WO2025222293A1 - Bacterial and fungal assemblage for use in agriculture - Google Patents

Abstract

The present technology generally relates to a bacterial-fungal assemblage for improving efficiency of phosphorus utilization by plants, wherein the bacterial-fungal assemblage comprises a fungus and bacteria, wherein the fungus is a phosphorus solubilizer fungus, a phosphorus mobilizer fungus or a phosphorus transporter fungus, and the bacteria are nitrogen fixing bacteria.

Description

BACTERIAL AND FUNGAL ASSEMBLAGE FOR USE IN AGRICULTURE

TECHNICAL FIELD

[001] The present technology relates to a bacterial and fungal assemblage for use in agriculture, in particular for the maintenance of plants.

BACKGROUND

[002] Bacteria and fungi can form a range of physical associations that depend on various modes of molecular communication for their development and functioning. These bacterial-fungal interactions often result in changes to the pathogenicity, the nutritional or symbiotic influence of one or both partners toward plants or animals. They can also result in unique contributions to biogeochemical cycles and biotechnological processes. Thus, the interactions between bacteria and fungi are of central importance to numerous biological questions in agriculture, forestry, environmental science, food production, and medicine.

[003] Phosphorus (P) is a crucial and often-limiting soil nutrient in nature, naturally confined to minerals, rocks, and oceanic deposits (Ruttenberg 2003). Total soil P content typically ranges from 100 ppm to 2000 ppm, of which only 10-15% is soluble P (Hinsinger 2001). Therefore, sufficient P is often not available to the plant from the soil as only inorganic orthophosphate (Pi, PO ) can be absorbed directly by the roots leading to the formation of Pi depletion zones (Vazquez et al. 2000, Schachtman et al. 1998). Hence, an adequate P application from an early stage of growth is essential for optimal crop production (Grant et al. 2001).

[004] Colonization with P. indica also impacted nodule formation by rhizobia. The number of nodules decreased slightly in the P. indica inoculated plant compared to the control (Bajaj et al. 2018). There was variable result about root growth promotion such as increase in the weight of roots after P. indica colonization (Bajaj et al. 2018) while no difference in root growth in other observation (Zhang et al. 2022).

[005] Soybean inoculation allows an average yield of 3.5 tons of grains ha ¹. without the need of nitrogen fertilizers. Methylobacterium spp. is classified within the Alphaproteobacteria as a gram- staining-negative, rod-shaped, pink-pigmented, strictly aerobic and facultative methylotroph. Able to grow using compounds containing only one carbon (Cl), such as methanol or methylamine, it grows at 28°C and can grow at up to 3% salinity in the presence of sodium chloride. Methylobacterium spp. can occupy different habitats (including soil, water, leaf surfaces, nodules, grains and air), and is found in more than 70 plant species, where they actively colonize the root as putative endophytes. M. symbioticum can fix atmospheric nitrogen and can also solubilize phosphorus.

[006] The genus Methylobacterium symbioticum comprises pink-pigmented facultative methylotrophic (PPFM) bacteria capable of synthesizing carotenoids and growing on single-carbon (Cl) reduced organic compounds, such as methanol and methylamine. These bacteria exhibit high phenotypic plasticity, allowing them to colonize diverse environments including soil, water, sediment, and various host plants as both endophytes and epiphytes. The frequency and distribution of plant colonization can be influenced by the plant's genotype and interactions with other microorganisms, potentially enhancing plant fitness.

[007] Cell protectants such as, polyvinlypyrrolidone (PVP, 1%), polyethylene glycol (PEG, 1%), gum arabic (0.8%) and sodium alginate (0.1%), adjuvants like xanthan gum (0.3%) and carboxymethyl cellulose (CMC, 0.1%), Tween 20 (0.05%) as a surfactant and potassium sorbate (0.2%) as a preservative, are normally used in the preparation of liquid inoculant formulation. For example, an inoculant containing the cell protectant, polyvinlypyrrolidone (2%), adjuvant xanthan gum (0.3%), Tween 20 (0.5%) as surfactant and potassium sorbate (0.2%) as preservative retained 1.76 x io¹⁰ CFU/ml of a bacterium at the end of 180 days of storage. These formulated liquid inoculants had both extended shelflife and viability.

[008] Nontoxic polymers Polyvinylpyrrolidone (PVP) may provide a favorable environment for the survival of bacteria in liquid formulation (Maitra et al. 2021).

[009] Gluconacetobacter diazotrophicus, an obligate endophyte, is incapable of surviving in soil without a plant host for more than two days. G. diazotrophicus is a nitrogen fixing bacterium that produces phytohormones, such as indole acetic and gibberellins. Upon inoculation, this bacterial species is able to stimulate the growth of row crops including com, cotton, rice soybeans, tomato, canola and sugarcane. The bacterium is able to gain entry into a host plant through the roots, stems, or leaves. Seed inoculation is a method of introducing the bacterium into the host. For example, inoculating 100 g of seeds with 10 m of a 10⁸ CFU mL ¹ bacterial culture in a phosphate saline buffer at a pH of 6.0 showed beneficial growth promotion activity. [010] There remains a need in the field of technology for compositions and methods to improve the overall health and growth of plants that alleviate some of the drawbacks of compositions and methods known in the field of technology.

SUMMARY

[Oi l] According to one aspect, the present technology relates to a bacterial -fungal assemblage for use in the field of agriculture.

[012] According to one aspect, the present technology relates to a bacterial -fungal assemblage for improving the efficiency of phosphorus utilization by plants. In some aspects, the technology described herein relates to a bacterial-fungal assemblage for improving efficiency of phosphorus utilization by plants, wherein the bacterial-fungal assemblage includes a fungus and bacteria, wherein the fungus is a phosphorus solubilizer fungus, a phosphorus mobilizer fungus or a phosphorus transporter fungus, and the bacteria are nitrogen fixing bacteria.

[013] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the bacterial-fungal assemblage of a chimera of at least one fungal organism and at least one bacterial organism.

[014] In some aspects, the bacterial are endophytic nitrogen fixing bacteria. In some aspects, the the bacteria are selected from Azospirillium spp., Azotobacter spp., Pseudomonas spp., Bacillus spp; Azoarcus spp., Achromobacter spp., Burkholderia spp., Gluconoacetobacter spp., Herbaspirillum spp., Klebsiella spp., and Serratia spp. In some aspects, the bacteria are nitrogen fixing alga. In some aspects, the bacteria are nitrogen fixing alga is Cyanobacterium. In some aspects, the bacteria are legume nodule formation bacteria. In some aspects, the legume nodule formation bacteria are selected from Sinorhizobium melilotii, Rhizobium legumninosarum biovar phaseoli, Rhizobium tropici, Rhizobium leguminosarum biovar trifolii, Mesorhizobium loti, Rhizobium leguminosarum biovar viceae, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium fredii, and Azorhizobium caulinodans .

[015] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the fungus is a fungus nematicide. In some aspects, the fungus nematicide is selected from Paecilomyces lilacinus, Colonostachys rosea, Pochonia spp Metarhizium spp., and Trichoderma spp.

[016] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the bacterial are bacteria nematicide.

[017] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the bacteria nematicide are selected from Bacillus spp. and Pastoria penetrans.

[018] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the fungus is a fungus antagonist.

[019] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the fungus antagonist is selected from Alternaria spp., Aspergillus spp., Candida spp., Fusarium spp., Penicillium spp., Pichia spp., Talaromyces spp., Trichoderma spp., Verticillium spp. and Chlonostachys spp.

