Seed treatment to induce bacterial biofilm formation

Abstract

The present disclosure provides compositions and methods for producing a crop plant or crop plant seed capable of inducing biofilm formation (e.g., biofilms comprising nitrogen-fixing bacteria) wherein the crop plant or seed is treated with a Table 1 compound (e.g., tannic acid).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 413,187, filed on October 4, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Background Art

[0002] Biofilms are essential for optimal colonization of host plants and aid in nitrogen fixation. Biofilms are typically seeded with "aggregates" embedded in a self-produced matrix of exopolymeric substances (EPS) containing polysaccharides, proteins, lipids and extracellular DNA. The matrix provides protection and nutrition to the bacteria and contributes to tolerance / resistance to antimicrobial compounds. Furthermore, biofilms enable efficient interactions through chemical communication (quorum sensing) to dynamically reshape soil bacterial communities, making biofilms one of the most successful modes of life on Earth. In some cases, biofilm formation is essential for successful bacterial colonization.

[0003] Bacterial biofilm formation also creates heterogeneity, including the establishment of stable nutrient gradients, pH, and redox conditions. More importantly, due to the reduced diffusion of oxygen across bacterial biofilms, free-living nitrogen-fixing bacteria (Azospirillum brasilen, Pseudomonas stutzeri, etc.) are able to fix nitrogen under natural aerobic conditions, as bacterial nitrogenase is protected from oxygen-induced damage due to the low oxygen concentration on the bacterial surface. Increased biofilm formation allows for enhanced interaction of plant roots with nitrogen-fixing bacteria, thereby allowing plants to take up nitrogen and grow efficiently even when inorganic nitrogen in the soil is reduced. Summary of the invention

[0004] In one aspect, the present disclosure provides a composition comprising a crop plant or a seed of a crop plant treated with a compound of Table 1 (e.g., tannic acid) or a salt thereof in an amount sufficient to induce biofilm formation, wherein the biofilm comprises nitrogen-fixing bacteria.

[0005] In some embodiments, the crop plant is a seedling. In certain embodiments, the root of the crop plant is treated with a compound of Table 1.

[0006] In some embodiments, the crop plant is a cereal crop, such as corn, wheat, rice, soybean, cotton, rapeseed or sugar cane. In certain embodiments, the crop plant is rice.

[0007] In another aspect, the present disclosure provides a method for producing a crop plant or crop plant seed that can induce biofilm formation, wherein the biofilm comprises nitrogen-fixing bacteria, the method comprising treating the crop plant or the seed with a compound of Table 1 (e.g., tannic acid). In some embodiments, the compound is apigenin. In some embodiments, the compound is tannic acid. In some embodiments, the compound is curcumin.

[0008] In some embodiments of the method, the method comprises treating the seed with a compound of Table 1 (e.g., tannic acid), and after the treatment, planting the seed in soil containing nitrogen-fixing bacteria. In some embodiments, the method further comprises planting the treated seed in soil. In some embodiments of the method, the method comprises treating the seed with a compound of Table 1 (e.g., tannic acid), and planting the seed in soil containing nitrogen-fixing bacteria, and then performing the treatment.

[0009] In some embodiments of the method, the method comprises treating a crop plant with a compound of Table 1 (e.g., tannic acid). In certain embodiments, the crop plant is a seedling, and after the treatment, the seedling is planted in soil containing nitrogen-fixing bacteria. In some embodiments, the method further comprises planting the treated seedling in soil.

[0010] In some embodiments, the seeds are treated with the compound by seed coating or seed injection. In some embodiments, the seed coating is selected from the group consisting of seed dressing, film coating, pelleting, and encrusting.

[0011] In some embodiments of the methods described herein, the crop plant is a cereal crop, such as corn, wheat, rice, soybean, cotton, rapeseed or sugar cane. In certain embodiments, the crop plant is rice.

[0012] In some embodiments of the method, the crop plant or the seed is planted under conditions of reduced inorganic nitrogen. In specific embodiments, the inorganic nitrogen content in the soil is less than 90%, 80%, 70%, 60% or 50% of the standard nitrogen content for crop plants. In certain embodiments, the nitrogen-fixing bacteria in the soil in which the crop plant or the seed grows show more biofilm formation than the control nitrogen-fixing bacteria in the soil in which the control plant is grown without the compound treatment.

[0013] In some embodiments, crop plants grown in soil containing reduced amounts of inorganic nitrogen assimilate at least twice the amount of atmospheric nitrogen assimilated by control plants grown in equivalent soil but whose seeds have not been treated with the compound.

[0014] In some embodiments, the biofilm area induced by the crop plant or the seed of the crop plant is larger than the biofilm area induced by the control crop plant or the control seed not treated with the compound of Table 1. In other embodiments, the biofilm contains a larger amount of nitrogen-fixing bacteria than the biofilm induced by the control crop plant or the control seed not treated with the compound of Table 1. In another embodiment, the biofilm produces more fixed nitrogen than the biofilm induced by the control crop plant or the control seed not treated with the compound of Table 1.

[0015] In another aspect, the present disclosure provides a method for selecting a compound that can induce biofilm formation, the method comprising: 1) treating the crop plant or the seed with a compound of Table 1; 2) comparing the amount of biofilm formation in the soil in which the crop plant or the seed grows with the amount of biofilm formation in the soil in which a control plant or control seed that is not treated with the compound of Table 1 grows; 3) selecting a compound for the crop plant or seed that can induce more biofilm formation compared to the control plant or control seed, wherein the biofilm comprises nitrogen-fixing bacteria.

[0016] In some embodiments, in step 3), the number of nitrogen-fixing bacteria contained in the biofilm induced by the compound is greater than the number of nitrogen-fixing bacteria contained in the biofilm in the soil containing the control crop plants or control seeds not treated with the compound of Table 1.

[0017] In another aspect, the present disclosure provides a method for selecting a crop plant or crop plant seed that can induce higher nitrogen assimilation relative to a control plant or control seed, the method comprising: 1) treating the crop plant or the seed with a compound of Table 1; 2) comparing the amount of nitrogen assimilated by the crop plant or the seed with the amount of nitrogen assimilated by the control crop plant or the control seed that was not treated with the compound of Table 1; 3) selecting the crop plant or the seed that has a higher amount of nitrogen assimilation compared to the control plant or the control seed.

