-3044 (NMI Accession No. V22 / 006355), Thozetella nivea DMTR-CTR-2359 (NMI Accession No. V22 / 003496), Trichoderma hamatan DMTR-CTR-US-73 (ATCC Accession No. PTA-127301), Trichderma langipile / spirals DMTR-CTR-1291 (NMI Accession No. V22 / 006354), Periconia macrospinosa AU- 7083 (NMI Accession No. V22 / 019796), Acrocalymma vagum US-738 (ATCC Accession No, PTA-127449), Acrocalymma vagum US-445 (ATCC Accession No. PTA-127444), Leptodonridium orchidicola US-210 (ATCC Accession No. PTA-127441), Leptodontidium orchidicola US-70 (ATCC Accession No. PTA-I27439), Leptadontidium orchidicola US- 1188 (ATCC Accession No. PTA-127640), Leptodontidiam orchidicola US-1202 (ATCC Accession No. PTA-127639), Clonostachys rosea US-114 (ATCC Accession No. PTA- 127443), Clonostachys rosea US-712 (ATCC Accession No. PTA-127446). Trichoderma hamatum US-724 (ATCC Accession No. PTA-127448), Trichderma langipile / spirals US- 77 (ATCC Accession No. PTA-I27440), and a mutant thereof having all identifying characteristics of the respective strain.

[0024] In yet other aspects, the disclosure relates to a mixture comprising at least two fungal strains selected from the group consisting of: Clohesyomyces aquaticus DMTR-CTR-7800 (NMI Accession No. V21 / 002328), Phialocephala fortinii s.l - Acephala applanata species complex (PAC) DMTR-CTR-7788 (NMI AcccssionNo. V21 / 002327), Darksidea zeta DMTR- CTR-6853 (NMI Accession No. V21W326), Darksidea zeta DMTR-CTR-4706 (NMI Accession No. V21 / 003117), Dartaidea sp. DMTR-CTR-360 (NMI Accession No. V21 / 003116), Acrocalymma vagum DMTR-CTR-11556 (NMI Accession No. V22 / 006357), Clonostachys rosea DMTR-CTR-US-173 (ATCC Accession No. PTA-127299), Clonastachys rosea DMTR-CTR-1081 (NMI Accession No, V22 / 003495), Leptodontidiion archidicala DMTR-CHM873 (NM) Acccssion No. V22 / (K)3497), Periconia sp. DMTR-CTR-6649 (NMI Accession No. V22 / 006356), Periconia macrospinosa DMTR-CTR-US-125 (ATCC Accession No. PTA-127300), Pericortia matrospiposa DMTR-dR-1852 (NMI Accession No. V22 / 006358), Phaeosphaeria luctuosa / vagans DMTR-CTR-3044 (NMI Accession No. V22 / 006355), Thozetella nivea DMTR-CTR-2359 (NMI Accession No. V22 / 003496). Trichoderma hamatum DMTR-CTR-UST73 (ATCC Accession No. PTA-127301), Trichoderma longipile / spirals DMTR-CTR-1291 (NMI Accession No. V22 / 006354), Periconia macrospinosa AU-7083 (NM1 Accession No. V22Z019796), Acroealymma vagum US-738 (ATCC Accession No. PTA-127449), Acrocalymma vagum US-445 (ATCC Accession: No. PTA-127444), Leptodantidiian orchulicola US-210 (ATCC Accession No. PTA-127441), Leptodontidium orchidicola US-70 (ATCC Accession No. PTA-I27439), Leptodontidium orchidicola US-1188 (ATCC Accession No, PTA-1.27640), Leptodonii<lium orchidicola US-1202 (ATCC Accession No. PTA-127639), Clonostachys rosea US-114 (ATCC Accession No. PTA-127443), Clonostadiys rosea US-712 (ATCC Accession No. PTA-127446). Trichoderma hamatum US-724 (ATCC Accession No. PTA-127448), Trichoderma longipile / spirale US-77 (ATCC Accession No. PTA-127440), and a mutant thereof having all identifying characteristics of the respective strain.

[0025] In certain aspects, the fungal strain is present at a concentration of at least 101CFU per millilitre or gram, at least 102CFU per millilitre or gram, at least 103CFU per millilitre or gram, at least 104CFU per millilitre or gram, at least 104CFU per millilitre or gram, or at least 105CFU per millilitre or gram. In one aspect, the fungal strain is present in the first composition at a concentration of at least 103CFU per millilitre or gram.

[0026] In some aspects, the fungal strata is present at a concentration of 102to 1012CFU / g,103to 1012CFU / g, 104to 1012CFU / g, 108to 1012CFU / g, 106to 1012CFU / g, 107to 1012CFU / g, 108to 1012CFU / g, or 109to 1012CFU / g.

[0027] In some aspects, the first composition and the second composition arc formulated independently fix application to a plant as a soil drench, in-furrow treatment, foliar application, or seed treatment In some aspects, the mixtures further comprise at least one auxiliary selected from the group consisting of extenders, solvent, diluents, emulsifiers, dispersants, binders, fixing agents, wetting agents, dyes, pigments, antifoams, preservatives, secondary thickeners, stickers, and combinations thereof.

[0028] In other aspects, the present disclosure provides a plant propagation material treated with a mixture disclosed herein in an amount of from 0.01 g to 10 kg per 100 kg of plant propagation material.

[0029] In certain aspects, the present disclosure provides a method of plant enhancement comprising applying to a plant, seedling, plant propagation material, or the locus surrounding the plant material an effective amount of a mixture disclosed herein to enhance at least one plant characteristic, wherein the plant characteristic is selected from the group consisting of accelerated seed germination, accelerated seedling emergence, improved seedling emergence, improved leaf formation, accelerated leaf formation, improved plant maturation, accelerated plant maturation, increased plant yield, increased plant growth, increased plant quality, increased plant health, increased fruit yield, increased fruit growth, increased fruit quality, improved root health, increased root nodule formation, plant healthy plant resistance to salt stress, plant resistance to heat stress, plant resistance to heavy metal stress, plant resistance to drought, and combinations thereof

[0030] In certain aspects, the first composition and / or the second composition are administered at a rate of 0.01 g to 10 kg per 100 kg of plant propagation material pre-planting and / or at a rate of 0.1-150 gallons per acre (0.935-1402.5 liters per hectare) post-planting to enhance the at least one plant characteristic.

[0031] In other aspects, the first composition and / or the second composition are administered at a rate of between 1 g / Ha and 1,000 g / Ha, between 10 g / Ha and 1,000 g / Ha, between 20 g / Ha and 1,000 g / Ha, between 30 g / Ha and 1 ,000 g / Ha, between 40 g / Ha and J ,000 g / Ha, between 50 g / Haand 1,000 g / Ha, between 60 g / Ha and 1,000 g / Ha, between 70 g / Ha and 1,000 g / Ha, between 80 g / Ha and 1,000 g / Ha, between 00 g / Ha and 1,000 g / Ha, between 100 g / Ha and 1,000 g / Ha, between 1 g / Ha and 500 g / Ha, between 10 g / Ha and 500 g / Ha, between 20 g / Ha arid 500 g / Ha, between 30 g / Ha and 500 g / Ha, between 40 g / Ha and 500 g / Ha, between 50 g / Ha and 500 g / Ha, between 60 g / Ha and 500 g / Ha, between 70 g / Ha and 500 g / Ha, between 80 g / Ha and 500 g / Ha, between 90 g / Ha and 500 g / Ha. or between 100 g / Ha and 500 g / Ha to enhance the at least one plant characteristic.

[0032] In other aspects, the first composition and / or the second composition are administered at a rate of between 10 CFU / seed and 10,000 CFU / seed, between 50 CFU / seed and 10,000 CFU / seed, between 100 CFU / seed and 10,000 CFU / seed, between 200 CFU / seed and 10,000 CFU / seed, between 300 CFU / seed and 10,000 CFU / seed, between 400 CFU / seed and 10,000 CFU / seed, between 500 CFU / seed and 10,000 CFU / seed, between 600 CFU / seed and 10,000 CFU / seed, between 700 CFU / seed and 10,000 CFU / seed, between 800 CFU / seed and 10,000 CFU / seed, between 900 CFU / seed and 10,000 CFU / seed, between 1,000 CFU / seed and 10,000 CFU / seed, between 1,500 CFU / seed and 10,000 CFU / seed, between 2,000 CFU / seed and 10,000 CFU / seed, between 2,500 CFU / seed and 10,000 CFU / seed, between 3,000 CFU / seed and 10,000 CFU / seed, between 3,500 CFU / seed and 10,000 CFU / seed, between: 4,000 CFU / seed and 10,000 CFU / seed, between 4,500 CFU / seed and 10,000 CFU / seed, or between 5,000 CFU / seed and 10,000 CFU / seed to enhance the at least one plant characteristic.

