CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/068,478, filed August 21, 2020, the contents of which are incorporated herein by reference in its entirety.

Biological control agents (BCAs) as well as biostimulants become more and more important in the area of plant protection, be it for combatting various bacterial, fungal or insect pests or for improving plant health. Although also viruses are available which can be used as biological control agents, mainly BCAs based on bacteria and fungi are used in this area. The most prominent form of biological control agents based on fungi are the asexual spores called conidia as well as blastospores, but also other fungal propagules may be promising agents, such as (micro) sclerotia, ascospores, basidiospores, chlamydospores or hyphal fragments.

Fungi belonging to the genus Trichoderma have long been known to exert a beneficial effect in plants either in being effective against plant diseases or in their property to improve plant health (Harman et al. (2004) Trichoderma species-opportunistic, avirulent plant symbionts. Nature Reviews Microbiology, 2, 43-56; Druzhinina et al. (2011) Trichoderma-. the genomics of opportunistic success. Nature Reviews Microbiology, 9, 749-759).

Bradyrhizobium japonicum is a species of legume-root nodulating, microsymbiotic nitrogen-fixing bacteria belonging to rhizobia. B. japonicum is frequently added to legume seed, in particular soybean, to improve crop yields.

Despite the beneficial properties of BCAs in plant protection and for improving plant health, there is still a need for even more beneficial solutions.

This technical problem has at least in part been solved by the present invention.

Accordingly, in a first embodiment, the present invention relates to a composition comprising at least one agriculturally useful fungal microorganism which is a strain belonging to the species Trichoderma asperellum, Trichoderma atroviride, or Trichoderma virens and an agriculturally useful bacterial microorganism which is of the species Bradyrhizobium japonicum or Bradyrhizobium elkani. Agriculturally useful in connection with the present invention denotes exerting a beneficial effect on plants. Such effects include a nematicidal, insecticidal, fungicidal, bactericidal effect as well as a beneficial effect on plant health and/or growth.

Trichoderma is a genus of fungi in the family Hypocreaceae that is present in all soils. Strains of many species in this genus have been characterized as opportunistic avirulent plant symbionts. Accordingly, several Trichoderma strains have been developed as biocontrol agents against fungal diseases of plants but also for improving plant health. Most biocontrol agents are from the species T. asperellum, T. harzianum, T. viride and T. hamatum. The biocontrol agent generally grows on the root surface, and so affects root disease in particular, but can also be effective against foliar diseases.

Trichoderma species useful in agriculture include /. asperellum, T. asperelloides, T. atroviride, T. fertile, T. gamsii, T. ghanense, T. hamatum, T. harzianum, T. koningii, T. longibrachiatum, T. ovalisporum, T. paucisporum, Trichoderma polysporum, T. reesei, T. stromaticum, T. theobromicola, T. virens and 7. viride. According to the present invention, the Trichoderma species used is selected from the group consisting of /. asperellum, T. atroviride, T. virens and T. viride.

More preferably, the Trichoderma species used is /. asperellum or /. atroviride. In another more preferred embodiment, the Trichoderma species used is /. viride. Most preferred are strains of the above Trichoderma species that have a beneficial effect on plant health, such as strains from /. atroviride and /. viride, and also /. virens.

Exemplary strains, belonging to the genus Trichoderma spp. are Trichoderma atroviride strain NMI No. V08/002387 (described in U.S. Patent No. 8,394,623 B2), strain NMI No. V08/002388, strain NMI No. V08/002389, strain NMI No. V08/002390, strain LC52 (e.g., SENTINEL® or TENET® from Agrimm Technologies Limited), strain CNCM 1-1237 (e.g., ESQUIVE® from Agrauxine, France), strain SCI (e.g., VINTEC® from Bi-PA or Belchim, described in International Application No. PCT/IT2008/000196), strain B77 (e.g., T77® from Andermatt Biocontrol or ECO-77® from Plant Health Products), strain LUI32 (e.g., TENET® from Agrimm Technologies Limited), strain IMI 206040/ATCC 20476 (e.g., BINAB® TF WP from BINAB Bio-Innovation AB, Sweden), strain Tl l/IMI 352941/CECT 20498 (e.g., TUSAL® from Certis), strain SKT-l/FERM P-16510 (e.g., ECOHOPE® from Kumiai Chemical Industry Co), strain SKT-2/FERM P-16511, strain SKT-3/FERM P- 17021, strain MUCL45632 (e.g., TANDEM® from Italpollina), strain WW10TC4/ATCC PTA 9707 (described in CA 2751694 Al), strain RR17Bc/ATCC PTA 9708, strain Fl l Bab/ATCC PTA 9709; strain TF280 (described in CN 107034146 A), strain OB-l/KCCM 11173P (described in WO 2012/124863 Al); Trichoderma harzianum strain KRL-AG2/ITEM 908/T-22/ATCC 20847 (e.g., TRIANUM-P® from Koppert or PLANTSHIELD® from BioWorks or TRICHO® D WP from Orius Biotecnologica), strain TH35 (e.g., ROOT-PRO® from Mycontrol), strain T-39 (e.g., TRICHODEX® and TRICHODERMA® 2000 from Mycontrol), strain DB 103 (e.g., T-GRO® 7456 from Dagutat Biolab, South Africa), strain DB 104 (e.g., ROMULUS® from Dagutat Biolab, South Africa), strain TSTh20/ ATCC PTA-10317 (described in Application EP 2478090 Al), strain ESALQ 1306 (e.g., TRICHODERMIL® from Koppert), Rifai strain KRL-AG2 (e.g., BW240® WP from BioWorks), strain T78 (e.g., OFFYOUGROW® TRIC from Microgaia Biotech), strain from Trichopel (Agrimm Technologies), strain RR17Bc/ATCC PTA 9708 (described in CA 2751694 Al), strain ThLml/NRRL 50846 (described in U.S. Patent Application Publication No. 2015/0033420 Al), strain IBLF 006 (e.g., ECOTRICH® WP and PREDATOX® SC from Ballagro Agro Tecnologia Ltda., Brazil), strain DSM 14944 (e.g., AGROGUARD® WG and Foliguard from Live Systems Technology S.A, Colombia), strain 21 (e.g., ROOTGARD® from Juanco SPS Ltd., Kenya), strain SF (e.g., BIO-TRICHO® from Agro-Organics, South Africa), strain IIHR-Th-2 (e.g., ECOSOM-TH® from Agri Life, India), strain MTCC5530 (described in U.S. Patent Application Publication No. 2012/0015806 Al); Trichoderma virens (also known as Gliocladium Virens') strain GL-21 (e.g., SOILGARD® by Certis, USA), strain Gl-21, strain G1-3/ATCC 58678 (e.g., QUICKROOTS® from Novozymes), strain DSM25764, strain G-41 (e.g., ROOTSHIELDPLUS® from BioWorks); Trichoderma asperellum strain B35 (Pietr et al., 1993, Zesz. Nauk. A R w Szczecinie 161: 125-137), Trichoderma viride strain TV1/MUCL 43093 (e.g., VIRISAN® from Isagro), strain MTCC5532 (described in U.S. Patent Application Publication No. 2012/0015806 Al), strain NRRL B-50520 (described in CN 104203871 A); Trichoderma polysporum strain IMI 206039/ATCC 20475/T-75 (e.g., BINAB® TF WP from BINAB BioInnovation AB, Sweden); Trichoderma stromaticum strain Ceplac 3550/ ALF 64 (TRICOVAB® from Ceplac, Brazil); Trichoderma asperellum strain kd (e.g., T-GRO® from Andermatt Biocontrol or ECO- T® from Plant Health Products), strain ICC 012/IMI 392716 (e.g., BIO-TAM® and REMEDIER® WP from Isagro Ricerca), strain BV10 (e.g., TRICHO-TURBO® from Biovalens), strain T34 (e.g., ASPERELLO® T34 Biocontrol from Biobest), strain T25/IMI 296237/CECT 20178 (e.g., TUSAL® from Certis), strain SKT-1 (e.g., ECOHOPE® from Kumiai Chemical Industry Co.), strain URM 5911/SF04 (e.g., QUALITY® WG from Laboratorio de BioControle Farroupilha Ltda, Patos de Minas-MG, Brazil), strain H22 (e.g., TRICHOTECH® WP from Dudutech); Trichoderma gamsii strain ICC 080 (e.g., BIO-TAM® and REMEDIER® WP from Isagro Ricerca), strain NRRL B-50520 (described in WO 2017/192117 Al); Trichoderma koningii strain SC164; Trichoderma hamatum strain TH382/ATCC 20765 (e.g., FLORAGARD® from Sellew Associates); Trichoderma fertile strain JM41R (e.g., TRICHOPLUS® from BASF); Trichoderma longibrachiatum strain Mkl/KV966 (described in WO 2015/126256A1).