[0018] In some embodiments, the amount of nitrogen assimilated by the crop plant or the seed is at least 0.1 times (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 times, 2 times, 3 times, 4 times or more) higher than the amount of nitrogen assimilated by the control crop plant or control seed not treated with the compound of Table 1.

[0019] In another aspect, the present disclosure provides a method for selecting crop plants having more tillers, tassels and / or ears and / or higher seed yield relative to control plants, the method comprising: 1) treating the crop plants with a compound of Table 1; 2) comparing the tiller, tassel and / or ear number, and / or seed yield with the number of control crop plants not treated with a compound of Table 1; 3) selecting the crop plants having more tillers, tassels and / or ears and / or higher seed yield compared to the control plants.

[0020] In some embodiments, the crop plant has at least 5% greater tiller, tassel and / or ear number and / or seed yield than a control crop plant not treated with a compound of Table 1.

[0021] In some embodiments of the above method, the crop plant or seed is grown under low nitrogen conditions. In certain embodiments, the crop plant is selected from the group consisting of corn, wheat, rice, soybean, cotton, rapeseed and sugarcane. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Workflow for chemical screening of biofilm inducers in nitrogen-fixing bacteria.

[0023] Figure 2 .Heatmap of the effects of 2800 compounds on biofilm formation.

[0024] Figure 3 .Chemical structures and hierarchical clustering of the top 30 biofilm inducers.

[0025] Figure 4 .Hit validation for biofilm induction in two nitrogen-fixing bacteria.

[0026] Figure 5 .Apigenin, tannic acid, and curcumin are general biofilm inducers of soil nitrogen-fixing organisms (diazotrophs).

[0027] Fig. 6A .Venn diagram showing the overlap of soil diazotrophs whose biofilms can be induced by apigenin and tannic acid.

[0028] Figure 6B .Venn diagram showing the overlap of soil diazotrophs whose biofilms can be induced by apigenin and curcumin.

[0029] Figure 6C .Venn diagram showing the overlap of soil diazotrophs whose biofilms can be induced by apigenin, tannic acid, and curcumin.

[0030] Fig. 7ARepresentative pictures of wheat plants grown in the presence of 100% N2 or 30% N2 and in the presence of added apigenin, tannic acid or curcumin (2 mL, 100 μM).

[0031] Figure 7B . Fig. 7A Quantification of grain yield of the plants shown in . DETAILED DESCRIPTION introduction

[0032] The present disclosure provides compositions and methods for treating crop plants (e.g., seedlings) or crop plant seeds with a compound of Table 1 (e.g., tannic acid) such that once the crop plants or seeds are planted, the compound diffuses into the soil and induces the formation of biofilms in nitrogen-fixing soil bacteria. The biofilm formed further protects the bacterial nitrogenase from the damaging effects of oxygen present in the soil and triggers its nitrogen-fixing activity while producing ammonium, which is easily taken up by the crop plants. The production of ammonium and the increased absorption of plants can reduce the use of inorganic nitrogen fertilizers, not only reducing the production costs of grains, but also reducing the harmful effects of inorganic nitrogen fertilizers on the environment. definition

[0033] Unless otherwise stated, the following terms used herein have the meanings given below.

[0034] As used herein, the terms "a", "an", or "the" include aspects of not only one component, but also more than one component. For example, unless expressly stated otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to the agent includes one or more agents known to those skilled in the art, and so forth.

[0035] As used herein, the terms "about" and "approximately" generally indicate an acceptable degree of error for a measured quantity, taking into account the nature or precision of the measurement. Typically, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given range or value. Any reference to about "X" specifically indicates that a value is at least X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 100X, 101X, 102X, 103X, 104X, 105X, 106X, 107X, 108X, 109X, 110X, 111X, 112X, 113X, 114X, 115X, 116X, 117X, 118X, 119X, 120X, 121X, 122X, 123X, 124X, 125X, 126X, 127X, 128X, 129X, 98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X. Thus, about "X" is intended to teach and provide written descriptive support to support a claim limitation, such as "0.98X."

[0036] As used herein, the terms "crop plants" and "crops" refer to plants that can be grown and harvested in large quantities for subsistence and / or profit. Crop plants can be grown on a large scale, typically in one location. Many crop plants are grown in agriculture or aquaculture. Typically, crop plants are harvested for food for humans or feed for livestock.

[0037] As used herein, the term "biofilm" refers to the accumulation of organisms on a surface (e.g., bacteria, archaea, fungi, molds, algae, or protozoa). A mature biofilm can include a community of microorganisms residing on a surface. In the present disclosure, biofilm formation refers to a biofilm formed by nitrogen-fixing bacteria. Plant and / or seed treatment

[0038] Plants (e.g., crop plants) or seeds can be treated with Table 1 compounds (e.g., tannic acid) or salts thereof. In some embodiments, the compound is a compound of Table 2. In a specific embodiment, the compound is selected from the following groups: kaempferol, tannic acid, phytol, γ-tocopherol, α-tocopherol, curcumin, orotic acid and apigenin. In some embodiments, the compound is tannic acid. In some embodiments, the compound is apigenin. In some embodiments, the compound is curcumin. In some embodiments, the treated plant is a seedling. For example, after treating the roots of a plant (e.g., a seedling) with a Table 1 compound (e.g., tannic acid), the plant (e.g., seedling) is planted in soil containing nitrogen-fixing bacteria. In another example, after the plant (e.g., seedling) is planted in soil containing nitrogen-fixing bacteria, the root of the plant (e.g., seedling) can be treated with a Table 1 compound (e.g., tannic acid). In some embodiments, the compound (e.g., tannic acid) can be injected into the root of the plant. In other embodiments, the roots of the plant can be immersed in a solution containing a compound (e.g., tannic acid) such that a sufficient amount of the compound remains on the roots to induce biofilm formation containing nitrogen-fixing bacteria. In other embodiments, plants (e.g., crop plants) or seeds treated with a compound (e.g., tannic acid) of Table 1 can also be treated with nitrogen-fixing organisms (such as rhizobia).