[020] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the bacteria are selected from Bacillus amyloliquefaciens , and B. suhtilis.

[021] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the fungus is a fungus insecticide.

[022] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the fungus insecticide is selected from Beauveria hassiana, Cordyceps fumosorosea, Akanthomyces muscarius, Metarhizium anisopliae, Purpureocillium lilacinum, and Trichoderma harzianum.

[023] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the bacterial are bacteria insecticide.

[024] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the bacteria insecticide are selected from Bacillus thuringiensis, Bacillus spp., Serratia entomophila, Streptomyces avermitilis, and Pseudomonas fluorescens . [025] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the fungus is selected from Ascomycetes, and Basidiomycetes.

[026] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the bacteria are selected from Alcaligenes spp., Acinetobacter spp., Arthrobacter spp., Azospirillum spp., Bacillus spp., Burkholderia spp., Enterobacter spp., Flavobacterium spp., Paenibacillus spp., and Pseudomonas spp.

[027] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the fungus is selected from Trametes versicolor, Stereum hirsutum, and Serendipita indica BS22. In some aspects, the fungus is Trametes versicolor. In some aspects, the fungus is WCieGW.

[028] In some aspects, the technology described herein relates to a bacterial -fungal assemblage, wherein the fungus is Stereum hirsutum. In some aspects, the fungus is P2A.

[029] In some aspects, the technology described herein relates to a bacterial-fungal assemblage, wherein the bacteria are selected from Gluconacetobacter diazotrophicus BS47 ,Methylobacterium symbioticum BS11 and Azospirillum brasilense BS66.

[030] In some aspects, the technology described herein relates to a method for improving health comprising the step of putting the plant in contact with the bacteria-fungal assemblage of the present technology. In some aspects, contact of the bacterial assemblage with the plant improves the solubilization and transfer of residual phosphorus and fixed air-nitrogen to the plant. In some aspects, the bacterial assemblage acts as a monocote/non-legume dicote biofertilizer biocomplement. In some aspects, the bacterial assemblage acts as a legume fertilizer bio-complement. In some aspects, the bacterial assemblage acts as a microbial nematicide. In some aspects, the bacterial assemblage acts as a biofungicide. In some aspects, the bacterial assemblage acts as a microbial bioremediation. In some aspects, the bacterial assemblage acts as a microbial assisted- phytoremediation. In some aspects, the bacterial assemblage acts as bio-insecticide. In some aspects, the bacterial assemblage improves phosphorus solubilization. In some aspects, the bacterial assemblage enhances induced-systemic-resistance (ISR) in plants. [031] In some aspects, the technology described herein relates to a method for improving the efficiency of nitrogen utilization by a plant, the method comprising putting the plant in contact with the bacterial-fungal assemblage as defined herein.

[032] In some aspects, the technology described herein relates to a method for improving the efficiency of nitrogen assimilation by a plant, wherein the bacterium transforms atmospheric nitrogen into inorganic compounds usable by plants or a fungus phosphate phosphorus transporter, the method comprising putting the plant in contact with the bacterial-fungal assemblage as defined herein.

[033] In some aspects, the technology described herein relates to a method for improving the efficiency of phosphorus utilization by a plant, the method comprising putting the plant in contact with the bacterial-fungal assemblage as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[034] All features of embodiments which are described in this disclosure are not mutually exclusive and can be combined with one another. For example, elements of one embodiment can be utilized in the other embodiments without further mention. A detailed description of specific embodiments is provided herein below with reference to the accompanying drawings in which:

[035] FIGs. 1A-1C are photographs (FIG. 1A and FIG. 1C) and agraph (FIG. IB) showing wheat plant responses to .S', hirsutum P2A. T. versicolor WCieGW, .S', indica BS22, AGTIV®IGNITE™ and G. diazotrophicus either alone- or dual-inoculated. Disinfected seeds were coated as follows: for fungi, seeds were coated with a homogenized mycelium solution prepared using a 10 mM MgSCfi, 0.1% PVP and 0.01% Tween 20 solution; for bacteria, seeds were coated with a bacterial suspension with an ODeoo of 1.2, in a solution of lOmM MgSC>4.7H2O with 1% PVP and 0.1% Tween 20. In the case of a mixture of both fungi and bacteria, an equal combination of the above-mentioned solutions was applied to the seeds. Afterwards, 4 coated seeds were planted in 2/1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada) without additional fertilizers (FIG. 1A) Wheat plants 21 days after inoculation; (FIG. IB) Root and shoot dry weights, mean values, and standard deviation of 11 plants are shown. (FIG. 1C) Plants roots are shown from control. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05). [036] FIGs. 2A-2C are photographs (FIG. 2A and FIG. 2C) and a graph (FIG. 2B) showing wheat plant responses to .S'. hirsutum P2A (Sh), T. versicolor ~WC16GW (Tv), and AT. symbioticum BS11 (Ms), either alone- or with a dual-inoculum seed coating. Disinfected seeds were coated as follows; for fungi, seeds were coated with a homogenized mycelium solution prepared using a 10 mM MgSC>4, 0.1% PVP and 0.01% Tween 20 solution; for bacteria, seeds were coated with a bacterial suspension having an ODeoo of 1.4, in a solution of lOmM MgSC .TFFO with 1% PVP and 0.1% Tween 20. In the controls seeds were coated with lOmM MgSC TITO, 1% PVP and 0.1% Tween 20. In the case of a mixture of both fungi and bacteria, an equal combination of the above-mentioned solutions was applied to the seeds. Afterwards, 4 coated seeds were planted in 2/1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada), without additional fertilizers (FIG. 2A) Wheat plants 21 days after inoculation; (FIG. 2B) Root and shoot dry weights, mean values, and standard deviation of 11 plants are shown. (FIG. 2C) Plant roots are shown from control. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[037] FIG. 3 is a photograph showing fungal colonization of T. versicolor WCieGW (Tv)- inoculated control and T. versicolor +M. symbioticum BS 11 (Ms)- inoculated roots of wheat plants, respectively, at 16 and 21 days after inoculation, which were grown on black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada), with WGA staining used to visualize fungal hyphae. Panel A: Root of Tv -inoculated wheat plant, fungi have colonized the root surface and penetrated the epidermal cell (ep) layer. Panel B: Root of 7V+ATs-inociilatcd wheat plant, hyphae have colonized a larger area of the root surface as compared to the TV-inoculated roots. Panel C: Root of Tv+Ms- inoculated wheat plant, shows hyphae branching and proliferation within the epidermal space then passed from one outer cortical layer. Roots were analyzed by fluorescence microscopy.

[038] FIGs. 4A-4D are photographs (Fig. 4A and FIG. 4C) and graphs (FIG. 4B and FIG. 4D) showing wheat plant responses to .S', hirsutum P2A (Sh), T. versicolor WCieGW (Tv) and A. brasilense BS66 (Ab). Disinfected seeds were coated as follows: for fungi, seeds were coated with a homogenized mycelium solution prepared using a 0.1% Gum xanthan and 0.01% Tween 20 solution; for bacteria, seeds were coated with a bacterial suspension having an OD600 of 1.4, in a solution of lOmM MgSC .TITO with 1% PVP and 0.1% Tween 20. In the case of a mixture of both fungi and bacteria, an equal combination of the above-mentioned solutions was applied to the seeds. Afterwards, 4 coated seeds were planted in black earth organic soil (Voila®) without additional fertilizers (FIG. 4A) Wheat plants 30 days after inoculation; (FIG. 4B) Shoot dry weights, mean values, and standard error of 8 plants are shown. (FIG. 4C) Plants are shown from control. (FIG. 4D) Length of roots (measured fresh, right after washing) mean values, and standard error of 7 plants are shown, Statistical analysis was performed in SPSS using One-Way ANOVA; Stars attop of the bars indicate statistically significant differences (P < 0.05).

