Methods of promoting plant health using microorganisms containing free and overexpressed enzymes

Abstract

Methods for stimulating plant growth and / or promoting plant health using free enzymes or recombinant microorganisms overexpressing enzymes are provided. Also provided are plant seeds coated with free enzymes or recombinant microorganisms overexpressing enzymes. Compositions comprising a fertilizer and an enzyme or a recombinant microorganism overexpressing an enzyme are provided. Also provided are modified enzymes having ACC deaminase activity, recombinant microorganisms expressing the modified enzymes, plant seeds treated with the modified enzymes or the recombinant microorganisms, and methods for stimulating plant growth and / or promoting plant health using the modified enzymes or the recombinant microorganisms.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 309,426, filed March 16, 2016, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention provides a method for stimulating plant growth and / or promoting plant health using recombinant microorganisms containing free or overexpressed enzymes. It also provides plant seeds treated with recombinant microorganisms containing free or overexpressed enzymes. A composition comprising fertilizer and recombinant microorganisms containing enzymes or overexpressed enzymes is provided. Furthermore, it provides modified enzymes having ACC deaminase activity, recombinant microorganisms expressing modified enzymes, plant seeds treated with modified enzymes or recombinant microorganisms, and methods for stimulating plant growth and / or promoting plant health using modified enzymes or recombinant microorganisms. Background Technology

[0004] The area surrounding the plant roots is called the rhizosphere. In the rhizosphere, bacteria, fungi, and other organisms compete for nutrients and bind to the plant's root structure. Both harmful and beneficial bacteria and fungi can occupy the rhizosphere. Bacteria, fungi, and the plant's roots are all affected by rhizosphere enzymes. Increasing soil concentration or treating plants with certain enzymes will have a beneficial effect on the overall population of beneficial soil bacteria and fungi, creating a healthier overall soil environment for plant growth, improving plant growth, and providing protection against certain bacterial and fungal pathogens. The environment surrounding the plant roots (rhizosphere) is a unique mixture of bacteria, fungi, nutrients, and roots, and its quality differs from that of natural soil. The symbiotic relationships between these organisms are unique and can be better altered by incorporating exogenous proteins.

[0005] Therefore, there is a need in the art for an efficient method for delivering enzymes and other proteins to plants. Furthermore, there is a need in the art to enhance plant responses to enzymes and provide benefits to growers. Summary of the Invention

[0006] This invention provides an enzyme. The enzyme comprises an amino acid sequence encoding an enzyme having 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) activity and a signal peptide. When the enzyme is expressed in a microorganism, the signal peptide leads to enzyme secretion. A recombinant microorganism expressing the enzyme is also provided. A formulation comprising the enzyme or recombinant microorganism and an agriculturally acceptable vector is also provided. Plant seeds treated with said enzyme, recombinant microorganism, or formulation are also provided.

[0007] An enzyme with ACC deaminase activity is provided. The amino acid sequence of this enzyme contains at least one amino acid substitution relative to the sequence of wild-type D-cysteine ​​desulfurase or ACC deaminase from Bacillus spp. Compared to ACC deaminase, the amino acid substitution results in increased ACC deaminase activity compared to the ACC deaminase activity of wild-type D-cysteine ​​desulfurase or ACC deaminase under the same conditions. A recombinant microorganism expressing the enzyme is also provided. A formulation comprising the enzyme or recombinant microorganism and an agriculturally acceptable vector is also provided. Plant seeds treated with the enzyme, recombinant microorganism, or formulation are also provided.

[0008] A method for stimulating plant growth and / or promoting plant health is provided. The method comprises applying any enzyme having ACC deaminase activity or a formulation containing such enzyme and an agriculturally acceptable carrier to a plant growth medium, a plant, plant seeds, or the area surrounding the plant or plant seeds.

[0009] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying any recombinant microorganism expressing an enzyme with ACC deaminase activity, or a formulation containing such a recombinant microorganism and an agriculturally acceptable carrier, to a plant growth medium, a plant, plant seeds, or the area surrounding the plant or plant seeds.

[0010] This provides yet another method for stimulating plant growth and / or promoting plant health. The method involves applying a free enzyme to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, non-cellulose-degrading dextranases, ACC deaminases, and any combination thereof.

[0011] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying two or more free enzymes to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The enzymes are independently selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, proteases, phytases, acid phosphatases, mannanases, pectins, glucans, and ACC deaminases.

[0012] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a free enzyme to the plant or plant seeds. The enzyme comprises a glucanase. Applying the enzyme to the plant seeds includes: (a) applying the enzyme to the plant seeds at the time of planting; or (b) coating the plant seeds with the enzyme.

[0013] This provides yet another method for stimulating plant growth and / or promoting plant health. The method involves applying a free enzyme to a plant growth medium, a plant, a plant seed, or a region surrounding the plant or plant seed. The enzyme comprises a dextranase. The method also includes applying an extended protein to a plant growth medium, a plant, a plant seed, or a region surrounding the plant or plant seed.

[0014] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a free enzyme to the plant or plant seeds. The enzyme includes phytase.

[0015] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying fertilizer and free enzymes to the plant growth medium, the area surrounding the plant or plant seeds, or applying them directly to the plant or plant seeds. The free enzymes include phytase.

[0016] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a recombinant microorganism to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganism expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in a wild-type microorganism of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, mannanases, pectins, proteases, phytases, acid phosphatases, and any combination thereof. The enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganism.

[0017] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a recombinant microorganism to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganism expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in a wild-type microorganism of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein also contains a signal peptide that causes the secretion of said enzyme or extended protein.

[0018] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying recombinant microorganisms to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganisms express enzymes or extended proteins, wherein the expression of the enzymes or extended proteins is increased compared to the expression levels of the enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions. The enzymes are selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, phytases, mannanases, pectins, acid phosphatases, and any combination thereof. The enzymes or extended proteins do not bind to the exospore wall of the recombinant Bacillus cereus family member.

[0019] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a recombinant microorganism to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganism expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in a wild-type microorganism of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, phytases, mannanases, pectins, acid phosphatases, and any combination thereof. The enzyme or extended protein is not part of the fusion protein.

[0020] Treated plant seeds are provided. The plant seeds are treated with a free enzyme. The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, mannanase, pectinase, lactonease, deacetylated chitosanase, protease, phytase, acid phosphatase, non-cellulose-degrading glucanase, ACC deaminase, and any combination thereof.

[0021] Treated plant seeds are provided. The plant seeds are treated with two or more free enzymes, wherein the enzymes are independently selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, mannanase, pectinase, protease, phytase, acid phosphatase, glucanase, and ACC deaminase.

[0022] The plant seeds were provided with a coating. The plant seeds were coated with a free enzyme. The enzyme contained glucanase.

[0023] Treated plant seeds are provided. The plant seeds are treated with free enzymes and extended proteins. The enzymes include glucanase.

[0024] Plant seeds were provided. The plant seeds were coated with recombinant vitamins. The recombinant microorganisms expressed enzymes or extended proteins, wherein the expression of the enzymes or extended proteins was increased compared to the expression levels of wild-type microorganisms of the same species under the same conditions. The enzymes were selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, phytases, mannanases, pectins, acid phosphatases, ACC deaminases, and any combination thereof. The enzymes or extended proteins were expressed during the vegetative growth of the recombinant microorganisms.

[0025] Another type of plant seed is provided. The plant seed is coated with a recombinant microorganism. The recombinant vitamin expresses an enzyme or extended protein, wherein the expression of the enzyme is increased compared to the expression level of the enzyme or extended protein in the same species of wild-type microorganism under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein also contains a signal peptide that causes the enzyme or extended protein to be secreted.