[0039] Plants (e.g., crop plants) or seeds can be treated with at least 0.1 mM of a compound of Table 1 (e.g., tannic acid). In some embodiments, plants (e.g., crop plants) or seeds can be treated with 0.1 mM to 500 mM (e.g., 1 mM to 500 mM, 5 mM to 500 mM, 10 mM to 500 mM, 20 mM to 500 mM, 40 mM to 500 mM, 60 mM to 500 mM, 80 mM to 500 mM, 100 mM to 500 mM, 150 mM to 500 mM, 200 mM to 500 mM, 250 mM to 500 mM, 300 mM to 500 mM, 350 mM to 500 mM, 400 mM to 500 mM, 450mM to 500mM, 0.1mM to 450mM, 0.1mM to 400mM, 0.1mM to 350mM, 0.1mM to 300mM, 0.1mM to 250mM, 0.1mM to 200mM, 0.1mM to 150mM, 0.1mM to 100mM, 0.1mM to 80mM, 0.1mM to 60mM, 0.1mM to 40mM, 0.1mM to 20mM, 0.1mM to 10mM, 0.1mM to 5mM, or 0.1mM to 1mM). In some embodiments, the treated plant (eg, seedling) or seed (eg, plant or seed coated with a compound of Table 1 (eg, tannic acid)) contains at least 0.01 mM of a compound of Table 1 (eg, tannic acid). In some embodiments, the treated plant (e.g., seedling) or seed (e.g., plant or seed coated with a compound of Table 1 (e.g., tannic acid)) contains 0.01 mM to 100 mM (e.g., 0.05 mM to 100 mM, 0.1 mM to 100 mM, 0.2 mM to 100 mM, 0.4 mM to 100 mM, 0.6 mM to 100 mM, 0.8 mM to 100 mM, 1 mM to 100 mM, 5 mM to 100 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 35 mM to 100 mM, 40 mM to 10 In some embodiments, the present invention relates to a compound of Table 1 (e.g., tannic acid) in an amount of 0.01 mM to 100 mM, 0.01 mM to 60 mM, 0.01 mM to 70 mM, 0.01 mM to 60 mM, 0.01 mM to 50 mM, 0.01 mM to 40 mM, 0.01 mM to 30 mM, 0.01 mM to 20 mM, 0.01 mM to 10 mM, 0.01 mM to 8 mM, 0.01 mM to 6 mM, 0.01 mM to 4 mM, 0.01 mM to 2 mM, or 0.01 mM to 1 mM).The amount or concentration of the compound (e.g., tannic acid) used to treat the plant or seed, or the amount or concentration of the compound (e.g., tannic acid) remaining on the surface of the plant or seed after treatment, will depend on the type of compound used, the solubility of the compound, the growth rate of the plant or seed, and the type of soil. In some embodiments, the compound (e.g., tannic acid) on the surface of the plant or seed can diffuse into the soil so that the soil can have a concentration of at least 1 μM, e.g., 1 to 10 μM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM) of the compound (e.g., tannic acid).

[0040] Various seed treatment techniques can be used to incorporate the compounds of Table 1 (e.g., tannic acid) into the seeds of plants (e.g., crop plants). In some embodiments, the seeds of the plants can be coated with the compound (e.g., tannic acid). In some embodiments, the compound (e.g., tannic acid) can be injected into the seeds.

[0041] Examples of techniques that can be used to coat seeds with compounds of Table 1 include, but are not limited to, seed dressing, film coating, granulation, and encrustation. Seed coating is the process of applying exogenous materials to the surface of seeds. In some embodiments, seed coating is used to change the physical properties of seeds and to deliver active ingredients (e.g., tannic acid). In some embodiments, one or more compounds of Table 1 (e.g., tannic acid) can be applied to the surface of seeds with the aid of an adhesive, and in some embodiments, a filler can serve as a carrier. Seed coating can cover a variety of forms from simple farm applications to complex industrial procedures. Although the processes used by farmers and industrial companies may be different, the principles are basically the same. In general, it includes seeds in a container (e.g., a rotating drum, a cement mixer), wherein a binder (e.g., an adhesive), a filler (bulking agent) (if necessary), and an active ingredient (e.g., tannic acid) are mixed. The filler can be a single or mixed component, most commonly peat, talc, and lime. These components can serve as compound carriers and, in some cases, can change the size, shape, and / or weight of the seeds. Some ingredients (such as alginate) can be used as fillers and binders at the same time. In some cases, biochar and chitosan are also considered fillers / carriers for seed coating. Adhesives, natural or synthetic polymers (such as methylcellulose, carboxymethylcellulose, gum arabic or polysaccharide gums) can usually be added during or at the end of the coating process to bind exogenous materials (such as tannic acid) and reduce the amount of dust in the final product. Some adhesives (e.g., gum arabic and xanthan gum) can also be used. Selecting the appropriate type and concentration of adhesives and fillers is essential for seed germination and plant development.

[0042] The classification of seed coating types is generally based on the weight, size, and grouping characteristics of the seeds. Types of seed coating include, but are not limited to, seed dressing, film coating, granulation, and encrustation. Seed dressing refers to sprinkling a small amount of finely ground solids on the surface of the seed. Film coating involves applying a thin layer of external material with little change in the shape, size, and weight of the seed. In some embodiments of film coating, a solution or suspension is applied to the seeds. In some embodiments, film coating allows for greater processing precision and minimizes dust generation. Compared to other seed coating types, in some embodiments, film coating interferes less with seed germination and releases the active ingredients more quickly.

[0043] Seed granulation involves applying fillers and liquid adhesives to the seeds, which may result in a significant increase in the weight and volume of the seeds. Granulation generally changes the seed morphology to a spherical or ovoid shape. If the original seed shape is still maintained, the term used for such seed coating is seed encrusting. Granulation and encrusting can increase the amount of active ingredients applied and improve seed handling and sowing, especially for irregularly shaped seeds.