[039] FIGs. 5A-5C are photographs (FIG. 5A and FIG. 5C) and a graph (FIG. 5B) showing wheat plant responses to .S'. hirsutum P2A (Sh), T. versicolor WCieGW (Tv), .S'. indica BS22 (Si) and Azospirillum brasilense BS66 (Ab) either alone- or with dual -inoculation. Disinfected seeds were coated as follows: for fungi, seeds were coated with a homogenized mycelium solution prepared using 10 mM MgSO4, 0.1% PVP and 0.01% Tween 20 solution; for bacteria, seeds were coated with a bacterial suspension having an OD₆oo of 1.2, in a solution of lOmM MgSO4.7H₂O with 1% PVP and 0.1% Tween 20. In the case of a mixture of both fungi and bacteria, an equal combination of the above-mentioned solutions was applied to the seeds. Afterwards, 4 coated seeds were planted in 2/1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada) without additional fertilizers. (FIG. 5 A) Wheat plants 21 days after inoculation. (FIG. 5B) Root and shoot dry weights, mean values, and standard deviation of 11 plants are shown. (FIG. 5C) Plants roots are shown from control. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[040] FIGs. 6A-6C are photographs (FIG. 6A and FIG. 6C) and a graph (FIG. 6B) showing soybean plant responses to .S', hirsutum P₂A, T. versicolor WCieGW and AL symbioticum BS11. Disinfected seeds were coated with a homogenized mycelium solution that was prepared by 0. 1% PVP and 0.05% tween 20 solution then were put in the black organic soil without additional fertilizers (FIG. 6A) Soybean plants 30 days after inoculation; (FIG. 6B) Shoot dry weights, mean values, and standard deviation of 8 plants were shown. (FIG. 6C) Root dry weights, mean values, and standard error of 8 plants were shown. Statistical analysis was performed in SPSS using One- Way ANOVA; Stars attop of the bars indicate statistically significant differences (P < 0.05).

[041] FIGs. 7A-7E is a graph (FIG. 7A) and photographs (FIGs. 7B-7E) showing soybean plant responses to .S', hirsutum P₂A, T. versicolor WCieGW and AL symbioticum BS11. Disinfected seeds were dressed in a homogenized mycelium solution that was prepared by 0.1% PVP and 0.01% tween 20 solution then were put in the black organic soil supplemented. (FIG. 7A) Number of nodules, mean values, and standard deviation of 8 plants were shown. (FIG. 7B) Nodules of Ms- inoculated plant. (FIG. 7C) Nodules of Tv-inoculated plant. (FIG. 7D) Nodules of Ms+Sh- inoculated plant. (FIG. 7E) Nodules of AN TV-inoculated plant. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[042] FIGs. 8A-8C are photographs (FIG. 8A and FIG. 8C) and a graph (FIG. 8B) soybean plant responses to inoculation of single or in consortium of .S', hirsutum P2A and T. versicolor WC i.,GW with Gluconacetobacter diazotrophicus. 20 disinfected seeds were coated with a 5 ml of single homogenized mycelium solution or 2.5+2.5 ml of mixing mycelium with G. diazotrophicus that was prepared by 0. 1% PVP and 0.05% tween 20 solution then were put in the soil mixture which mentioned above without additional fertilizers (FIG. 8 A) Soybean plants 30 days after inoculation; (FIG. 8B) Shoot dry weights, mean values, and standard deviation of 10 plants were shown. (FIG. 8C) Root dry weights, mean values, and standard deviation of 10 plants were shown. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[043] FIGs. 9A-9G is a graph (FIG. 9A) and photographs (FIGs. 9B-9G) showing soybean plant responses to Stereum hirsutum, Trametes versicolor WCieGW and Gluconacetobacter diazotrophicus (Gd). Disinfected seeds were dressed in a homogenized mycelium solution that was prepared by 0.1% PVP and 0.01% tween 20 solution then were put in the black organic soil supplemented. (FIG. 9A) Number of nodules, mean values, and standard deviation of 10 plants were shown. (FIG. 9B) Nodules of mock-inoculated plant. (FIG. 9C) Nodules of TV-inoculated plant. (FIG. 9D) Nodules of .S'/?-inoculatcd plant. (FIG. 9E) Nodules of Gd+ TV-inoculated plant. (FIG. 9F) Nodules of GT .S'/?-inociilatcd plant. (FIG. 9G) Nodules of G -inoculated plant. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P = 0.05).

[044] FIGs. 10A-10C are photographs (FIG. 10A and FIG. 10C) and a graph (FIG. 10B) showing Soybean plant responses to inculcation of single or in consortium of .S', hirsutum P2A and T. versicolor WCieGW with Azospirillum brasilense. 20 disinfected seeds were coated with a 5 ml of single homogenized mycelium solution or 2.5+2.5 ml of mixing mycelium with A. brasilense that was prepared by 0. 1% PVP and 0.05% tween 20 solution then were put in the soil mixture which mentioned above without additional fertilizers (FIG. 10A) Soybean plants 30 days after inoculation; (FIG. 10B) Shoot dry weights, mean values, and standard deviation of 10 plants were shown. (FIG. 10C) Root dry weights, mean values, and standard deviation of 10 plants were shown. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P = 0.05).

[045] FIGs. 11A-11G is a graph (FIG. 11A) and photographs (FIGs. 1 IB-11G) showing soybean plant responses to .S'. hirsutum, T. versicolor and Azospirillum brasilense BS66. Disinfected seeds were dressed in a homogenized mycelium solution that was prepared by 0.1% PVP and 0.01% tween 20 solution then were put in the black organic soil supplemented. (FIG. 11 A) Number of nodules, mean values, and standard deviation of 10 plants were shown. (FIG. 11B) Nodules of mock -inoculated plant. (FIG. 11C) Nodules of Tv-inoculated plant. (FIG. 11D) Nodules of Sh- inoculated plant. (FIG. HE) Nodules of Ab + v-inoculated plant. (FIG. 1 IF) Nodules of Ab+Sh- inoculated plant. (FIG. 11G) Nodules of Aft-inoculated plant. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P = 0.05).

DETAILED DESCRIPTION

[046] The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving” and variations thereof herein, is meant to encompass the items listed thereafter as well as, optionally, additional items. In the following description, the same numerical references refer to similar elements.

[047] The present investigators have designed bacterial-fungal assemblages (e.g., a bacteriumfungus chimera) for improving the overall health of plants. As used herein, the expression “bacterial-fungal assemblage” relates to a group of microbes (i.e., bacteria and fungi) that are expected to co-occur. In some embodiments, the present investigators have designed bacterial- fungal assemblages for improving the solubilization and transfer of residual phosphorus and fixed air-nitrogen to plants.

[048] The term “endophyte” is used herein to describe a microorganism inhabiting the interior of plants, irrespective of the function in association with the plant. Endophytes constitute a major part of hidden fungal diversity inside almost all plants. Fungal endophytes also can use recalcitrant substrates because of their wide-ranging enzymatic capabilities, and their ability to translocate nutrients through their hyphae. Controlled cultivation of plants in symbiosis with endophytic fungi might help in reducing the quantity of fertilizers and pesticides that need to be expended in sustainable agriculture.

