Compositions and methods for enhancing plant growth

Abstract

The present technology relates to a method for enhancing seed germination and / or growth of a plant, the method comprising contacting seeds and / or roots of the plant with a starter fertilizer and a composition comprising a fungal endophyte.

Description

COMPOSITIONS AND METHODS FOR ENHANCING PLANT GROWTHTECHNICAL FIELD

[0001] The present technology relates to compositions and methods using them for enhancing plant growth.BACKGROUND

[0002] The study of mutualistic relationships between plants and fungi in natural ecosystems has become a key focus in plant science and agricultural production. Endophytes represent a significant part of the hidden fungal diversity present in nearly all plant species. In particular, grasses have a close, symbiotic relationship with a diverse community of root-associated endophytic fungi.

[0003] Nearly all terrestrial plants are believed to form symbiotic relationships with microorganisms. Plant-friendly fungi, including beneficial endophytes and mycorrhizal fungi, play crucial roles in supporting biomass growth plant biomass by enhancing root development, extracting inorganic nutrients — primarily phosphorus (P) — that would otherwise be inaccessible to plants, and providing protection against pathogens and pests. In return, plants supply these fungi with energy-rich carbohydrates produced through photosynthesis. Such symbiosis promotes plant growth and health, even under challenging conditions such as nutrient-poor soils, stress, and pest pressure.

[0004] There remains a need in agriculture to develop improved ways in which to enhance growth of plants starting as early on in the life cycle as possible.

[0005] Beneficial endophytes are microorganisms that reside on the surface or within plant tissues without harming their hosts. Unlike mycorrhizal fungi, they offer practical and economic advantages, as they can be propagated in axenic culture. With their extensive enzymatic capabilities, fungal endophytes can utilize recalcitrant substrates and transport nutrients throughtheir hyphae. Cultivating plants in controlled symbiosis with endophytic fungi may help reduce the need for fertilizers and pesticides in agriculture.

[0006] Compositions comprising fungal endophytes that have the capacity to efficiently convey soluble phosphate from various inorganic and / or chemical fertilizers to the plant's root system through its hyphal networks after plant root colonization may therefore help address the issue of improving plant growth at the earliest stages of the life cycle.SUMMARY

[0007] From one aspect, there is provided a method for enhancing seed germination and / or growth of a plant, comprising contacting a seed of the plant with a starter fertilizer and a composition comprising a fungal endophyte.

[0008] In certain embodiments, the method accelerates the development and resilience of transplants, promoting faster establishment in soil.

[0009] In certain embodiments, the starter fertilizer is a chemical fertilizer comprising phosphorus and / or and a chemical fertilizer comprising nitrogen.

[0010] In certain embodiments, the chemical fertilizer comprising phosphorus is selected from diammonium phosphate (DAP), mono-calcium phosphate (CP), single superphosphate (SSP), triple superphosphate (TSP), and monoammonium phosphate (MAP).

[0011] In certain embodiments, the chemical fertilizer comprising nitrogen is selected from diammonium phosphate (DAP) and monoammonium phosphate (MAP).

[0012] In certain embodiments, the fungal endophyte is Trametes versicolor and / or Stereum hirsutum.

[0013] In certain embodiments, the composition improves nutrient absorption by promoting root growth and expanding root surface area, leading to higher phosphorus uptake efficiency and improve overall plant health and yield.

[0014] In certain embodiments, the contacting of the seed with the starter fertilizer and the composition comprising the fungal endophyte improves overall biomass growth, yield and drought tolerance of the plant by fostering water retention and root vigor.

[0015] In certain embodiments, the method enhances soil health by reducing the amount of synthetic fertilizer needed.

[0016] In certain embodiments, the contacting application occurs through root-dip inoculation.

[0017] In certain embodiments, the fungal endophyte inoculum is formulated within granular biochar beads.

[0018] In certain embodiments, the method enhances nodule formation in leguminous plants, leading to increased nitrogen fixation and improved overall plant health.

[0019] In certain embodiments, the method / fungal endophyte inoculation increases the concentration of nutrients in the plant selected from phosphorus (P), iron (Fe), sulfur (S), calcium (Ca), copper (Cu), sodium (Na), manganese (Mn), and combinations thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For a better understanding of the present technology, as well as other aspects and further features thereof, reference is made to the following description and drawings, in which:

[0021] Figure 1 shows wheat plant responses to growth in soil with CP supplementation after inoculation with Stereum hirsutum (5 / 2-P2A) and Trametes versicolor (Tv-WCieGW), wherein disinfected seeds were inoculated with a homogenized mycelium solution prepared in 0.1% Gum xanthan and 0.01% Tween™ 20 solution, then inoculated seeds were put in black earth organic soil (Voila®) supplemented with 0.8 g / Kg mono calcium phosphate (CP). (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard error of 7 plants are shown. (C) Roots are shown from fungal treatments groups. (D) Mock-inoculated plant. (E) 5 / 2-inoculated plant. (F) Tv-WCieGW-inoculated plant. Statistical analysis was performed in SPSSusing One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0022] Figure 2 shows wheat plant responses to growth in soil with TSP supplementation after inoculation with Stereum hirsulum-W and Trametes versicolor-WCieGW , wherein disinfected seeds were inoculated with a homogenized mycelium solution that was prepared in 0.1% Gum xanthan and 0.01% Tween™ 20 solution, then inoculated seeds were put in black earth organic soil (Voila®) supplemented with 1.1 g / kg TSP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard error of 7 plants are shown. (C) Roots are shown from fungal treatments groups. (D) Mock-inoculated plant. (E) 5'A-P2A-inoculated plant. (F) Tv-WCieGW-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).

[0023] Figure 3 shows wheat plant responses to growth in soil with SSP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW , wherein disinfected seeds were inoculated with a homogenized mycelium solution that was prepared in 0.1% Gum xanthan and 0.01% Tween™ 20 solution, then inoculated seeds were put in black earth organic soil (Voila®) supplemented with 0.84 g / Kg SSP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard error of 7 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05). (D-F) Fungal colonization of 7v-WC16GW-inoculated roots of wheat plants 5 days after inoculation in black organic soil (Voila®) supplemented with 0.84 g / kg SSP. (G, H, I) Fungal colonization of Sh-P - inoculated roots of wheat plants 5 days after inoculation in black organic soil (Voila®) supplemented with 0.84 g / Kg SSP.