[0033] In other aspects, the first composition and the second composition in the mixture are applied simultaneously or subsequently.

[0034] In some aspects, the plant is a legume selected from the group consisting of alfalfa, clover, peas, cowpeas, beans, mung beans, lentils, lupins, mesquite, carob, soybeans, peanuts* tamarind, wisteria, siratro, plants from the Lespedeza genus, Genistoid legumes, serradella, and combinations thereof.

[0035] In other aspects, the plant is a pasture crop or cover crop selected from the group consisting of Lucarne (aka alfalfa), arrow leaf clover, Balansa clover, chicory, plantain, phalaris, cocksfoot, fescue, prairie grass, Warrego summer grass, Italian rye grass, perennial rye grass, Biserrula, Serradella, Gland clover, Bladder clover, switchgrass, radish, medic, buckwheat, cow pea, lablab, sunn hemp, sunflower, tillage radish, and subterranean clover.

[0036] In yet other aspects, the plant is selected from the group consisting of wheat, rice, corn (maize), canola, rye, oats, barley, sorghum, millet, flax, hemp, Jute, sugarcane, and cotton. In one aspect, the plant is a millet selected from the group consisting of finger millet (Eleusine coracana), foxtail millet (Setaria italica ), browntop millet (Urochloa ramose), pearl millet (Pennisetian glaucum ), Japanese millet, / barnyard millet (Echinochloa esculenta), little millet (Panicum, sumatrense), broomcorn millet / proso millet (Panicum miliaceum), Kodo millet (Paspalum scrobiculatum), fonio millet (Digitaria exilis), guinea millet (Brachteria deflexa), great millet (Sorghan bicolor), Sonoran millet (Panteum hirticande), Polish millet (Digitaria sanguinalis), adlay millet (Coix Iacrytna-jobi), and Taiwan oil millet (Spodiopogon formosanus).

[0037] In one aspect, the present disclosure provides a bioorganic soil conditioner comprising a mixture disclosed herein.

[0038] In other aspects, the present disclosure provides a method of increasing organic carbon in a soil, comprising applying to a plant, seedling, plant propagation material, or the locus surrounding the plant material an effective amount of a mixture disclosed herein, wherein the mixture is in an amount effective to increase organic carbon in the soil compared to a non- inoculated control soil.

[0039] In certain aspects, the present disclosure provides a method for sequestering atmospheric carbon for storage as organic carbon in a soil, comprising applying to a planty seedling, plant propagation material, or the locus surrounding the plant material an effective amount of a mixture disclosed herein, wherein the mixture is m an amount eftective to increase sequestered atmospheric carbon in the soil compared to a non-inoculated control soil.

[0040] In other aspects, the present disclosure provides a kit-of-parts comprising the first composition and the second composition of the mixture, as defined herein, in a spatially separated arrangement, and optionally at least one auxiliary, wherein a combination of the first composition and the second composition exhibits synergy. In yet other aspects, the present disclosure provides the use of a mixture disclosed herein for plant enhancement, increasingorganic carbon in a soil or sequestering atmospheric carbon for storage as organic carbon in a soil.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FlGs. 1A and 1B depict the eompaxibiltiy of Bradyrhizobium japanicum CB1809 growth with that of Thozetella nivea DMTR-CTR-2359 (NM1 Accession No, V22 / 003496) and Leptodontidium orchidicola DMTR-CTR-4873 (NMI Accession No, V22 / 003497), respectively.

[0043] FIG. 2 depicts the compatibility of Bradyrhizobium japanicum US-B-1237 (NRRL Accession No. B-68330) growth with that of Leptodontidium orchidicola US-210 (ATCC Accession No. PTA-127441).

[0044] FIG.3 depicts siderophore production measured in nineteen bacterial strains including Pseudomonas argentinensisAU-B-13 (Accession No. V23 / 018893). Pseudomonas lini AU-B- 44 (Accession No. V23 / 018895), and Pseudomonas azatoformans AU-B-130 (Accession No. V23 / 018897).

[0045] FIG. 4 depicts the average phosphate solubilization indices for nineteen bacterial strains including Pseudomonas lini AU-B-44 (Accession No. V23 / 018895) and Pseudomonas azotoformans AU-B-130 (Accession No. V23 / 018897).

[0046] FIG. 5 depicts the nitrogen, fixing capacity of nineteen bacterial strains including Kosakonia (Enterobacter) oryzae AU-B-27 (Accession No. V23 / 018894), Azotobacter chroocoecum AU-B-122 (Accession No. V23 / 018896), Pseudomonas argentinensis AU-B-13 (Accession No. V23 / O18893). and Sphingomonas sp. AU-B-172 (Accession. No. V23 / 018898).

[0047] FIG. 6 depicts iudole-3-acctic add production in nineteen bacterial strains including Kosakonia (Enterobacter) orysae AU-B-27 (Accession No. V23 / 018894) and Sphingomonas sp. AU.B-172 (Accession No. V23 / 018898).

[0048] DETAILED DESCRIPTION

[0049] As used herein and m the appended claims, the singular forms “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0050] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the passible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that cach intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits arc also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0051] A “mutant” is a strain haying all the identifying characteristics of the NMI, NRRL, of ATCC Accession Numbers as indicated in this text and can be identified as having a genome that hybridizes under conditions of high stringency to the genome of the NMI, NRRL or ATCC Accession Numbers.

[0052] “Hybridization” refers tb a reaction in which one ormore polynucleotides react to form acomplex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing Strand, or any combination of these. Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 X SSC or a solution of equivalent ionic strength / temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 X SSC, and a high stringency hybridization reaction is generally performed at about 60 °C to 1 X SSC.

[0053] A mutant of the indicated NMI, NRRL, or ATCC Accession Number may also be defined as a strain having a genomic sequence that is greater than 85%, more preferably greater than 90% or more preferably greater than 95% sequence identity to the genome of the indicated NRRL or ATCC Accession Number. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%. 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. for example, those described in Current Protocols to Molecular Biology (F. M. Ansubel et al., eds., 1987). NRRL is the abbreviation for the Agricultural Research Service Culture Collection, an international depositary authority tor the purposes of deposing microorganism strains under the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, having the address National Center for Agricultural Utilization Research, Agricultural Research service, U.S. Department of Agriculture, 1815 North university Street, Peroira, Illinois 61604 USA.

[0054] ATCC is the abbreviation for the American Type Culture Collection, an international depositary authority for the purposes of deposing microorganism strains under the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, having the address ATCC Patent Depository, 10801 University Blvd., Manassas, VA 10110 USA.

[0055] NMI is the abbreviation for the National Measurement Institute, an international depositary: authority for the purposes of deposing microorganism strains trader the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, having the address 1 / 153 Bertie Street, Port Melbourne, Victoria 3207, Australia.

[0056] As used herein, the teams “mixture” “activecompoundcombination” and “compound combination” are interchangeable and. refer to a combination of at least two agricultural agents for application to a plant, plant part, plant propagation material, or a locus where a plant is grown.

[0057] The term “plant propagation material” is to be understood to denote all the generative parts of the plant such as seeds and vegetative plant material such as cuttings and tubers (e, g. potatoes), which can be used for the multiplication of the plant This includes seeds, roots, fruits, tubers , bulbs, rhizomes, shoots, sprouts and other parts of plants, including seedlings arid young plants, which are to be transplanted after germination or after emergence from soil.

[0058] The term “auxiliary” as used herein refers to an inert ingredient commonly used in agricultural compositions. Examples of auxiliaries include, but are not limited to, extenders, solvents, diluents, emulsifiers, dispersants, binders, fixing agents, wetting agents, dyes, pigments, antifoams, preservatives, secondary thickeners, and stickers.

[0059] A “soil conditioner” is to be understood to denote a mixture of substances or a. blend that can be added directly to soil to improve soil characteristics or can be added to an agricultural or fertilizer composition, which, in turn, is added to a soil. The soil conditioner can be applied to any type of soil, including black cotton soil, saline soil, medium to high saline soil, yellow soil, sandy soil, loamy soil, alluvial soil (delta soil), Iava soil, topsoil, and subsoil that can be used in crop / plant production.

[0060] By artificially controlling aspects of the microbial cell culturing process such as the organic carbon feed, oxygen levels, pH, and fight, the culturing process differs from the culturing process that microbial cells experiences in nature. Through artificial control of aspects of the culturing process and intervening in the culturing process with contamination control methods, the microbial cell culture produced as a whole and used in the described inventive compositions differs from the culture that results from a microbial cell culturing process that occurs in nature.