[0044] Depending on the type of coating, specific equipment needs to be considered. The rotating disc is the most common equipment used for seed coating (e.g., pelleting, shelling, seed dressing, and film coating). It usually consists of an inclined disc rotating at a slow speed, to which the material is gradually added, then size graded (screened and sifted), and then dried. Film coating and shelling can also be performed using a fluidized bed or spouted bed, which is a cylindrical device in which the seeds are kept in suspension by a constant vertical / bottom-up stream of hot air while the coating material is sprayed. The warm air flow allows the water to evaporate. Another equipment used for most types of seed coating is a rotary coater or rotor stator, which is a cylindrical drum with two rotating base discs, one concave, whose rotation causes the seeds to move steadily along the drum wall, and the other smaller, which allows the liquid / slurry coating to be atomized and sprayed onto the rotating seed mass.

[0045] Additional descriptions of seed coating and the materials and equipment required can be found, for example, in Pedrini et al., Trends Plant Sci. 22(2):106-116, 2017; Rocha et al., Front Plant Sci. 10:1357, 2019; Ma, Biotechnol Adv 37(7):107423, 2019; and Ehsanfar and Modarres-Sanavy, Commun Agric Appl Biol Sci. 70(3):225-9, 2005.

[0046] In some embodiments, the treated plant (e.g., seedling) or seed (e.g., plant or seed coated with a compound of Table 1 (e.g., tannic acid)) is not in soil. In some embodiments, the treated plant (e.g., seedling) or seed (e.g., plant or seed coated with a compound of Table 1 (e.g., tannic acid)) is in a container (e.g., a bag, box, etc.). In some embodiments, the treated plant (e.g., seedling) or seed (e.g., plant or seed coated with a compound of Table 1 (e.g., tannic acid)) contains an amount of a compound of Table 1 (e.g., tannic acid) that is greater than the amount of the compound (if any) in soil. In some embodiments, the treated plant (e.g., seedling) or seed (e.g., plant or seed coated with a compound of Table 1 (e.g., tannic acid) contains an amount of a compound of Table 1 (e.g., tannic acid) that is greater than the amount of the compound (if any) in soil, and the treated plant (e.g., seedling) or seed is not in soil.

[0047] In some embodiments, in addition to treating (e.g., coating) the seeds with a compound of Table 1 (e.g., tannic acid), the seeds may be treated with a nitrogen-fixing bacterium (e.g., Glucanoacetobacter diazotrophicus). In some embodiments, in addition to treating (e.g., coating) the seeds with a compound of Table 1 (e.g., tannic acid), the seeds may be treated with a nitrogen-fixing organism (e.g., Rhizobium). In some embodiments, in addition to treating (e.g., coating) the seeds with a compound of Table 1 (e.g., tannic acid), the seeds may be treated with a nitrogen-fixing bacterium (e.g., Glucanoacetobacter diazotrophicus) and a nitrogen-fixing organism (e.g., Rhizobium).

[0048] In other embodiments, the compound of Table 1 (e.g., tannic acid) can be added to soil containing nitrogen-fixing bacteria (e.g., diazo-gluconoacetobacter) to induce biofilm formation. In certain embodiments, after adding the compound of Table 1 (e.g., tannic acid) to soil containing nitrogen-fixing bacteria (e.g., diazo-gluconoacetobacter), plants (e.g., seedlings) or seeds (e.g., plants or seeds coated with the compound of Table 1 (e.g., tannic acid)) are planted in the soil. In certain embodiments, after planting plants (e.g., seedlings) or seeds (e.g., plants or seeds coated with the compound of Table 1 (e.g., tannic acid)) in the soil, the compound of Table 1 (e.g., tannic acid) can be added to soil containing nitrogen-fixing bacteria (e.g., diazo-gluconoacetobacter). In some embodiments, the compound can be added to the soil as a solution containing the desired compound concentration. In other embodiments, the compound can be added to the soil as a solid (e.g., in powder form or granular form). Whether the compound is added to the soil as a solution or a solid, the compound should be thoroughly and evenly mixed in the soil containing the nitrogen-fixing bacteria. In other embodiments, nitrogen-fixing organisms (eg, rhizobia) may also be added to the soil containing the nitrogen-fixing bacteria.

[0049] In other embodiments, the treatment can be carried out in the form of any type of soil application, such as in-ditch application, drip irrigation application, soil mixing application, root irrigation application, sprinkler irrigation, microinjection or particle application. In some embodiments, the treatment is carried out in the soil before seed germination, and / or in the soil in contact with the plant root, or the treatment carried out at the plant expected growth place. In some embodiments, the treatment is repeated. In some embodiments, repetition can refer to at least twice, at least three times, at least four times or even at least five times before sowing / planting and / or during plant germination and / or growth.

[0050] In some embodiments, the methods of the present disclosure further comprise simultaneously or sequentially applying at least one other plant protection agent, such as a nematicide, an insecticide, a bactericide, a miticide, a fungicide, or other agent that promotes or improves plant health. plant