[0026] Another type of plant seed is provided. The plant seed is coated with a recombinant microorganism. The recombinant vitamin expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in the same species of wild-type microorganism under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein does not bind to the exospore wall of the recombinant Bacillus cereus family member.

[0027] Another type of plant seed is provided. The plant seed is coated with a recombinant microorganism. The recombinant vitamin expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in the same species of wild-type microorganism under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein is not part of the fusion protein.

[0028] A composition is provided. The composition comprises fertilizer and an enzyme or extended protein. The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof.

[0029] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucans, ACC deaminases, and any combination thereof. The enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganisms.

[0030] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucans, ACC deaminases, and any combination thereof. The enzyme or extended protein also comprises a signal peptide that causes the secretion of the enzyme or extended protein.

[0031] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucans, ACC deaminases, and any combination thereof. The enzyme or extended protein does not bind to the exospore wall of the recombinant Bacillus cereus family member.

[0032] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucans, ACC deaminases, and any combination thereof. The enzyme or extended protein is not part of the fusion protein.

[0033] The features of the invention are further defined in the appended claims and a list of embodiments is provided below in the section entitled "Embodiments". Other objects and features will be apparent in part and pointed out in part below.

[0034] definition

[0035] When the articles “a,” “one,” “a,” “the,” and “the” are used in this text, they mean “at least one” or “one or more” unless otherwise stated.

[0036] As used herein, the term "members of the Bacillus cereus family" refers to any species of the genus of Bacillus capable of producing an outer spore wall. Therefore, the Bacillus cereus family of bacteria includes species such as *Bacillus anthracis*, *Bacillus cereus*, *Bacillus thuringiensis*, *Bacillus mycosis*, *Bacillus pseudomycosis*, *Bacillus samanii*, *Bacillus gaemokensis*, *Bacillus weihenstephensis*, and *Bacillus toyoiensis*. Members of the Bacillus cereus family are also referred to in this field as "Bacillus cereus in a broad sense."

[0037] The terms “composition” and “formulation” are used interchangeably herein to refer to a mixture of two or more chemical or biological materials (e.g., a mixture of enzymes and agriculturally acceptable carriers or a mixture of recombinant microorganisms and agriculturally acceptable carriers).

[0038] The terms “contains,” “includes,” and “have” are intended to be inclusive and mean that there may be other elements besides the listed elements.

[0039] As used herein, the term "foliation" in relation to the application of enzymes or recombinant microorganisms to plants refers to the application of enzymes or recombinant microorganisms to one or more above-ground parts of a plant, including the stem, leaves, fruit, flower, or other exposed above-ground parts of the plant.

[0040] As used herein, the term "free enzyme" refers to an enzyme preparation that is substantially free of intact cells. The term "free enzyme" includes, but is not limited to, crude cell extracts containing enzymes, partially purified, substantially purified, or purified enzymes. Free enzymes may optionally be immobilized on a chemical matrix or carrier to allow controlled enzyme release. Free enzyme preparations preferably do not contain enzymes bound to the outer spore wall of members of the Bacillus cereus family. Free enzymes also preferably do not contain enzymes bound to the outer spore wall of intact Bacillus cereus family spores.

[0041] As used herein, the term "fusion protein" refers to a protein having a polypeptide sequence comprising sequences derived from two or more separate proteins. Fusion proteins can be generated by linking all or part of a nucleic acid molecule encoding a first polypeptide with all or part of a nucleic acid molecule encoding a second polypeptide to produce a nucleic acid sequence that, upon expression, produces a single polypeptide having the functional properties derived from each of the original proteins.

[0042] As used in this article, the term "germination rate" refers to the number of seeds that germinate within a specific time period. For example, a germination rate of 85% means that 85 out of 100 seeds germinate within a given time period.

[0043] As used herein, the term "glucanase" refers to any enzyme capable of hydrolyzing glycosidic bonds. The term "non-cellulose-degrading glucanase" as used herein refers to any glucanase whose primary enzymatic activity does not involve cellulose or cellulose subunits as substrates. Non-cellulose-degrading glucans preferably do not use cellulose as a substrate.

[0044] As used in this article, “immobilization” refers to the binding of an enzyme to a matrix or support, which allows the enzyme to remain on or be released from the matrix or support for a controlled period of time, rather than dissipating into the environment in an uncontrolled manner.

[0045] The terms “natural sequence,” “natural amino acid sequence,” “wild-type sequence,” and “wild-type amino acid sequence” are used interchangeably in this document to refer to amino acid sequences present in naturally occurring proteins.

[0046] As used in this article, the term "overexpression" in recombinant microorganisms refers to recombinant microorganisms that have been modified to increase the level of protein (e.g., enzyme) expression in the recombinant microorganisms compared to the expression level of the same protein in wild-type microorganisms of the same species under the same conditions.

[0047] "Plant growth medium" includes any material that can support plant growth.

[0048] The terms “promote plant growth” and “stimulate plant growth” are used interchangeably herein and refer to the ability to enhance or increase at least one plant’s height, weight, leaf size, root size, fruit size, or stem size, and / or increase plant protein production and / or improve crop yield.

[0049] The term “promoting plant health” refers to any beneficial effects on plant health, including but not limited to increased germination rate, increased synchronous germination, reduced susceptibility to pathogens, reduced susceptibility to environmental stresses (e.g., drought, flood, heat, freezing, salt, heavy metals, low pH, high pH, ​​or any combination thereof), increased crop yield, increased root nodules, and increased nutrient uptake and / or nutrient content (e.g., increased sugar uptake or sugar content, or increased protein uptake or protein content).

[0050] The term "rhizosphere" is used interchangeably with "root zone" to refer to the section of soil surrounding and influenced by the roots of a plant.

[0051] As used herein, the term "partially purified" means that the crude formulation of an enzyme (e.g., cell lysate) has undergone a process to remove at least some non-enzymatic components (e.g., waste proteins, dead cell material, excess water, and / or unwanted cell debris). In a partially purified enzyme formulation, the enzyme preferably comprises at least 1% of the total protein content of the formulation, more preferably at least 3% of the total protein content of the formulation, and even more preferably greater than 5% of the total protein content of the formulation.

[0052] As used herein, the term "substantially purified" means that the enzyme preparation has undergone a process to remove a large number of non-enzymatic components (e.g., waste proteins, dead cell material, excess water, and / or unwanted cell debris). In substantially purified enzyme preparations, the enzyme preferably contains more than 30% of the total protein content of the preparation, more preferably more than about 40% of the total protein content of the preparation, and even more preferably more than 50% of the total protein content of the preparation.

[0053] As used herein, the term "synergistic effective amount" refers to the amount of a first substance (e.g., a first enzyme) that, when used in combination with a second substance (e.g., a second enzyme), produces a biological effect greater than the sum of the biological effects of the respective first and second substances when used alone. Detailed Implementation

[0054] This invention generally relates to methods for stimulating plant growth and / or promoting plant health. The method includes applying recombinant bacteria containing free enzymes, extended proteins, and overexpressed enzymes to plant seeds, plants, or areas surrounding plant seeds. This invention also relates to seeds treated or coated with recombinant bacteria containing free enzymes or overexpressed enzymes. This invention further relates to compositions comprising fertilizers and recombinant bacteria containing enzymes or overexpressed enzymes. The use of recombinant bacteria containing free enzymes or overexpressed enzymes to deliver enzymes to plants to allow for a brief burst of enzyme activity, which in turn provides a safe and transient effect on the plant, with limited residual material remaining on harvestable plant material. Alternatively, where a longer-lasting effect is required, the free enzyme can be immobilized on a substrate or support to provide controlled release of the enzyme.

[0055] I. Enzyme and Extended Protein Sequence

[0056] For ease of reference, exemplary sequences of wild-type and modified ACC deaminases, as well as sequences of other enzymes and extended proteins that may be used with the methods, seeds, and compositions described herein, are shown below.