Preferably, the Trichoderma strains are selected from the group consisting of Trichoderma atroviride strain CNCM 1-1237 (e.g., ESQUIVE® WP from Agrauxine, FR); Trichoderma atroviride strain SCI, having Accession No. CBS 122089, WO 2009/116106 and U.S. Patent No. 8,431,120 (from Bi-PA); Trichoderma atroviride strain 77B (T77® from Andermatt Biocontrol); Trichoderma atroviride strain LU132 (e.g., SENTINEL® from Agrimm Technologies Limited); Trichoderma asperellum strain B35 (Pietr et al., 1993, Zesz. Nauk. A R w Szczecinie 161: 125-137). Particularly preferred are Trichoderma atroviride strain CNCM 1-1237 and Trichoderma asperellum strain B35 deposited under accession number DSM 33245.

As described before, Bradyrhizobium japonicum and also B. elkani live as symbionts in leguminous plants, in particular soybean. Strains belonging to any of these two species include Bradyrhizobium japonicum (e.g., VAULT. RTM.® from BASF Corp., USA), Bradyrhizobium japonicum 532c isolated from Wisconsin field (Nitragin 61A152; Can. J. Plant. Sci. 70, 661-666, 1990; e.g., in RHIZOFLO.RTM. ®, HISTICK.RTM. ®, HICOAT.RTM. ® SUPER from BASF Agricultural Specialties Ltd., Canada), Bradyrhizobium japonicum E- 109 variant of strain USDA 138 (INTA E109, SEMIA 5085; Eur. J. Soil Biol. 45, 28-35, 2009; Biol. Fertil. Soils 47, 81-89, 2011; the complete genome sequence of which is available at NCBI GenBank under the accession number CPO1O313), Bradyrhizobium japonicum G49 (MSDJ G49; C. R. Acad. Agric. Fr. 73, 163-171, 1987); Bradyrhizobium japonicum strains deposited at SEMIA known from Appl. Environ. Microbiol. 73(8), 2635, 2007: SEMIA 566 isolated from North American inoculant in 1966 and used in Brazilian commercial inoculants from 1966 to 1978, SEMIA 586 originally isolated in Maryland, USA, in 1961 but received from Australia in 1966 and used in Brazilian inoculants in 1977 (CB 1809, USDA 136, Nitragin 61A136, RCR 3407), SEMIA 5079 a natural variant of SEMIA 566 used in commercial inoculants since 1992 (CPAC 15; e.g., GELFIX® 5 or ADHERE® 60 from BASF Agricultural Specialties Ltd., Brazil), Bradyrhizobium japonicum SEMIA 5080 a natural variant of SEMIA 586 used in commercial inoculants since 1992 (CPAC 7; e.g., GELFIX® 5 or ADHERE® 60 from BASF Agricultural Specialties Ltd., Brazil); Bradyrhizobium japonicum TA- 11 (TA11 NOD. sup. +) (NRRL B-18466; U.S. Pat. No. 5,021,076; Appl. Environ. Microbiol. 56, 2399-2403, 1990; e.g., VAULT.RTM. ® NP, from BASF Corp., USA), Bradyrhizobium japonicum strains deposited at USDA known from U.S. Patent No. 7,262,151 and Appl. Environ. Microbiol. 60, 940-94, 1994: USDA 3 isolated from GLYCINE® MAX in Virginia (USA) in 1914, USDA 31 (=Nitragin 61A164) od Serogroup 31 isolated from GLYCINE® MAX in Wisconsin (USA) in 1941, USDA 76 isolated from plant passage of strain USDA 74 (Serogroup 76) which has been isolated from G. MAX in California (USA) in 1956, USDA 110 (=IITA 2121, SEMIA 5032, RCR 3427, ARS 1-110 and Nitragin 61A89; Serogroup 110) isolated from G. MAX in Florida in 1959, USDA 121 isolated from G. MAX in Ohio (USA) in 1965 (Crop Science 26(5), 911-916, 1986); Bradyrhizobium japonicum WB74 (e.g., ECO- RHIZ SOYA® from Plant Health Products (Pty) Ltd, South Africa; or Soybean inoculant from Stimuplant CC, South Africa), Bradyrhizobium elkanii SEMIA 587 (Appl. Environ. Microbiol. 73(8), 2635, 2007; e.g., GELFIX® 5 from BASF Agricultural Specialties Ltd., Brazil), Bradyrhizobium elkanii SEMIA 5019 (=29W; Appl. Environ. Microbiol. 73(8), 2635, 2007; e.g., GELFIX® 5 from BASF Agricultural Specialties Ltd., Brazil), Bradyrhizobium elkanii USDA 76, Bradyrhizobium elkanii USDA 948. Bradyrhizobium elkanii USDA 3254, Bradyrhizobium elkanii U-1301 and U-1302 (e.g., NITRAGIN.RTM. ® OPTIMIZE from Novozymes Bio As S.A., Brazil, or NLITRASEC® for soybean from LAGE y Cia, Brazil).

As can be seen in the examples, strains of the species T. asperellum, T. atroviride and Trichoderma viride and when combined with Bradyrhizobium japonicum exert a superior beneficial effect in plants which manifests mainly in improved plant health.

Specifically, Trichoderma asperellum, such as strain B35, when applied in combination as a seed treatment, enhanced soybean growth and development. As can be seen in the appended examples, compared to standard nitrogen fixing Bradyrhizobium inoculant control, the plants treated with Bradyrhizobium and Trichoderma asperellum exhibited highest plant height, leaf surface area that resulted in increased shoot biomass at V6 growth stage (BBCH17 107). Additionally, the combination of a Trichoderma strain with B. japonicum advanced the development of plants to reproductive stages faster than the respective control. More plants were at R7 (BBCH 79 709) stage compared to control plants. As soybean plants solely rely upon biologically fixed nitrogen for their N needs, nodulation is an important feature for Bradyrhizobium to colonize the roots and fix atmospheric nitrogen. In this study we recorded an increased number of nodules per plant in the treatment of Bradyrhizobium + Trichoderma asperellum as compared to treatment with B. japonicum alone, that resulted in higher nitrogen in the plants in addition to an increased content of potassium, Zinc, Mn, Cu and B (Table 3). Without wishing to be bound by any scientific theory, the inventors believe that the increased nitrogen uptake could be explained by the increased root biomass that provided additional surface area for the increased nodules as evident from the nodule number per plant. The increased N uptake was also evident from the highest chlorophyll content from the treated plants (Table 2).

A similar study with strain T1 (Trichoderma atroviride) surprisingly revealed several similarities in plant growth promotion effects between T1 and B35. A consistent increase in plant height, root biomass and chlorophyll content were noticed when T1 was applied together with Bradyrhizobium base compared to the B35 treatments (Table 3). Hence our studies strongly indicate the ability of Trichoderma strains to positively influence growth and yield of soybeans.

Said agriculturally useful fungal or bacterial microorganisms embraces not only the isolated, pure Trichoderma and Bradyrhizobium strains, but also cultures of such isolated strains, including their suspensions in a whole broth culture or, if applicable, as a metabolite-containing supernatant or a purified metabolite obtained from a whole broth culture of the strain.

“Whole broth culture” refers to a liquid culture containing both cells and media.

“Supernatant” refers to the liquid broth remaining when cells in a whole broth culture are separated by centrifugation, filtration, sedimentation, or other means well known in the art.