[0051] The compositions and methods of the present invention can be used to modify any plant, including monocots and dicots, cereals, trees and vegetable crops, to improve its ability to interact with nitrogen-fixing bacteria in the soil. In a specific embodiment, the plant is a crop species, such as corn, wheat, rice, soybean, cotton, rapeseed or sugarcane. In some embodiments, the crop plant is a cereal crop. Available crops include but are not limited to cereals, rapeseed, beans, hay, etc. A non-limiting list of useful cereals includes rice (e.g., Oryza sativa, Zizani spp.), wheat (e.g., Triticum aestivum), barley (e.g., Hordeum vulgare), oats (e.g., Avenasativa), rye (e.g., Secale cereal), triticale (e.g., Triticosecales spp.), corn (e.g., Zea mays), sorghum (Sorghum spp.), millet (e.g., Digitaria, Echinochloa, Eleusine, Panicum, Setaria, Pennisetum, spp.), canary seed (e.g., Phalaris canariensis), teff (e.g., Eragrostis spp.), abyssinica), and Job's Tears (e.g., Coix lacryma-jobi). In a specific embodiment, the plant is rice, such as Oryza sativa. A non-limiting list of oilseeds includes soybeans (e.g., Glycine spp.), peanuts (e.g., Arachis hypogaea), rapeseed and mustard (e.g., Brassica spp., Brassica napus), sunflowers (e.g., Helianthus annuus), safflowers (e.g., Carthamus spp.), and flax (e.g., Linum spp.).A non-limiting list of legumes includes pinto beans (e.g., Phaseolus vulgaris), lima beans (e.g., Phaseolus lunatus), black beans (e.g., Phaseolus mung), red beans (e.g., Phaseolus angularis), chickpeas (e.g., Cicer arietinum), field, green and yellow peas (e.g., Pisum spp.), lentils (e.g., Lens spp.), broad beans (e.g., Vicia faba), and other legumes including Dolichos, Cajanus, Vigna, Pachyrhizus, Tetragonolobus genera. A non-limiting list of hay and forage plants includes grasses such as meadow foxtail (e.g., Alopecurus pratensis), brome (e.g., Brome spp.), orchard grass (e.g., Dactylis glomerata), fescue (e.g., Festuca spp.), ryegrass (e.g., Lolium spp.), canary grass (e.g., Phalaris arundinacea), Kentucky bluegrass (e.g., Poa pratensis), Timothy (e.g., Phleum pretense), and redtop grass (e.g., Agropyron spp.), and legumes such as alfalfa and yellow clover (e.g., Medicago spp.). spp.), alfalfa (Medicago sativa), clover (Trifolium spp.), birdsgoot trefoil (e.g., Lotus corniculatus), and vetch (e.g., Vicia spp.). Other useful plants include buckwheat, tobacco, hemp, sugar beet, and amaranth. In some embodiments, the plant is a shrub, such as cotton (e.g., Gossypium hirsutum, Gossypium barbadense). In some embodiments, the plant is a grass, such as sugar cane (e.g., Saccharum officinarum).

[0052] In some embodiments, the plant is a tree. Any tree can be modified with the current method, including angiosperms and gymnosperms. A non-limiting list of trees includes, for example, cycads, ginkgo, conifers (e.g., araucaria, cedar, cypress, douglas fir, fir, hemlock, juniper, larch, pine, podocarpus, redwood, spruce, yew), monocots (e.g., palm, agave, aloe, dracaena, spiral pine, yucca) and dicots (e.g., birch, elm, holly, magnolia, maple, oak, poplar, ash and willow). In a specific embodiment, the tree is a poplar (e.g., cottonwood, poplar, balsam poplar), such as Populus alba, Populus grandidentata, Populus tremula, Populus tremuloides, Populus deltoids, Populus fremontii, Populus nigra, Populus angustifolia, Populus balsamifera, Populus trichocarpa, or Populus heterophylla.

[0053] In some embodiments, the plant is a vegetable. Vegetables that may be used include, but are not limited to, arugula (Erucasativa), beets (Beta vulgaris vulgaris), cabbage (Brassica rapa), broccoli (Brassicooleracea), kohlrabi (Brassic oleraca), cabbage (Brassican oleracea), celery (Apium graveolens), endive (Cichorium intybus), Chinese mallow (Malva vertilillata), chrysanthemum coronarium, kale (Brassica oleracea), purslane (Portulaca oleraca), wild lettuce (Valerianela locusta), house lepidium sativum, dandelion (Taraxacum officinale), dill (Anethum gravelolens), endive (Cichorium endivia), grapes (Vitis), plantain (Plantago major), kale (Brassic oleracea), sheep’s lettuce (Valerianella locusta), ground mustard (Barbarea verna), lettuce (Lactuca sativa), mustard greens (Sinapis alba), Chinese cabbage (Brassica rapa), apricot (Tetragonia tetragonioides), pea (Pisum sativum), poke (Phytolacca Americana), red chicory (Cichorium intybus), sorrel (Rumex acetosa), sauerkraut (Brassic oleracea), spinach (Spinacia oleracea), rainbow purslane (Portulaca oleracea), chard (Beta vulgariscicla), turnip (Brassia rapa), watercress (Nasturtium officinale), water spinach (Ipomoeaaquatic), and lamb’s horn (Achillea millefolium). Also included are fruits and flowers such as gourds, squash, avocados, bell peppers, cucumbers, eggplants, sweet peppers, tomatoes, herbs, zucchini, artichokes, broccoli, capers, and cauliflower. Assessment of biofilm formation

[0054] Any of a variety of assays can be used to assess the ability of plants or seeds treated with a compound of Table 1 (e.g., tannic acid) or a salt thereof to induce biofilm formation. For example, biofilm produced by nitrogen-fixing bacteria (e.g., Gluconobacter diazoacetobacter) can be assessed (i.e., quantified) by incubating crop plants (e.g., seedlings) or seeds treated with the compound with nitrogen-fixing bacteria in wells of a microtiter plate, removing the culture from the plate, washing the wells, adding a developing solution (e.g., crystal violet), rinsing and drying the plate, then adding ethanol and measuring the absorbance at, e.g., 540 nm.

[0055] In some embodiments, nitrogen-fixing bacteria expressing markers such as fluorescent proteins (e.g., mCherry) can be used to assess biofilm formation. In some embodiments, for example in bacteria transformed with gumDpro::GFP, the bacteria may also express marker components of the biofilm. Dual labeling in such bacteria allows for separate visualization of the bacteria and their biofilm development.

[0056] For example, the nitrogen fixation activity of bacteria can be assessed using the acetylene reduction assay (ARA), in which the bacteria are cultured in the presence of acetylene gas and the conversion of acetylene to ethylene is measured, for example, by gas chromatography.

[0057] Plants and seeds treated with a compound of Table 1 (e.g., tannic acid) can also be evaluated in a variety of ways. For example, the treated plants or seeds can be grown in the presence of fluorescently labeled nitrogen-fixing bacteria, and the bacteria can be determined to be attached to plant root hairs, or to the root surface or present within plant tissues. Plants can also be evaluated by determining the number of tillers and / or seed yield. In some embodiments, by, for example, 15 The plants or seeds are grown in the presence of N2 gas and then the absorbed N2 in the plant leaves is measured, for example, using mass spectrometry. 15 N levels to assess nitrogen assimilation fixed by bacteria in soil.