[0061] Fungal Strains Increasing Carbon Sequestration in the SoO

[0062] The mixtures and active compound combinations disclosed heroin comprise a first composition comprising a fungal attain. In certain aspects, the fungal strain increases carbon sequestration in the soil In one aspect, the fungal strain comprises a nuclear ribosomal internal transcribed Spacer 2Q.TS2) sequence, that comprises a nuclear ribosomal internal transcribed spacer 2 (1TS2) sequence that is at feast 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-22 (see Table 1).

[0063] In one aspect, the mixture comprises a fungal strain of the genus Acroculymma comprising a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that comprises a nucleotide sequence that is at least 95% identical, at least 96% identical, at least 97%. identical, at least 98% identical, or al least 99% identical to the nucleotide sequence of SEQ ID NO: 1, 13, or 14.

[0064] In another aspect, the mixture comprises a fungal strain of the genus Clonostachys comprising a nuclear ribosomal internal transcribed spacer 2 (1TS2) sequence that comprises a nucleotide sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 2, 3, 17, or 18.

[0065] In another aspect, the mixture comprises a fungal strain of the genus Leptodontidium comprising a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that comprises a nucleotide sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 4, 15, 16, 21, or 22.

[0066] In another aspect, the mixture comprises a fungal strain of the genus Periconia comprising a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that comprises a nucleotide sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 5, 6. 7, or 12.

[0067] In another aspect, the mixture comprises a fungal strain of the genus Phaeosphaeria comprising a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that comprises a nucleotide sequence that is at least 95% identical at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 8.

[0068] In another aspect, the mixture comprises a fungal strain of the genus Thozetella comprising a nuclear ribosomal internal transcribed spacer 2 (1TS2) sequence that comprises a nucleotide sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 9.

[0069] In another aspect, the mixture comprises a fungal strain of the genus Trichoderma comprising a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that comprises a nucleotide sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO; 10, 11, 19, or 20.

[0070] Table 1

[0071]

[0072]

[0073]

[0074] Deposited Microbial Strains

[0075] Biological deposits of each of the fungal strains listed in Table 2 and several bacterial strains were made on the dates shown at the American Type Culture Collection (ATCC*), located at 10801 University Blvd., Manassas. VA 20110. USA, the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria. Illinois, USA, or the National Measurement Institute (NMI), 1 / 153 Bertie Street, Port Melbourne, Victoria 3207, Australia, under the provisions of the Budapest Treaty and assigned by each International Depositary Authorily (IDA) the accession numbers indicated. Upon issuance of a patent, all restrictions upon the deposits will be irrevocably removed. The deposits are intended to meet the requirements of 37 CFR §§ 1.801-1.809, The deposits will be maintained in the IDAs for a period of 30 years, or 5 years after the last request. or for the effective, enforceable life of the patent, whichever is longer, and will be replaced, if necessary, during that period; and the requirements of 37 CFR §§ 1,801- 1.809 are met

[0076] Table 2

[0077]

[0078] Mixtures of Fungi and Rhizobial Bacteria

[0079] Non-limiting examples of rhizobial bacteria that can be used in the compositions, mixtures, and methods of the disclosure include those assigned to the order Rhizobiales other than Agrobacterium bacterial strains comprising the taxonomic families Rhizobiaceae (e.g. Rhizobium spp., Sinorhizobium spp.); Phyllobacteriaceae (e.g. Mesorhizobium spp., Phyllobacterium spp.); Brucellaceac (e.g. Ochrobactrum spp.); Bradyrhizobiaceae (e.g. Bradyrkizobium spp,), and Xanthobacteraceae (e.g. spp.).

[0080] In some aspects, the rhizobial bacteria belong to the genus Sinorhizobium. to one aspect, the rhizobial bacteria belong to one or more of the following species. Sinorhizobium fredii, Sinorhizobium medicae and Sinorhizobium meliloti . In another aspect, the rhizobial bacteria are one or more of: Sinorhizobium melloti Rm1021, Sinorhizobium (Ensifer) meliloti strain RBD1. Sinorhizobium medicae strain NRRL Accession No. X78, Sinorhizobium meliloti strain NRRL Accession No, X79, Sinorhizobium frediiCCBAU114, and Sinorhizobium fredii USDA 205.

[0081] In certain aspects, the rhizobial bacteria belong to the genus Bradyrhizobium, to one aspect, the rhizobial bacteria belong to one or more of the following species: Bradyrhizobium diazoefflciens . Bradyrhizobium japonicum , Bradyrhizobium elkanii, Bradyrhizobium canariense, Bradyrhizobium denitrificans Bradyrhizobium iriamotense, Bradyrhizobium jicamae, Bradyrhizobium liaoningense , Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense. In another aspect, the rhizobial bacteria are one or more of Bradyrhizobium elkanii SEMIA 501, Bradyrhizobium elkanii SEM1A 587, Bradyrhizobium elkanii SEMIA 5019, Bradyrhizobium japonicum NRRL B-50586 (also deposited as NRRL B- 59565), Bradyrhizobium japonicum NRRL B-50587 (also deposited as NRRL B- 59566), Bradyrhizobium japonicum NRRL B-50588 (also deposited as NRRL B-

[0082] 59567), Bradyrhizobium japonicum NRRL B-50589 (also deposited as NRRL B-

[0083] 59568), Bradyrhizobium japonicum NRRL B-50590 (also deposited as NRRL B- 59569), Bradyrhizobium japonicum NRRL B-50591 (also deposited as NRRL B- 59570), Bradyrhizobium japonicum NRRL B-50592 (also deposited as NRRL B- 59571), Bradyrhizobium japonicum NRRL B-50593 (also deposited as NRRL B- 59572), Bradyrhizobium japonicum NRRL B-50594 (also deposited as NRRL B-

[0084] 50493), Bradyrhizobium japonicum NRRL B -50608, Bradyrhizobium japonicum NRRL B- 50609, Bradyrhizobium japonicum NRRL B -50610, Bradyrhizobium japonicum NRRL B- 50611, Bradyrhizobium japonicum NRRL B -50612, Bradyrhizobium japonicum NRRL B- 50726, Bradyrhizobium japonicum NRRL B -50727, Bradyrhizobium japonicum NRRL B- 50728, Bradyrhizobium japonicum NRRL B -50729, Bradyrhizobium japonicum NRRL B-

[0085] 50730, Bradyrhizobium japonicum SEMIA 566, Bradyrhizobium japonicum SEMIA 5079, Bradyrhizobium japonicum SEMIA 5080, Bradyrhizobium japonicum USDA 6, Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 122, Bradyrhizobium japonicum USDA 123, Bradyrhizobium japonicum USDA 127, Bradyrhizobium japonicum USDA 129 and Bradyrhizobium japonicum USDA 532C. In one aspect, the rhizobial. bacteria is SIGNUM* ( Bradyrhizobium japonicum). In another aspect, the rhizobial bacteria is RHIZOLIQ TOP* ( Bradyrhizobium japonicum). In another aspect, the rhizobial bacteria is LAUNCHER™ (Bradyrhizobium japonicum).

[0086] In other aspects, the rhizobial bacteria belong to the genus Mesorhizobium In one aspect, the rhizobial bacteria belong to one or more of the following species; Mesorhizobium loti, Mesorhizobium huakii, and Mesorhizobiu ciceri. In another aspect, the rhizobial bacteria are one or more of: Mesorhizobium sp. WSM1271, Mesorhizobium sp, WSM1497, Mesorhizobium ciceri CC1192., Mesorhizobium huakii, Mesorhizobium loti CC829, Mesorhizobium loti SU343, Mesorhizobium loti ML542G, Mesorhizobium loti ML4404 , and Mesorhizobium ciceri CC1653. In one aspect, the rhizobial bacteria is CHICKPEA OSMIUM™ OSMOPROTECTOR Mesorhizobium ciceri).