The term “metabolite” refers to any compound, substance or byproduct of a fermentation of a microorganism that has plant growth promoting and/or fungicidal activity.

A sample of a Trichoderma asperellum strain named B35 which is suitable in the present invention has been deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) located at InhoffenstraBe 7B, 38124 Braunschweig, Germany, under the Budapest Treaty on August 27, 2019, and has been assigned the following depository designation: DSM 33245.

A sample of a Trichoderma atroviride strain named T1 which is suitable in the present invention has been deposited with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur), under the Budapest Treaty, and has been assigned the following depository designation: CNCM 1-1237.

Said agriculturally useful bacterial microorganism is selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, that can nodulate agricultural and pastoral crops, such as Alfalfa, Beans, Clover, lotus, lupines, peanuts, kudzu, and rooibos.

In a preferred embodiment, said agriculturally useful bacterial microorganism is of the species Bradyrhizobium japonicum.

It is most preferred that said agriculturally useful bacterial microorganism is Bradyrhizobium japonicum strain E109. Bradyrhizobium japonicum E-109 is a variant of strain USDA 138 (INTA E109, SEMIA 5085; Eur. J. Soil Biol. 45, 28-35, 2009; Biol. Fertil. Soils 47, 81-89, 2011). INTA is the National Institute of Agricultural Technology in Argentina, and E109 is deposited in the strain collection (la Coleccion BPCV) of INTA’s Institute of Agricultural Microbiology and Zoology.

In a preferred embodiment, said agriculturally useful fungal microorganism is present in the form of spores.

Fungal spores as within the scope of the present invention comprise asexual spores, such as conidia as well as blastospores, but also other fungal propagules such as ascospores, basidiospores, chlamydospores. (Micro)Sclerotia, although not being spores in the strict sense, may also be used within the scope of the invention. Preferably, the spores are conidia. Conidia are a kind of spores asexually formed by many fungal microorganisms useful in agriculture, e.g., of the genus Purpureocillium, Isaria, Metarhizium, Beauveria, Trichoderma. Conidia include but are not limited to aleurispores, anellospores, arthrospores, phialospores and pycnidiospores.

The fungal microorganism producing spores and acting as biological control agent and/or plant growth promoter is cultivated or fermented according to methods known in the art or as described in this application on an appropriate substrate, e.g., by submerged fermentation or solid-state fermentation, e.g., using a device and method as disclosed in WO 2005/012478 or WO 1999/057239. Although specific fungal propagules such as microsclerotia (see e.g., Jackson and Jaronski (2009); Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects; Mycological Research 113, pp. 842-850) may be produced by liquid fermentation techniques, it is preferred that the dormant structures or organs according to the present invention are produced by solid-state fermentation. Solid-state fermentation techniques are well known in the art (for an overview see Gowthaman et al., 2001. Appl Mycol Biotechnol (1), p. 305-352).

The composition may additionally comprise at least one auxiliary selected from the group consisting of extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost protectants, thickeners, colorants and adjuvants. These may be added as long as they are compatible with both active ingredients, or, for sequential application, with at least one active ingredient. The resulting compositions are referred to as formulations.

Accordingly, in one aspect of the present invention such formulations, and application forms prepared from them, are provided as crop protection agents and/or plant health promoting agents, such as drench, drip and spray liquors as well as seed treatment formulations, comprising the composition of the invention. The application forms may comprise further crop protection agents and/or plant health promoting agents, and/or activity-enhancing adjuvants such as penetrants, examples being vegetable oils such as, for example, rapeseed oil, sunflower oil, mineral oils such as, for example, liquid paraffins, alkyl esters of vegetable fatty acids, such as rapeseed oil or soybean oil methyl esters, or alkanol alkoxylates, and/or spreaders such as, for example, alkylsiloxanes and/or salts, examples being organic or inorganic ammonium or phosphonium salts, examples being ammonium sulphate or diammonium hydrogen phosphate, and/or retention promoters such as dioctyl sulphosuccinate or hydroxypropylguar polymers and/or humectants such as glycerol and/or fertilizers such as ammonium, potassium or phosphorous fertilizers, for example.

Examples of typical formulations include water-soluble liquids (SL), emulsifiable concentrates (EC), emulsions in water (EW), suspension concentrates (SC, SE, FS, OD), water-dispersible granules (WG), granules (GR) and capsule concentrates (CS); these and other possible types of formulation are described, for example, by Crop Life International and in Pesticide Specifications, Manual on Development and Use of FAO and WHO Specifications for Pesticides, FAO Plant Production and Protection Papers - 173, prepared by the FAO/WHO Joint Meeting on Pesticide Specifications, 2004, ISBN: 9251048576. The formulations may comprise active agrochemical compounds other than one or more active compounds of the invention.

An adjuvant in this context is a component which enhances the biological effect of the formulation, without the component itself having a biological effect. Examples of adjuvants are agents which promote the retention, spreading, attachment to the leaf surface, or penetration.

These formulations are produced in a known manner, for example by mixing the active compounds with auxiliaries such as, for example, extenders, solvents and/or solid carriers and/or further auxiliaries, such as, for example, surfactants. The formulations are prepared either in suitable plants or else before or during the application.

Suitable for use as auxiliaries are substances which are suitable for imparting to the formulation of the active compound or the application forms prepared from these formulations (such as, e.g., usable crop protection agents, such as spray liquors or seed dressings) particular properties such as certain physical, technical and/or biological properties.

Suitable extenders are, for example, water, polar and nonpolar organic chemical liquids, for example from the classes of the aromatic and non-aromatic hydrocarbons (such as paraffins, alkylbenzenes, alkylnaphthalenes, chlorobenzenes), the alcohols and polyols (which, if appropriate, may also be substituted, etherified and/or esterified), the ketones (such as acetone, cyclohexanone), esters (including fats and oils) and (poly)ethers, the unsubstituted and substituted amines, amides, lactams (such as N-alkylpyrrolidones) and lactones, the sulphones and sulphoxides (such as dimethyl sulphoxide).

If the extender used is water, it is also possible to employ, for example, organic solvents as auxiliary solvents as long as they are compatible with both active ingredients according to the present invention.

A carrier may be any carrier suitable for formulating fungal spores. Suitable carriers are in particular: for example, ammonium salts and ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals, such as finely divided silica, alumina and natural or synthetic silicates, resins, waxes and/or solid fertilizers. Mixtures of such carriers may likewise be used. Carriers suitable for granules include the following: for example, crushed and fractionated natural minerals such as calcite, marble, pumice, sepiolite, dolomite, and also synthetic granules of inorganic and organic meals, and also granules of organic material such as sawdust, paper, coconut shells, maize cobs and tobacco stalks.

Carriers useful for liquid compositions include plant oils, mineral oils, polyethylenglycols (PEGs) and their derivatives, sugar surfactants, liquid sugars and derivatives, silicon derived liquids (such as organo-modified trisiloxane), ethoxylated sorbitan, sorbitan esters (such as sorbitan monolaurate), alcohol ethoxylates and/or propoxylates, glycerin derivatives (such as glycerin triacetate), oil ethoxylates and propoxylates (such as soybean oil ethoxylates), polymers and block-co -polymers (such as polyalkylene oxide co-polymers), vegetable oils and many others.

Liquefied gaseous extenders or solvents may also be used. Particularly suitable are those extenders or carriers which at standard temperature and under standard pressure are gaseous, examples being aerosol propellants, such as halogenated hydrocarbons, and also butane, propane, nitrogen and carbon dioxide.

Examples of emulsifiers and/or foam-formers, dispersants or wetting agents having ionic or nonionic properties, or mixtures of these surface-active substances, are salts of polyacrylic acid, salts of lignosulphonic acid, salts of phenolsulphonic acid or naphthalenesulphonic acid, polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, with substituted phenols (preferably alkylphenols or arylphenols), salts of sulpho succinic esters, taurine derivatives (preferably alkyltaurates), phosphoric esters of polyethoxylated alcohols or phenols, fatty acid esters of polyols, and derivatives of the compounds containing sulphates, sulphonates and phosphates, examples being alkylaryl polyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates, protein hydrolysates, lignin- sulphite waste liquors and methylcellulose. The presence of a surface-active substance is advantageous if one of the active compounds and/or one of the inert carriers is not soluble in water and if application takes place in water. Preferred emulsifiers are alkylaryl polyglycol ethers.