[049] The term “chimera” refers to an organism that comprises cells or tissues from two or more genetically distinct individuals. This can occur naturally or be created in the laboratory by combining cells from different organisms. Because chimeras comprise cells with different genetic backgrounds, they can have unique physical characteristics and may exhibit unexpected traits or behaviors.

[050] Trametes versicolor WCieGW is an endophytic phosphorus-transporting fungi, which is also known by the name Trametes versicolor WCieGW.

[051] Stereum hirsutum P2A is an endophytic phosphorus-transporting fungi, which is also known by the name also known as Stereum hirsutum P2A.

[052] Trametes versicolor WCieGW is an endophytic Basidiomycetes phosphorus-transporting fungus of the order Poly por ales . This fungus was initially isolated in Giessen, Germany (Table 1), and the DNA sequences of the 18S and ITS including 5.8S ribosomal RNA are shown in Table 2 (SEQ ID NOs: 1-3).

[053] Stereum hirsutum P2A is an endophytic Basidiomycetes phosphorus-transporting fungus of the order Russulales. This fungus was initially isolated in Giessen, Germany (Table 1), and the DNA sequence of the 18S and ITS including 5.8S ribosomal RNA are shown in Table 2 (SEQ ID NO:).

Table 1: Fungal Isolate information.

Table 2: DNA sequences from Fungal Isolates.

[054] “Phosphorus” as used herein, includes any compound that comprises phosphorus. [055] According to a first embodiment, the present technology relates to a bacterial -fungal assemblage that acts as a monocote/non-legume dicote biofertilizer bio-complement. In some instances, the fungus of the bacterial-fungal assemblage is a posphorus solubilizer/mobilizer/transporter fungus and the bacteria of the bacterial-fungal assemblage is a free-living nitrogen fixing hetrotrophic bacteria (e.g. Azospirillium spp., Azotobacter spp., Pseudomonas spp., Bacillus spp.). In some other instances, the fungus of the bacterial-fungal assemblage is a phosphorus solubilizer/mobilizer/transporter fungus and the bacteria of the bacterial-fungal assemblage is an associative/endophytic nitrogen fixing bacteria (e.g. Azoarcus spp. , Achromobacter spp. , Burkholderia spp. , Gluconoacetobacter spp. , Herbaspirillum spp., Klebsiella spp. , and Serratia spp.). In some instances, the fungus of the bacterial-fungal assemblage is a phosphorus solubilizer/mobilizer/transporter fungus and the bacteria of the bacterial -fungal assemblage is a nitrogen fixing (alga) Cyanobacterium (e.g. Nostoc spp.; Anabaena spp.).

[056] Nitrogenous fertilizer production accounts for approximately 2% of global energy consumption. The energy used to manufacture nitrogen fertilizer releases over 1 gigatonne of CO2 - 5% of global emissions. Moreover, high level of residual P on much agricultural land due to the cumulative input of P fertilizer, namely in the period of 1965-2007 in Europe (1.115 kg.hc ') that is not accessible to plants.

[057] Phosphorus mobilizer fungi could solubilize and transfer residual phosphorus to plants and could transport endohyphal/epihyphal nitrogen-fixing bacteria in the rhizosphere/endosphere.

[058] Fungi colonize plant roots and transport phosphorus by external hyphae and deliver hyphal bacteria to the root; and bacteria enable plants to use fixed air-nitrogen directly inside the root. Advantages of the assemblage according to this embodiment include, but are not limited to, reduction of chemical fertilizer consumption; increase the chemical fertilizer absorption; and increase N-fixing efficiency by providing carbon and energy to free living bacteria.

[059] In one embodiment, the present technology relates to a bacterial -fungal assemblage that acts as a legume fertilizer bio-complement. In some instances, the fungus of the bacterial-fungal assemblage is a phosphorus solubilizer/mobilizer/transporter fungus, and the bacteria of the bacterial-fungal assemblage is a legume nodule formation bacterium such as for examples those identified in Table 3.

Table 3 : Nodulating bacteria and associated crop

Crop Nodulating Bacterium

Alfalfa Sinorhizobium meliloti

Beans Rhizobium legumninosarum biovar phaseoli, and Rhizobium tropici

Clover Rhizobium leguminosarum biovar trifolii

Lotus Mesorhizobium loti

Peas Rhizobium leguminosarum biovar viceae

Soybean Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium fredii

Sesbania Azorhizobium caulinodans

[060] Bradyrhizobium are competitive with indigenous soil strains at the point of placement in the soil but have limited mobility and so are incapable of sustaining high populations throughout the developing root system, therefore superior inoculant rhizobia applied to soybeans usually occupy only 5 to 20% of nodules. The bacterial-fungal assemblage of the present technology allows to provide simultaneous delivery of phosphorus and nitrogen to the crop and allows to overcome the limited mobility of legume nodule formation bacteria. Some advantages of the bacterial -fungal assemblage of the present embodiment include, but are not limited to, the large-scale axenic cultivation of microbial consortia, reduction of chemical fertilizer consumption; increase chemical fertilizer absorption; increase the yield of legume crops, as the fungus could provide an alternative source of carbon and energy for fixing nitrogen. Examples of crops with which the bacterial-fungal assemblage include, but are not limited to, com, cotton, peanut, grass, soybean, sugarbeet, potatoes, vegetables.

[061] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts as a microbial nematicide. In some instances, the fungus of the bacterial -fungal assemblage is a Fungi nematicide (e.g. Paecilomyces lilacinus, Colonostachys rosea, Pochonia spp.; Metarhizium spp., Trichoderma spp.). In some instances, the bacteria of the bacterial-fungal assemblage is a bacterium nematicide (Bacillus spp.; Pastoria penetrans). In some instances, Meloidogyne spp. species as a root-knot nematode pathogen of different crops and suppression of soybean cyst nematode Heterodera glycines. P. lilacinus could colonize the surface of epidermal cells as well as the internal cells of the epidermis and cortex. P. lilacinus parasitize eggs and root- knot nematode females. Endospores of the bacterium P. penetrans attach to the cuticle of a juvenile nematode.

[062] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts as a biofungicide. In some instances, the fungus of the bacterial -fungal assemblage is a fungal antagonist (e.g. Alternaria spp., Aspergillus spp., Candida spp., Fusarium spp., Penicillium spp., Pichia spp., Talaromyces spp., Trichoderma spp., and Verticillium spp. Chlonostachys spp.). In some instances, the bacterial of the bacterial-fungal assemblage is a bacterial antagonist (e.g. Bacillus amyloliquefaciens ; B. subtilis). The bacterial-fungal assemblage according to this embodiment may be used for seed rot and seedling pre/post-emergence damping- off caused by Rhizoctonia solanil Fusarium spp./ Pythium s\yF phyiophiora spp. Fungus colonizes and protect roots at the susceptible seedling stage, with defense mechanisms induced/produced by the hyphae and the associated bacterium. Examples of crops with which the bacterial-fungal assemblage may be used include, but are not limited to, turfgrasses, vegetables, and flowers cotton, com, soybean and cereals.

[063] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts as a microbial bioremediation. Polycyclic Aromatic Hydrocarbons (PAHs) are relatively non-volatile and of low solubility in soil and water. PAHs are moderately to highly carcinogenic for humans. Major sources of PAHs to the aquatic and soil environments include creosote-treated products (up to 2000 t/yr), spills of petroleum products (76 t/yr), metallurgical and coking plants (4 t/yr), and deposition of atmospheric PAHs. In some instances, the fungal of the bacterial -fungal assemblage is free living fungus.