[0087] In other aspects, the rhizobial bacteria belong to the genus Rhizobium. In one aspect, the rhizobial bacteria belong to one or more of the following species: Rhizobium leguminosarium, Rhizobium tropici, Rhizobium phaseoli, Rhizobium fredii, and Rhizobium etli In another aspect, the rhizobial bacteria are one or more of: Rhizobium sp, NGR234, Rhizobium leguminosarum Madison, R. leguminosarum USDA2370, R. leguminosarum USDA2408, R. leguminosarum USDA2668, R. leguminosarum 2370G, R leguminosarum 2370LBA, R. leguminosarum 2048G, R. leguminosarum 2048LBA, R. leguminosarum bv . phaseoli. R. leguminosarum bv. phaseoli 2668G, R. leguminosarum bv. phaseoli 2668LBA, R. leguminosarum RL542C, R. leguminosarum bv, viciae. R. leguminosarum bv. trifollii , Rhizobium etli USDA 9032, and R. etli by, Phaseoli. In one aspect, the rhizobial bacteria is PEA / LENTIL OSMIUM™ OSMOPROTECTOR (Rhizobium leguminosarum bv. viciae)

[0088] In some aspects, the rhizobial bacteria include but are not limited to Rhizobium sp.,

[0089] Rhizobium sp. NGR234, Rhizobium leguminosarum Madison, R. leguminosarum USDA2370, R. leguminosarum USDA2408, R leguminosarum USDA2668, R. leguminosarum 2370G. R. leguminosarum 2370LBA, R. leguminosarum 2048G, R. leguminosarum 2048LBA, R. leguminosarum bv. phaseoli R. leguminosarum bv. phaseoli 2668G, R leguminosarum bv. phaseoli 2668LBA, R. leguminosarum RL542C, R leguminosarum bv, viciae. R. leguminosarum bv. trifolii Rhizobium etli USDA 9032, R. elti bv. phaseoli Rhizobium tropici, Mesorhizobium sp. Mesorhizobium loti ML542O, M, ioti ML4404, Sinorhizobium sp., Sinorhizobium meliloti SD630, S, meliloti USDA1002, Sinorhizobium fredii USDA205, S fredii SF542G, S fredii SF4404, S fredii SM542C. Bradyrhizobium sp., Bradyrhizobium japonicum USDA 6, B. japonicum USDA 110

[0090] In certain aspects, the present disclosure provides a mixture comprising a first composition comprising a fungal strain selected from any one of the following:

[0091] In some aspects, the mixture comprises the fungal strain in a first composition and rhizobial bacteria in a second composition wherein the rhizobial bacteria arc members of one of the following species: (

[0092] In certain aspects, the mixture is or

[0093] In other aspects, the mixture is or

[0094] In other aspects, the mixture is or

[0095] In other aspects, the mixture is or

[0096] In other aspects, the mixture is or

[0097] In other aspects, the mixture is or

[0098] In other aspects, the mixture is or In other aspects, the mixture is

[0099] In other aspects, the mixture is

[0100] In other aspects, the mixture is

[0101] In other aspects, the mixture is

[0102] In other aspects, the mixture is

[0103] In other aspects, the mixture is

[0104] In other aspects, the mixture is

[0105] In other aspects, the mixture is

[0106] In other aspects, the mixture is

[0107] In other aspects, the mixture is

[0108] In other aspects, the mixture is

[0109] In other aspects, the mixture is

[0110] In other aspects, the mixture is

[0111] In certain aspects, the ratio by weight of fungal cells to rhizobial bacterial cells is from In one aspect, the ratio by weight of fungal cells to rhizobial bacterial cells is from 1 :500 to 500:1. In another aspect, the ratio by weight of fungal cells to rhizobial bacterial cells is from 1:100 to 100:1.

[0112] Mixtures of Fungi and Arbuscular Mycorrhizal Fungi

[0113] In certain aspects, the present disclosure provides a mixture comprising a first composition comprising a fungal strain selected from any one of the following:

[0114] In some aspects, the mixture comprises the fungal strain in a first composition and arbuscular mycorrhizal fungi (AMF) in a second composition wherein the AMF are members of one of the following species:

[0115] In certain aspects, the mixture is

[0116] In other aspects, the mixture is

[0117] In other aspects, the mixture is

[0118] In other aspects, the mixture is

[0119] In other aspects, the mixture is

[0120] In other aspects, the mixture is

[0121] In other aspects, the mixture is

[0122] In other aspects, the mixture is

[0123] In other aspects, the mixture is In other aspects, the mixture is

[0124] In other aspects, the mixture is

[0125] In other aspects. the mixture Is

[0126] In other aspects, the mixture is

[0127] In other aspects, the mixture is

[0128] In other aspects, the mixture is

[0129] In other aspects, the mixture is

[0130] In other aspects, the mixture is

[0131] In other aspects, the mixture is

[0132] In other aspects, the mixture is

[0133] In other aspects, the mixture is

[0134] In certain aspects, the ratio by weight of fungal cells to AMF cells is from 1:500 to In one aspect, the ratio by weight of fungal cells to AMF cells is from 1:500 to 500:1. In another aspect, the ratio by weight of fungal cells to AMF cells is from 1:100 to 100:1. Mixtures of Fungi and Free-Living Nitrogen-Fixing Bacteria and / or Phosphate- Solubilizing Bacteria

[0135] In certain aspects, the present disclosure provides a mixture comprising a first composition comprising a fungal strain selected from any one of the following: In some aspects, the mixture comprises the fungal strain in a first composition and free- living nitrogen-fixing bacteria in a second composition wherein the free-living nitrogen-fixing bacteria are members of one of the following species:

[0136] In certain aspects, the mixture is

[0137] IN other aspects, the mixture is

[0138]

[0139] Ih other aspects, the mixture is

[0140] In other aspects, the mixture is

[0141] In other aspects, the mixture is

[0142] In other aspects, the mixture is

[0143] In other aspects, the mixture is

[0144]

[0145] In other aspects, the mixture is

[0146] In other aspects, the mixture is

[0147] In certain aspects, the ratio by weight of fungal cells to freoliving nitrogen-flxing bacteria cells is from , , , , . In one aspect, the ratio by weight of fungal cells to Oree-living nitfogen-fixing bacteria cells is from 1:500 to 500:1. In another aspect, the ratio by weight of fungal cells to freo-living nitrogen* fixing bacteria cells is from .

[0148] Kit of Parts

[0149] According to one embodiment, individual components of the composition according to the invention such as parts of « kit or parts of abinaiy mixture may be mixed by the user himself in a spray tank or any other kind of vessel used for applications (c.g., seed treater drums, seed pelleting machinery, knapsack sprayer) and further auxiliaries may be added, if appropriate. Consequently, one embodiment of the invention is a kit for preparing an agricultural composition, the kit comprising the individual component in a mixture comprising: a) a first composition comprising a fungal strain: and b) a second composition comprising rhizobial bacteria, an arbuscular mycorrhizal fungus (AMF), or free-living nhrogen-fixing bacteria, as defined herein, in a spatially separated arrangement.

[0150] Moreover, the kit of parts according to the present invention can additionally comprise at least one auxiliary selected from the group consisting of extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost proterxante, thickenersand a^uvants. Thus, at least one auxiliary can be present either in the fungal strain component of the kit of parts or in the rhizobial bacteria, aft arbuscular mycorrhizal fungus (AMF), or free-living nitrOgen- fixing bacteria of the kit of parts being spatially separated or in both components.

[0151] Methods of Application

[0152] As used herein, the ternu “inoculate,” “apply,” “treat,” and “deploy” are used interchangeably asarethdrassociatednouns(i.e., “inoculation, “application,” “trctement,”and “deployment”). We inoculation of the plant with the microbial species may be achieved by any suitable means such as direct addition to the soil and / or plant. roots and / or to soil proximal to plant rpots, or may be achieved by an initial microbial inoculation of any propagation material, seeds, seedlings and / or immature plants of the crop plant prior to placement of the seed, seedling or immature plant in the soil within which the plant will grow; The inoetdation of the microbial species may also be achieved by direct addition to a culti vated soil prior to sowing seeds or planting seedling that are coated or partially coaled with microbial species such that the microbes will become associated with, or grow proximal to, or grow into the roots of a crop plant as the crop matures.

[0153] By means of the inoculation, the microbes are deliberately encouraged to become established in the soil and / or grow proximal to, or grow into the roots of a plant (i,e., become associated with) that is a crop plant, wherein it would be understood the microbe may exist and grow in the soil or exist within the plant, or m both simultaneously. In aspects of the invention wherein the treatments or methods rely on the inoculation of a plant with a microbe, it would be understood the microbe need only be associated with the plant for parte of the microbe's lifecycle and that the microbe may survi ve in the soil in the absence of a plant host or host crop plant. In other embodiments, the inoculation may be considered a semi-permanent inoculation to a plot of so il that is cultivated, such that the microbe is deployed to said plot of soil and is retained by the soil as the crops are rotated, evert in the absence of crops for periods of time. lit some embodiments, the soil is inoculated with the microbial species. The soil may be inoculated with the microbial species prior to planting the plant, for example before, during, or after tilling the soil in preparation for planting. In other embodiments, the soil may be inoculated with the microbial species after the plant has been planted. In some embodiment, the soil is inoctdated with the microbial species by planting i« the soil plants that have been inoculated with the microbial species.

[0154] According to certain aspects of the inventimi, a find composition comprising a fungal strain is applied in combination with a second composition comprising rhizobtal bacteria, an arbuscular mycorrhizal fungus (AMF), or free-living nitrogen-fixing bacteria. “In combination” in context shall mean foal the first composition and the second, composition are applied to a plant, seedling, plant propagation material, or the locus surrounding the plant material simultaneously or with a time Span of no more than 14 days, preferably no more than 7 days or 3 days.