[0057] AD-cysteine ​​desulfatase and ACC deaminase

[0058] For ease of reference, Table 1 below provides descriptions of exemplary D-cysteine ​​desulfurases and 1-aminocyclopropane-1-carboxylic acid deaminases (ACC deaminases), along with their SEQ ID NOs. Table 2 below provides the corresponding amino acid sequences for the nucleotide sequences listed in Table 1. As explained in more detail below, mutations in certain amino acids in wild-type D-cysteine ​​desulfurases or ACC deaminases can result in increased ACC deaminase activity compared to the wild-type enzyme under the same conditions.

[0059] In Table 1, SEQ ID NOs 1-3 and 111 are nucleotide sequences of wild-type enzymes exhibiting ACC deaminase and D-cysteine ​​desulfatase activities, and SEQ ID NOs 4-6 and 112 are nucleotide sequences encoding the corresponding forms of these enzymes, having two amino acid substitutions relative to the wild-type sequence, resulting in increased ACC deaminase activity. Thus, for example, SEQ ID NO: 1 provides the nucleotide sequence of a wild-type enzyme, and SEQ ID NO: 4 provides the nucleotide sequence of the same enzyme, wherein the nucleotide sequence has been altered to encode an enzyme with two amino acid substitutions relative to the enzyme encoded by SEQ ID NO: 1. Similarly, SEQ ID NO: 2 provides the nucleotide sequence of a wild-type enzyme, and SEQ ID NO: 5 provides the nucleotide sequence of the same enzyme, wherein the nucleotide sequence has been altered to encode an enzyme with two amino acid substitutions relative to the enzyme encoded by SEQ ID NO: 2. Likewise, SEQ ID NO: 3 is the wild-type sequence, and SEQ ID NO: 6 provides the corresponding altered sequence.

[0060] In Table 2, SEQ ID NOs 7-9 and 113 are the amino acid sequences of the wild-type enzymes, exhibiting ACC deaminase and D-cysteine ​​desulfatase activities, and SEQ ID NOs 10-12 and 114 are the corresponding amino acid sequences of these enzymes, having two amino acid substitutions relative to the wild-type sequence, which result in increased enzyme activity. Therefore, SEQ ID NO: 7 is the wild-type sequence, and SEQ ID NO: 10 provides the amino acid sequence of the same enzyme with two amino acid substitutions relative to the wild-type sequence. SEQ ID NOs 8 and 11, 9 and 12, and 113 and 114 are related to each other in the same manner. The substituted amino acids are shown as SEQ ID NOs 10-12 and 114 in Table 2 and are indicated in bold and underlined text.

[0061] Table 1. Nucleotide sequences of d-cysteine ​​desulfurase and ACC deaminase

[0062]

[0063] Table 2. Amino acid sequences of d-cysteine ​​desulfurase and ACC deaminase

[0064]

[0065]

[0066]

[0067] B. Phospholipase

[0068] For ease of reference, the descriptions of exemplary phospholipase amino acid sequences and their SEQ ID NOs are shown in Table 3 below.

[0069] Table 3. Amino acid sequences of phospholipases

[0070]

[0071] The native amino acid sequences of the phospholipases of SEQ ID NO. 13, 14, and 15 include the N-terminal signal peptide sequence MKKKVLALAAAITLVAPLQSVAFA (SEQ ID NO: 49) immediately preceding the first amino acid of SEQ ID NO. 13, 14, and 15. This signal peptide is not included in SEQ ID NO. 13, 14, or 15. However, the signal peptide of SEQ ID NO: 49 or another signal peptide may optionally be included at the N-terminus of the phospholipase of any of SEQ ID NO. 13, 14, and 15 or at the N-terminus of any other enzyme or extended protein described herein.

[0072] The native amino acid sequence of the lipase in SEQ ID NO:16 includes an N-terminal signal peptide MKGKLLKGVLSLGVGLGALYSGTSAQAE (SEQ ID NO:50) immediately preceding the first amino acid of SEQ ID NO:16. This signal peptide is not included in SEQ ID NO:16. The signal peptide of SEQ ID NO:50 or another signal peptide may optionally be included at the N-terminus of the phospholipase in SEQ ID NO:16 or at the N-terminus of any other enzyme or extended protein as described herein.

[0073] The native amino acid sequence of the lipase in SEQ ID NO:17 includes an N-terminal signal peptide MKKKVLALAAAITVVAPLQSVAFA (SEQ ID NO:51) immediately preceding the first amino acid of SEQ ID NO:17. This signal peptide is not included in SEQ ID NO:17. The signal peptide of SEQ ID NO:51 or another signal peptide may optionally be included at the N-terminus of the phospholipase in SEQ ID NO:17 or at the N-terminus of any other enzyme or extended protein as described herein.

[0074] The native amino acid sequence of the lipase of SEQ ID NO:18 includes an N-terminal signal peptide MKRKICKALICATLATSLWAGASTKVYAW (SEQ ID NO:52) immediately preceding the first amino acid of SEQ ID NO:18. This signal peptide is not included in SEQ ID NO:18. The signal peptide of SEQ ID NO:52 or another signal peptide may optionally be included at the N-terminus of the phospholipase of SEQ ID NO:18 or at the N-terminus of any other enzyme or extended protein as described herein.

[0075] The native amino acid sequence of the lipase in SEQ ID NO:19 includes an N-terminal signal peptide MLAGPLAAALPARATTGTPAFLHGVASGD (SEQ ID NO:53) immediately preceding the first amino acid of SEQ ID NO:19. This signal peptide is not included in SEQ ID NO:19. The signal peptide of SEQ ID NO:53 or another signal peptide may optionally be included at the N-terminus of the phospholipase in SEQ ID NO:19 or at the N-terminus of any other enzyme or extended protein as described herein.

[0076] The native amino acid sequence of the lipase in SEQ ID NO:115 includes an N-terminal signal peptide MKKKVLALAAAITLVAPLQNVAFA (SEQ ID NO:135) immediately preceding the first amino acid of SEQ ID NO:115. This signal peptide is not included in SEQ ID NO:115. The signal peptide of SEQ ID NO:135 or another signal peptide may optionally be included at the N-terminus of the phospholipase in SEQ ID NO:115 or at the N-terminus of any other enzyme or extended protein as described herein.

[0077] C. Lipase

[0078] For ease of reference, the description of the exemplary lipase amino acid sequence and its SEQ ID NO are shown in Table 4 below.

[0079] Table 4. Amino acid sequences of lipases

[0080]

[0081] The native amino acid sequence of the lipase of SEQ ID NO:21 includes an N-terminal signal peptide MKFVKRRIIALVTILMLSVTSLFALQPSAKA (SEQ ID NO:54) immediately preceding the first amino acid of SEQ ID NO:21. This signal peptide is not included in SEQ ID NO:21. However, the signal peptide of SEQ ID NO:54 or another signal peptide may optionally be included at the N-terminus of the phospholipase of SEQ ID NO:21 or at the N-terminus of any other enzyme or extended protein as described herein.

[0082] The native amino acid sequence of the lipase of SEQ ID NO:118 includes an N-terminal signal peptide, MARTMRSRVVAGAVACAMSIAPFAGTTAVMTLATTHAAMAATAP (SEQ ID NO:137), immediately preceding the first amino acid of SEQ ID NO:118. This signal peptide is not included in SEQ ID NO:118. However, the signal peptide of SEQ ID NO:137 or another signal peptide may optionally be included at the N-terminus of the phospholipase of SEQ ID NO:118 or at the N-terminus of any other enzyme or extended protein as described herein.