The composition according to the invention may further comprise at least one substance selected from the group of surfactants, rheology modifiers, antifoaming agents, antioxidants and dyes.

Non-ionic and/or anionic surfactants are all substances of this type which can customarily be employed in agrochemical agents. Possible nonionic surfactants are selected from the groups of polyethylene oxide-polypropylene oxide block copolymers, ethoxylated mono-, di- and/or triglycerides where ethoxylated castor oil or ethoxylated vegetable oils may be mentioned by way of example, polyethylene glycol ethers of branched or linear alcohols, reaction products of fatty acids or fatty acid alcohols with ethylene oxide and/or propylene oxide, furthermore branched or linear alkylaryl ethoxylates, where polyethylene oxide- sorbitan fatty acid esters may be mentioned by way of example. Out of the examples mentioned above selected classes can be optionally phosphated and neutralized with bases. Possible anionic surfactants are all substances of this type which can customarily be employed in agrochemical agents. Alkali metal, alkaline earth metal and ammonium salts of alkylsulphonic or alkylphosphoric acids as well as alkylarylsulphonic or alkylarylphosphoric acids are preferred. A further preferred group of anionic surfactants or dispersing aids are alkali metal, alkaline earth metal and ammonium salts of polystyrenesulphonic acids, salts of polyvinylsulphonic acids, salts of alkylnaphthalene sulphonic acids, salts of naphthalenesulphonic acid-formaldehyde condensation products, salts of condensation products of naphthalenesulphonic acid, phenolsulphonic acid and formaldehyde, and salts of ligno sulphonic acid. A further preferred group of anionic surfactants or dispersing aids are alkali metal, alkaline earth metal and ammonium salts of sarcosinates or taurates. Suitable ranges of surfactants in the liquid preparation according to the invention comprise 0-20%, preferably 0-15%, more preferably 0.5-10%.

Rheology modifiers, also known as thickener, anti-caking agent, viscosity modifier or structuring agent, may be added to the present formulation, e.g., in order to prevent (irreversible) sedimentation. Rheology modifiers are preferably derived from minerals. These rheological control agents provide long term stability when the formulation is at rest or in storage. Suitable compounds are rheological modifier selected from the group consisting of hydrophobic and hydrophilic fumed and precipitated silica particles, gelling clays including bentonite, hectorite, laponite, attapulgite, sepiolite, smectite, or hydrophobically/organophilic modified bentonite. Suitable ranges of rheology modifier in the liquid preparation according to the invention comprise 0-10%, preferably 0-7%, more preferably 0.5-5%.

Other commonly used rheology modifiers are polymeric rheology modifiers, such as cellulose derivatives, xanthan and polyacrylates. Examples of cellulose rheology modifiers include hydroxypropyl cellulose of different molecular weight (e.g., KLUCEL® H, G, L, E). Examples of xanthan rheology modifiers include medium to larger molecular weight naturally-drived xanthan polymers with or without modification (e.g., KELZAN® CC or KELZAN® S). Example of polyacrylate -based rheology modifiers are the medium to larger molecular weight polyacrylates or its (or for example, partially-hydrolyzed polyacrylamide) with or without modifications (e.g., HYSORB®).

As far as not otherwise defined, % in the present application refers to wt.-%.

Further auxiliaries that may be present in the formulations and in the application forms derived from them include colorants such as inorganic pigments, examples being iron oxide, titanium oxide, Prussian Blue, and organic dyes, such as alizarin dyes, azo dyes and metal phthalocyanine dyes, and nutrients and trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

Stabilizers, such as low-temperature stabilizers, preservatives, antioxidants, light stabilizers or other agents which improve chemical and/or physical stability may also be present. Additionally, present may be foam-formers or defoamers.

Furthermore, the formulations and application forms derived from them may also comprise, as additional auxiliaries, stickers such as carboxymethylcellulose, natural and synthetic polymers in powder, granule or latex form, such as gum arabic, polyvinyl alcohol, polyvinyl acetate, polysaccharides such as starch and derivatives thereof, e.g., amylose, amylopectin, maltodextrin, cellulose and derivative thereof, and also natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids. Further possible auxiliaries include mineral and vegetable oils.

There may possibly be further auxiliaries present in the formulations and the application forms derived from them. Examples of such additives include fragrances, protective colloids, binders, adhesives, thickeners, thixotropic substances, penetrants, retention promoters, stabilizers, sequestrants, complexing agents, humectants and spreaders. Generally speaking, the active compounds may be combined with any solid or liquid additive commonly used for formulation purposes.

Suitable retention promoters include all those substances which reduce the dynamic surface tension, such as dioctyl sulphosuccinate, or increase the viscoelasticity, such as hydroxypropylguar polymers, for example.

Suitable penetrants in the present context include all those substances which are typically used in order to enhance the penetration of active agrochemical compounds into plants. Penetrants in this context are defined in that, from the (generally aqueous) application liquor and/or from the spray coating, they are able to penetrate the cuticle of the plant and thereby increase the mobility of the active compounds in the cuticle. This property can be determined using the method described in the literature (Baur et al., 1997, Pesticide Science 51, 131-152). Examples include alcohol alkoxylates such as coconut fatty ethoxylate (10) or isotridecyl ethoxylate (12), fatty acid esters such as rapeseed or soybean oil methyl esters, fatty amine alkoxylates such as tallowamine ethoxylate (15), or ammonium and/or phosphonium salts such as ammonium sulphate or diammonium hydrogen phosphate, for example.

The composition preferably comprises between 0.00000001% and 95% by weight of Trichoderma spores, with particular preference, between 0.01% and 75% by weight, and between 0.00000001% and 95% and between 0.01% and 75% wt. % of Bradyrhizobium particles, more preferably between 0.5% and 90% based on the weight of the formulation. The content of the active compound is defined as the sum of the at least one specified fungal microorganism which is a strain belonging to herein described Trichoderma species and the at least one specified bacterial microorganism of the herein described Bradyrhizobium species.

The composition according to the present invention may be applied in any desired manner, such as in the form of a seed coating, soil drench, and/or directly in-furrow and/or as a foliar spray and applied either pre-emergence, post-emergence or both. In other words, the composition can be applied to the seed, the plant or to harvested fruits and vegetables or to the soil wherein the plant is growing or wherein it is desired to grow (plant’s locus of growth).

The composition may additionally comprise at least one further plant protection agent. Such additional plant protection agent may be an agent used as a fungicide, bactericide, insecticide, nematicide or acaricide or as an agent for promoting plant health and be of synthetic or natural origin. For example, the composition may be combined with at least one further plant protection agent which may be a bacterial or fungal microorganism. Suitable examples include Bacillus spp., such as B. velezensis, B. pumilus, B. amyloliquefaciens, B. subtilis and B. megaterium. Alternatively, the composition may be combined with at least one synthetic plant protection agent. Suitable examples include prothioconazole, metalaxyl, mefenoxam, fluoxastrobin, tebuconazole, ipconazole, metconazole, cyproconazole, epoxiconazole, propiconazole, azoxystrobin, pyraclostrobin, picoxystrobin, benzovindiflupyr, fluxapyroxad and chlorothalonil as fungicides and, thiodicarb, imidocloprid, carbendazim and thiram as insecticides. The present invention also relates to a seed treated and/or coated with the composition according to the invention. For example, said seed, following treatment with the composition of the invention, is subjected to a film-coating process in order to prevent dust abrasion of the seed. Both active ingredients according to the invention have strong interactions with plants and inhabit the plant or the soil or both. Accordingly, treating seed with the composition according to the invention is particular advantageous to ensure early colonization of the plant or its surroundings in the soil, in particular the rhizosphere.