[064] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts as a microbial assisted-phytoremediation. As an example, arsenic can be taken up by plants as arsenate (AsO/' ) from aerobic soil through phosphorus transporters and as arsenite (AsOs³ ) from flooded soil through silicon (Si) transporters. Despite the competitive behavior of As with inorganic phosphate (Pi), Serendipita indica, a fungal species from the Serendipitaceae family, may associate with plants and promote their growth by reducing As bioavailability (i.e., adsorption, accumulation, and precipitation by the fungus) in the rhizosphere. Another study showed that fungal colonization enhances the arsenic bioaccumulation factor in the root. High concentrations of As in substrates also significantly impacts plants. It tends to decrease shoot and root dry weight, chlorophyll, and P and Mg uptake in plants. In some instances, the fungal of the bacterial -fungal assemblage is fungus endophyte.

[065] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts as a bio-insecticide. Root crop pests can cause significant damage to crops and lead to reduced yields and economic losses for farmers. The damage caused by root crop pests can also make crops more susceptible to other diseases and pests, further compounding the problem. In some instances, the fungus of the bacterial -fungal assemblage is a fungi insecticide (e.g. Beauveria bassiana, Cordyceps fumosorosea; Akanthomyces muscarius, Metarhizium anisopliae, Purpureocillium lilacinum, Trichoderma harzianum) . In some instances, the bacteria of the bacterial -fungal assemblage is a bacteria insecticide (e.g. Bacillus thuringiensis, Bacillus spp., Serratia entomophila, Streptomyces avermitilis, Pseudomonas fluorescens). The use of the chimera of the present application as an insecticide could provide a more effective and sustainable approach to pest control than using a single type of insecticide. Moreover, this could work by targeting different parts of the insect's biology, so using them in combination may provide a broader spectrum of pest control and also reduce the likelihood of pests developing resistance to a single type of insecticide, which can become a major problem with prolonged use of a single type.

[066] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts as an assistant in phosphorus solubilization. In some instances, the bacterial- fungal assemblage is to be applied to tropical soils where there is very strong binding to aluminum and iron. In some instances, the fungus of the bacterial-fungal assemblage is a fungus phosphorus mobilizer (e.g. Ascomycetes, Basidiomycetes). In some instances, the bacteria of the bacterial- fungal assemblage is a bacterium phosphorus solubilizer (e.g. Alcaligenes spp., Acinetobacter spp., Arthrobacter spp., Azospirillum spp., Bacillus spp., Burkholderia spp., Enterobacter spp., Flavobacterium spp., Paenibacillus spp., Pseudomonas spp.). Some advantages of the bacterial- fungal assemblage of the embodiment include, but are not limited to, improvement of the efficiency of phosphorus utilization by plants, leading to increased crop yields, and since each partner in the chimera produces different enzymes, it can provide a more complete and effective phosphorus solubilization process.

[067] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts to empower beneficial fungi. In some instances, the fungus of the bacterial- fungal assemblage is a fungus that acts as biofertilizer or biocontrol agents (e.g. Piriformospora spp., Trametes spp., Trichoderma spp., Clonostachys spp). In some instances, the bacteria of the bacterial -fungal assemblage is bacteria that act as nitrogen-fixing or Cyanobacteria (e.g. Azospirillium spp., Nostoc spp.). Some advantages of the bacterial-fungal assemblage of the embodiment include but are not limited to, the production of compounds that provide nutrients to the fungus, such as amino acids, vitamins, and other organic molecules. This can help the fungus grow and reproduce more efficiently, allowing it to better compete with other organisms in its environment. Additionally, the bacterium can help the fungus access nutrients that might otherwise be unavailable. Lastly, the bacterium may help the fungus avoid predation or infection by other organisms.

[068] According to one embodiment, the present technology relates to a bacterial and fungal assemblage that acts to enhance induced-systemic-resistance (ISR) in plants. In some instances, the fungus of the bacterial-fingal assemblage is a fungus that acts as biofertilizer or biocontrol agents (e.g. Piriformospora spp., Trametes spp. Trichoderma spp., Stereum spp., Clonostachys spp). In some instances, the bacteria of the bacterial -fungal assemblage are bacteria, such as Bacillus spp. or Cyanobacteria (e.g. Nostoc spp.). Some advantages of the bacterial-fungal assemblage of the embodiment include, but are not limited to, providing higher protection for plants against pests and diseases, and it may reduce the need for chemical pesticides or other interventions.

[069] According to one embodiment, the present technology relates to a bacterial-fungal assemblage that acts in pathogen inactivation. In some instances, the fungus of the bacterial-fungal assemblage is a fungi pathogens (e.g. Rhizoctonia solani and Fusarium oxysporum). In some instances, the bacteria of the bacterial-fungal assemblage is endohyphal bacteria. Fusarium oxysporum is a plant pathogenic fungus that can cause a range of diseases in crops, including wilting, root rot, and vascular wilt. Collimonas fungivorans is an endohyphal bacterium that lives inside the hyphae of Fusarium oxysporum. When present, C. fungivorans can help to reduce the pathogenicity of the fungus by producing compounds that can inhibit the production of toxins and reduce damage to plant tissues.

EXAMPLES

[070] The examples below are given so as to illustrate the practice of various embodiments of the present disclosure. They are not intended to limit or define the entire scope of this disclosure. It should be appreciated that the disclosure is not limited to the particular embodiments described and illustrated herein but includes all modifications and variations falling within the scope of the disclosure as defined in the appended embodiments. Example 1 - Materials and Methods

[071] Fungal growth condition: .S' hirsutum P2A and T. versicolor WCieGW were grown in MYP broth (Malt extract 10.0g; Yeast extract 2.0g; Proteose peptone 1.5g; Sucrose 10.0g; Glucose 5.0g; KC1 0.5g; Na₂HPO₄ 0.5g; KH₂PO₄ 0.5g; K₂HPO₄ 0.5g; pH: 7.24) for three weeks at 28°C at the MaxQ™ 8000 (Japan) shaker in the dark.

[072] Bacterial growth condition: hrasilense BS66 culture in a medium that induces PHB accumulation, in a medium with a high carbon-to-nitrogen (C/N) ratio. The high C:N medium contained (g I ¹) contains D-fructose (6.67), MgSO₄ (0.2), NaCl (0.1), CaCl₂ (0.02), K₂HPO, (6.0), KH₂PO₄ (4.0), Yeast extract (0. 1), pH: 6.8. The initial OD 540 of approximately 0.05 (about 10⁷ c.f.u. ml ¹); therefore, incubated on a rotary shaker (150 r.p.m.) at 30° C for 24 h /A. hrasilense BS66 culture in medium at 28 overnight, Cells were harvested by centrifugation (10 min at 8142g) and re-suspended in lOmM MgSO4.7H2O solution with OD600=1.5. Methylobacterium symbioticum were grown on Jayasuriya's medium (KaHPO₄ 1.74 g; NaH₂ PO₄ H₂0 1.38 g; (NH4)₂SO₄ 0.5 g; MgSO₄ 7 H₂0 0.2 g; CaC12-2 H20 0.025 mg; FeC12.4 H20 3.5 mg, 0.5 ml of a trace element, KNO3 (0.2~o w/v), methanol (0.5~v/v), pH 7.0) and produces pink color. Pure cultures of Gluconacetobacter diazotrophicus were cultivated in SYP medium, which contained (in g/L): 10 sucrose, 3 yeast extract, 1 K2HPO4, and 3 KFLPCh, with a final pH of 6.2.