[0083] The native amino acid sequence of the lipase in SEQ ID NO:119 includes an N-terminal signal peptide, MGIFDYKNLGTEGSKTLFADAMA (SEQ ID NO:138), immediately preceding the first amino acid of SEQ ID NO:119. This signal peptide is not included in SEQ ID NO:119. However, the signal peptide of SEQ ID NO:138 or another signal peptide may optionally be included at the N-terminus of the phospholipase in SEQ ID NO:119 or at the N-terminus of any other enzyme or extended protein as described herein.

[0084] D. Xylanase

[0085] For ease of reference, the description of the exemplary xylanase amino acid sequence and its SEQ ID NO are shown in Table 5 below.

[0086] Table 5. Amino acid sequence of xylanase

[0087]

[0088] The native amino acid sequence of the xylanase of SEQ ID NO:22 includes an N-terminal signal peptide MCENLEMLNLSLAKTYKDYFKIGAAVTA (SEQ ID NO:55) immediately preceding the first amino acid of SEQ ID NO:22. This signal peptide is not included in SEQ ID NO:22. However, the signal peptide of SEQ ID NO:55 or another signal peptide may optionally be included at the N-terminus of the xylanase of SEQ ID NO:22 or at the N-terminus of any other enzyme or extended protein as described herein.

[0089] The native amino acid sequence of the xylanase of SEQ ID NO:23 includes an N-terminal signal peptide MFKFKKNFLVGLSAALMSISLFSATASA (SEQ ID NO:56) immediately preceding the first amino acid of SEQ ID NO:23. This signal peptide is not included in SEQ ID NO:23. However, the signal peptide of SEQ ID NO:56 or another signal peptide may optionally be included at the N-terminus of the xylanase of SEQ ID NO:23 or at the N-terminus of any other enzyme or extended protein as described herein.

[0090] The native amino acid sequence of the xylanase of SEQ ID NO:24 includes an N-terminal signal peptide MRKKCSVCLWILVLLLSCLSGKSAYA (SEQ ID NO:57) immediately preceding the first amino acid of SEQ ID NO:24. This signal peptide is not included in SEQ ID NO:24. However, the signal peptide of SEQ ID NO:57 or another signal peptide may optionally be included at the N-terminus of the xylanase of SEQ ID NO:24 or at the N-terminus of any other enzyme or extended protein as described herein.

[0091] The native amino acid sequence of the xylanase of SEQ ID NO:25 includes an N-terminal signal peptide MKLKKKMLTLLLTASMSFGLFGATSSA (SEQ ID NO:58) immediately preceding the first amino acid of SEQ ID NO:25. This signal peptide is not included in SEQ ID NO:25. However, the signal peptide of SEQ ID NO:58 or another signal peptide may optionally be included at the N-terminus of the xylanase of SEQ ID NO:25 or at the N-terminus of any other enzyme or extended protein as described herein.

[0092] E. xylosidase

[0093] For ease of reference, the description of the exemplary xylosidase amino acid sequence and its SEQ ID NO are shown in Table 6 below.

[0094] Table 6. Amino acid sequence of xylosidase

[0095]

[0096] F. lactonease

[0097] For ease of reference, the descriptions of exemplary lactonease amino acid sequences and their SEQ ID NOs are shown in Table 7 below.

[0098] Table 7. Amino acid sequences of lactoneases

[0099] enzymes SEQ ID NO. of the amino acid sequence. Lactonease (AiiA), Bacillus thuringiensis strain B184 27 Lactonease (AiiA), Bacillus pseudomycetamol strain B30 28

[0100] G. Deacetylated chitosanase

[0101] For ease of reference, the description of the exemplary deacetylated chitosan polysaccharide enzyme amino acid sequence and its SEQ ID NO are shown in Table 8 below.

[0102] Table 8. Amino acid sequence of deacetylated chitosan polysaccharide enzyme

[0103]

[0104] The native amino acid sequence of the deacetylated chitosanase of SEQ ID NO:29 includes an N-terminal signal peptide MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA (SEQ ID NO:59) immediately preceding the first amino acid of SEQ ID NO:29. This signal peptide is not included in SEQ ID NO:29. However, the signal peptide of SEQ ID NO:59 or another signal peptide may optionally be included at the N-terminus of the deacetylated chitosanase of SEQ ID NO:29 or at the N-terminus of any other enzyme or extended protein as described herein.

[0105] The native amino acid sequence of the deacetylated chitosanase of SEQ ID NO:124 includes an N-terminal signal peptide MHSQHRTARIALAVVLTAIPASLATAGVGYASTQASTAVK (SEQ ID NO:139) immediately preceding the first amino acid of SEQ ID NO:124. This signal peptide is not included in SEQ ID NO:124. However, the signal peptide of SEQ ID NO:139 or another signal peptide may optionally be included at the N-terminus of the deacetylated chitosanase of SEQ ID NO:124 or at the N-terminus of any other enzyme or extended protein as described herein.

[0106] H. glucanase

[0107] For ease of reference, the description of the exemplary dextranase amino acid sequence and its SEQ ID NO are shown in Table 9 below.

[0108] Table 9. Amino acid sequence of glucanase

[0109]

[0110]

[0111] The native amino acid sequence of the dextranase of SEQ ID NO:42 includes an N-terminal signal peptide MKRSISIFITCLLITLLTMGGMIASPASA (SEQ ID NO:60) immediately preceding the first amino acid of SEQ ID NO:42. This signal peptide is not included in SEQ ID NO:42. However, the signal peptide of SEQ ID NO:60 or another signal peptide may optionally be included at the N-terminus of the dextranase of SEQ ID NO:42 or at the N-terminus of any other enzyme or extended protein as described herein.

[0112] The native amino acid sequence of the dextranase of SEQ ID NO:43 includes an N-terminal signal peptide MPYLKRVLLLLVTGLFMSLFAVTATASA (SEQ ID NO:61) immediately preceding the first amino acid of SEQ ID NO:43. This signal peptide is not included in SEQ ID NO:43. However, the signal peptide of SEQ ID NO:61 or another signal peptide may optionally be included at the N-terminus of the dextranase of SEQ ID NO:43 or at the N-terminus of any other enzyme or extended protein as described herein.

[0113] The native amino acid sequence of the dextranase of SEQ ID NO:44 includes an N-terminal signal peptide MKRSQTSEKRYRQRVLSLFLAVVMLASIGLLPTSKVQA (SEQ ID NO:62) immediately preceding the first amino acid of SEQ ID NO:44. This signal peptide is not included in SEQ ID NO:44. However, the signal peptide of SEQ ID NO:62 or another signal peptide may optionally be included at the N-terminus of the dextranase of SEQ ID NO:44 or at the N-terminus of any other enzyme or extended protein as described herein.

[0114] The native amino acid sequence of the dextranase of SEQ ID NO:45 includes an N-terminal signal peptide MKPSHFTEKRFMKKVLGLFLVVVMLASVGVLPTSKVQA (SEQ ID NO:63) immediately preceding the first amino acid of SEQ ID NO:45. This signal peptide is not included in SEQ ID NO:45. However, the signal peptide of SEQ ID NO:63 or another signal peptide may optionally be included at the N-terminus of the dextranase of SEQ ID NO:45 or at the N-terminus of any other enzyme or extended protein as described herein.

[0115] The native amino acid sequence of the dextranase of SEQ ID NO:125 includes an N-terminal signal peptide MFKKWKKFGISSLALVLVAAVAFTGWSAKASA (SEQ ID NO:140) immediately preceding the first amino acid of SEQ ID NO:125. This signal peptide is not included in SEQ ID NO:125. However, the signal peptide of SEQ ID NO:140 or another signal peptide may optionally be included at the N-terminus of the dextranase of SEQ ID NO:125 or at the N-terminus of any other enzyme or extended protein as described herein.