[0024] Figure 4 shows wheat plant responses to growth in soil with MAP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW , wherein disinfected seeds were dressed in a homogenized mycelium solution that was prepared in 0.1% Gum xanthan and 0.01% Tween™ 20 solution, then inoculated seeds were put in black earth organic soil (Voila®) supplemented with 0.8 g / kg MAP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard error of 7 plants are shown. (C) Roots are shown fromfungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA;Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0025] Figure 5 shows wheat plant responses to growth in soil with DAP supplementation after inoculation with S. hirsutum-P . and T. versicolor-WCieGW , wherein wheat seeds were soaked in approximately 0.9 g / ml of crushed mycelium for 2 h and subsequently transferred to heat sterilized soil with 1 g / Kg of DAP. (A) Wheat plants 2 weeks after inoculation. (B) Shoot dry weights, mean values, and standard error of 31 plants are shown. (C) Root dry weights, mean values, and standard error of 31 plants are shown. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0026] Figure 6 shows wheat plant responses to growth in soil with SSP supplementation after inoculation with S. hirsulum-W and T. versicolor-WCieGW , wherein seeds were soaked in 1 g / 1.5 ml of crushed mycelium for 1 h and subsequently transferred to heat sterilized soil with 0.84 g / Kg of SSP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard deviation of 8 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0027] Figure 7 shows wheat plant responses to growth in soil with CP supplementation after inoculation with S. hirsulum-W and T. versicolor-WCieGW , wherein seeds were soaked in 1 g / 1.5 ml of crushed mycelium for 1 h and subsequently transferred to heat sterilized soil with additional 0.84 g / kg of CP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard deviation of 8 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0028] Figure 8 shows wheat plant responses to growth in soil with SSP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW, wherein disinfected seeds were inoculated with 20 pl of a homogenized mycelium solution containing 1 g of mycelium in 2 ml of 0.1% PVP and 0.01% Tween™ 20, then placed in soil supplemented with 0.84 g / kg SSP. (A) Wheat plants 8 days after inoculation. (B) Root and shoot dry weights, mean values, and standarderror of 8 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0029] Figure 9 shows wheat plant responses to growth in soil with CP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW, wherein disinfected seeds were inoculated with 20 pl of a homogenized mycelium solution (1g of mycelium in 2 ml of 0.1% PVP and 0.01% Tween™ 20), then placed in soil supplemented with 0.8 g / kg CP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard deviation of 8 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0030] Figure 10 shows wheat plant responses to growth in soil with TSP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW, wherein disinfected seeds were inoculated with 20 pl of a homogenized mycelium solution (1 g of mycelium in 2 ml of 0.1% PVP and 0.01% Tween™ 20), then placed in soil supplemented with 1.1 g / kg TSP. (A) Wheat plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard deviation of 8 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0031] Figure 11 shows wheat plant responses to growth in soil with DAP supplementation after root-dip inoculation with S. hirsutum-P , wherein 4-day-old wheat seedlings were dip-inoculated with approximately 1 g / ml of crushed mycelium for 2 h, and subsequently transferred to heat sterilized soil supplemented with 1 g / kg of DAP. (A) Wheat plants 2 weeks after inoculation. (B) Root and shoot dry weights, mean values, and standard deviation of 6 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One- Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0032] Figure 12 shows wheat plant responses to growth in soil with CP supplementation after root-dip inoculation with S. hirsutum-P , wherein 4-day-old wheat seedlings were dip-inoculatedwith approximately 1 g / ml of crushed mycelium for 2 h, and subsequently transferred to heat sterilized soil supplemented with 0.5 g / kg of CP. (A) Wheat plants 2 weeks after inoculation. (B) Root and shoot dry weights, mean values, and standard deviation of 6 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One- Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0033] Figure 13 shows wheat plant responses to growth in soil with DAP supplementation after root-dip inoculation with T. versicolor-WCieGW, wherein 4-day-old wheat seedlings were dip- inoculated with approximately 1 g / ml of crushed mycelium for 2 h, and subsequently transferred to heat sterilized soil supplemented with 1 g / kg of DAP. (A) Wheat plants 16 days after inoculation; (B) Root and shoot dry weights, mean values, and standard error of 7 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA.

[0034] Figure 14 shows the fungal colonization of wheat plant roots grown in soil with DAP supplementation after root-dip inoculation with T. versicolor-WCieGW or a mock-inoculated control, using WGA staining to visualize fungal hyphae. (A) Root of mock-inoculated control wheat plant, root cap zone (rc) of wheat showed autofluorescence signal but free of fungus; and meristem zone (me), elongation zone (el) and maturation zone (ma) are free of hyphae. (B) Root of Tv-WCieGW-inoculated wheat plant, root cap zone (rc), meristem zone (me), elongation zone (el) are free of hyphae, except maturation zone (ma) which are heavily colonized by fungus mycelium. Roots were analyzed by fluorescence microscopy.

[0035] Figure 15 shows soybean plant responses after inoculation with S. hirsutum-P and T. versicolor-WCieGW when grown in soil without additional fertilizers, wherein disinfected seeds were inoculated with a homogenized mycelium solution prepared in 0.1% Gum xanthan and 0.05% Tween™ 20 solution, after which inoculated seeds were placed in black organic earth soil (Voila®) without additional fertilizer. (A) Soybean plants 15 days after inoculation. (B) Root and shoot dry weights, mean values, and standard error of 8 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0036] Figure 16 shows soybean plant responses to growth in soil with TSP supplementation after inoculation with Stereum sp. and Trametes sp., wherein disinfected seeds were inoculated with a homogenized mycelium solution prepared in 0.1% Gum xanthan, 0.1% PVP and 0.01% Tween™ 20 solution, after which inoculated seeds were placed in black organic earth soil (Voila®) supplemented with 0.8 g / kg TSP. (A) Soybean plants 15 days after inoculation; (B) root and shoot dry weights, mean values, and standard error of 7 plants were shown. (C) Roots are shown from fungal treatments groups (D) Statistical analysis of number of nodules, mean values, and standard error of 8 plants are shown. (E) Nodules are shown on roots from the fungal treatment groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P = 0.05).

[0037] Figure 17 shows soybean plant responses to growth in soil with MAP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW , wherein disinfected seeds were inoculated with a homogenized mycelium solution prepared in 0.1% PVP and 0.01% Tween™-20 solution, after which seeds were placed in black earth organic soil (Voila®) + Oil Dri® (Oil-Dri, Canada) with 0.8 g / kg MAP. Soybean plants 32 days after inoculation. (B) Statistical analysis of root and shoot dry weights, mean values, and standard error of 6 plants are shown. (C) Roots are shown from fungal treatments groups. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at top of the bars indicate statistically significant differences (P < 0.05).