[0155] The inventive compositions are suitable for application to any seed of any plant variety which is used in agriculture, in greenhouses, in forests or m horticulture and viticulture, tn particular, this is the seed of cereals (Such as wheat, barley, rye, triticale, sorghum / mUlet arid oats), maize, cotton, soya beans, rice, potatoes, sunflower, bean, coffee, beet (for example sugar beet and fodder beet), peanut, oilseed rape, poppy, olive, coconut, cocoa, sugar cane, tobacco, vegetables (such as tomato, cucumbers, onions and lettuce), turf and ornamentals (see also below). The treatment of the seed of cereals (such as wheat, barley, rye, triticale and oats), maize and rice is of particular significance.

[0156] As also described herein, the treatment of transgenic seed with the Inventive active compound combinations or compositions is of particular significance. This relates to the seed of plants containing at least one heterologous gene which enables the expression of a polypeptide or protein having insecticidal properties. The heterologous gene in transgenic seed can originate, for example, from microorganisms of the species Bacillus, Rhisabwm, Psettdomonas, Serratia, Tritihaderma, Clavibacter, Glomus or Gliacladban. This heterologous gene preferably originates from Bacillus sp., in which case the gene product is effective against the European maize borer and / or the Western maize rootworm. The heterologous gene more preferably originates from BacOlux itoringiensls. In the context of the present invention, the inventive composition is applied to the seed alone or fa a suitable formulation. Preferably, the seed is treated in a state in which it fa sufficiently stable for nd damage to occur in the course of treatment. In general, the seed can be treated at any time between harvest and sowing. It fa customary to use seed which has been separated from the plant and freed from cobs. shells, stalks, coats, hairs or the flesh of the fruits. For example, it is possible to use seed which has been harvested, cleaned and dried down to a moisture content of less than 15% by weight Alternatively, it is also possible to use seed which, after drying, for example, has been treated with water and then dried again.

[0157] When treating the seed, care must generally be taken that tire amount of the inventive composition applied to the seed and / or the amount of farther additives is selected such that the germination of the seed is not impaired, or that the suiting plant is not damaged. This has to be borne in mind in particular in the case of active ingredients which can have phytotoxic effects at certain application rates.

[0158] The inventive compositions can be applied direcfly, i>e., without containing any other components and without having been diluted, fa general, it is preferable to apply the compositions to the seed fa the form of a suitable formulation. Suitable formulations and methods for seed treatment are known to those skilled in the art and are described, for example, in the following documents: US 4572.417. US 4,245,432, US 4,808,430, US 5,876,739, US 2003 / 0176428, WO 2002 / 080675, and WO 2002 / 028186.

[0159] The active compound combinations usable in accordance with the invention can be convened to the customary seed dressing formulations. such as solutions, emulsions, suspensions, powders, foams, slurries or other coaling compositions for seed, and also ULV formulations.

[0160] These formulations are prepared in a known manner, by mixfag the active compound combinations with customary additives, for example customary extenders and also solvents or diluents, dyes, wetting agents, dispersants, emulsifiers, amifoams, preservatives, secondary thickeners; adhesives, gibberetims and also water.

[0161] Useful dyes which may be present in the seed dressing formulations usable in accordance with the invention are all dyes which are customary far such purposes. It is possible to use either pigments, which arc sparingly soluble in water, or dyes, which sue soluble in water. Examples Include the dyes know by the names Rhodamine B, CJL Pigment Red, 112 and -CX Solvent Red L

[0162] Useful wetting agents which may be present in the seed dressing formulations usable in accordance with the invention are all substances which promote wetting and which are conventionally used for the formulation of active agrochemical ingredients, Preference is given to using alkyl naphthalcnesulphonatcs. such as diisopropyl or diisobutyl napiithalenesulphonates.

[0163] Useful dispersants and / or emulsifiers which may be present in the seed dressing formulations usable in accordance with (he invention are all nonionic, anionic and cationic dispersants conventionally used tor the formulation of active agrochemical ingredients. Usable with preference arc nonionic or anionic dispersants or mixtures of nonionic or anionic dispersants. Suitable nonionic dispersants include especially ethylene oxide / propylcnc oxide block polymers, alkylphcnol polyglycol ethers and tristryrylphenol polyglycol ether, and the phosphated or sulphated derivatives thereof Suitable anionic dispersants are especially lignosulphonates, polyacrylic acid salts and arylsuiphonatedbrmaldchyde condensates.

[0164] Antifoams which may be present in the seed dressing formulations usable in accordance with (he invention are al) foam-inhibiting substances conventionally used for the formulation of active agrochemical ingredients. Silicone antifoams and magnesium stearate can be used with preference.

[0165] Preservatives which may be present in the seed dressing formulations usable in accordance with the invention are all substances usable for such purposes in agrochemical compositions. Examples include dichlorophcne and benzyl alcohol hemiformal.

[0166] Secondary thickeners which may be present in too seed dressing formulations usable in accordance with the invention arc all substances usable for such purposes in agrochemical compositions. Preferred examples include cellulose derivatives, acrylic acid derivatives, xanthan, modified clays and finely divided silica.

[0167] Adhesives which may bepresenl id the seed dressing formulations usable in accordance with the invention are all customary binders usable in seed dressing products. IWcrtcd examples Include polyvinylpyrrolidone, polyvinyl acetate^ polyvinyl alcohol and ty lose.

[0168] The seed dressing fonnulations usable in ac Sc3oKrdance with the invention can be used, either directly or after previously having been diluted with water, for the treatment of a wide range of different seed, including the seed of transgenic plants.

[0169] For treatment of seed with the seed dressing formulations usable in accordance With the invention, dr the preparations prepared therefrom by adding water, all mixing units usable customarily for the seed diming are useful Specifically, the procedure in the seed dressing is to place the seed into a mixer, to add the particular desired amount of seed dressing formulations, either as such or after prior dilution with water* and to mix everything until the formulation is distributed homogeneously on the seed. If appropriate, this is followed by a drying process.

[0170] Analysis of Organic Carbon in the Soft

[0171] In certain aspects, the disclosed mixtures increase organic carbon in the soil Soil organic C (SOC) concentration in mineral soils (0-10 cm depth) varies from <0,2% to >11.6% C; above this concentration, a soil is classified as a ‘peat' soil, for example, in peatlands or tundra lands.

[0172] Soft organic C in mineral soils contains a range of organic substances at various stages of dewnnposition such as plant materials - both produced aboveground (straw, litter) and belowground (roots, root exudates), fungal hyphae, soil fauna, and microbid biomass and their products. Organic compounds include lipids, proteins, carbohydrates, quinones, and their derivatives; Major functional groups include alkyl C (10-45 ppm), N-alkyi and methoxyl C (45-60 ppm), O-alkyl C (60-110 ppm), aromatic C (110-145 ppm), phenolic C (145-165 ppm), and amide add carboxyl C (165-215 ppm), as identified in13C NMR Spectra of SOC (Almeida el al.2021 ), These are also grouped as aromatic, aliphatic and polysaccharide groups.

[0173] Since SOC consists of different C substances, which turnover (decompose) at different rates, persist in soil for different periods, stabilize with minerals with different mechanisms, and contribute to bio-physico-chemical functions in separate ways (Chenu et al, 2015), Most components of SOC are separated by chemical oxidation (acids, alkali, oxidants), biological (decomposition rates, microbial respiration), and physical methods. Of these, physical methods are preferred because these methods cause minimum disturbance, disruption, and alteration of SOC substances in soil. The physical methods are based on density, size, and sedimentation of soil, and justification of these methods are given by Poeplau et ah (2018).

[0174] Density, size, and sedimentation procedures broadly fractionate SOC or SOM in three groups, fPOC or fPOM, aggregate occluded particulate organic C (oPOC, or oPOM) and silt-belay size associated or fine mineral-associated organic C (MAOC or MAOM). Dissolved organic C, DOC or DOM are also measured and plays a significant role in MAOM formation although it accounts for <2% of total Plant C or SOC.