[0116] I. Protease

[0117] For ease of reference, the descriptions of the amino acid sequences of exemplary proteases and their SEQ ID NOs are shown in Table 10 below.

[0118] Table 10. Amino acid sequence of proteases

[0119]

[0120] The native amino acid sequence of the protease of SEQ ID NO:47 includes an N-terminal signal peptide MKKGIIRFLLVSFVLFFALSTGITGVQA (SEQ ID NO:64) immediately preceding the first amino acid of SEQ ID NO:47. This signal peptide is not included in SEQ ID NO:47. However, the signal peptide of SEQ ID NO:64 or another signal peptide may optionally be included at the N-terminus of the protease of SEQ ID NO:47 or at the N-terminus of any other enzyme or extended protein as described herein.

[0121] The native amino acid sequence of the protease of SEQ ID NO:127 includes an N-terminal signal peptide MVVFSKTAALVLGLSTAVSA (SEQ ID NO:141) immediately preceding the first amino acid of SEQ ID NO:127. This signal peptide is not included in SEQ ID NO:127. However, the signal peptide of SEQ ID NO:141 or another signal peptide may optionally be included at the N-terminus of the protease of SEQ ID NO:127 or at the N-terminus of any other enzyme or extended protein as described herein.

[0122] J. Mannanase

[0123] For ease of reference, the description of the exemplary mannanase amino acid sequence and its SEQ ID NO are shown in Table 11 below.

[0124] Table 11. Amino acid sequence of mannanase

[0125] enzymes SEQ ID NO. of the amino acid sequence. Mannanase, Bacillus 128

[0126] The native amino acid sequence of the mannanase of SEQ ID NO:128 includes an N-terminal signal peptide MAKLQKGTILTVIAALMFVILGSAAPKA (SEQ ID NO:142) immediately preceding the first amino acid of SEQ ID NO:128. This signal peptide is not included in SEQ ID NO:128. However, the signal peptide of SEQ ID NO:142 or another signal peptide may optionally be included at the N-terminus of the mannanase of SEQ ID NO:128 or at the N-terminus of any other enzyme or extended protein as described herein.

[0127] K. pectinase

[0128] For ease of reference, the description of the exemplary pectinase amino acid sequence and its SEQ ID NO are shown in Table 12 below.

[0129] Table 12. Amino acid sequence of pectinase

[0130] Enzyme (SEQ ID NO) SEQ ID NO. of the amino acid sequence. Pectinase, Aspergillus japonicus 129

[0131] The native amino acid sequence of the pectinase of SEQ ID NO:129 includes an N-terminal signal peptide MPSAKPLFCLATLAGAALAAP (SEQ ID NO:143) immediately preceding the first amino acid of SEQ ID NO:129. This signal peptide is not included in SEQ ID NO:129. However, the signal peptide of SEQ ID NO:143 or another signal peptide may optionally be included at the N-terminus of the pectinase of SEQ ID NO:129 or at the N-terminus of any other enzyme or extended protein as described herein.

[0132] L. acid phosphatase

[0133] For ease of reference, the descriptions of exemplary acid phosphatases and their SEQ ID NOs are shown in Table 13 below.

[0134] Table 13. Amino acid sequences of acid phosphatases

[0135] enzymes SEQ ID NO. of the amino acid sequence. Acid phosphatase, wheat 130 Acid phosphatase, wheat 131

[0136] The native amino acid sequence of the acid phosphatase of SEQ ID NO:130 includes an N-terminal signal peptide MARGSMAAVLAVLAVAALRCAPAAA (SEQ ID NO:144) immediately preceding the first amino acid of SEQ ID NO:130. This signal peptide is not included in SEQ ID NO:130. However, the signal peptide of SEQ ID NO:144 or another signal peptide may optionally be included at the N-terminus of the acid phosphatase of SEQ ID NO:130 or at the N-terminus of any other enzyme or extended protein as described herein.

[0137] The native amino acid sequence of the acid phosphatase of SEQ ID NO:131 includes an N-terminal signal peptide MRGLGFAALSLHVLLCLANGVSSRRTSSYV (SEQ ID NO:145) immediately preceding the first amino acid of SEQ ID NO:131. This signal peptide is not included in SEQ ID NO:131. However, the signal peptide of SEQ ID NO:145 or another signal peptide may optionally be included at the N-terminus of the acid phosphatase of SEQ ID NO:131 or at the N-terminus of any other enzyme or extended protein as described herein.

[0138] M. phytase

[0139] For ease of reference, the description of exemplary phytase amino acid sequences and their SEQ ID NOs are shown in Table 14 below.

[0140] Table 14. Amino acid sequences of phytase

[0141] enzymes SEQ ID NO. of the amino acid sequence. Phytase, wheat 132 Phytase, wheat 133 Phytase, wheat 134

[0142] The native amino acid sequence of the phytase of SEQ ID NO:132 includes an N-terminal signal peptide MWWGSLRLLLLLAAAVAA (SEQ ID NO:146) immediately preceding the first amino acid of SEQ ID NO:132. This signal peptide is not included in SEQ ID NO:132. However, the signal peptide of SEQ ID NO:146 or another signal peptide may optionally be included at the N-terminus of the phytase of SEQ ID NO:132 or at the N-terminus of any other enzyme or extended protein as described herein.

[0143] The native amino acid sequence of the phytase of SEQ ID NO:133 includes a signal peptide MWWGSLRLLLLLAAAVAA (SEQ ID NO:146) immediately preceding the first amino acid of the sequence at the N-terminus. This signal peptide is not included in SEQ ID NO:133. However, the signal peptide of SEQ ID NO:146 or another signal peptide may optionally be included at the N-terminus of the phytase of SEQ ID NO:133 or at the N-terminus of any other enzyme or extended protein as described herein.

[0144] The native amino acid sequence of the phytase of SEQ ID NO:134 includes a signal peptide MGIWRGSLPLLLLAA (SEQ ID NO:147) immediately preceding the first amino acid of the sequence at the N-terminus. This signal peptide is not included in SEQ ID NO:134. However, the signal peptide of SEQ ID NO:147 or another signal peptide may optionally be included at the N-terminus of the phytase of SEQ ID NO:134 or at the N-terminus of any other enzyme or extended protein as described herein.

[0145] N. extended protein

[0146] For ease of reference, the description of the exemplary extended protein amino acid sequence and its SEQ ID NO are shown in Table 15 below.

[0147] Table 15. Amino acid sequence of the extended protein

[0148]

[0149] The native amino acid sequence of the extended protein of SEQ ID NO:74 includes an N-terminal signal peptide MKKIMSAFVGMVLLTIFCFSPQASA (SEQ ID NO:68) immediately preceding the first amino acid of SEQ ID NO:74. This signal peptide is not included in SEQ ID NO:74. However, the signal peptide of SEQ ID NO:74 or another signal peptide may optionally be included at the N-terminus of the protease of SEQ ID NO:74, the N-terminus of any enzyme described herein, or the N-terminus of another extended protein.

[0150] O. Mutations that increase enzyme activity

[0151] In any enzyme described herein, including free enzymes and enzymes expressed by recombinant microorganisms, the enzyme may contain at least one amino acid substitution relative to the wild-type sequence of the same enzyme, and wherein the amino acid substitution results in increased enzyme activity compared to the enzyme activity of the wild-type enzyme under the same conditions.