[073] Plant cultivars, germination of seeds and growth condition: OAC Bruton is an indeterminate large-seeded food-grade soybean [Glycine max (L.) Merr.] cultivar with high yield potential, high seed protein concentration, and resistance to soybean cyst nematode (SCN). OAC Bruton is developed and recommended for soybean growing areas in southwestern Ontario with 2950 or greater crop heat units. OAC Bruton is classified as a maturity group 1 (MG1) cultivar with a relative maturity of 1.8. Kernels of soybean, OAC Bruton (seed size: 3900 seeds/kg; germination: 92%) were sterilized with 1% (v/v) NaOCl for 6 min, then rinsed with sterilized water 3 times for 3 min. Kernels of wheat cv. Hard Red Spring was sterilized with 3% (v/v) NaOCl for 1 h and 70% (v/v) ethanol 70% for 1 min, then rinsed with sterilized water 3 times for 5 min. Then seeds were grown in small pots in the lab. The pot was containing 2: 1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada). The growth condition in the Jiffy 4004781 Hydro Grow light, was 16 h photoperiod, 22/22°C day/night. [074] Seed inoculation, Seeds dressing (coating) method: Kernels of wheat cv. Hard Red Spring were sterilized with 3% (v/v) NaOCl for 1 h and 70% (v/v) ethanol for 1 min, then rinsed with sterilized water 3 times for 5 min. 110 disinfected seeds were coated with 2 ml of a homogenized mycelium solution that was prepared by shaking 3g of mycelium in 10ml of 0.1% PVP (polyvinylpyrrolidone) and 0.01% tween 20 solution in the pots containing heat sterilized (121°C for 5 min at 15 psi) soil with the combinations mentioned above. Moreover, sterile wheat seeds were inoculated in 2 ml of fresh bacteria (24 hours old) solution for 20 min, while 0.1% PVP + lOmM MgSC>4.7H2O solution was used as a control. For a mix of fungus and bacteria, 1 ml of bacteria solution plus 1 ml of fungus solution were used.

[075] For the (M. symbioticum experiment) soybean seeds were grown on in small pots in the lab. The pot was containing 2: 1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada). The growth condition in the Jiffy 4004781 Hydro Grow light, was 16 h photoperiod, 22/22 °C day/night.

[076] For the (Gluconacetobacter diazotrophicus experiment) soybean seeds were grown on in small pots in the lab. The pot was containing 1 : 1 : 1 : 1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada), perlite and clay. The growth condition in the Jiffy 4004781 Hydro Grow light, was 16 h photoperiod, 22/22°C day/night.

[077] or Azospirillum brasilense soybean seeds were grown on in small pots in the lab. The pot was containing 1: 1: 1: 1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada), perlite and clay. The growth condition in the Jiffy 4004781 Hydro Grow light, was 16 h photoperiod, 22/22°C day/night.

[078] Four pre-inoculated seeds were sown in individual plastic pots, each with a 7 cm top diameter, a 5 cm bottom diameter, and a height of 8 cm. These pots received a daily watering of 30 mb of distilled water. Two seedlings were retained in each small pot, and the plants were harvested 30 days post-planting. Subsequently, the fresh root length was measured immediately after washing. The root and shoot dry weights were determined by weighing plant samples after 24 hours at 65 degrees Celsius.

[079] Seeds coating method (M. symbioticum BS11 experiment): OAC Bruton seeds of each treatment were coated with 4ml of a homogenized mycelium solution that was prepared by shaking 8- 14g of mycelium in 12-14ml of 1% PVP and 0.01% tween 20 solution for 2h then transferred in the pots containing heat sterilized (121 °C for 5 min at 15 psi) soil with the combinations mentioned above. M. symbioticum culture media mentioned above at 28 overnight. After culturing and overnight, cells were harvested by centrifugation (10 min at 8142g) and re-suspended in lOmM MgSO4.7H2O solution with OD600=1.1. The cell protectants and surfactants such as polyvinylpyrrolidone (PVP, 1%) and Tween 20 (0.1%) were added to the suspension. Subsequently, sterile soybean seeds were inoculated in 4 ml of the bacteria solution for 20 min, while lOmM MgSO4.7H2O + PVP, 1% and Tween 20 (0.1%) solution was used as a control. For a mixture of fungus and bacteria, 2 ml of the bacterial solution was combined with 2 ml of a fungus solution, which was prepared by homogenizing 8 grams of fungus in 12 ml of sterile distilled water with the addition of PVP at a concentration of 0.1%. 4 seeds in each plastic pot (IL). Then two will be removed. The pots were watered every day with 50 mL of sterile pure water. The number of pods were calculated after 30 days. Plants dried in an oven at 65 degrees Celsius for 24 hours to measure root and shoot dry weight.

[080] Seeds coating method (Gluconacetobacter diazotrophicus experiment): OAC Bruton seeds of each treatment were coated with 4ml of a homogenized mycelium solution that was prepared by shaking 10-11g of mycelium in 10ml of 1% PVP, lOmM MgSCLTFLO and 0.01% tween 20 solution for 2h then transferred in the pots containing heat sterilized (121 °C for 5 min at 15 psi) soil with the combinations mentioned above. M. symbioticum culture media mentioned above at 28 and rpm 180 overnight. After 2 days, cells were harvested by centrifugation (10 min at 8142g) and re-suspended in lOmM MgSCLTFLO solution with OD600=1.8. The cell protectants and surfactants such as polyvinylpyrrolidone (PVP, 1%) and Tween 20 (0.1%) were added to the suspension. Subsequently, sterile soybean seeds were inoculated in 4 ml of the bacteria solution for 20 min, while lOmM MgSCLJFLO + PVP, 1% and Tween 20 (0.1%) solution was used as a control. For a mixture of fungus and bacteria, 2.5 ml of the bacterial solution was combined with 2.5 ml of a fungus solution, which was prepared by homogenizing 10 grams of fungus in 10 ml of sterile distilled water with the addition of PVP at a concentration of 0. 1%. 4 seeds in each plastic pot (IL) then two will be removed. The pots were watered every day with 50 mL of sterile pure water. The number of pods were calculated after 30 days. Plants dried in an oven at 65 degrees Celsius for 24 hours to measure root and shoot dry weight.

[081] Seeds coating method (Azospirillum brasilense experiment): The liquid medium was initially placed in a shaker incubator set to 28°C at 180 rpm for 3 days. After this period, it was transferred to a second shaker incubator at 23 °C with a reduced speed of 100 rpm, where it was incubated until the inoculum became visibly cloudy. Five treatment including Control, .S' hirsutum (Sh), T. versicolor (Tv), A. brasiliense (Ab) + Sh, and Ab + Tv were applied with 5 replications of each. OAC Bruton seeds of each treatment were coated with 12 ml of a homogenized mycelium solution that was prepared by shaking 4g of mycelium in 12- 14ml of 0.1% PVP and 0.01% tween 20 solution for 2h then transferred in the pots containing heat sterilized (121 °C for 5 min at 15 psi) soil with the combinations mentioned above. A. brasiliense culture media mentioned above at 28°C overnight. After culturing and overnight, cells were harvested by centrifugation (10 min at 8142g) and re-suspended in lOmM MgSC .VFFO solution with OD600=1.68. The cell protectants and surfactants such as polyvinylpyrrolidone (PVP, 0.1%) and Tween 20 (0.01%) were added to the suspension. Subsequently, sterile soybean seeds were inoculated in 5 ml of the bacteria solution (for 20 min, while lOmM MgSC TFFO + PVP 0.1% and Tween 20 (0.01%) solution was used as a control. For a mixture of fungus and bacteria, 2.5 ml of the bacterial solution was combined with 2.5 ml of a fungus solution, which was prepared by homogenizing 8 grams of fungus in 12 ml of sterile distilled water with the addition of PVP at a concentration of 0.1 %. Each pot watered before putting the seeds, then 4 seeds in each plastic pot (IL). After germination, two will be removed. The pots were watered every day with 50 m of sterile pure water. The number of pods were calculated after 21 days. Plants dried in an oven at 65 degrees Celsius for 24 hours to measure root and shoot dry weight.