[0175] The stabilized MAOM are separated from labile organic matter using density and size fractionation procedure (Poeplau etal. 20 J 8; Mayer ei a|. 2022; Rodrigues el al. 2022). The organic matter that floats in the heavy density liquid, either sodium iodide (NAI) solution or sodium polytungstate (SPT, or ) al 1.8 Mg m-3(1.8 g cm4) -or <1.8 Mg m-3density soil organic matter arc considered as 1POM, After separation of fPOM, occluded particulate organic matter within aggregates, oPOM and MAOM arc Usually dispersed either using ultrasonic energy or sodium hexametaphosphate solution to disperse the soil particles, In the former, ultrasonic energy, -400 - 500 J mL-1is applied to the soil in the SPT solution (1.8 Mg m-3) contained in a ternperature-controlled container. Excess oPOM is separated, and the remaining soil is sieved through 53 μm sieve to collect < 53 μm as MAOM. The >53 μm fraction remained on the sieve to considered as the organic matter in the sand-size fraction, usually contains small amount of organic C and could be added to the fPOM fraction, This to circumvented when after separation of fPOM, excess SPT is washed from the remaining: soil and the soil is dispersed in sodium hexametaphosphatc and stoved through 53 μm stove to collect < 53 μm as MAOM. The >53 μm fraction remained on the sieve is considered as the organic matter occluded in the sand-size aggregates or aggregate C since sand fraction as such contains only small amount of organic carbon. Thus, SOC is separated into three fractions: fPOM, oPOM, and MAOM. Obviously, dissolved organic matter (DOM) to either lost or added to the MAOM fraction although in most mineral soils, DOM constitutes <2% of SOC (Poeplau et al 2018), For practical purposes and routine SOC fractionation, SOM cap be separated into >53μm POM (fPOM + oPOM) and < 53 μm MAOM (Uvallec et al 2019),

[0176] The MAOM fraction provides the long-term storage of SOC (Kleber et al 2015; Hemingway et al 2019). However, MAOM is subject to the C saturation of fine silt+clay (<53 μm) or fine mineral fraction, which to dependent on silt+clay contents (Feng et al, 2013) and their mineralogy, Fe and Al (hydro-)oxidcs, specific surface area, soil architecture, nature of organic C inputs, especially their C and N contents, and soil pH. Once the C saturation of mineral fraction is achieved, further SOC sequestration for the long-term storage as MAOM to not likely to occur although the potential turnover through C mineralization and fresh C addition may still be required (Mayer et al. 2022; Rodrigues et al. 2022). From the boundary line approach, Feng et al (2013) estimated that the alHclay size fraction (<2 pm) may store 84+1 g C kg- 1 silt+clay size fraction in 2:1 clay dominant (smectite, illite, vermiculite) soil, and 43±1 g C kg-1 silt+clay in 1:1 day dominant (kaolinite) soil. This provides a ‘rule of thumb’ estimate to identify a soil, in which SOC may be sequestered long-term in the MAOM fraction. It to worth noting hero that the 20-53 μm MAOM fraction may contain silt-size micro* aggregates which may have faster turnover rate than the <20 μm MAOM.

[0177] Further, organic carbon may not uniformly cover the surface of the fine mineral fraction. For example, Schweizer et al. (2021) found that day surfaces of the soil containing low clay contents (5-18%) had twice as much organic C in the MAOM than the high day soils. It is sobering to note that long-term field experiments have shown that it is less likely that SOC will be sequestered in the stabilized MAOM fraction if this fraction is already saturated (Mayer et al. 2022; Rodrigues et at, 2022), and further C inputs will be stabilized in the oPOM fraction (occluded in aggregates) or remain (POM. However, oPOM is readily lost when the soil is disturbed. It is not known whether there is a saturation limit for the oPOM fraction. (POM can be increased in soil indefinitely although this fraction is affected by global warming, add quantity and quality of continuous C inputs more than the other SOM fractions (Lugato el ak 2021 ; Rocei et al. 2021 ),

[0178] SOC fractionation protocols vary widely. Poeplau et al (2018) compared two protocols id: use at the time of the study, Details of these protocols and a preferred protocol are given by

[0179] Poeplau et al. (2018). In conclusion, they found that no SOC fraction identified file rapid turnover rate component, that particle-size separation was better for separating the (POM than that the oPOM incorporated into aggregates, and the separation of silt* clay-size fraction from the sand-size fraction was the most effective protocol in identifying fractions of different turnover rates. Admittedly, since microbial inoculants in the rhizosphere may be involved in aggregation (Mugerwa and McGee 2017; Buss et al, 2021), and, therefore, soil structure, it is recommended that in such situations oPOM separation may be desirable to detect the effect of inoculants in C accumulation in the rhizosphere of the microbial inoculated plants. However, as stated above, since turnover rate of oPOM is uncertain, and subject to disturbance, it should not be considered for long-term C sequestration in soil.

[0180] Planta

[0181] The mixtures and the compositions of the invention can be applied to any plants or plant parts.

[0182] Plants mean all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants may be plants which can be obtained by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods, including the genetically modified plants (GMO or transgenic plants) and the plant cultivars which are protectable and non-protectable by plant breeders* rights.

[0183] In some embodiments, the plant is either amonocotyledonous plant (aka a monocot) or a dicotyledonous plant (aka a dieot) “. Monocotyledon,” “ monocolyledpnous,” or “monocot” are understood to mean grass and grass-like flowering plants, the seeds of which typically containonly one embryonic leaf, or cotyledon. “Dicotyledon, “or “dicotyledonous,” or “dicot ” ate understood to mean plants the seeds of which typically have two embryonic leaves, or cotyledons.

[0184] “Genetically modified plants” (“GMO" of “transgenic plants”) am plants of which a heterologous gene has been stably integrated into the genome. The expression heterologous gene** essentially means a gene which is provided or assembled outside the plant and when introduced in the nuclear, chleroplastic or mitochondrial genome. This gene gives the transformed plant new or improved agronomic or other properties by expressing a protein or polypeptide of interest or by downregulating or silencing other genef s) which are present in the plant (using for example, antisense technology, cosuppression technology, RNA interference - RN Al - technology or microRNA - miRNA - technology). A heterologous gene that is located in the genome is also called a transgene. A transgene that is defined by its particular location in the planl genome is Called a transformation or transgenic event.

[0185] “Plant cultivars” are understood to mean plants which have new properties ( “trails”) and. have been obtained by conventional breeding, by mutagenesis or by recombinant DNA techniques. They can be cultivars, varieties, bio- or genotypes.

[0186] “Plant parts” arcundcrstood to mean all parte and organs of plants above and below the ground, such as shoots, leaves, noodles, stalks, stems, flowers, fruit bodies, fruits, seeds, toots, tubers and rhizomes. The plant parts also include harvested material and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, slips and seeds.

[0187] Plants which can be treated in accordance with the methods of the invention include the following; cotton, flax, grapevine, fruit, vegetables, such as Rosaeeae sp. (far example pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds and peaches, tod soft fruits such as strawberries), Ribesioidtie sp., Jugtandaceae sp„ Betylaceae sp.> Anacardiaceae sp.. Fagaceae Mpraceae sp., Oleaceae sp.. Actintdticeae sp.. Lauraceae sp.. Musaceae sp. (for example banana trees tod plantations), Rubiaceae sp. (for example (fofthe), Theaceae sp„ Sterctdiceae sp„ Rutoc«ae sp. (for example lemons, oranges and grapefruit); Solcmaceae sp. (for example tomatoes), Litiaceae sp., Asteraccae sp. (for example lettuce), Umbettiferae sp>, Cruciferae sp.. Ckenopodifteeae sp., CucMrbitoceae Sp. (for example cucumber), Alliaeeae xp. (for example ledk, onion), Papitionactoe sp. (for example peas); major crop plants, such as Gramirieae sp. (for example maize, turf, cereals such as wheat, rye, rice, barley, oats, millet tod triticale), Asteracdae sp, (fcr example sunflower), Brassicaceae sp. (for example white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radishes, and oilseed rape, mustard, horseradish and cress), Fajbacae sp. (for example bean, peanuts), Papttionaceae sp. ifor example soya bean), Sotonaeeae sp (for example potatoes), Chenopodiacene sp. (for example sugar beet, fodder beet, Swiss chard, beetroot): useful plants and omamenlal plants for gardens and wooded areas; and genetically modified varieties of each of these plants,

[0188] In some aspects, the plants or plant parts are cover crops, Cover crops include but are not limited to ryegrass, clover, red clover, white clover, crimson clover, annual medics, annual ryegrass, Italian ryegrass, canola, fine fescue, Kentucky bluegrass, orchard grass, and other grasses.

[0189] In other aspects, the plants or plant parts are grasses such as switchgrass, tall fescue, meadow fescue, perennial ryegrass, Italian ryegrass, orchardgrass, guinea grass, foxtail millet, peari millet, Bahia grass and Miscanthus

[0190] Synergistic Effects

[0191] In a preferred embodiment, the mixtures / composttions according to the invention have, as an improvement of the application profile, synergistic effects. These synergistic effects can be observed, for example, when applying the first composition and the second comprnMm togeliier; however, they can frequently also be observed when the components arc applied at different times (splitting). It is also possible to apply the individual compositions in a plurality of portions (sequential application); for example pre-emergence applications followed by post- emergence applications or early post-emergence applications followed by medium or late post- emergence applications. Preference is gi ven here to the joint or almost simultaneous, application of the active compounds of the nuxturcs / compositions according to the inventipn.