For the purposes of the present invention, the composition of the invention is applied alone or in a suitable formulation to the seed. The seed is preferably treated in a condition in which its stability is such that no damage occurs in the course of the treatment. Generally speaking, the seed may be treated at any point in time between harvesting and sowing. Typically, seed is used which has been separated from the plant and has had cobs, hulls, stems, husks, hair or pulp removed. Thus, for example, seed may be used that has been harvested, cleaned and dried to a moisture content of less than 15% by weight. Alternatively, seed can also be used that after drying has been treated with water, for example, and then dried again.

The combinations which can be used in accordance with the invention may be converted into the customary seed-dressing formulations, such as solutions, emulsions, suspensions, powders, foams, slurries or other coating compositions for seed, and also ULV formulations.

These formulations are prepared in a known manner, by mixing composition with customary adjuvants, such as, for example, customary extenders and also solvents or diluents, colorants, wetters, dispersants, emulsifiers, antifoams, preservatives, secondary thickeners, stickers, gibberellins, and also water.

Colorants which may be present in the seed-dressing formulations which can be used in accordance with the invention include all colorants which are customary for such purposes. In this context it is possible to use not only pigments, which are of low solubility in water, but also water-soluble dyes. Examples include the colorants known under the designations Rhodamin B, C.I. Pigment Red 112 and C.I. Solvent Red 1. Wetters which may be present in the seed-dressing formulations which can be used in accordance with the invention include all of the substances which promote wetting, and which are customary in the formulation of active agrochemical ingredients. Use may be made preferably of alkylnaphthalenesulphonates, such as diisopropyl- or diisobutyl-naphthalenesulphonates.

Dispersants and/or emulsifiers which may be present in the seed-dressing formulations which can be used in accordance with the invention include all of the nonionic, anionic and cationic dispersants that are customary in the formulation of active agrochemical ingredients. Use may be made preferably of nonionic or anionic dispersants or of mixtures of nonionic or anionic dispersants. Suitable nonionic dispersants are, in particular, ethylene oxide-propylene oxide block polymers, alkylphenol polyglycol ethers and also tristryrylphenol polyglycol ethers, and the phosphated or sulphated derivatives of these. Suitable anionic dispersants are, in particular, lignosulphonates, salts of polyacrylic acid, and arylsulphonate-formaldehyde condensates.

Antifoams which may be present in the seed-dressing formulations which can be used in accordance with the invention include all of the foam inhibitors that are customary in the formulation of active agrochemical ingredients. Use may be made preferably of silicone antifoams and magnesium stearate.

Preservatives which may be present in the seed-dressing formulations which can be used in accordance with the invention include all of the substances which can be employed for such purposes in agrochemical compositions. Examples include dichlorophen and benzyl alcohol hemiformal.

Secondary thickeners which may be present in the seed-dressing formulations which can be used in accordance with the invention include all substances which can be used for such purposes in agrochemical compositions. Those contemplated with preference include cellulose derivatives, acrylic acid derivatives, xanthan, modified clays and highly disperse silica.

Stickers which may be present in the seed-dressing formulations which can be used in accordance with the invention include all customary binders which can be used in seed-dressing products. Preferred mention may be made of polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol and tylose.

Gibberellins which may be present in the seed-dressing formulations which can be used in accordance with the invention include preferably the gibberellins Al, A3 (= gibberellic acid), A4 and A7, with gibberellic acid being used with particular preference. The gibberellins are known (cf. R. Wegler, “Chemie der Pflanzenschutz- und Schadlingsbekampfungsmittel”, Volume 2, Springer Verlag, 1970, pp. 401-412).

The seed-dressing formulations which can be used in accordance with the invention may be used, either directly or after prior dilution with water, to treat seed of any of a wide variety of types. Accordingly, the concentrates or the preparations obtainable from them by dilution with water may be employed to dress the seed of cereals, such as wheat, barley, rye, oats and triticale, and also the seed of maize, rice, oilseed rape, peas, beans, cotton, sunflowers and beets, or else the seed of any of a very wide variety of vegetables. The seed-dressing formulations which can be used in accordance with the invention, or their diluted preparations, may also be used to dress seed of transgenic plants. In that case, additional synergistic effects may occur in interaction with the substances formed through expression.

For the treatment of seed with the seed-dressing formulations which can be used in accordance with the invention, or with the preparations produced from them by addition of water, suitable mixing equipment includes all such equipment which can typically be employed for seed dressing. More particularly, the procedure when carrying out seed dressing is to place the seed in a mixer, to add the particular desired amount of seed-dressing formulations, either as such or following dilution with water beforehand, and to carry out mixing until the distribution of the formulation on the seed is uniform. This may be followed by a drying operation.

The application rate of the seed-dressing formulations which can be used in accordance with the invention may be varied within a relatively wide range. It is guided by the particular amount of the at least one fungal microorganism and the at least one bacterial microorganism in the formulations, and by the seed. The application rates in the case of the composition are situated generally at between 0.001 and 50 g per kilogram of seed, preferably between 0.01 and 15 g per kilogram of seed.

The composition according to the invention, in combination with good plant tolerance and favourable toxicity to warm-blooded animals and being tolerated well by the environment, are suitable for protecting plants and plant organs, for increasing harvest yields, for improving the quality of the harvested material in agriculture, in horticulture, in animal husbandry, in forests, in gardens and leisure facilities and in protection of stored products and of materials. According to the invention all leguminous plants and plant parts can be treated. By plants is meant all plants and plant populations such as desirable and undesirable wild plants, cultivars and plant varieties (whether or not protectable by plant variety or plant breeder’s rights). Cultivars and plant varieties can be plants obtained by conventional propagation and breeding methods which can be assisted or supplemented by one or more biotechnological methods such as by use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers or by bioengineering and genetic engineering methods. By plant parts is meant all above ground and below ground parts and organs of plants such as shoot, leaf, blossom and root, whereby for example leaves, needles, stems, branches, blossoms, fruiting bodies, fruits and seed as well as roots, corms and rhizomes are listed. Crops and vegetative and generative propagating material, for example cuttings, corms, rhizomes, runners and seeds also belong to plant parts.

Furthermore, the present invention relates to a method for promoting or improving plant health and/or plant growth of agricultural plants wherein the plants, the plant propagules, the seed of the plants and/or the locus where the plants are growing or are intended to grow are treated with an effective amount of a composition according to the present invention. Said composition may also be a combination.

If not mentioned otherwise, the expression “combination” stands for the various combinations of the agriculturally useful fungal microorganism which is a strain belonging to the herein described Trichoderma species and the agriculturally useful bacterial microorganism of the herein described Bradyrhizobium species and optionally the at least one further plant protection agent, in a solo- formulation, in a single “ready-mix” form, in a combined spray mixture composed from solo- formulations, such as a “tank-mix”, and especially in a combined use of the single active ingredients when applied in a sequential manner, i.e., one after the other within a reasonably short period, such as a few hours or days, e.g., 2 hours to 7 days. The order of applying the composition according to the present invention is not essential for working the present invention. Accordingly, the term “combination” also encompasses the presence of the at least one fungal microorganism and the at least one bacterial microorganism, and optionally the at least one further plant protection agent on or in a plant to be treated or its surrounding, habitat or storage space, e.g., after simultaneously or consecutively applying the at least one fungal microorganism and the at least one bacterial microorganism, and optionally the at least one further plant protection agent to a plant its surrounding, habitat or storage space.

If both microorganisms, and optionally the at least one further plant protection agent are employed or used in a sequential manner, it is preferred to treat the plants or plant parts (which includes seeds and plants emerging from the seed), harvested fruits and vegetables according to the following method: Firstly applying the a Trichoderma species on the plant or plant parts, in particular seeds, and secondly applying the Bradyrhizobium japonicum to the same plant or plant parts or the surrounding soil. The time periods between the first and the second application within a (crop) growing cycle may vary and depend on the effect to be achieved.

If not mentioned otherwise the treatment of plants or plant parts (which includes seeds and plants emerging from the seed), harvested fruits and vegetables with the composition according to the invention is carried out directly or by action on their surroundings, habitat or storage space using customary treatment methods, for example dipping, spraying, atomizing, irrigating, evaporating, dusting, fogging, broadcasting, foaming, painting, spreading-on, watering (drenching), drip irrigating. It is furthermore possible to apply any of the microorganisms, and optionally the at least one further plant protection agent as solo-formulation or combined- formulations by the ultra- low volume method, or to inject the composition according to the present invention as a composition or as sole-formulations into the soil (in-furrow).