[0038] Figure 18 shows soybean plant responses to growth in soil with MAP supplementation after inoculation with S. hirsutum-P and T. versicolor-WCieGW , wherein disinfected seeds were inoculated with a homogenized mycelium solution prepared using 0.1% PVP and 0.01% Tween™ 20 solution, after which seeds were placed in black earth organic soil (Voila®) supplemented with 0.8 g / kg MAP. (A) Statistical analysis of number of nodules, mean values, and standard error of 8 plants are shown. (B) Nodules are shown on roots from fungal inoculated plants. Statistical analysis was performed in SPSS using One-Way ANOVA; Stars at the top of the bars indicate statistically significant differences (P < 0.05).

[0039] Figure 19 shows wheat plant responses to inoculation with Trametes versicolor WCieGW, Stereum hirsutum P2A and Serendipita indica BS22 via mycelium biochar beads or via root-dip inoculation, wherein 4-day-old wheat seedlings were either inoculated with mycelium biocharbeads (1 ml / g crushed mycelium / beads; 2 beads per plant) or mycelium dip-inoculated with approximately 1 g / ml of crushed mycelium (2 h), and subsequently transferred to heat sterilized soil. (A) Wheat plants 4 weeks after inoculation. (B) Root and shoot dry weights, mean values and standard deviation of 12 plants are shown. (C) Roots are shown from fungal treatments groups. 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

[0040] The present technology is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology may be implemented, or all the features that may be added to the instant technology. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which variations and additions do not depart from the present technology. Hence, the following description is intended to illustrate some particular embodiments of the technology, and not to exhaustively specify all permutations, combinations and variations thereof.

[0041] As used herein, the singular form “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0042] The recitation herein of numerical ranges by endpoints is intended to include all numbers subsumed within that range (e.g., a recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0043] The term “about” is used herein explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and / or measurement conditions for suchgiven value. For example, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0044] The expression “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0045] As used herein, the term “comprise” is used in its non-limiting sense to mean that items following the word “comprise” are included, but items not specifically mentioned are not excluded.

[0046] For the purposes of the present application, the term “endophyte” is used herein to refer to any microorganism that lives within plant tissues, regardless of its specific function in relation to the plant.

[0047] The term “starter fertilizer” is used to describe a small, concentrated amount of essential nutrient(s) that may be applied near plant seeds or seedlings during planting or transplanting. Soluble phosphate and soluble nitrogen provides a readily available source of phosphorus and nitrogen, respectively, both of which are critical nutrients that support rapid plant growth, stronger root development, and enhanced flower production. Fertilizers comprising soluble nitrogen and / or soluble phosphate may serve as effective starter fertilizers for plants.

[0048] For the purposes of the present application, the terms “phosphorus / P” as used herein includes any compound that contains phosphorus, including but not limited to compounds containing “phosphate / P2O5” .

[0049] For the purposes of the present application, the term “inorganic fertilizer” is used herein to refer to fertilizers comprising nutrient components that are obtained from rock and / or minerals, which are formed by physical and / or chemical processes (such as, but not limited to, rockphosphate). For the purposes of the present application, the term “synthetic fertilizer” or “chemical fertilizer”, is used herein to refer to fertilizers comprising concentrated nutrient components in specific ratios, which are manufactured through chemical processes.

[0050] Methods of Application of Starter Fertilizer to Soil. The starter fertilizer(s) of the present technology may be applied to the soil in various ways, including by: Uniform mixing, whereby the starter fertilizer is evenly distributed throughout the soil in order to ensure consistent nutrient availability, resulting in what is referred to herein as “uniformly fertilized soil”; Targeted placement or “in-furrow application”, wherein the fertilizer is placed from between about 2 cm and about 30 cm below the seed bed (in the furrow), in order to optimize nutrient uptake during early plant growth.

[0051] Methods of Application of Fungal Endophytes to Seeds and / or Plants. The fungal endophyte(s) of the present technology are used as a “fungal inoculant” / “fungal inoculum” for application to seeds and or plants, which may be applied to said seeds and / or plants in a variety of ways, such as, but not limited to: by “Root-Dip inoculation” of seedlings, wherein the seedling roots are dipped in a fungal inoculum solution and then planted in uniformly fertilized soil; by “Seed inoculation” of plant seeds, wherein seeds are initially inoculated with a fungal inoculum, and subsequently sown into soil that is either uniformly fertilized soil, or soil that has a targeted placement of fertilizer (i.e. the fertilizer is applied “in-furrow”); by “granular application”, wherein Biochar beads / granules comprising the fungal inoculant are placed near the roots of seedlings in uniformly fertilized soil.

[0052] Formulations. The fungal inoculum comprising the fungal endophyte(s) used to inoculate plant seeds and / or the roots of seedlings, may be formulated in a variety of ways, such as but not limited to: As a liquid; in freeze-dried form; and in granule or bead form. Different formulations of fungal inoculum may be contemplated for their specific advantages and properties. For example, a freeze-dried formulation may preserve fungal viability and extend shelf life. A biochar-based granular formulation may be designed for controlled and sustained release.

[0053] A non-limiting example of a liquid formulation of fungal inoculum that may be used with the present technology is prepared from crushed fungal mycelium, incorporating 0.1% xanthan gum, 0.1% polyvinylpyrrolidone (PVP), and 0.01% Tween™ 20, to enhance consistency and application.

[0054] Trametes versicolor WCieGW is an endophytic Basidiomycetes phosphorus-transporting fungus of the order Polyporales. This fungus was initially isolated in Giessen, Germany (see 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).

[0055] Stereum hirsutum P2A is an endophytic Basidiomycetes phosphorus-transporting fungus of the order Russulales. This fungus was initially isolated in Giessen, Germany (see Table 1). The DNA sequence of the 18S and ITS and including 5.8S ribosomal RNA, are shown in Table 2 (SEQ ID NO:4).Table 1: Fungal Isolate information.Table 2: DNA sequences from ribosomal RNA genes of Fungal Isolates.