[0152] II. Modified enzymes with ACC deaminase activity

[0153] Modified 1-aminocyclopropane-1-carboxylic acid ester (ACC) deaminases are provided. ACC deaminases and D-cysteine ​​desulfurases (DCDs) generally have similar amino acid sequences and can have overlapping enzymatic activities, acting on 1-aminocyclopropane-1-carboxylic acid ester (ACC) and D-cysteine ​​as substrates. Some enzymes possess only one of these activities, while others can function as both ACC deaminases and D-cysteine ​​desulfurases. ACC deaminases cleave ACC into ammonia and α-ketobutyrate, while D-cysteine ​​desulfurases convert D-cysteine ​​to pyruvate, H₂S, and ammonia. ACC is a direct precursor to ethylene, and if present at high levels, it can cause undesirable effects in plants.

[0154] Therefore, enzymes with increased ACC deaminase activity would be beneficial for use in agriculture to reduce ACC levels, thereby reducing ethylene levels. The application of ACC deaminases in plant growth media, plants, plant seeds, or the area surrounding plants or seeds can stimulate plant growth, promote plant health (e.g., through increased nutrient uptake), and slow fruit ripening. These effects, in turn, lead to increased yield, early-season vigor, and plant resistance to early-season stress. ACC deaminases can also protect plants from pathogens and abiotic stresses.

[0155] As explained in more detail below, enzymes exhibiting D-cysteine ​​desulfatase and / or ACC deaminase activity can be mutated to increase their ACC deaminase activity. Furthermore, enzymes with ACC deaminase activity can be modified to include a signal peptide that leads to enzyme secretion when expressed in microorganisms, thereby allowing for easier production and purification of the enzyme. Such modifications (mutation and addition of the signal peptide) can be used alone or in combination with each other. All plants produce ACC and respond to ethylene, therefore such modified ACC deaminases have broad applicability.

[0156] The amino acid sequences of the three wild-type enzymes are provided above in Table 2 as SEQ ID NO. 7–9 and 113. The sequences of the corresponding forms of these wild-type enzymes with two amino acid substitutions that result in increased ACC deaminase activity are provided above in Table 2 as SEQ ID NO. 10–12 and 114.

[0157] Naturally occurring ACC deaminases are not secreted proteins. ACC deaminases have been found in many types of microorganisms, including bacteria of the phyla Bacteroidetes, Firmicutes, and Actinobacteria, as well as bacteria of the genera *Pseudomonas*, *Bacillus*, *Rhizobium*, *Brachyrhizobium*, and many others. However, the ACC deaminases found in these bacteria are intracellular and are subject to limited exposure to the substrate ACC from their colonizing host plants.

[0158] This article provides a modified ACC deaminase containing a signal peptide that causes the ACC deaminase to be secreted from the microorganism in which it is expressed. This ACC deaminase can be expressed in a microorganism and then applied to a plant growth medium, a plant, a plant seed, or the area surrounding a plant or plant seed. The ACC deaminase is secreted by the microorganism in contact with its substrate. Therefore, the secreted ACC deaminase can stimulate plant growth and / or promote plant health.

[0159] The enzyme is provided. The enzyme contains the amino acid sequence encoding an enzyme with 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity and a signal peptide that leads to the secretion of the enzyme when expressed in a microorganism.

[0160] Enzymes with ACC deaminase activity can include enzymes derived from Bacillus bacteria.

[0161] Alternatively, one or more amino acid substitutions may be introduced into the amino acid sequence of ACC deaminase to increase enzyme activity.

[0162] An enzyme with ACC deaminase activity is provided. The amino acid sequence of this enzyme contains at least one amino acid substitution relative to the sequence of wild-type D-cysteine ​​desulfurase or ACC deaminase from Bacillus spp. This amino acid substitution results in increased ACC deaminase activity compared to that of wild-type D-cysteine ​​desulfurase or ACC deaminase under the same conditions.

[0163] Enzymes containing at least one amino acid substitution may also contain a signal peptide, which, when expressed in a microorganism, leads to the secretion of the enzyme.

[0164] For any enzyme with ACC deaminase activity, the microorganism expressing the enzyme may include Bacillus, Pseudomonas, Rhizobium, Bacillus-like bacteria, Lysinobacillus, Paracoccus, Intermediate Rhizobium, Short Rhizobium, Acinetobacter, Arthrobacter, Azotobacter, Azospirillum, Pink pigment facultative methyltrophic bacteria, mycorrhizal fungi, Gastromycosis fungi, Trichoderma fungi, Kruvorella fungi, Glycotric fungi, or any combination thereof.

[0165] For example, microorganisms can include Bacillus, Lysine Bacillus, Pseudomonas, Bacillus-like bacteria, or any combination thereof.

[0166] For any enzyme with ACC deaminase activity, the enzyme may contain Bacillus thuringiensis enzyme or Bacillus pseudomycoidase.

[0167] The enzyme may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0168] The enzyme may contain an amino acid sequence that has at least 75% identity with any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0169] The enzyme may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0170] The enzyme may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0171] The enzyme may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0172] The enzyme may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0173] The enzyme may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0174] The enzyme may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO. 7–9 and 113, wherein the enzyme has ACC deaminase activity.

[0175] The sequence of the enzyme relative to the wild-type D-cysteine ​​desulfurase or ACC deaminase may contain two amino acid substitutions, wherein the amino acid substitutions result in an increase in ACC deaminase activity compared to the wild-type enzyme under the same conditions.

[0176] For example, the amino acid sequence of the enzyme may include a threonine residue at position 290 of SEQ ID NO:7 being replaced by a glutamic acid residue and a serine residue at position 317 of SEQ ID NO:7 being replaced by a leucine residue.

[0177] The amino acid sequence of the enzyme may include the substitution of a threonine residue at position 290 of SEQ ID NO:8 with a glutamic acid residue and the substitution of a serine residue at position 317 of SEQ ID NO:8 with a leucine residue.

[0178] The amino acid sequence of the enzyme may include the substitution of a threonine residue at position 290 of SEQ ID NO:9 with a glutamic acid residue and the substitution of a serine residue at position 317 of SEQ ID NO:9 with a leucine residue.

[0179] The amino acid sequence of the enzyme may include a threonine residue at position 290 of SEQ ID NO:113 replaced by a glutamic acid residue and a serine residue at position 317 of SEQ ID NO:113 replaced by a leucine residue.

[0180] The enzyme may include any one of SEQ ID NO. 10, 11, 12 or 14.

[0181] If an enzyme with ACC deaminase activity contains a signal peptide but does not contain any amino acid substitutions relative to the sequence of wild-type D-cysteine ​​desulfurase or ACC deaminase, then the ACC deaminase contains an amino acid sequence that is 100% identical to any one of SEQ ID NO. 7–9 and 113.

[0182] Signal peptides that can be used to modify enzymes with ACC deaminase activity are further described in Part XII below.

[0183] III. Recombinant bacteria expressing modified enzymes with ACC deaminase activity and bacteria containing or expressing modified enzymes. Recombinant bacterial preparations

[0184] Recombinant microorganisms expressing any of the enzymes described in Part II above are also provided.

[0185] In any recombinant microorganism expressing the enzyme as described in Part II above, the expression of the enzyme is preferably increased compared to the expression level of the enzyme in the same species of wild-type microorganism under the same conditions.

[0186] Suitable microorganisms for enzyme expression are described in Part XIII below.

[0187] Preparations comprising an agriculturally acceptable carrier and any modified enzymes described in Part II above, or recombinant microorganisms expressing any modified enzymes, are also provided. Suitable carriers and additional formulation components that may be used in such preparations are described in Part XVI below.

[0188] IV. Methods to stimulate plant growth and / or promote plant health

[0189] A method for stimulating plant growth and / or promoting plant health is provided. As described in more detail below, the method includes applying an enzyme, an extended protein, or a recombinant microbial enzyme expressing an enzyme or an extended protein to a plant growth medium, a plant, a plant seed, or a region surrounding the plant or plant seed.