[0058] In some embodiments, the number of tillers / tassels / ears and / or seed yield of plants produced using the compositions and methods of the present invention (involving treatment of plants or seeds with a compound of Table 1 (e.g., tannic acid)) is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or more compared to plants produced from untreated plants or seeds. In some embodiments, the biofilm formation induced by plants produced using the compositions and methods of the present invention (involving treatment of plants or seeds with a compound of Table 1 (e.g., tannic acid)) is increased by at least about 0.1 (i.e., an increase of about 10%), 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1-fold, 2-fold, 3-fold, 4-fold or more compared to plants produced from untreated plants or seeds. In some embodiments, plants produced using the compositions and methods of the invention involving treating plants or seeds with a compound of Table 1 (e.g., tannic acid) induce an increase in nitrogen assimilation by at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1-fold, 2-fold, 3-fold, 4-fold or more when grown under low nitrogen conditions compared to plants produced from untreated plants or seeds.

[0059] Because plants produced using the compositions and methods of the present invention (involving plants or seeds treated with compounds of Table 1 (e.g., tannic acid)) have increased assimilation of nitrogen-fixing bacteria, such plants can assimilate sufficient nitrogen to achieve high yields even when inorganic nitrogen levels in the soil are low. As used herein, "reduced" or "low" or "minimum" inorganic "nitrogen conditions" or "nitrogen levels" refer to inorganic nitrogen levels (e.g., levels resulting from fertilization) that are lower than levels typically used for crop plants or recommended for crop plants. For example, for rice plants, inorganic nitrogen levels of less than 50 ppm, such as about 25 ppm, can be used. In some embodiments, the inorganic nitrogen level is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% lower than normal or recommended levels. In some embodiments, plants and seeds treated with a compound of Table 1 (e.g., tannic acid) can be planted in soil containing less than 50 ppm, less than 45 ppm, less than 40 ppm, less than 35 ppm, less than 30 ppm, less than 25 ppm, less than 20 ppm, less than 15 ppm, less than 10 ppm, or less than 5 ppm of inorganic nitrogen levels.

[0060] In some embodiments of the methods described herein, the crop plant or the seed of the crop plant can induce a larger biofilm area than the biofilm induced by the control crop plant or the control seed that is not treated with the compound of Table 1 (e.g., tannic acid) (e.g., the biofilm area is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%). In certain embodiments, the biofilm contains more nitrogen-fixing bacteria than the biofilm induced by the control crop plant or the control seed that is not treated with the compound of Table 1 (e.g., tannic acid) (e.g., the number of bacterial cells in the biofilm is greater; for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%). Biofilms can be quantified by cell imaging and / or automatic cell counting. For example, optical and confocal microscopes can be used to count bacterial cells and determine the total biofilm volume. Instruments (such as automatic cell counters and flow cytometers) can also be used to quantify biofilms. Other methods and techniques for measuring and analyzing biofilms are described in the art, for example, see Wilson et al., Res Rev J Eng Technol. 6(4), 2017.

[0061] In some embodiments of the methods described herein, the biofilm induced by the treated plants or seeds described herein can produce more fixed nitrogen than the biofilm induced by the control crop plants or control seeds that were not treated with the compounds of Table 1 (e.g., tannic acid). Methods and techniques for measuring nitrogen fixation are available in the art, such as 15 N isotope dilution, 15 N natural abundance, acetylene reduction assay, microbial bioassay, ureide content measurement, etc. These methods and techniques are described, for example, in Fonseca-Lopez et al., Cienc. Tecnol. Agropecuaria 21(1), 2020. Example

[0062] The present invention will be described in more detail by specific examples. The examples provided below are only for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce substantially the same result. Identifying chemicals that induce biofilm formation

[0063] A chemical library screening was performed to evaluate the ability of different flavonoids to induce biofilm formation in nitrogen-fixing soil bacteria. The chemical library consisted of 2,800 natural compounds (TargetMol L6000 - Natural Compound Library). The bacterium diazo-fixing Gluconobacter acetobacter was used. The workflow of the chemical screening was as follows Figure 1As shown. Briefly, diazo-fixing gluconobacter was cultured overnight at 28°C in modified ATCC medium (0.5% yeast extract, 0.3% peptone, 1.5% sucrose, 1.5% mannitol). The culture was diluted 1:50 in fresh ATCC medium and the culture was grown to OD600=0.4. The culture was precipitated and the supernatant removed. The precipitate was washed three times with sterile water and then suspended with exudate from 3-day-old Kitaake rice germination seedlings until the final OD600=0.01.

[0064] Secretions for biofilm assays were collected by germinating 50 Kitaake seeds in 25 ml sterile milli-Q water in the dark at 28 ° C for 3 days. 2 μL of compounds (10 mM) from the chemical library were added to 198 μL of bacteria plus Kitaake secretions in each well of a sterile 96-well plate (Corning 3595) to a final concentration of 100 μM for each compound. The 96-well plate was shaken (150 rpm, 28 ° C) for 3 days. After incubation, the floating cells were discarded. 200 μL of crystal violet solution (0.2% crystal violet, 2% ethanol in water) was added to the wells of each plate, and the plate was shaken at 28 ° C (150 rpm, 30 minutes). The solution in each well was discarded, the plate was rinsed with water 3-4 times and naturally air-dried. 200 μL of 95% ethanol was added to each well to dissolve the crystal violet, shaken at 28°C (150 rpm, 15 min) and the absorbance was measured at 540 nm (Biotek Synergy Mx plate reader).