[082] Statistical analysis: First data were checked for normal distribution, then one-way ANOVA was performed using IBM SPSS version 29. The significance of the treatment effects was judged by the magnitude of the F-value (P < 0.05). Then Fishers protected LSD was performed for separation of means.

[083] Here, the compatibility of three endophytic phosphorus-transporting fungi (including Trametes versicolor WCieGW, Stereum hirsutum P2A and Serendipita indica BS22) with three strains of free-living nitrogen fixing bacteria (including Gluconacetobacter diazotrophicus BS47, Methylobacterium symbioticum BS11 and Azospirillum brasilense BS66) has been evaluated. A successful combination would be a promising candidate for making bacterial-fungal assemblages for use as a monocot/non-legume dicot biofertilizer.

Example 1 - Wheat seed coating by combined inoculums including Trametes versicolor WCieGW (“Tv”) and Gluconacetobacter diazotrophicus BS47 (“Gd”) showed beneficial activity in spring wheat root and shoot growth promotion [084] To evaluate the compatibility of seed dual-inoculum coating in spring wheat seedlings growth promotion, among G. diazotrophicus BS47 and three selected Basidiomycetes endophytes (T. versicolor WCieGW, Stereum hirsutum P2A, Serendipita indica), with AGTIV®IGNITE™ as positive control, an equal volume of each inoculum were combined and applied as a seed dressing. Dual seed coatings of T. versicolor WCieGW and G. diazotrophicus BS47 showed significant beneficial activity in spring wheat seedlings root and shoot growth promotion as compared to mock -inoculated plants. Moreover, the shoots dry weights were elevated 15% and 12% respectively in the Tv+Gd dual-inoculated plants as compared to singly TV-inoculated plants and Gd-inoculated plants (FIGs. 1A, IB and 1C). Alternative combinations did not prove to be more effective than each individual inoculum on its own.

Example 2 - Methylobacterium symbioticum BS11 (“Ms ”) showed beneficial activity in promotion of spring wheat shoot growth; seed coating by dual inoculants of S. hirsutum P2A and M. symhioticum BS11 inoculants significantly boosted these parameters as compared to plants inoculated solely with M. symbioticum.

[085] To evaluate the activity of a free-living nitrogen fixation bacteria AT. symbioticum BS 11 on spring wheat or with co-inoculation seed coating including each basidiomycetes endophyte of T. versicolor WCieGW or .S', hirsutum P2A, four-day-old wheat seedlings were inoculated with single or combined inoculum of strains. While there was no significant increase in root or shoot dry weight when compared to mock inoculated plants, the combined application of .S', hirsutum P2A and AT symbioticum BS11 to inoculants significantly boosted these parameters as compared to plants inoculated solely with AT symbioticum BS11 (FIGs. 1A, IB and 1C). Furthermore, WGA staining of the roots germinated from seeds inoculated with Tv +Ms revealed a notable expansion in the colonization of the root cells by T. versicolor hyphae as compared to TV-inoculated seeds (FIGs. 2A and B). Additionally, in TV +Ms -inoculated seeds, it was observed that hyphal proliferation occurred within the root epidermal space, followed by penetration through subepidermal cells to ultimately reach the outer cortical layer (Fig. 2C). WGA staining revealed the colonization patterns of T. versicolor (Tv) and its interaction with AT symbioticum (Ms) in wheat roots. In Tv-inoculated roots, fungal hyphae were detected on the root surface and had penetrated the epidermal cell layer (Fig. 3, panel A). Co-inoculation with Tv and Ms led to more extensive colonization of the root surface compared to Tv alone (Fig. 3 panel B). Additionally, in the Tv+Ms treatment, hyphae showed increased branching and proliferation within the epidermal region and progressed into the outer cortical layer, indicating enhanced root colonization dynamics (Fig. 3 panel C). Example 3 - Wheat seed coating with dual inoculants of Azospirillum brasilense BS66 and Trametes versicolor WCifjW significantly boosted growth parameters as compared to mock- inoculated plants or plants inoculated solely with A. brasilense BS66.

[086] To evaluate the compatibility of dual-microbial inoculation among A. brasilense BS66 and two selected Basidiomycetes endophytes (T. versicolor WC16GW, Stereum hirsutum P2A) for the promotion of spring wheat seedlings growth, an equal volume of each inoculum were combined and applied as a seed dressing. Dual seed coating of T. versicolorWC^GW and A. brasilense BS66 showed significant beneficial activity with an increase in spring wheat seedlings shoot dry weight (18%>) as compared to mock-inoculated plants (FIGs. 4A-4C). Moreover, shoot length was elevated 16% in the T. versicolor WCieGW and A. brasilense BS66 dual-inoculated plants as compared to mock-inoculated plants. In the second experiment, while there was no significant increase in root or shoot dry weight when compared to mock-inoculated plants, the plants inoculated with the combined seed-coating of .S', hirsutum P2A and A. brasilense BS66 showed a significant boost in these parameters as compared to plants inoculated solely with A. brasilense BS66 (FIGs. 5A-5C).

Example 4: Trametes versicolor WCigGW demonstrated a positive effect on promoting soybean nodule formation. However, seed soaking with the dual inoculants ofT. versicolor WCi GW or S. hirsutum P2A along with M. symbioticum BS11 did not result in any significant improvement in growth parameters compared to mock-inoculated plants.

[087] To evaluate the activity of a free-living nitrogen fixation bacteria, M. symbioticum BS11 on soybean alone or with co-inoculation seed soaking including each basidiomycetes endophyte of T. versicolor P2A or .S', hirsutum P2A. While there was not a significant increase in root or shoot dry weight when compared to mock inoculated plants (FIGs. 6A-6C). The single application of T. versicolor WCieGW inoculant significantly boosted number of nodules compared to mock inoculated plants (FIGs. 7A-7C) 30 days after inoculation.

Example 5: Consortium of Trametes versicolor WCigGW and Gluconacetobacter diazotrophicus BS47 demonstrated a beneficial effect on promoting soybean root dry weight compared to other biological inoculants. [088] To assess the biological activity of free-living nitrogen fixation bacterium G. diazotrophicus strain BS47, soybean seeds were soaked in bacterial solution alone or mixing with two endophytic basidiomycetes of T. versicolor WCieGW or Stereum hiorsutum P2A. After 30 days, plants which inoculated with T. versicolor WCieGW (TV) and G. diazotrophicus BS47 (Gd) increase in biomass (FIG. 8). Root dry weights were elevated significantly by 44% in Tv+Gd inoculation to mock-inoculated plants (FIGs. 8A-9B). Visual analysis of the roots confirmed an increase in biomass upon bacterial inoculation (FIG. 8C). Moreover, the co-application of Tv+Gd inoculant significantly boosted the number of nodules compared to mock inoculated plants (FIGs. 9A-9G) 30 days after inoculation.

Example 6: Consortium ofT. versicolor WCieGW and A. hrasilense strain BS66 demonstrated a positive effect on promoting soybean nodule formation. Moreover, T. versicolor WCi GW demonstrated a significant beneficial effect on promoting soybean root dry weight compared to other biological inoculants.