[0190] The application of enzymes or extended proteins or recombinant bacteria preferably results in a higher level of delivery of enzymes or extended proteins to the plant growth medium, plant seeds or the area of ​​the plant or plant seed plant than the level of enzymes or extended proteins that can actually be found in the plant growth medium, plant seeds or the area of ​​the plant or plant seed plant.

[0191] A. Modified enzymes with ACC deaminase activity

[0192] A method for stimulating plant growth and / or promoting plant health is provided. The method comprises applying any enzyme having ACC deaminase activity as described in Part II above to a plant growth medium, a plant, a plant seed, or a region surrounding the plant or plant seed. Alternatively, the method may comprise applying a preparation containing an agriculturally acceptable carrier and any enzyme having ACC deaminase activity as described in Part II above to a plant growth medium, a plant, a plant seed, or a region surrounding the plant or plant seed.

[0193] Another method for stimulating plant growth and / or promoting plant health is provided. This method comprises applying a recombinant microorganism expressing an enzyme with ACC deaminase activity as described in Part III above to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed. Alternatively, the method may comprise applying a formulation containing an agriculturally acceptable carrier and a recombinant microorganism expressing an enzyme with ACC deaminase activity as described in Part III above to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0194] For example, the method may include applying any enzyme with ACC deaminase activity as described in Part II above to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0195] The method may include applying a free enzyme to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0196] The method may include applying any of the recombinant microorganisms described in Part III above to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0197] For example, the effects of any enzyme with ACC deaminase activity described in this section or another part of this document on plants can be tested by measurements of increased root mass, increased plant height, increased yield, increased nodulation, altered germination, altered seed germination, and delayed fruit ripening.

[0198] B. Phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, mannanase, pectinase, acid phosphatase, phytase, ACC deaminase, and extended protein

[0199] 1. Free enzymes

[0200] As described in more detail below, a method for stimulating plant growth and / or promoting plant health is provided, which relates to the use of phospholipases, lipases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectinases, acid phosphatases, phytases, ACC deaminases, and / or extended proteins and / or recombinant bacteria expressing such enzymes or extended proteins.

[0201] A method for stimulating plant growth and / or promoting plant health is provided. The method includes applying a free enzyme to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, non-cellulose-degrading dextranases, ACC deaminases, and any combination thereof.

[0202] The enzyme is preferably selected from phospholipase, lipase, xylanase, xylosidase, mannanase, pectinase, lactonease, deacetylated chitosanase, protease, phytase, acid phosphatase, ACC deaminase, and any combination thereof.

[0203] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying two or more free enzymes to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The enzymes are independently selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, proteases, phytases, acid phosphatases, mannanases, pectins, glucans, and ACC deaminases.

[0204] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a free enzyme to the plant or plant seeds. The enzyme comprises a glucanase. Applying the enzyme to the plant seeds includes: (a) applying the enzyme to the plant seeds at the time of planting; or (b) coating the plant seeds with the enzyme.

[0205] In methods including applying a free enzyme to a plant or plant seed, wherein the enzyme comprises a glucanase, the method may include coating the plant seed with a seed coating formulation comprising the enzyme and an agriculturally acceptable carrier.

[0206] In methods including the application of a free enzyme to a plant or plant seed, wherein the enzyme comprises a glucanase, the method may further include applying the enzyme or an expansile protein to a plant growth medium or a region surrounding the plant or plant seed. For example, the method may include applying the enzyme or an expansile protein to a plant growth medium. The method may include applying both the enzyme and the expansile protein to a plant growth medium.

[0207] Another method for stimulating plant growth and / or promoting plant health is also provided. This method involves applying a free enzyme to a plant growth medium, the plant, the plant seed, or the area surrounding the plant or plant seed. The enzyme comprises a dextranase. The method also includes applying an extended protein to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or plant seed.

[0208] A method comprising applying a free enzyme and an expansive protein, or applying an enzyme or an expansive protein to plant seeds, includes: (a) applying the enzyme or expansive protein to the plant seeds at the time of planting; or (b) coating the plant seeds with the enzyme or expansive protein. For example, the method may include coating the plant seeds with a seed coating formulation comprising an agriculturally acceptable carrier and an enzyme, an expansive protein, or both an enzyme and an expansive protein.

[0209] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying a free enzyme to the plant or plant seeds. The enzyme includes phytase.

[0210] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying fertilizer and free enzymes to the plant growth medium, the area surrounding the plant or plant seeds, or applying them directly to the plant or plant seeds. The free enzymes include phytase.

[0211] 2. Recombinant microorganisms

[0212] A method for stimulating plant growth and / or promoting plant health is provided. The method includes applying a recombinant microorganism to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganism expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in a wild-type microorganism of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, mannanases, pectins, proteases, phytases, acid phosphatases, and any combination thereof. The enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganism.

[0213] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying recombinant microorganisms to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganisms express enzymes or extended proteins, wherein the expression of the enzymes or extended proteins is increased compared to the expression levels of the enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions. The enzymes are selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectinases, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzymes or extended proteins also contain a signal peptide that causes the secretion of the enzymes or extended proteins.

[0214] Another method for stimulating plant growth and / or promoting plant health is provided. This method involves applying recombinant microorganisms to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganisms express enzymes or extended proteins, wherein the expression of the enzymes or extended proteins is increased compared to the expression levels of the enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions. The enzymes are selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, phytases, mannanases, pectins, acid phosphatases, and any combination thereof. The enzymes or extended proteins do not bind to the exospore wall of the recombinant Bacillus cereus family member.

[0215] Another method for stimulating plant growth and / or promoting plant health is also provided. This method involves applying recombinant microorganisms to a plant growth medium, a plant, plant seeds, or a region surrounding the plant or plant seeds. The recombinant microorganisms express enzymes or extended proteins, wherein the expression of the enzymes or extended proteins is increased compared to the expression levels of the enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions. The enzymes are selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, phytases, mannanases, pectins, acid phosphatases, and any combination thereof. The enzymes or extended proteins are not components of the fusion protein.

[0216] In any method, enzymes or extended proteins can be expressed during the vegetative growth of recombinant microorganisms.

[0217] If an enzyme or extended protein is expressed during the vegetative growth of a recombinant microorganism, the recombinant microorganism can be a spore-forming microorganism.

[0218] In any method other than one in which the enzyme is not a component of the fusion protein, the enzyme or extended protein may further include a signal peptide that causes the secretion of the enzyme or extended protein. Suitable signal peptides are described in Section XII below.

[0219] In any method, the enzyme or extended protein suitably does not bind to the outer spore wall of recombinant Bacillus cereus family members.

[0220] In any method, the enzyme or protein suitably does not bind to the outer spore wall of intact Bacillus cereus family members.

[0221] In any method other than those involving the use of signal peptides, enzymes or extended proteins are suitably not components of fusion proteins.

[0222] C. Pathways for the delivery of enzymes, extended proteins, and / or recombinant microorganisms to plants.

[0223] In any of the methods described herein, the method may include applying an enzyme or recombinant microorganism to a plant growth medium. For example, the enzyme or recombinant microorganism may be applied in furrows or may be included in a soil conditioner. Alternatively or additionally, the enzyme or recombinant microorganism may be impregnated onto dried granules, vermiculite or other substrates, plastic polymers, peat moss, or potting mixes prior to application to the plant growth medium. The enzyme or recombinant microorganism may also be applied to the plant growth medium via water, drip irrigation, seed solution application to the soil, or broadcast drying application to the soil.

[0224] Plant growth media may contain fertilizers or consist primarily of fertilizers. The mixture of fertilizers and enzymes or recombinant microorganisms is then applied to the soil or another plant growth medium using standard fertilization methods, including furrow application, 2×2 application, broadcast application, fertilizer impregnation, drip irrigation, topdressing, etc.