[0056] In some aspects, the present technology may improve seed germination and / or plant growth in com, wheat, canola, soybean, potato, and vegetables, by applying fungal endophytes to the seed and / or plant roots: prior to planting in soil containing starter fertilizer, or simultaneously at the time of planting in conjunction with starter fertilizer. The starter fertilizer may be applied to the soil and mixed uniformly throughout, referred to as “uniformly fertilized soil”, or the starter fertilizer may be applied to the soil “in furrow”, fertilizing the soil within the furrow (corn, wheat, canola, soybean, potato, vegetable).

[0057] In some aspects, the present technology may provide for a liquid formulation comprising a fungal endophyte and a precisely calibrated level of soluble phosphate for seed and / or seedling inoculation (of cereals, vegetables or legumes).

[0058] In some aspects, the present technology may enhance the development of transplants and / or may improve nutrient absorption in a hydroponic system with a significant concentration of inorganic fertilizer.

[0059] In some aspects, the present technology may simultaneously incorporate fungal endophytes in a granular form alongside chemical fertilizer(s) comprising phosphorus and / or nitrogen. Such an application may be useful within the agricultural field.

[0060] In some aspects, the present technology may reduce the level of applied commercial P needed by about 15% to 30%, through increasing the efficiency of P uptake, which promotes fasterroot development (and thus resilience to abiotic stresses), and provides higher yields as a result of more P being absorbed by the plant.EXAMPLES

[0061] Enhancement of wheat seed and seedling growth through the activity of two endophytic Basidiomycetes fungi: Stereum hirsutum (Sh-P2A) and Trametes versicolor (Tv-WCieGW), in soils enriched with different types of starter fertilizers comprising phosphorus: Diammonium phosphate (DAP), Mono-calcium phosphate (CP), Single superphosphate (SSP), Triple superphosphate (TSP), or Monoammonium phosphate (MAP).

[0062] Application Techniques: Seed inoculation, in-furrow treatment and root-dip inoculation.

[0063] The enhancement of wheat growth was assessed following the application of strains of basidiomycetes endophytes, Trametes versicolor (Tv-WCieGW) and Stereum hirsutum (5 / 2-P2A) to wheat seedlings by root-dip inoculation, to wheat seeds by seed inoculation. The evaluation was conducted across various type of phosphate within the soil enriched with 0.9 g / Kg1of P2O5, as diammonium phosphate (DAP), mono-calcium phosphate (CP), single superphosphate (SSP), triple superphosphate (TSP), and monoammonium phosphate (MAP). The mycelium inoculum was applied by, the root-dip inoculation technique, at a concentration of 1 g / ml; In contrast, the seed inoculation technique employed the lowest concentration (0.1 g / ml) of mycelium, involving 1.36 kg of dry weight mycelium (equivalent to 3 kg of wet weight) for the inoculation of 80 kg of spring wheat seeds per acre.

[0064] In soil supplemented with all four varieties of soluble phosphate; DAP, SSP, TSP and MAP, the application of either one or both fungal strains, using at least one of the application methods, led to a significant increase in growth of either the plant roots or the shoots.

[0065] For root-dip inoculation, both Tv-WCieGW and Sh-P2A demonstrated a significant enhancement of root dry weight when applied in DAP-enriched soil. For seed inoculation, Tv- WCieGW exhibited a significant impact on root growth in SSP-enriched soil and on shoot growth in CP-enriched soil, and both Tv-WCieGW and 5 / 2-P2A significantly improved root dry weight inMAP-enriched soil. Notably, the most significant increase in root growth was achieved through the in-furrow application of 5 / 2-P2A in SSP- and CP-enriched soil, respectively.

[0066] Fungal growth conditions: T. versicolor WCieGW and S. hirsutum-P^ were obtained from Giessen University, Germany. Fungi were grown in MYP broth (Malt extract 10.0 g; Yeast extract 2.0 g; Proteose peptone 1.5 g; Sucrose 10.0 g; Glucose 5.0 g; KC1 0.5 g; ISfeHPC 0.5 g; KH2PO4 0.5g; K2HPO40.5 g; pH: 7.24) for three weeks at 28°C on a MaxQ™ 8000 (Japan) shaker in the dark.

[0067] Germination of seeds: Seeds 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 three times for 5 min.

[0068] Plant growth conditions: Wheat seeds were grown in small pots containing 2: 1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada). A Jiffy 4004781 Hydro Grow light was used for a 16 h photoperiod, at 22 / 22°C day / night.

[0069] Seed inoculation method: Seeds 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 three times for 5 min. 110 disinfected seeds were inoculated with 3 ml of a homogenized mycelium solution that was prepared by shaking 1 g of mycelium in 1.5 ml or 4 g of mycelium in 50 ml of 0.1% Gum xanthan / 0.1% PVP and 0.01% Tween™ 20 solution for 2 h (For the DAP experiments - 1 g of mycelium in 1 ml of 5% com syrup (Crown®) solution for 2 h). Then the inoculated seeds were put in the pots containing heat sterilized (at 121 °C for 5 min at 15 psi) soil with the soil components previously described above. A standard application rate of 0.9 g / kg of P2O5 was established for each individual fertilizer comprising phosphate. Two seeds were included in each plastic pot (7 cm opening diameter, 5 cm bottom diameter and 8 cm height). The pots were watered daily with 30 mL sterile pure water. Plants were harvested 15 to 30 days after planting. Roots (dry weight) and shoots (dry weight) were subsequently measured by weighting plant samples.

[0070] In-furrow treatment: Seeds 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 three times for 5 min. Disinfected seeds were inoculated with 20 pl of a homogenized mycelium solution (preparedby shaking 1 g of mycelium in 2 ml of 0.1% PVP and 0.01% Tween™ 20 solution). Then inoculated seeds were transferred to the pots containing heat sterilized (at 121 °C for 5 min at 15 psi) soil with the soil components previously described above. A standard application rate of 0.9 g / kg of P2O5 was established for each individual chemical fertilizer. Two seeds were included in each plastic pot (7 cm opening diameter, 5 cm bottom diameter and 8 cm height). The pots were watered daily with 30 mL sterile pure water. Plants were harvested 15-30 days after planting. Roots (dry weight) and shoots (dry weight) were subsequently measured by weighting plant samples.