[089] To assess the biological activity of free-living nitrogen fixation bacteria A. brasilense strain BS66, soybean seeds were soaked in bacterial solution alone or mixing with two endophytic basidiomycetes of T. versicolor WCieGW or Stereum hiorsutum P2A. After 30 days, plants which inoculated with T. versicolor WCieGW (TV) increased in biomass (FIG. 10A). Root dry weights were elevated significantly in TV inoculation to mock-inoculated plants (FIGs. 10A-10B). Visual analysis of the roots confirmed an increase in biomass upon bacterial inoculation (FIG. 10C). Moreover, the single application of T. versicolor WCieGW or .S', hiorsutum P2A and consortium of T. versicolor WCieGW and A. brasilense significantly boosted number of nodules compared to mock inoculated plants (FIGs. 11A-11C) 30 days after inoculation.

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Claims

CLAIMS:

1. A bacterial -fungal assemblage for improving efficiency of phosphorus utilization by plants, wherein the bacterial-fungal assemblage comprises a fungus and bacteria, wherein the fungus is a phosphorus solubilizer fungus, a phosphorus mobilizer fungus or a phosphorus transporter fungus, and the bacteria are nitrogen fixing bacteria.

2. The bacterial -fungal assemblage of claim 1, wherein the bacterial -fungal assemblage of a chimera of at least one fungal organism and at least one bacterial organism.

3. The bacterial -fungal assemblage of claim 1 or 2, wherein the bacterial are endophytic nitrogen fixing bacteria.

4. The bacterial -fungal assemblage of claim 1 or 2, wherein the bacteria are selected from Azospirillium spp., Azotobacter spp., Pseudomonas spp., Bacillus spp; Azoarcus spp. , Achromobacter spp., Burkholderia spp., Gluconoacetobacter spp. , Herbaspirillum spp., Klebsiella spp., and Serratia spp., Methylobacterium spp.,

5. The bacterial -fungal assemblage of claim 1 or 2, wherein the bacteria are nitrogen fixing alga.

6. The bacterial -fungal assemblage of claim 5, wherein the bacteria are nitrogen fixing alga is Cyanobacterium.

7. The bacterial -fungal assemblage of claim 1 or 2, wherein the bacteria are legume nodule formation bacteria.

8. The bacterial -fungal assemblage of claim 7, wherein the legume nodule formation bacteria are selected from Sinorhizobium meliloti, Rhizobium leguminosarum biovar phaseoli, Rhizobium tropici, Rhizobium leguminosarum biovar trifolii, Mesorhizobium loti, Rhizobium leguminosarum biovar viceae, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium fredii, and Azorhizobium caulinodans.

9. The bacterial-fungal assemblage of any one of claims 1 to 8, wherein the fungus is a fungus nematicide.

10. The bacterial-fungal assemblage of claim 9, wherein the fungus nematicide is selected from Paecilomyces lilacinus, Colonostachys rosea, Pochonia spp. ; Metarhizium spp., and Trichoderma spp.

11. The bacterial -fungal assemblage of claim 8 or 9, wherein the bacterial are bacteria nematicide.

12. The bacterial -fungal assemblage of claim 11, wherein the bacteria nematicide are selected from Bacillus spp. and Pastoria penetrans.

13. The bacterial -fungal assemblage of any one of claims 1 to 8, wherein the fungus is a fungus antagonist.

14. The bacterial-fungal assemblage of claim 13, wherein the fungus antagonist is selected from Alternaria spp., Aspergillus spp., Candida spp., Fusarium spp., Penicillium spp., Pichia spp., Talaromyces spp., Trichoderma spp., and Verticillium spp. Chlonostachys spp.

15. The bacterial -fungal assemblage of claim 14, wherein the bacteria are selected from Bacillus amyloliquefaciens, and B. suhtilis.

16. The bacterial-fungal assemblage of any one of claims 1 to 8, wherein the fungus is a fungus insecticide.

17. The bacterial-fungal assemblage of claim 16, wherein the fungus insecticide is selected from Beauveria hassiana, Cordyceps fumosorosea; Akanthomyces muscarius, Metarhizium anisopliae, Purpureocillium lilacinum, and Trichoderma harzianum.

18. The bacterial -fungal assemblage of claim 16 or 17, wherein the bacterial are bacteria insecticide.

19. The bacterial -fungal assemblage of claim 19, wherein the bacteria insecticide are selected from Bacillus thuringiensis, Bacillus spp., Serratia entomophila, Streptomyces avermitilis, and Pseudomonas fluorescens.

20. The bacterial -fungal assemblage of any one of claims 1 to 8, wherein the fungus is selected from Ascomycetes, and Basidiomycetes .

21. The bacterial-fungal assemblage of any one of claims 1 to 8, wherein the bacteria are selected from Alcaligenes spp., Acinetobacter spp., Arthrobacter spp., Azospirillum spp., Bacillus spp., Burkholderia spp., Enterobacter spp., Flavobacterium spp., Paenibacillus spp., and Pseudomonas spp.

22. The bacterial -fungal assemblage of any one of claims 1 to 8, wherein the fungus is selected from Trametes versicolor, Stereum hirsutum, and Serendipita indica BS22.

23. The bacterial -fungal assemblage of claim 22, wherein the fungus is Trametes versicolor.

24. The bacterial -fungal assemblage of claim 23, wherein the fungus is WCieGW.

25. The bacterial -fungal assemblage of claim 22, wherein the fungus is Stereum hirsutum.

26. The bacterial -fungal assemblage of claim 24, wherein the fungus is P2A.

27. The bacterial-fungal assemblage of any one of claims 1 to 8, wherein the bacteria are selected from Gluconacetobacter diazotrophicus BS47, Methylobacterium symbioticum BS 11 and Azospirillum brasilense BS66.

28. A method for improving health of a plant, the method comprising putting the plant in contact with a bacterial -fungal assemblage as defined in any one of claims 1 to 27.

29. The method of claim 28, wherein contact of the bacterial assemblage with the plant improves the solubilization and transfer of residual phosphorus and fixed air-nitrogen to the plant.

30. The method of claim 28, wherein the bacterial assemblage acts as a monocote/non-legume dicote biofertilizer bio-complement.

31. The method of claim 28, wherein the bacterial assemblage acts as a legume fertilizer biocomplement.

32. The method of claim 28, wherein the bacterial assemblage acts as a microbial nematicide.

33. The method of claim 28, wherein the bacterial assemblage acts as a biofungicide.

34. The method of claim 28, wherein the bacterial assemblage acts as a microbial bioremediation.

35. The method of claim 28, wherein the bacterial assemblage acts as a microbial assisted- phytoremediation .

36. The method of claim 28, wherein the bacterial assemblage acts as bio-insecticide.

37. The method of claim 28, wherein the bacterial assemblage improves phosphorus solubilization.

38. The method of claim 28, wherein the bacterial assemblage enhances induced-systemic- resistance (ISR) in plants.

39. A method for improving the efficiency of nitrogen utilization by a plant, the method comprising putting the plant in contact with the bacterial-fungal assemblage as defined in any one of claims 1 to 28.

40. A method for improving the efficiency of nitrogen assimilation by a plant, wherein the bacterium transforms atmospheric nitrogen into inorganic compounds usable by plants or a fungus phosphate phosphorus transporter, the method comprising putting the plant in contact with the bacterial -fungal assemblage as defined in any one of claims 1 to 28.

41. A method for improving the efficiency of phosphorus utilization by a plant, the method comprising putting the plant in contact with the bacterial-fungal assemblage as defined in any one of claims 1 to 28.