[0192] The synergistic effects permit a reduction of the application rates of the individual active compounds, a higher efficacy at the same application rate, an extension of the period of application and / or a reduction in the number of individual applications required and— as a result for the user-plant, soil, and microbial systems which are more advantageous economically and ecologically,

[0193] The present invention is further illustratied by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporaiedherein by reference in their entirety for all purposes. EXAMPLES

[0194] Example L Growth Compatibility of Thozetella nivea DMTR-CTR-2359 (NMI Accession No. V22 / 003496) and Leptodontidium orchidicola DMTR-CTR-4873 (NMI Accession No. V22 / 003497) with Bradyrhizobium japonicum CB1809

[0195] Bradyrhizobium japonicum CB 1809 has been shown io enhance soybean nodulation in New South Wales, Australia (see Roughley,R. I, et al. (1995). Autecology of Bradyrhizobium japonicum in soybean-rice rotations. Plant and Soil, 176(1), 7—14). An experiment was conducted to determine the compatibility of this rhiwbial Strain with DMTR- CTR-2359 (NMI Accession No. V22 / 003496) DMTR-CTR-

[0196] 4873 (NMI Accession No. V22 / 003497).

[0197] Htigs from agar cultures of each fungal strain were placed on PDA-100ppm streptomycin plates. Rhizobia were grown by streaking a culture from a CB1809 on PDA plates.

[0198] For control plates (see flic top and bottom rows of FlGs. 1A and 1B), the PDA pities were split into two halves by drawing a line down the middle and labeling each tide. Two agar plugs of fungal mycelia were transferred from the leading edge of the colony to opposite edges of the fresh plate oir rhirobial sells wore streaked on opposite sides of the fresh plate.

[0199] For interaction plates (see the middle rows of FIGs. 1A and 1B), one agar plug of fungal mycelia was transferred from the leading edge of the target colony to one edge of the piste, arid then the bacterial isolate was Streaked up to the other edge of half the plate. The PDA plates were incubated in the dark at 25°C for 21 days and then photographed to image fungal and rhizobial growth.

[0200] Bradyrhizobium japonicum CB1809 growth was compatible with the fungal growth of both Thozetella nivea DMTR-CTR-2359 (NMI Accession No. V22 / 003496) (FIG. 1A) arid Leptodoaridium orchidicola DMTR-CTR-4873 (NMI Accession No. V22 / 003497) (FIG.1 B).

[0201] Example 2. Determination of a Synergistic Effect

[0202] The synergistic activity of the mixtures of microalgae and biological control agent described herein can be determined as follows. A synergistic effect is present when the activity of the mixture exceeds the total of the activities of the microaigac and biological control agent when, applied individually. The expected activity for a given combination of two agents can be calculated as follows (cf> Colby, S. R, “Calculating Synergistic and Antagonistic Responses of Biological control agent Combinations;” Weeds, 1967, 15; 20-22):

[0203] If • X is the activity when agent A is applied at an application rate of m gallons / acre (or liters / hectare),

[0204] • Y is the activity when agent B is applied at an application rate of n gallons / acre (or liters / hectare),

[0205] • E is the activity when the active compounds A and B are applied at application rales of m and n gallons / acre (or liters / hectare), respectively,

[0206] • Then

[0207] If the actual activity exceeds the calculated value, then the activity of the combination is supdr additive, i.e., a synergistic effect exists. In this case, the efficacy which was actually observed must be greater than the value for the expected efficacy (E) calculated from the above- mentioned formula.

[0208] Example 3- Synergistic Root Modulation with Bradyrhizobium japonicum US-B-1237 (NRRL Accession No. B-68330)

[0209] An experiment was conducted to evaluate the effect of co-inoculation of soybeans with

[0210] Bradyrhizobium japonicum US-B-1237 (NRRL Accession No. B-68330) and each of Trichoderma longipile / spirale DMTR-CTR-1291 (NMI Accession. No. V22 / 006354). Leptodontidium orchidicola DMTR-CTR-4873 (NMI Accession No. V22 / 003497), Acrocalymna vagum DMTR-CTR-l 1556 (NMI Accession No. V22 / 006357), and Leptodontidium orchidicola US-210 (ATCC Accession No, ETA-12744 I).

[0211] As a preliminary matter, the compatibility of each of the fungal strains with

[0212] Bradyrhizobium japonicum US-B-1237 (NRRL Accession No. B-68330) was confirmed as described in Example L Each of the fungal strains was found to be compatible with

[0213] Bradyrhizobium japonicum US-B-1237 (NRRL Accession No. B-68330). FIG. 2 presents a photograph of representative results with Leptodontidium orchidicola US-210 (ATCC Accession No. PTA-127441).

[0214] Soybean seeds were inoculated with the number of colony forming units (CPU) indicated in Table 3. Soybean seeds were inoculated with Bradyrhizobium japonicum US-B* 1237 (NRRL Accession No, B-68330) alone, each of the fungal strains atone, and combinations of Bradyrhizobium japonicum US-B-1237 (NRRL Accession No, B-68330) and each of the fungal strains. Untreated soybean seeds were planted as controls. Seeds were planted nt-A sterile synthetic soil mix and grown m agreenhouse.

[0215] After five weeks the plants were harvested and the root nodules were counted. All values represent the average of at least three replicates. The number of nodules reported represents the difference between the a verage number of nodules observed in ihie treated group from that observed in the untreated control group. In this experiment, no nodulation was observed in the untreated control plants.

[0216] If the actual number of nodules exceeds the calculated value, then the activity of the combination is super additive, Le., a synergistic effect exists; In this case, the efficacy (i.e., the nodulation) which was actually observed must be greater than the value for the expected efficacy (8) calculated from the formula in Example 2.

[0217] The restilts shown in Table 3 dearly indicate a synergistic effect resulting from the combination treatment of soybean plants with

[0218] Table 3

[0219] Example 4. Siderophore Produetiou with Bacterial Strains

[0220] Siderophore production was tested by growing bacteria on Chrome Azurol S (CAS) agar media. Upon siderophore production by microbe CAS media color changes from blue to orange, Production of iron-chelating siderophores by tile bacteria was observed after growing the cultures on the CAS media (ie., iron-selective media) for 14 days. The resulting halo diameters were mewcod for each bacterial strain with all measurements take With three independent replicates, A total of 19 bacterial strains were evaluated for siderophore production.

[0221] As shown in FIG.3, fifteen of the nineteen tested bacteria produced siderophores with some having higher production , The species with the highest siderophore producti on (i .e vwith hate diameters ≥ 3 mm) were and another strain designated as Strain K. Eleven other strains produced siderophores to a lesser degree (i.c.<, with halo diameters ≤ 2mm).

[0222] Example 5 Phosphate Solubilization with Bacterial Strains

[0223] Phosphate solubilization was tested by growing bacteria on Pikovskaya (PVK) media. A dear halo is formed upon solubilization of phosphate. The PVK. assay was executed by •(potting single bacterial colonies onto PVK and growing for 22 days. Assays were performed with three replicates of each bacterial strain. To determine the phosphate solubilization index (PSI) for each strain, ameasurement of the ratio between colony diameter anrizone of clearance produced when the bacteria solubilize phosphate was measured. Eleven of lheninetcen bacterial strains tested were able to solubilize phosphate to some degree. The strongest solubilizers were ) with a PSI of 2.5 and 2.9 respectively. All others strains had a lower PSI and therefore lower ability to solubilize phosphate (see FIG.4).

[0224] Example 6. Nitrogen Fixation Capacity with Bacterial Strains

[0225] Nitrogen is a key component of amino acids, which form the building blocks of plant proteins and enzymes. lherefore, its acquisition is important for healthy crops. Currently, fotmets often address a lack of nitrogen with synthetic fertilizers, bed poor fertilizer management can lead to downstream pollution (e.g. water eutrophication). Increased crop yield and soil carbon sequestration can be achieved by combining a nitrogen-fixing bacteria with a fungal inoculant, which could also decrease the need for synthetic fertilizers and improve crop management strategics,

[0226] Nitrogen-fixing potential of We bacterial strains was. assessed by growing Bacteria on Ashby’s mannitol agar. This media selects for nitrogen-fixing bacteria that use mannitol as a carbon source and atmospheric nitrogen nitrogen source. Nitrogen-fixing ability was assessed by comparing the growth of all organisms on each plate with a scoring system of 0 to 5. A score of 0 was assigned to the worst performer (!,&., smallest colony) on the plate while a score qf 5 was assigned to the best performer (i.ewlargest colony) on the plate.

[0227] The nitrogen-fixing potential of the bacteria was assessed by spotting a single colony of each of six species onto one Ashby’s mannitol agar plate and growing them for 22 days wilh throe replicate plates scored. Bacterial species were evaluated against each other on the same plates.