[0071] Root-dip inoculation: Roots of 4-day old wheat seedlings were inoculated by dipping them in a homogenized mycelium solution that was prepared by shaking 15 g of mycelium in 15 ml 0.01% Tween™ 20 solution and incubated for 2 h. Inoculated seedlings were transferred to pots containing heat sterilized (at 121 °C for 5 min at 15 psi) soil with the soil components previously described above, which were supplemented with 1 g / kg of Diammonium phosphate. Subsequently six seedlings were planted in six pots (one seedling in each plastic pot of 7 cm opening diameter, 5 cm bottom diameter and 8 cm height). The potted seedlings were watered daily with 40 mL sterile pure water. Plants were harvested 15 days after planting. Roots (dry weight) and shoots (dry weight) were subsequently measured by weighting plant samples.

[0072] Granule inoculation'. Fungal crushed mycelium was prepared in a 1 : 1 weight / volume ratio of 0.1% PVP and 0.01% Tween™ 20 solution. Subsequently, 10 ml of the homogenized mycelium solution was combined with 10 g of beads and allowed to mix overnight. 10 g of beads mixed with 0.1% PVP and 0.01% Tween™ 20 solution was used as a control. To inoculate the seedlings, two fungal -formulated biochar-beads each of Tv-WCieGW, Sh-P A, and Serendipita indica B S22 were placed near the wheat (4-day old) seedling roots during planting. Additionally, two un-inoculated beads were placed near the roots as a control.

[0073] To compare with the root-dip inoculation method, roots of 4-day old wheat seedlings were inoculated in a homogenized mycelium solution that was prepared by shaking 10 g of mycelium in 10 ml 0.1% PVP and 0.01% Tween™ 20 solution and incubated for 2 h. Inoculated seedlings were transferred to pots containing heat sterilized (at 121 °C for 5 min at 15 psi) soil with the soil components previously described above. Subsequently, 12 seedlings were planted in six pots (two seedling in each plastic pot of 10 cm opening diameter, 5.8 cm bottom diameter, and 9 cm height).The potted seedlings were watered daily with 40 mL sterile pure water. Plants were harvested 27 days after planting. Root (dry weight) and shoot (dry weight) were subsequently measured by weighting plant samples.

[0074] 3L pots greenhouse experiment: Seeds 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 three times for 5 min. First inoculation: 100 disinfected seeds were inoculated in a homogenized mycelium solution that was prepared by shaking 11 to 15g of mycelium in 10 ml of 0.1% PVP and 0.01% Tween™ 20, then 5 to 7 ml of the inoculum were mixed with 100 seeds for 3 h. Inoculated seeds were transferred to pots containing non-sterilized soil with the soil components containing 4:2:2:4:2 of clay (Liaflor®), potting soil, black earth organic soil (Voila®), perlite, and Oil Dri® (Oil-Dri, Canada). A standard application rate of 0.9 g / kg of P2O5 was established for MAP and DAP. Subsequently, seeds were planted in 50 pots (with ten seeds in each plastic 3L pot of 19 cm opening diameter, 14 cm bottom diameter, and 15 cm height). The second inoculation was performed one month after planting. Accordingly, 1 ml of homogenized mycelium solution, which was prepared by shaking 23 g of mycelium in 11 ml of 0.1% PVP and 0.01% Tween™ 20, was added to each plant. The pots were watered daily with 100 mL tap water. Plants were harvested 43 days after planting. The number of tillers per plant, number of headings per plant, heading weight per treatment, and average 1000 grain weight (“TGW”) were subsequently measured.

[0075] Statistical analysis: Data were first 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). A Fishers protected LSD was subsequently performed for separation of means.

[0076] Microscopically observation of the fungal root colonization: Hyphae in inoculated wheat root segments were stained with the chitin-specific binding wheat germ agglutinin (WGA), which is a specific plant lectin that strongly binds to chitin, conjugated with Alexa Flour® 647 (Thermo Fisher Scientific, USA). Root material was fixed and stored in a solution containing 20% (v / v) chloroform, 80% (v / v) ethanol, and 0.15% (w / v) trichloroacetic acid for days. Before staining, fixed roots were washed three times for 5 min with deionized water, then treated with 10% (w / v) potassium hydroxide by boiling for 30 sec, and then rinsed three times for 5 min with phosphate buffered saline (PBS: KC1, 0.2 g; KH2PO4, 0.2 g; Na2HPO4, 1.15; H2O, 1 1; pH 7.4). The rootfragments were then incubated in 5 ml PBS containing 10 pg WGA Alexa Flour® 647 ml’1and 0.02% (v / v) Silwet L-77. During the incubation, roots were stained by vacuum infiltration three times for 1 min and kept in staining solution for 10 min. After washing with 1 x PBS buffer, roots were incubated in 20 pM propidium iodide solution (Sigma- Aldrich, USA) in PBS for 10 min; then roots were analyzed by confocal laser scanning microscope Leica TCS SP8).Example 1 - Seed inoculation - Stereum hirsutum P2A and Trametes versicolor WCieGW show beneficial activity in spring wheat by seed inoculation in soil fertilized with monoammonium phosphate (MAP), mono-calcium phosphate (CP), and triple super phosphate (TSP).

[0077] To assess the improvement on growth promotion activity of S. hirsutumAp.S and T. versicolor-WCieGW in the presence of chemical fertilizers comprising P, disinfected seeds were inoculated with 3 ml of a homogenized mycelium solution that was prepared by shaking 4 g of mycelium in 50 ml of 0.1% Gum xanthan and 0.01% Tween™ 20 solution and were planted in the pots containing heat sterilized (at 121 °C for 5 min at 15 psi) fertilized-soil. In the MAP- and CP- fertilized soil, after 15 days, Tv-WCieGW-inoculated plants showed an overall increase in root dry weight (Fig. 4 & 1) and shoot dry weight by 5A-P2A-inoculated plant in TSP-fertilized soil (Fig. 2). There was no noticeable increase in overall biomass growth in SSP-fertilized soil by both fungi (Fig. 3). Root and shoot dry weights were elevated 34% and 15%, respectively, in T. versicolor- WCieGW inoculated plants in the MAP-fertilized soil as compared to mock-inoculated plants (Fig. 4). Root dry weights were significantly elevated by 19% in the CP-fertilized soil in T. versicolor- WCieGW inoculated plants (Fig. 1).Example 2 - Seed inoculation - Stereum hirsulum-WA and Trametes versicolor WCieGW show beneficial activity in spring wheat by seed inoculation method in soil fertilized with diammonium phosphate (DAP), mono-calcium phosphate (CP) and single super phosphate (SSP)