The term “plant to be treated” encompasses every part of a plant including its root system and the material - e.g., soil or nutrition medium - which is in a radius of at least 10 cm, 20 cm, 30 cm around the caulis or bole of a plant to be treated or which is at least 10 cm, 20 cm, 30 cm around the root system of said plant to be treated, respectively.

The amount of the fungal microorganism which is used or employed in combination with the bacterial microorganism, optionally in the presence of a further plant protection agent, depends on the final formulation as well as size or type of the plant, plant parts, seeds, harvested fruits and vegetables to be treated. Usually, the fungal microorganism to be employed or used according to the invention is present in about 2% to about 80% (w/w), preferably in about 5% to about 75% (w/w), more preferably about 10% to about 70% (w/w) e.g., for soil application or in about 10% to about 70% (w/w) e.g., for seed dressings of its solo-formulation or combined-formulation with the bacterial microorganism, and optionally the further plant protection agent. The above values in particular apply to soil applications, but also to seed treatment applications.

In a preferred embodiment the fungal microorganism or e.g., its spores are present in a solo- formulation or the combined formulation in a concentration of at least 10 ⁵ colony forming units per gram preparation (e.g., cells/g preparation, spores/g preparation), such as 10 ⁵ - 10 ¹² cfu/g, preferably 10 ⁶ - 10 ¹¹ cfu/g, more preferably 10 ⁷ - IO ¹⁰ cfu/g and most preferably 10 ⁹ - IO ¹⁰ cfu/g at the time point of applying the fungal microorganism on a plant or plant parts such as seeds, fruits or vegetables. For seed treatment, the amount of fungal spores usually range between 10 ⁴ to 10 ⁹ cfu/seed, preferably between 10 ⁵ and 10 ⁸ cfu/seed, more preferably between 10 ⁶ and 10 ⁷ cfu/seed. For soil applications, the amount of fungal spores usually ranges between 10 ⁸ and 10 ¹⁷ cfu/plant, which depends on the crop plant and the respective growth stage of the plant. Preferably, the amount of fungal spores in soil applications ranges between 10 ¹⁰ and 10 ¹⁶ cfu/plant. Also references to the concentration of fungal microorganisms in form of, e.g., spores or cells - when discussing ratios between the amount of a preparation of fungal microorganisms and the amount of bacterial microorganism - are made in view of the time point when both agents are applied on a plant or plant parts such as seeds, fruits or vegetables.

The amount of the bacterial microorganism which is used or employed in combination with the fungal microorganism, optionally in the presence of a further plant protection agent, equally depends on the final formulation as well as size or type of the plant, plant parts, seeds, harvested fruits and vegetables to be treated. Usually, the bacterial microorganism to be employed or used according to the invention is present in about 2% to about 80% (w/w), preferably in about 5% to about 75% (w/w), more preferably about 10% to about 70% (w/w) of its solo-formulation or combined-formulation with the fungal microorganism, and optionally the further plant protection agent.

In a preferred embodiment, the bacterial microorganism or e.g., its spores are present in a solo- formulation or the combined-formulation in a concentration of at least 10 ⁵ colony forming units per gram preparation (e.g., cells/g preparation, spores/g preparation), such as 10 ⁵ - 10 ¹² cfu/g, preferably 10 ⁶ - 10 ¹¹ cfu/g, more preferably 10 ⁷ - 10 ¹⁰ cfu/g and most preferably 10 ⁹ - 10 ¹⁰ cfu/g at the time point of applying the bacterial microorganism on a plant or plant parts such as seeds, fruits or vegetables. For seed treatment, the amount of bacterial microorganism usually ranges between 10 ⁴ to 10 ⁸ cfu/seed, preferably between 10 ⁵ and 5 x 10 ⁷ cfu/seed, more preferably between 5 x 10 ⁵ and 2 x 10 ⁷ cfu/seed. For soil applications, the amount of bacterial microorganism usually ranges between 10 ⁸ and 10 ¹⁷ cfu/plant, which depends on the crop plant and the respective growth stage of the plant. Preferably, the amount of bacterial microorganism in soil applications ranges between 10 ¹⁰ and 10 ¹⁶ cfu/plant. Also references to the concentration of bacterial microorganisms in form of, e.g., spores or cells - when discussing ratios between the amount of a preparation of bacterial microorganisms and the amount of fungal microorganism - are made in view of the time point when both agents are applied on a plant or plant parts such as seeds, fruits or vegetables.

The at least one fungal microorganism and at least one bacterial microorganism, and if present also the additional plant protection agent may be used or employed in a synergistic weight ratio. The skilled person is able to find out the synergistic weight ratios for the present invention by routine methods. The skilled person understands that these ratios refer to the ratio within a combined- formulation as well as to the calculative ratio of the at least one fungal microorganism described herein and the specified at least one bacterial microorganism when both components are applied as monoformulations to a plant to be treated. The skilled person can calculate this ratio by simple mathematics since the volume and the amount of the fungal microorganism and bacterial microorganism, respectively, in a mono-formulation is known to the skilled person.

The ratio can be calculated based on the amount of the at least one fungal microorganism, at the time point of applying said component of a combination according to the invention to a plant or plant part and the amount of bacterial microorganism simultaneously or sequentially to a plant or plant part.

The application of the at least one fungal microorganism and the at least one bacterial microorganism according to the present invention to a plant or a plant part can take place simultaneously or at different times as long as both components are present on or in the plant after the application(s).

In particular, in one embodiment the synergistic weight ratio of the at least one fungal microorganism/spore preparation and the at least one bacterial microorganism/spore preparation lies in the range of 1:1000 to 1000:1, preferably in the range of 1:500 to 500:1, all in cfu, more preferably in the range of 1 :500 to 300: 1 , even more preferably in the range of 100: 1 to 1 : 100. It is most preferred that the ratio is from 25:1 to 1:25 such as from 10:1 to 1:10 or from 4:1 to 1:4. It has to be noted that these ratio ranges refer to the number of fungal cells/spores fungal microorganism put into perspective with the according number of cells/spores of the at least one bacterial microorganism. For example, a ratio of 100:1 means 100 cfu of cells/spores of a fungal microorganism and 1 cfu of cells/spores of the bacterial microorganism (either as a solo formulation, a combined formulation or by separate applications to plants so that the combination is formed on the plant).

Preferably, in particular where the fungal microorganism is spores of a Trichoderma asperellum strain, in particular the B35 strain, and where the bacterial microorganism is a Bradyrhizobium strain, in particular the E109 strain, the ratio is between 1:0.5 and 1:100.

The cell/spore concentration of preparations can be determined by applying methods known in the art.

In one embodiment of the present invention, the concentration of both active ingredients after dispersal to a plant or in the field is at least 50 g/ha, such as 50 - 7500 g/ha, 50 - 2500 g/ha, 50 - 1500 g/ha; at least 250 g/ha (hectare), at least 500 g/ha or at least 800 g/ha.

The application rate of composition to be employed or used according to the present invention may vary. The skilled person is able to find the appropriate application rate by way of routine experiments.

Depending on the plant species or plant cultivars, their location and growth conditions (soils, climate, vegetation period, diet), using or employing the composition according to the present invention the treatment according to the invention may also result in super-additive (“synergistic”) effects. Thus, for example, by using or employing inventive composition in the treatment according to the invention, reduced application rates and/or a widening of the activity spectrum and/or an increase in the activity better plant growth, increased tolerance to high or low temperatures, increased tolerance to drought or to water or soil salt content, increased flowering performance, easier harvesting, accelerated maturation, higher harvest yields, bigger fruits, larger plant height, greener leaf color, earlier flowering, higher quality and/or a higher nutritional value of the harvested products, higher sugar concentration within the fruits, better storage stability and/or processability of the harvested products are possible, which exceed the effects which were actually to be expected.