[0065] Chemical screens identified positive and negative regulators of biofilm formation (e.g., biofilms containing Gluconobacter diazotrophicola) ( Figure 2). Heat maps were generated using the MORPHEUS software for the average of 4 biological replicates for each compound. For biofilm inducers, 7.36% (206 out of 2800) of the compounds increased biofilm formation by more than 1.5 times compared to the DMSO control, and 2.75% (77 out of 2800) of the compounds increased biofilm formation by more than 2 times compared to the DMSO control. 1.21% (34 out of 2800) of the compounds inhibited biofilm formation to less than 50% compared to the DMSO control. More importantly, the identified top compounds had a stronger induction effect on biofilm formation than the flavonoid apigenin (Table 1), suggesting that they are more effective in protecting bacterial nitrogenase and increasing biological nitrogen fixation. Of the top biofilm inducers identified, 33.3% (10 out of 30) belonged to the flavonoid / chloroketone class of compounds, which further confirmed the important role of flavonoids in biofilm formation and biological nitrogen fixation of nitrogen-fixing bacteria. Other biofilm inducers identified belong to classes such as curcuminoids, terpenoids, quinones, and anthrone / flavonoids ( Figure 3 ). Table 1

[0066] The high proportion of phenol groups in biofilm inducers suggests that phenol units are a common backbone structure ( Figure 3 Table 2 shows natural compounds that induce biofilm formation (e.g., biofilms containing Acetobacter diazogluconobacter) (compared to the effect of DMSO). Some of the identified compounds are very low cost. For example, tannic acid increased biofilm formation 4.3-fold at a cost of 0.04% of the cost of apigenin (Table 2); curcumin increased biofilm formation 4.3-fold at a cost of 0.2% of the cost of apigenin. Table 2

[0067] The compounds were tested for their effects on biofilm formation in two well-known nitrogen-fixing bacteria: Gluconobacter diazotrophicus and Burkholderia vietnamiensi. Kaempferol, tannic acid, phytol, gamma-tocopherol, alpha-tocopherol, curcumin, and orotic acid showed a promoting effect in both tested nitrogen-fixing bacteria ( Figure 4). Soil nitrogen-fixing bacteria were isolated as follows: Root segments (5-10 cm below ground) were harvested from 16-week-old rice plants grown under 22.5 ppm nitrogen conditions (nitrogen-limited conditions). Three separate roots were combined and ground in a mortar and pestle after vortexing to remove adhering soil particles. The tissue was filtered through 2 layers of cheesecloth and suspended in 50 ml of sterile water. 10% of the original solution was added. -4 The dilutions were inoculated in Jensen's nitrogen-free medium containing 1.5% agar and incubated at 28°C for 7 days. Independent colonies were picked and grown in fresh Jensen's nitrogen-free medium for secondary selection. 80 random colonies that survived the secondary selection were tested for biofilm formation in the presence of apigenin, tannic acid or curcumin.

[0068] like Figure 5 As shown, apigenin and tannic acid are biofilm inducers of various nitrogen-fixing bacteria. In 71.2% (57 of 80) of the tested nitrogen-fixing bacteria, apigenin promoted biofilm formation by at least 20% more than the DMSO control. Tannic acid showed a positive effect on 68.8% (55 of 80) of the tested bacteria. Curcumin had a narrower effect on biofilm induction, with only 47.5% (38 of 80) of the nitrogen-fixing bacteria producing more biofilm.

[0069] Fig. 6A In the figure, a Venn diagram shows the overlap of soil diazotrophs whose biofilms are induced by both apigenin and tannic acid. Of the bacteria that were induced to form biofilms by apigenin, 78.9% (45 of 57) were also induced by tannic acid. Of the bacteria that were induced to form biofilms by tannic acid, 81.8% (45 of 55) were also induced by apigenin. Of the bacteria that were induced to form biofilms by tannic acid, 54.4% (31 of 57) were also induced by curcumin ( Figure 6B ). Among the bacteria that tannic acid could induce to form biofilm, 81.6% (31 out of 38 species) could also be induced by apigenin. Given that various soil nitrogen-fixing organisms act similarly to tannic acid and apigenin in forming biofilm, tannic acid would be a cheap alternative to apigenin in seed treatment (e.g., seed coating) to increase biological nitrogen fixation. Tannic acid has been used as a safe and environmentally friendly food additive and is included in the EU list of food flavorings. In agricultural practice, tannic acid can replace safer fungicides to control fungi (such as Fusarium graminearum (Forrer et al., 2014) and Penicillium digitatum (Zhu et al., 2019)). Similar to apigenin, tannic acid is also known for its antibacterial activity against antipathogens (Ekambaram et al., 2016). Effects of Chemicals on Common Wheat (Triticum aestivum)

[0070] Seeds of hexaploid common wheat (Triticum aestivum) were germinated in the laboratory and then transferred to pots filled with commercial soil. The seeds were grown under two different nitrogen concentrations. Seeds grown under 100% N2 used a solution containing 140 ppm N2, while seeds grown under 30% N2 used a solution containing 42 ppm N2. Fig. 7A and 7B As shown, one week after the seedlings were transplanted into pots filled with soil, 2 mL of a solution containing 100 μM apigenin, tannic acid, or curcumin was added to the soil. References 1. Ekambaram, SP, Perumal, SS, and Balakrishnan, A. (2016) Scope of Hydrolysable Tannins as Possible Antimicrobial Agents. Phyther. Res., 30, 1035–1045. 2. Forrer, H. R., Musa, T., Schwab, F., Jenny, E., Bucheli, T. D., Wettstein, F. E., and Vogelgsang, S. (2014) Fusarium head blight control and prevention of mycotoxin contamination in wheat with botanicals and tannic acid. Toxins (Basel), 6, 830–849. 3. Zhu, C., Lei, M., Andargie, M., Zeng, J., and Li, J. (2019) Antifungal activity and mechanism of action of tannicacid against Penicillium digitatum. Physiol. Mol. Plant Pathol., 107, 46–50. 4. Yan, D., Tajima, H., Cline, L. C., Fong, R. Y., Ottaviani, J. I., Shapiro, H. Y., Blumwald, E. (2022). Genetic modification of flavone biosynthesis in rice enhances biofilm formation of soil diazotrophic bacteria and biological nitrogen fixation.

[0071] Although the above invention has been described in detail by way of illustration and example for clear understanding, it will be appreciated by those skilled in the art that certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated herein by reference in its entirety, just as each reference is incorporated herein by reference alone.