[0078] To assess the phosphorus mobilization activity of 5 / 2-P2A and Tv-WCieGW, seeds were soaked in the fungi inoculum for 1 h to 2 h in different experiments. In DAP- and CP- fertilized soil, after 15 days, plants inoculated with 5 / 2-P2A showed an overall increase in biomass growth (Fig. 5 and 7) and Tv-WCieGW-inoculated plants in SSP - and CP-fertilized soil (Fig. 6 and 7). Root and shoot dry weights were elevated 14% in 5 / 2-P2A inoculated plants in the DAP -fertilized soil as compared to mock-inoculated plants (Fig. 5B). Root dry weights were significantly elevated(30% and 23%, respectively) in the SSP - and CP-fertilized soil as compared to mock-inoculated plants (Fig. 6B and 7B). Visual analysis of the roots also confirmed an increase in biomass upon fungal colonization in DAP, SSP and CP (Fig. 5C and 6C and 7C).Example 3 - In- furrow treatment - Stereum hirsutum P2A and Trametes versicolor WCieGW show beneficial activity in spring wheat by seed inoculation method in soil fertilized with mono-calcium phosphate (CP), single super phosphate (SSP) and triple super phosphate (TSP).To assess the phosphorus mobilization activity of 5 / 2-P2A and Zv-WCieGW, seeds were inoculated in the fungi inoculum for 2 h in different experiments. Then seeds were planted 5cm above a layer of starter fertilizer in the furrow. The significant increase in root and should growth be achieved through 5 / 2-P2A inoculated seeds through the in-furrow application in SSP-, TSP- and CP-enriched soil, respectively (Fig. 8 - 10). Root and shoot dry weights were elevated 21% and 76% in 5 / 2-P2A inoculated plants in the SSP-fertilized soil as compared to mock-inoculated plants (Fig. 8B). Root and shoot dry weights were significantly elevated (14% and 47%, respectively) in the CP- and (32% and 37%) in TSP-fertilized soil as compared to mock-inoculated plants (Fig. 9B and 10B). Visual analysis of the roots also confirmed an increase in biomass growth upon fungal colonization (Fig. 8C, 9C and 10C).Example 4 - Root dip-inoculation method - S. hirsutum-V^R shows beneficial activity in spring wheat in soil fertilized with diammonium phosphate (DAP) and mono-calcium phosphate (CP).

[0079] To assess the phosphorus mobilization activity of 5 / 2-P2A, the roots of 4-day-old wheat seedlings were root dip-inoculated in crushed mycelia from three-week-old liquid cultures. In DAP- and CP- fertilized soil, after 15 days, plants inoculated with 5 / 2-P2A showed an increase in biomass growth (Fig. 11A and 12A). Root and shoot dry weights were elevated 31% and 20%, respectively, in the DAP-fertilized soil as compared to mock-inoculated plants (Fig. 1 IB). Root and shoot dry weights were significantly elevated in 5 / 2-P2A inoculated plants (by 20% and 23%, respectively) in the CP-fertilized soil as compared to mock-inoculated plants (Fig. 12B). Visualanalysis of the roots also confirmed an increase in biomass growth upon Sh- P2A fungal colonization in DAP and CP fertilized soil (Fig. 11C and 12C),Example 5 - T. versicolor-XNCisGXN shows beneficial activity in spring wheat in soil fertilized with diammonium phosphate (DAP).

[0080] To assess the phosphorus mobilization activity of Tv-WCieGW, 4-day-old wheat seedlings were dip-inoculated in crushed mycelia from three-week-old liquid cultures. In the DAP-fertilized soil after 15 days, plants inoculated with Tv-WCieGW showed an overall increase in biomass growth (Fig. 13 A). Both root and shoot dry weights were elevated 27% in the DAP -fertilized soil compared to mock-inoculated plants (Fig. 13B). Visual analysis of the roots also confirmed an increase in biomass upon fungal colonization (Fig. 13C). To trace the endophytic growth of the fungal strains in wheat, root colonization patterns were analyzed by staining with the chitinspecific dye Wheat Germ Agglutinin (WGA)-Alexa Fluor 647. The visualization of hyphae by fluorescent microscopy revealed that the T. versicolor-WCieGW strain endophy tically colonize wheat roots. Heavy fungal colonization of the root cap and meristem zone with an extracellular and intracellular network of Tv-WCieGW hyphae is visible (Fig. 14B). Moreover, the root maturation zone was colonized with extra- and intraradical hyphae (Fig. 14B), while noninoculated roots were free of fungal hyphae (Fig. 14A).

[0081] Soybeans

[0082] Enhancement of soybean (OAC Bruton) seedling nodulation through the activity of the endophytic Basidiomycetes Fungi (SI1-P2A) and Trametes versicolor (Tv-WCieGW), in soils enriched with different types of starter fertilizers comprising phosphorus. Types of starter fertilizers utilized: Single superphosphate (SSP), Triple superphosphate (TSP). Application Techniques Employed: Seed inoculation.

[0083] The enhancement of soybean growth using two strains of basidiomycetes endophytes, Trametes versicolor ( / V-WCieGW) and Stereum hirsutum (5 / 2-P2A), was assessed following seeding. The evaluation was conducted across soils that were enriched with 0.9- g / Kg1of P2O5 assingle superphosphate (SSP), triple superphosphate (TSP), and a control (without additional phosphorus). In the seed inoculation method, a concentration of 1.36 kilograms of dry weight (equivalent to 3 kilograms of wet weight) of mycelium was used, which could be generalized for the inoculation of 32k of soybean seeds per acre. Every experimental treatment was individually conducted using four plastic pots, and within each pot, two plants were placed for 15 days. In soil supplemented with SSP and TSP, neither the application of a single Tv / Sh, nor of both Tv+Sh via the seed inoculation method, led to a significant increase in growth of either the roots or the shoots. However, notably, in soil that was enriched with TSP, there was a significant increase in the number of nodules on the plants that were inoculated with Tv+Sh. However, both Tv and Sh significantly improved root dry weight in the control soil without additional phosphorus.

[0084] Fungal growth conditions'. T. versicolor-WCvsGW and S. hirsulum-W.S were obtained from Giessen University, Germany. Fungi were grown in MYP broth (Malt extract 10.0 g; Yeast extract 2.0 g; Proteose peptone 1.5 g; Sucrose 10.0 g; Glucose 5.0 g; KC1 0.5 g; Na2HPO4 0.5 g; KH2PO4 0.5 g; K2HPO40.5 g; pH: 7.24) for three weeks at 28°C on a MaxQ™ 8000 (Japan) shaker in the dark.