At certain application rates of the inventive composition in the treatment according to the invention may also have a strengthening effect in plants. The defense system of the plant against attack by unwanted phytop athogenic fungi and/ or microorganisms and/or viruses is mobilized. Plantstrengthening (resistance-inducing) substances are to be understood as meaning, in the present context, those substances or combinations of substances which are capable of stimulating the defense system of plants in such a way that, when subsequently inoculated with unwanted phytopathogenic fungi and/or microorganisms and/or viruses, the treated plants display a substantial degree of resistance to these phytopathogenic fungi and/or microorganisms and/or viruses. Thus, by using or employing composition according to the present invention in the treatment according to the invention, plants can be protected against attack by the abovementioned pathogens within a certain period of time after the treatment. The period of time within which protection is effected generally extends from 1 to 10 days, preferably 1 to 7 days, after the treatment of the plants with the active compounds.

The composition according to the present invention may be used for any plant. However, due to the characteristics of the bacterial microorganism as symbiont in leguminous plants, the agricultural plant preferably is a legume. Exemplary legumes include soybean, pea, lentil, beans, lupins and peanuts. Especially preferred is soybean.

Plants and plant cultivars which are more preferably treated according to the invention are resistant against one or more biotic stresses, i.e., said plants show a better defense against animal and microbial pests, such as against nematodes, insects, mites, phytopathogenic fungi, bacteria, viruses and/or viroids.

Plants and plant cultivars which may alternatively or in addition be treated according to the invention are those plants which are resistant to one or more abiotic stresses, i.e., that already exhibit an increased plant health with respect to stress tolerance. Abiotic stress conditions may include, for example, drought, cold temperature exposure, heat exposure, osmotic stress, flooding, increased soil salinity, increased mineral exposure, ozone exposure, high light exposure, limited availability of nitrogen nutrients, limited availability of phosphorus nutrients, shade avoidance. Preferably, the treatment of these plants and cultivars with the composition of the present invention additionally increases the overall plant health (cf. above).

Plants and plant cultivars which may also be treated according to the invention, are those plants characterized by enhanced yield characteristics i.e., that already exhibit an increased plant health with respect to this feature. Increased yield in said plants can be the result of, for example, improved plant physiology, growth and development, such as water use efficiency, water retention efficiency, improved nitrogen use, enhanced carbon assimilation, improved photosynthesis, increased germination efficiency and accelerated maturation. Yield can furthermore be affected by improved plant architecture (under stress and non-stress conditions), including but not limited to, early flowering, flowering control for hybrid seed production, seedling vigor, plant size, internode number and distance, root growth, seed size, fruit size, pod size, pod or ear number, seed number per pod or ear, seed mass, enhanced seed filling, reduced seed dispersal, reduced pod dehiscence and lodging resistance. Further yield traits include seed composition, such as carbohydrate content, protein content, oil content and composition, nutritional value, reduction in anti-nutritional compounds, improved processability and better storage stability. Preferably, the treatment of these plants and cultivars with the composition of the present invention additionally increases the overall plant health (cf. above).

Plants that may be treated according to the invention are hybrid plants that already express the characteristic of heterosis or hybrid vigor which results in generally higher yield, vigor, health and resistance towards biotic and abiotic stress factors. Such plants are typically made by crossing an inbred male-sterile parent line (the female parent) with another inbred male-fertile parent line (the male parent). Hybrid seed is typically harvested from the male sterile plants and sold to growers. Male sterile plants can sometimes (e.g., in corn) be produced by detasseling, i.e., the mechanical removal of the male reproductive organs (or males flowers) but, more typically, male sterility is the result of genetic determinants in the plant genome. In that case, and especially when seed is the desired product to be harvested from the hybrid plants it is typically useful to ensure that male fertility in the hybrid plants is fully restored. This can be accomplished by ensuring that the male parents have appropriate fertility restorer genes which are capable of restoring the male fertility in hybrid plants that contain the genetic determinants responsible for male- sterility. Genetic determinants for male sterility may be located in the cytoplasm. Examples of cytoplasmic male sterility (CMS) were for instance described in Brassica species. However, genetic determinants for male sterility can also be located in the nuclear genome. Male sterile plants can also be obtained by plant biotechnology methods such as genetic engineering. A particularly useful means of obtaining male-sterile plants is described in WO 89/10396 in which, for example, a ribonuclease such as bamase is selectively expressed in the tapetum cells in the stamens. Fertility can then be restored by expression in the tapetum cells of a ribonuclease inhibitor such as barstar.

Plants or plant cultivars (obtained by plant biotechnology methods such as genetic engineering) which may be treated according to the invention are herbicide-tolerant plants, i.e., plants made tolerant to one or more given herbicides. Such plants can be obtained either by genetic transformation, or by selection of plants containing a mutation imparting such herbicide tolerance.

Herbicide-tolerant plants are for example glyphosate-tolerant plants, i.e., plants made tolerant to the herbicide glyphosate or salts thereof. Plants can be made tolerant to glyphosate through different means. For example, glyphosate-tolerant plants can be obtained by transforming the plant with a gene encoding the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Examples of such EPSPS genes are the AroA gene (mutant CT7) of the bacterium Salmonella typhimurium, the CP4 gene of the bacterium Agrobacterium sp., the genes encoding a Petunia EPSPS, a Tomato EPSPS, or an Eleusine EPSPS. It can also be a mutated EPSPS. Glyphosate-tolerant plants can also be obtained by expressing a gene that encodes a glyphosate oxido-reductase enzyme. Glyphosate-tolerant plants can also be obtained by expressing a gene that encodes a glyphosate acetyl transferase enzyme. Glyphosate-tolerant plants can also be obtained by selecting plants containing naturally-occurring mutations of the above-mentioned genes.

Other herbicide resistant plants are for example plants that are made tolerant to herbicides inhibiting the enzyme glutamine synthase, such as bialaphos, phosphinothricin or glufosinate. Such plants can be obtained by expressing an enzyme detoxifying the herbicide or a mutant glutamine synthase enzyme that is resistant to inhibition. One such efficient detoxifying enzyme is an enzyme encoding a phosphinothricin acetyltransferase (such as the bar or pat protein from Streptomyces species). Plants expressing an exogenous phosphinothricin acetyltransferase are also described.

Further herbicide-tolerant plants are also plants that are made tolerant to the herbicides inhibiting the enzyme hydroxyphenylpyruvatedioxygenase (HPPD). Hydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. Plants tolerant to HPPD-inhibitors can be transformed with a gene encoding a naturally-occurring resistant HPPD enzyme, or a gene encoding a mutated HPPD enzyme. Tolerance to HPPD-inhibitors can also be obtained by transforming plants with genes encoding certain enzymes enabling the formation of homogentisate despite the inhibition of the native HPPD enzyme by the HPPD-inhibitor. Tolerance of plants to HPPD inhibitors can also be improved by transforming plants with a gene encoding an enzyme prephenate dehydrogenase in addition to a gene encoding an HPPD- tolerant enzyme. Still further herbicide resistant plants are plants that are made tolerant to acetolactate synthase (ALS) inhibitors. Known ALS -inhibitors include, for example, sulfonylurea, imidazolinone, triazolopyrimidines, pyrimidinyoxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinone herbicides. Different mutations in the ALS enzyme (also known as acetohydroxyacid synthase, AHAS) are known to confer tolerance to different herbicides and groups of herbicides. The production of sulfonylurea-tolerant plants and imidazolinone-tolerant plants is described in WO 1996/033270. Other imidazolinone-tolerant plants are also described. Further sulfonylurea- and imidazolinone- tolerant plants are also described in for example WO 2007/024782.

In one embodiment, promoting or improving plant health comprises and/or manifests in improved stress tolerance, less dead basal leaves, greener leaf color, pigment content, photosynthetic activity and enhanced plant vigor; or wherein promoting or improving plant growth comprises or manifests in tillering increase, increase in plant height, bigger leaf blade, larger leaf surface area, stronger tillers, earlier flowering, early grain maturity, less plant verse (lodging), increased shoot growth, increased plant stand and early and better germination, earlier emergence, improved crop yield, improved total vegetative weight or whole plant biomass, improved protein content, improved oil content, improved starch content, improved root growth, improved root surface area, volume, root number, increased root weight, increased plant biomass and/or improved root effectiveness, improved shoot weight and improved fruit weight, improved solubilization of nutrients in the soil and an increase in water uptake. Improved solubilization may manifest in improved availability of N, P, K, Ca, Mg, S, Mn, Fe, B, Zn, Cu.