[0228] Out of the nineteen bacteria assessed, all species grew on Ashby’s mannitol agar media to vmying degrees. The Species with the best growth on Ashby’s media included Kosokoma (i,e., scores of 4 or greater; sec FIG.5). These strains showed large and mucoid growth on the plate, indicating favorable nitrogen-fixing potential; Example 7. Nitrogen Fixation Capacity whit Bacterial Strains

[0229] Plant hormones play a crucial role in the continued growth and development of plants. Of these hormones, the auxin class is integral; The bacterial strain's ability to produce indole- 3-acetic acid (IAA), the most common plant hormone in the auxin class, was evaluated,

[0230] The production of IAA was determined by growing liquid cultures of bacterial species in nutrient broth for seven days in darkness. Supernatants were then collected and added to Salkowski reagent (1:1 ratio). Thus provided a colorimetric detection method and spectral measurement for approximate concentrations. Three technical replicates were evaluated for each measurement.

[0231] IAA production in the bacterial strains is shown in FIG. 6. All the tested bacteria produced IAA at different levels with most producing greater than 5 μg / ml The highest bacterial producers of the IAA plant hormone were All Other bacterial strains produced between 1- 10 μg / ml of lAA.

[0232] All headings arc for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0233] While the invention has been described in connection with specific embodiments thereof, it willbc understood that it is capable of further modifications and tins application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within know or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0234] INCORPORATION BY REFERENCE

[0235] Alt references, articles, publications, patents, patent publications, and patent applications cited herein within the above text and / or cited below arc incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. REFERENCES

Claims

CLAIMSWhat is claimed is:

1. A mixture comprising: a) a first composition comprising a fungal strain belonging to a genus Selected ftom the group consisting. of:andb) a second composition comprising rhizobial bacteria selected from the group consisting of:(also known as , andcombinations thereof; wherein: a combination of the first composition and the second composition exhibits synergy.

2. The mixture of Claim 1 wherein the rhizobial bacteria comprise3. The mixture of Claim 3, wherein the rhizobial bacteria comprise4. The mixture of Claim 1 , wherein the rfiizobial bacteria comprise(also known as ).

5. The mixture of Claim 4, wherein the rhizobial bacteria comprise6. A mixture comprising: a) a first composition comprising a ftmgal strain belonging to a genus selected from the group consisting of:. : andb) a second composition comprising an arbuscular mycorrhizal fungus (AMF) selected from the group consisting of:and combinations thereof; wherein a combination of the first composition and the second composition exhibits synergy.

7. A mixture comprisin g: a) a first composition comprising a fungal strain belonging to a genus Selected from the group consistingof:; andb) a second composition comprisirig free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria belonging to a genus selected from the group consisting of;and combinations thereof;wherein a combination of the first composition and the second composition exhibits synergy.

8. The mixture of Claim 4, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria are selected from the group consisting of:and combinations thereof.

9. The mixture of Claim 8, wherein the free-living nitrogen-fixing bacteria arid / or phosphate-solubilizing bacteria comprise10. The mixture of Claim 9, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise,11. The mixture of Claim 8, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise12. The mixture of Claim 11, wherein the free-li ving nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise13, The mixture df Claim 8, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise14. The mixture of Claim 13, wherein the free-living nitrogen-fixing bacteria and / cr phosphate-solubilizing bacteria comprise15. The mixture of Claim 8, wherein the free-li ving ni irogen-jfixin^ bacteria and / or phosphate-solubilizing bacteria compriseThe mixture of Claim 15, wherein the free-living nitrogen-fixingbacieria and / or phosphate-solubilizing bacteria comprise17, The mixture of Claim 8, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise18. The mixture of Claim 17, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise19. The mixture of Claim 8, wherein the free-living nitrogen-fixing bacteria and / or phosphate-solubilizing bacteria comprise20. The mixture of Claim 19. wherein the free-living nifrogen-fixing bacteria and / w phosphate-solubilizing bacteria comprise21. The mixture of anyone of Claims 1 to20, wherein the fungal strain has a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that is at least 90% identical to the nucleotide sequence of any one of SEQ ID NOs; 1 -22.

22. The mixture of any one of Claims 1 to 21, wherein the fungal strain is selected from the group consisting of:, and a mutant thereof having all identifying characteristics of the re^ectivc strain,23. The mixture o f an y one of Claims 1 to 22, wherein (he first composition and the second composition are formulated independently for application to a plant as a soil drench, in-furrow treatment foliar application, or seed treatment.

24. A mixture comprising at least two fungal strains selected from the group consisting of:and a mutant thereof having all identifying diaractcristics of the respective strain.

25. The mixture of any one of Claims 1 to 24, further comprising at least one auxiliary selected from the group consisting of extenders, solvents, diluents, emulsifiers,dispersants, binders, fixing agents, wetting agents, dyes, pigments, antifoams, preservatives, secondary thickeners, stickers, and combinations thereof.

26. A plant propagation material treated with the mixture of any one of Claims 1 to25 in ah amount of from 0.01 g to 10 kg per 100 kg of plant propagation material.

27. A bioorganic soil conditioner comprising the mixture of any one of Claims 1 to25.

28. A kit-of-parts comprising the first composition and the second composition of tlie mixture, defined in any one of Claims 1 to 25, in a spatially separated arrangement, and optionally at least one auxiliary, wherein a combination of the first composition and the second composition exhibits synergy.

29. A method of plant enhancement comprising applying to a plant, seedling, plant propagation material, or the locus surrounding the plant material an effective amount of the mixture of any one of Claims 1 to 25 to enhance at least oneplant characteristic, wherein the plant characteristic is selected from the group consisting of accelerated seed germination, accelerated seeching emergence, improved seedling emergence, improved leaf formation, accelerated leaf formation, improved plant maturation, accelerated plant rnmuration, increased plant y ield, increased plant growth, increased plant quality, increased plant health, increased fruit yield, increased fruit growth, increased fruit quality, improved root health, increased root nodule formation, plait health, plant resistance to salt stress, plant resistance to heat stress, plant resistance to heavy metal stress, ptant resistance to drought, and combinations thereof30. The method of Claim 29, wherein the first composition and / or the second composition are administered at a rate of 0.01 g to 10 kg per 100 kg of plant propagation material pre-planting and / or at a rate of 0.1-150 gallons per acre (0,935-1402.5 liters pct hectare) post-planting to enhance the at least one plant characteristic.

31. The method of Claims 29 or 30, wherein the first composition and the second composition in the mixture are applied simultaneously or subsequently.

32. The method of any one of Claims 29 to 31 wherein the plarti is a legume selected from the group consisting of alfatfe, cloven peas, cowpeas, beans, mung beans, lentils, lupins, mesquite, card), soybeans, peanuts, tamarind, wisteria, siratro, plants from the Lespedeza genus, Gemstoid legumes, serradella, and combinations thereof.

33. The method of any one of Claims 29 to 31, wherein the plant is a pasture crop or cover crop selected from the group consisting of Lucerne (aka alfalfa), arrow leaf clover, Balansa clover, chicory, plantain, Phalaris. cocksfoot, fescue, prairie grass, Warregp summer grass, Italian rye grass, perennial rye grass, Biserrate, Serradella, Gland clover, Bladder clover, switchgyass, radish, medic, buckwheat, cow pea, lablab, surm hemp, sunflower, tillage radish, and subterranean clover.

34. The method of any one of Claims 29 to 31, wherein the plant is Selected from the group consisting of wheat, rice; com (maize), canola, rye, oats, barley, sorghum, millet, flax, hemp, jute, sugarcane, and cotton.

35. A method of increasing organic carbon m a soil, comprising applying to a plant, seedling, plant propagation material, or the locus surrounding the plant material an effective amount of the mixture of any oneof Claims 1 to 25, wherein the mixture is in an amount effective to increase organic carbon in the soil compared to a pan-inoculated control soil .

36. A method for sequestering atmospheric carbon for storage as organic carbon in a soil, comprising applying io a plant, seedling, plant propagation material, or the locus stirrounding the plamrnaterial an effective amount of the mixture of any one of Claims 1 to 25, wherein the mixture is m an amount effective to increase sequestered atmospheric carbon in M soil compared to anon-inoculated control soil.

37. Use of the mixture of any one of Claims 1 to 25 for plant enhancement, increasing organic carbon in a Mh or sequestering atmospheric carbon for storage as Organic carbon in a soil.

38. The method or use ufany one of claims 29 to 37, therein the mixture increases organic carbon in the soil in the stable forms of aggregate carbon faction (AggC), aggregate oecltidedparticiilate organic C (oPOC), and / or mineral-associated organic carbon (MAOC),

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