[0085] Germination of seeds'. Soybean seeds, OAC Bruton (seed size: 3900 seeds / kg; germination: 92%) were sterilized with 1% (v / v) NaOCl for 6 min, then rinsed with sterilized water three times for 3 min.

[0086] Plant growth conditions’. Soybean seeds , OAC Bruton, were grown in small pots containing 2: 1 black earth organic soil (Voila®) and Oil Dri® (Oil-Dri, Canada). A Jiffy 4004781 Hydro Grow light was used for a 16 h photoperiod, at 22 / 22°C day / night.

[0087] Seed inoculation method: 17 disinfected soybean seeds were inoculated with 2 ml of a homogenized mycelium solution that was prepared by shaking 3.4 g of mycelium of single or 1.7 g of each fungus in mixture (f Tv+Sh”) in 50 ml of 0.1% Gum xanthan and 0.01% Tween™ 20 solution. The inoculated seeds were then transferred to pots containing heat sterilized (at 121°C for 5 min at 15 psi) soil having the soil components previously described above. A standard application rate of 0.9 g / kg of P2O5 was established for each individual fertilizer. Two seeds wereincluded in each plastic pot (7 cm opening diameter, 5 cm bottom diameter and 8 cm height). The pots were watered daily with 30 mL sterile pure water. Plants were harvested 15 days after planting. Roots (dry weight) and shoots (dry weight) were subsequently measured by weighting plant samples.

[0088] Statistical analysis: Data were first 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). Fishers protected LSD was subsequently performed for separation of means.

[0089] Microscopically observation of the fungal root colonization: Hyphae in inoculated soybean root segments were stained with the chitin-specific binding wheat germ agglutinin (WGA, is a specific plant lectin that strongly binds to chitin) conjugated with Alexa Flour® 647 (Thermo Fisher Scientific, US). Root material was fixed and stored in a solution containing 20% (v / v) chloroform 80% (v / v) ethanol and 0.15% (w / v) trichloroacetic acid for days. Before staining fixed roots were washed three times for 5 min with deionized water, then treated with 10% (w / v) potassium hydroxide by boiling for 30 sec, and then rinsed 3 times for 5 min with phosphate buffered saline (PBS: KC1, 0.2 g; KH2PO4, 0.2 g; Na2HPO4, 1.15; H2O, 1 1; pH 7.4). Then the root fragments were incubated in 5 ml PBS containing 10 pg WGA Alexa Flour 647 mF1and 0.02% (v / v) Silwet L-77. During the incubation, the roots were stained using vacuum infiltration three times for one min and kept in staining solution for 10 min. After washing with 1 x PBS buffer, the roots were incubated in 20 pM propidium iodide solution (Sigma-Aldrich, USA) in PBS for 10 min; then roots were analysed by confocal laser scanning microscope Leica TCS SP8).Example 6 - Stereum hirsulumdp.S and Trametes versicolor-WC sGW show beneficial activity in soybeans using the seed inoculation method in soil fertilized with triple super phosphate (TSP).

[0090] The improvement of growth promotion activity of soybeans by S. hirsulum-M\ and / or T. versicolor-WCieGW used in the presence of chemical fertilizers comprising phosphorus, and without additional fertilizer was examined. Inoculated seeds were transferred to pots containing heat sterilized soil. In TSP- fertilized soil, after 15 days, plants inoculated with both “Tv+Sh” showed an overall increase in the number of nodules (Fig. 16D-E). There was no noticeableincrease in biomass growth in TSP- fertilized soil by either fungi (Fig. 16). Nevertheless, root dry weights were significantly elevated by 75% and 84%, respectively, in the Tv- and 571-inoculated plants as compared to mock-inoculated plants in soil without additional fertilizer (Fig. 15B). Visual analysis of the roots also confirmed an increase in biomass growth upon fungal colonization (Fig. 15C and 16C).Example 7 - Enhancement of soybean (OAC Bruton) seedling nodulation through the activity of Stereum hirsutum-P 2A and Trametes versicolor-WCjgGW in soil enriched with mono-ammonium phosphate (MAP) as starter fertilizer.

[0091] To assess the increased number of nodules of soybean roots after inoculation with S. hirsulum-WA and / or T. versicolor-WCvsGW in the presence of chemical fertilizers comprising phosphorus, inoculated seeds were transferred to pots containing heat-sterilized soil. In MAP- fertilized soil, after 32 days the plants inoculated with both Tv-WCieGW and 5A-P2A showed an overall increase in the number of nodules (Fig. 18D-E). However there was no noticeable increase in biomass growth in the plants inoculated with both Tv-WCieGW and 5A-P2A in the MAP- fertilized soil (Fig. 17). The number of nodules was significantly elevated in the Tv-WCieGW inoculated plants compared to the mock-inoculated control, representing twice the count observed in the control group (Fig. 18).Example 8 - Wheat P content under MAP or SSP soil phosphate treatment and / V-WCieGW and Sh-WA inoculation using seed inoculation method.Method for phosphorus and microelements AnalysisThe plant samples were pooled, with shoots and roots separated for analysis. The separated samples were dried, ground, and digested using either nitric acid (HNO3) or a nitric acid-perchloric acid (HNO3-HCIO4) mixture. The concentrations of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were determined in the digested solutions using Microwave-Induced Plasma Atomic Emission Spectrometry (MIP-AES).Method for Nitrogen AnalysisThis method determines total nitrogen (%N) in soil, sediment, plant, or biological samples using flash combustion. A weighed sample is placed in a tin or silver capsule and introduced into a high- temperature combustion chamber (1020°C to 1800-2000°C). Combustion occurs in the presence of chromium (III) oxide and silver-cobalt oxide catalysts, with purified oxygen added to enhance the reaction.During combustion:• Carbon (C) converts to carbon dioxide (CO2).• Nitrogen (N) converts to nitrogen gas (N2) and nitrogen oxides (NOx).The gases are carried through a reduction furnace with helium, where NOx is reduced to N2 by copper wires. Water vapor is removed using magnesium perchlorate, and CO2 is optionally trapped using EMASorb if only N2 analysis is required.The remaining N2 and / or CO2 are separated via chromatographic columns and quantified using a Thermal Conductivity Detector (TCD). The TCD signal, combined with the sample weight, calculates the nitrogen percentage in the material.