In a preferred embodiment, plant growth refers to root growth, root size, root architecture, root weight, fruit weight, shoot weight, leaf surface area, plant biomass, plant height, nodule size and weight, crop yield, and/or plant nutrient uptake.

In another preferred embodiment, said treating comprises treating seed.

Alternatively, or in addition, said treatment is carried out in the soil prior to germination of a seed and/or in the soil in contact with a seed or root of said plant or where a plant is intended to grow.

The treatment may be carried out as often as necessary in order to achieve the desired effect. However, it is preferred that the treatment is carried out at least twice. The method according to the invention may further comprise applying, simultaneously or sequentially, at least one further plant protection agent as described elsewhere in this application.

The method according to the present invention may be applied to any plant. However, due to the characteristics of the bacterial microorganism as symbiont in leguminous plants, the agricultural plant preferably is a legume. Exemplary legumes include soybean, pea, lentil, beans, lupins and peanuts. Especially preferred is soybean.

The present invention also relates to the use of the composition according to the invention for promoting or improving plant health and/or plant growth of agricultural plants.

Finally, the invention relates to a kit-of-parts comprising at least one agriculturally useful fungal microorganism which is a strain belonging to the species Trichoderma atroviride, Trichoderma asperellum or Trichoderma virens and an agriculturally useful bacterial microorganism which is of the species Bradyrhizobium japonicum or Bradyrhizobium elkani, all as described herein, in a spatially separated arrangement.

The examples illustrate the present invention without limiting its application in any manner.

Materials and Methods:

Two large pot experiments were conducted for evaluating the plant growth promotion effects of the combination of Bradyrhizobium and Trichoderma strains (Trichoderma asperellum strain B35 and Trichoderma atroviride strain Tl). Large plastic tubes (minifields) with a dimension of 13 cubic feet volume were used as pots for these studies. Minifields provide an opportunity to grow soybeans in a field plot like set up where plants are communicating with each other and grow in deep soil profile. Briefly, each minifield tube is filled with a soil mixture composed of field soil mixed with artificial soil. The pots were placed in a temperature and humidity-controlled greenhouse with ~29-33°C day and ~20°C night temperature, 16/8 photoperiod, 52-59%RH and 350Klux light intensity. 24 soybean seeds were prepared with three treatments (Bradyrhizobium (treatment 1), Trichoderma (treatment 2) and Bradyrhizobium + Trichoderma (treatment 3), all with an underlying chemical base treatment) with two biological replications per treatment. The minifields were applied with water until saturation in the beginning and thereafter water was applied every other day and the frequency increased as crop grew. The pots were also applied with standard fertilizer solution (Peters professional water soluble fertilizer 20-20-20). Once the seeds were dropped in the small holes in the pots, Bradyrhizobium japonicum E109 strain diluted solution was drenched on the seeds at 2 pl/seed mimicking on-farm seed treatment for treatments 1 and 3. The pots in treatment 2 with Trichoderma did not receive any Bradyrhizobium.

A commercial soybean variety treated with a chemical plant protection package was used for the studies.

At the end of the experiment at ~7.5 weeks after sowing, when plants were at R3 (B35) and -R7 stage (Tl), plants were hand harvested and measurements were taken on a per plant basis, and the average of 12 plants per minifield represented one biological replicate. The study with B35 was terminated when control plants were approximately at R3 (BBCH 71 701) growth stage. The study with Tl was planted on the same pots again and study was terminated at R7 growth stage.

Measurement of Trichoderma or Bradyrhizobium Concentration

For Trichoderma, the spore concentration in cfu was measured using serial dilution technique for conidial units/g of formulation or conidial units/seed using seed washes prepared in sterile water and a dispersing agent like Tween based on e.g., Gams, W. and Bissett, J. (1998. Morphology and Identification of Trichoderma, pp. 3-34. In: Kubicek, C. P. and Harmann, G E. (Eds.). Trichoderma and Gliocladium. Volume I: Basic Biology, Taxonomy and Genetics. Taylor and Francis, London, UK, 278 pp).

The concentration of Bradyrhizobium was measured using a serial dilution technique with suitable growth medium based on Vincent et al., 1970 or Penna et al., 2011.

The number of colony forming unit was calculated by using the formula:

_ Colony Count _

((Sample volume added to plate) x (dilution factor)) according to Vincent J, Thompson J, Donovan K. (Death of root-nodule bacteria on drying. Aust J Agricult Res. 1962;13:258-270) or Penna et al. (A simple method to evaluate the number of bradyrhizobia on soybean seeds and its implication on inoculant quality control [published correction appears in AMB Express. 2011 Sep 01 ; 1:25] . AMB Express. 2011 ; 1(1):21).

Measurements:

Plant height was measured from the base of the plants to the apical bud using a standard ruler. The leaf surface area was measured using a LiCOR leaf area meter. Chlorophyll content was measured by assessing the Nitrogen Balance Index (NBI) of leaves using Dualex meter from Decagon Devices. The total number of nodules per plant were counted in an area of 5Xcm upper root surface counting primary and secondary nodules. The total shoot and root biomass were determined by drying the plants in a forced air oven for two weeks and weighing the dry mass using a scale. After the dry weight measurements, shoot samples and leaves were used for analyzing the plant elemental composition by standard methods known in the art (e.g., nitrogen assay: http://www.elementar.de/en/products/nprotein-analysis/rapid- n-exceed.html; other nutrients: Havlin, J.L., and P.N. Soltanpour. 1980. A nitric acid plant tissue digest method for use with inductively coupled plasma spectrometry. Comm. Soil Sci. Plant Anal. l l(10):969-980.

Example 1: Improved Growth Parameters and Nutrient Uptake for Combination of Trichoderma asperellum B35 and B. japonicum E109

The experiments were conducted as described in the Materials & Methods section above. Compared to the treatment with Bradyrhizobium only, the combination of Bradyrhozobium japonicum strain El 09 and Trichoderma asperellum strain B35 resulted in the highest plant height, shoot dry weight, root dry weight, leaf surface area, leaf chlorophyll, nitrogen balance index (NBI), and nodule number (Table 1). Importantly, more plants were at advanced reproductive stage than for the single treatment as well as in plants inoculated with Trichoderma only. In addition to the growth parameters according to Table 1, plants treated with both Bradyrhizobium and Trichoderma showed increased uptake of several plant growth essential nutrients including N, K, S, Zn, Mn, B and Cu as shown in Table 2.

Table 1: Growth parameters measured form study using Trichoderma asperellum B35 or B. japonicum E109 as single treatment or as combined treatment. Seeds used were treated with a chemical base package as commercially available.

Example 2: Improved Growth Parameters and Nutrient Uptake for Combination of Trichoderma atroviride T1 and B.japonicum E109

The minifields used in Example 1 with pre-existing levels of Bradyrhizobium were used to repeat the studies with Trichoderma atroviride strain Tl. In this case the combination of T1 and E109 improved plant height, chlorophyll content, root biomass and nutrient uptake as compared to single treatments (see Table 3). No improvements in leaf area and shoot biomass were noticed, which could be because the pots had variable amounts of native Bradyhizobia. Compared to the study according to Example 1, the plants took longer to grow to the R7 stage, where most of the plants started senescence and were in the process of transferring nutrients to developing seeds. Nevertheless, the increased nutrient uptake (see Table 4) is thought to be, at least in part, caused by the increased root biomass. The nutrient benefit resulting from the combination of Bradyrhiozbium + Tl can be clearly explained by 6% increase in N as well as 16% increased NBI. This indicates that the plants can supply additional nutrients to the developing pods.

Table 3: Growth parameters measured form study using Trichoderma atroviridae T1 or B. japonicum E109 as single treatment or as combined treatment.

Table 4: Nutrient uptake measured in study of Trichoderma atroviride T1 in combination with Bradyrhizobium E109