Claims

1. A composition comprising a crop plant or a seed of a crop plant treated with a compound of Table 1 in an amount sufficient to induce biofilm formation, wherein the biofilm comprises nitrogen-fixing bacteria.

2. The composition of claim 1, wherein the crop plants are seedlings.

3. The composition according to any one of claims 1 to 2, wherein the roots of the crop plants are treated with a compound of Table 1.

4. The composition of claim 1, wherein the compound is tannic acid.

5. The composition according to any one of claims 1 to 4, wherein the crop plant is a cereal crop.

6. The composition according to any one of claims 1 to 5, wherein the crop plant is selected from the group consisting of corn, wheat, rice, soybean, cotton, rapeseed and sugarcane.

7. The composition according to claim 6, wherein the crop plant is rice.

8. A method for producing a crop plant or a seed of the crop plant capable of inducing biofilm formation, the method comprising treating the crop plant or the seed with a compound of Table 1, wherein the biofilm comprises nitrogen-fixing bacteria.

9. The method of claim 8, wherein the method comprises treating the seeds with a compound of Table 1 and, after the treatment, planting the seeds in soil containing the nitrogen-fixing bacteria.

10. The method of claim 9, further comprising planting the treated seeds in soil.

11. The method of claim 8, wherein the method comprises treating crop plants with a compound of Table 1.

12. The method of claim 11, wherein the crop plants are seedlings, and after the treatment, the seedlings are planted in soil containing the nitrogen-fixing bacteria.

13. The method of claim 11, wherein the crop plants are seedlings and the seedlings are planted in soil containing nitrogen-fixing bacteria prior to the treatment.

14. The method of claim 12, further comprising planting the treated seedlings in soil.

15. The method according to any one of claims 8 to 14, wherein the compound is tannic acid.

16. The method according to any one of claims 8 to 15, wherein the seeds are treated with the compound by seed coating or seed injection.

17. The method of claim 16, wherein the seed coating is selected from the group consisting of seed dressing, film coating, pelleting and encrusting.

18. The method according to any one of claims 8 to 17, wherein the crop plant is a cereal crop.

19. The method according to any one of claims 8 to 18, wherein the crop plant is selected from the group consisting of corn, wheat, rice, soybean, cotton, rapeseed and sugar cane.

20. The method of claim 19, wherein the crop plant is rice.

21. The method according to any one of claims 8 to 20, wherein the crop plant or the seed is planted under conditions of reduced inorganic nitrogen.

22. The method of claim 21, wherein the inorganic nitrogen content in the soil is less than 90%, 80%, 70%, 60% or 50% of the standard nitrogen content for crop plants.

23. The method of any one of claims 8 to 22, wherein the nitrogen-fixing bacteria in the soil in which the crop plants or seeds are grown exhibit more biofilm formation than control nitrogen-fixing bacteria in soil in which control plants or control seeds are grown that have not been treated with the compound.

24. The method of any one of claims 8 to 23, wherein the amount of atmospheric nitrogen assimilated by crop plants grown in soil containing reduced amounts of inorganic nitrogen is at least twice the amount assimilated by control plants grown in equivalent soil but not treated with the compound.

25. The method according to any one of claims 8 to 24, wherein the crop plant or the seed of the crop plant induces a biofilm area that is larger than the biofilm area induced by a control seed or a control crop plant that is not treated with a compound of Table 1.

26. The method of any one of claims 8 to 25, wherein the biofilm contains a greater amount of nitrogen-fixing bacteria than a biofilm induced by a control seed or control crop plant not treated with a compound of Table 1.

27. The method of any one of claims 8 to 26, wherein the biofilm produces more fixed nitrogen than a biofilm induced by a control seed or control crop plant not treated with a compound of Table 1.

28. A method for selecting a compound capable of inducing biofilm formation, the method comprising: 1) treating the crop plants or the seeds with the compound in Table 1; 2) comparing the amount of biofilm formed in the soil where the crop plants or seeds grow with the amount of biofilm formed in the soil where control plants or control seeds that are not treated with the compound of Table 1 grow; 3) selecting a compound for use in crop plants or seeds that induces more biofilm formation compared to control plants or control seeds, Wherein the biofilm comprises nitrogen-fixing bacteria.

29. The method according to claim 28, wherein in step 3), the number of nitrogen-fixing bacteria contained in the biofilm induced by the compound is greater than the number of nitrogen-fixing bacteria contained in the biofilm in the soil where the control crop plants or control seeds are located that have not been treated with the compound of Table 1.

30. A method for selecting crop plants or crop plant seeds capable of inducing higher nitrogen assimilation relative to control plants or control seeds, the method comprising: 1) treating the crop plants or the seeds with the compound in Table 1; 2) comparing the amount of nitrogen assimilated by the crop plant or the seed with the amount of nitrogen assimilated by the control crop plant or the control seed that has not been treated with the compound of Table 1; 3) Selecting the crop plants or the seeds having a higher nitrogen assimilation amount compared to the control plants or control seeds.

31. The method of claim 30, wherein the amount of nitrogen assimilated by the crop plant or the seed is at least 0.1 times greater than the amount of nitrogen assimilated by the control crop plant or control seed not treated with a compound of Table 1.

32. A method for selecting crop plants having more tillers, tassels and / or ears and / or higher seed yield relative to control plants, the method comprising: 1) treating the crop plants with the compound in Table 1; 2) comparing the number of tillers, tassels and / or ears, and / or seed yield with that of control crop plants not treated with the compound of Table 1; 3) Selecting said crop plants having more tillers, tassels and / or ears and / or higher seed yield compared to said control plants.

33. The method of claim 32, wherein the tiller, tassel and / or ear number and / or seed yield of the crop plant is at least 5% greater than that of the control crop plant not treated with a compound of Table 1.

34. according to the method described in any one of claims 28 to 33, wherein the crop plants or seeds are grown under low nitrogen conditions.

35. The method according to any one of claims 28 to 34, wherein the crop plant is selected from the group consisting of corn, wheat, rice, soybean, cotton, rapeseed and sugar cane.

Images