[0092] To evaluate the effect of two different fungal endophyte in soil supplemented with MAP or SSP phosphate fertilizer during wheat-fungi interaction on plant growth parameters, the total phosphorus content of the shoot was determined using acid digests containing HNO3 or HNO3 / HCI. The significant increase in wheat seedling root growth observed 15 days after Tv- WCieGW- and 5A-P2A-seed inoculation aligns with the rise in shoot phosphorus (P) content under SSP- or MAP-enriched soil. Furthermore, root inoculation increased the concentration of several microelements, including iron (Fe), sulfur (S), calcium (Ca), copper (Cu), sodium (Na), and manganese (Mn) (see Table 3, in mg / kg), in wheat shoot tissues 15 days post-inoculation.Table 3: Effect of Stereum hirsulum- ^ and Trametes versicolor- Ci6G seed inoculation of wheat on wheat shoots’ phosphorus (P), iron (Fe), sulfur (S), calcium (Ca), copper (Cu), sodium (Na), and manganese (Mn) content (mg / kg), when grown in soil supplemented with MAP or SSP. The concentration of applied inoculum corresponds to an inoculation rate of 80,000 seeds per acre. Each sample represents a composite of eight plant shoot tissues.Example 9 - Biochar bead formulation (granules) of Trametes versicolor WCieGW, Stereum hirsutum P2A, and Serendipita indica BS22 show beneficial activity in spring wheat as compared to the root-dip inoculation method.

[0093] To assess the efficacy of a mycelium biochar-beads formulation of each Tv-WCieGW, Sh- P2A, or S. indica BS22, 4-day-old wheat seedlings were planted with two mycelium biochar-beads around the rhizosphere or dip-inoculated. After 27 days, plants inoculated with the biochar beads formulation of 7'v-WCieGW, 5 / 2-P2A, and S. indica BS22 showed an overall increase in biomass (Fig. 19A). Shoot dry weights were elevated significantly in all three bead-formulated strain treatments and in 7V-dip inoculated plants compared to mock-inoculated plants (Fig. 19B). Moreover, root dry weights were significantly increased in 571-beads and Tv-inoculated plants (Fig. 19B). Visual analysis of the roots also confirmed an increase in biomass upon fungal colonization (Fig. 19C).Example 10: Effects of Trametes versicolor WCieGW and S. hirsutum P2A on yield parameters under MAP and DAP phosphate regimes.

[0094] Co-application of P (in the form of MAP or DAP) and Tv- WCieGW significantly increased the average of TGW compared to the mock inoculated plants (Table 4). Plants inoculated with Sh- P2A showed higher numbers of tillers per plant in the MAP treatment group. The number of tillers per plant was increased by 17.3% (Sh + MAP) and the average TGW was increased by 10.0% (Tv + MAP) (See Table 4).Table 4: Effect of Trametes versicolor WCGGW and Stereum hirsutum P2A inoculation on growth and yield parameters in wheat using two different types of soluble phosphate fertilizers (MAP or DAP). Values shown are the means of ten pots (with 5 plants per pot). *Statistically significant differences between colonized plants and control.Example 11 - Soybean N & P content without soil additional phosphate treatment and Tv- WCieGW and Sh-p.\ inoculation using seed inoculation method.To evaluate the effect of two different fungal endophytes in soil without additional phosphate fertilizer during soybean-fungi interaction on plant growth parameters, the total phosphorus content of the shoot was determined with acid digests containing HNO3 or HNO3 / HCI and the total nitrogen content by % CNH / O dry combustion method . The increase in the number of nodulations observed after Tv-WCieGW- and 5'A-P2A-seed inoculation aligns with the rise in root and shoot nitrogen (N) and phosphorus (P) content under no additional P fertilizer in the soil.Table 5: Effect of Stereum hirsutum-P^ and Trametes versicolor-^ CI6GW seed inoculation on soybean root shoot phosphorus (P) and nitrogen (N) contents (mg / kg) in soil without additional phosphate fertilizer. Each sample represents a composite of eight plant shoot tissues.

[0095] The above description is given by way of example, and not limitation. Given the above disclosure, one skilled in the art could devise variations that are within the scope of technology disclosed herein. Further, the various features of the embodiments disclosed herein may be used alone or in varying combinations with each other, and are not intended to be limited to the specific combination(s) described herein.

Claims

WHAT IS CLAIMED IS:

1. A method for enhancing seed germination and / or growth of a plant, comprising contacting a seed of the plant with a starter fertilizer and a composition comprising a fungal endophyte.

2. The method of claim 1, wherein the method accelerates the development and resilience of transplants, promoting faster establishment in soil.

3. The method of claim 1 or 2, wherein the starter fertilizer is a chemical fertilizer comprising phosphorus and / or and a chemical fertilizer comprising nitrogen.

4. The method of claim 3, wherein the chemical fertilizer comprising phosphorus is selected from diammonium phosphate (DAP), mono-calcium phosphate (CP), single superphosphate (SSP), triple superphosphate (TSP), and monoammonium phosphate (MAP).

5. The method of claim 3 or 4, wherein the chemical fertilizer comprising nitrogen is selected from diammonium phosphate (DAP) and monoammonium phosphate (MAP).

6. The method of any one of claims 1 to 5, wherein the fungal endophyte is Trametes versicolor and / or Stereum hirsutum.

7. The method of any one of claims 1 to 6, wherein the composition improves nutrient absorption by promoting root growth and expanding root surface area, leading to higher phosphorus uptake efficiency and improve overall plant health and yield.

8. The method of any one of claims 1 to 7, wherein the contacting of the seed with the starter fertilizer and the composition comprising the fungal endophyte improves overall biomass growth, yield and drought tolerance of the plant by fostering water retention and root vigor.

9. The method of any one of claims 1 to 8, wherein the method enhances soil health by reducing the amount of synthetic fertilizer needed.

10. The method of any one of claims 1 to 9, wherein the contacting application occurs through root-dip inoculation.

11. The method of any one of claims 1 to 10, wherein the fungal endophyte inoculum is formulated within granular biochar beads.

12. The method of any one of claims 1 to 11, wherein the method enhances nodule formation in leguminous plants, leading to increased nitrogen fixation and improved overall plant health.

13. The method of any one of claims 1 to 12, wherein the method / fungal endophyte inoculation increases the concentration of nutrients in the plant selected from phosphorus (P), iron (Fe), sulfur (S), calcium (Ca), copper (Cu), sodium (Na), manganese (Mn), and combinations thereof.

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