[0225] In any of the methods described herein, the method may include applying an enzyme, an extended protein, or a recombinant microorganism to a plant.

[0226] In any of the methods described herein, the method may include applying an enzyme, an extended protein, or a recombinant microorganism to plant roots.

[0227] In any of the methods described herein, the method may include applying an enzyme, an extended protein, or a recombinant microorganism to a leaf.

[0228] In any of the methods described herein, the method may include applying an enzyme, an extended protein, or a recombinant microorganism to a plant seed.

[0229] If the method includes applying an enzyme, an extended protein, or a recombinant microorganism to plant seeds, applying the enzyme, extended protein, or recombinant microorganism to plant seeds may include: (a) applying the enzyme, extended protein, or recombinant microorganism to plant seeds at the time of planting; or (b) coating plant seeds with the enzyme, extended protein, or recombinant microorganism.

[0230] For example, the method may include coating plant seeds with a seed coating agent comprising: an agriculturally acceptable carrier and enzyme, an extended protein, a recombinant microorganism, or a combination thereof.

[0231] V. Plant seeds

[0232] Plant seeds treated with enzymes, extended proteins, or recombinant microorganisms expressing enzymes or extended proteins are also provided.

[0233] A. Plant seeds treated with a modified enzyme possessing ACC deaminase activity

[0234] Treated plant seeds are provided. The plant seeds are treated with any enzyme having ACC deaminase activity as described in Part II above. Alternatively, the plant seeds are treated with a formulation as described in Part II above, comprising any enzyme having ACC deaminase activity and an agriculturally acceptable carrier.

[0235] Another type of plant seed is provided, which is treated with any recombinant microorganism expressing ACC deaminase activity as described in Part III above. Alternatively, the plant seed is treated with a formulation containing any recombinant microorganism expressing an enzyme with ACC deaminase activity as described in Part III above.

[0236] B. Plant seeds treated with enzymes or recombinant microorganisms

[0237] Plant seeds treated with enzymes, extended proteins, or recombinant bacteria are provided.

[0238] 1. Free enzymes

[0239] Treated plant seeds are provided. The plant seeds are treated with a free enzyme. The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, mannanase, pectinase, lactonease, deacetylated chitosanase, protease, phytase, acid phosphatase, non-cellulose-degrading glucanase, ACC deaminase, and any combination thereof.

[0240] The enzyme is preferably selected from phospholipase, lipase, xylanase, xylosidase, mannanase, pectinase, lactonease, deacetylated chitosanase, protease, phytase, acid phosphatase, ACC deaminase, and any combination thereof.

[0241] Another type of treated plant seed is provided, which is treated with two or more free enzymes, wherein the enzymes are independently selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, mannanase, pectinase, protease, phytase, acid phosphatase, glucanase, and ACC deaminase.

[0242] Treated plant seeds are provided. The plant seeds are treated with free enzymes and extended proteins. The enzymes include glucanase.

[0243] The plant seeds were provided with a coating. The plant seeds were coated with a free enzyme. The enzyme contained glucanase.

[0244] 2. Recombinant microorganisms

[0245] Plant seeds were provided. The plant seeds were coated with recombinant microorganisms. The recombinant microorganisms expressed enzymes or extended proteins, wherein the expression level of the enzymes or extended proteins was increased compared to the expression level of wild-type microorganisms of the same species under the same conditions. The enzymes were selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, phytases, mannanases, pectins, acid phosphatases, ACC deaminases, and any combination thereof. The enzymes or extended proteins were expressed during the vegetative growth of the recombinant microorganisms.

[0246] Another type of plant seed is provided. The plant seed is coated with a recombinant microorganism. The recombinant microorganism expresses an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in the same species of wild-type microorganism under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein also contains a signal peptide that leads to the secretion of the enzyme or extended protein.

[0247] Another type of plant seed is provided. The plant seed is coated with a recombinant microorganism. The recombinant microorganism expresses an enzyme or extended protein, wherein the expression level of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in the same species of wild-type microorganism under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein does not bind to the exospore wall of the recombinant Bacillus cereus family member.

[0248] Another type of plant seed was provided. The plant seed was coated with a recombinant microorganism. The recombinant microorganism expressed an enzyme or extended protein, wherein the expression level of the enzyme or extended protein was increased compared to the expression level of the enzyme or extended protein in the same species of wild-type microorganism under the same conditions. The enzyme was selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein was not part of the fusion protein.

[0249] In any seed, enzymes or extended proteins can be expressed during the vegetative growth of recombinant microorganisms.

[0250] If the enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganism, the recombinant microorganism can be a spore-forming microorganism.

[0251] In any seed except those in which the enzyme is not a component of the fusion protein, the enzyme or expansive protein may also contain a signal peptide that causes the secretion of the enzyme or expansive protein. Suitable signal peptides are described in Part XII below.

[0252] In any seed, the enzyme or expansin suitably does not bind to the exospore wall of recombinant Bacillus cereus family members.

[0253] In any seed, the enzyme or expansin is suitably not bound to the exospore wall of the intact Bacillus cereus family members.

[0254] In any seed except those involving the use of signal peptides, enzymes or extended proteins are suitably not components of fusion proteins.

[0255] C. Enclosed plant seeds

[0256] For any plant seed, it can be coated with enzymes, recombinant microorganisms, extended proteins, or any combination thereof.

[0257] For example, plant seeds can be coated with enzymes and extended proteins.

[0258] Any plant seed can be coated with a seed coating formulation comprising an enzyme, recombinant microorganism, extended protein, or any combination thereof and an agriculturally acceptable carrier.

[0259] VI. Composition

[0260] It provides recombinant microorganisms containing fertilizer and enzymes or extended proteins or overexpressed enzymes or extended proteins.

[0261] A. Enzymes

[0262] A composition is provided. The composition comprises fertilizer and an enzyme or extended protein. The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof.

[0263] The enzyme preferably contains a stress enzyme.

[0264] B. Recombinant microorganisms

[0265] A composition is provided. The composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, glucans, proteases, mannanases, pectins, phytases, acid phosphatases, ACC deaminases, and any combination thereof. The enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganisms.

[0266] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucans, ACC deaminases, and any combination thereof. The enzyme or extended protein also contains a signal peptide that causes the secretion of the enzyme or extended protein.

[0267] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectinases, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucanases, ACC deaminases, and any combination thereof. The enzyme or extended protein does not bind to the exospore wall of the recombinant Bacillus cereus family member.

[0268] Another composition is provided. This composition comprises fertilizer and recombinant microorganisms. The recombinant microorganisms express an enzyme or extended protein, wherein the expression of the enzyme or extended protein is increased compared to the expression level of the enzyme or extended protein in wild-type microorganisms of the same species under the same conditions. The enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectins, deacetylated chitosanases, proteases, acid phosphatases, phytases, glucans, ACC deaminases, and any combination thereof. The enzyme or extended protein is not part of the fusion protein.

[0269] In any composition, enzymes or extended proteins can be expressed during the vegetative growth of recombinant microorganisms.

[0270] If the enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganism, the recombinant microorganism can be a spore-forming microorganism.

[0271] In any composition other than those in which the enzyme is not part of the fusion protein, the enzyme or extended protein may also contain a signal peptide that causes the enzyme or extended protein to be secreted. Suitable signal peptides are described in Section XII below.

[0272] In any composition, the enzyme or extended protein suitably does not bind to the outer spore wall of recombinant Bacillus cereus family members.

[0273] In any composition, the enzyme or extended protein suitably does not bind to the outer spore wall of intact Bacillus cereus family members.

[0274] In any composition except those involving the use of signal peptides, enzymes or extended proteins are suitably not components of the fusion protein.

[0275] C. Carriers and other agricultural chemicals

[0276] In any composition, the composition may also contain an agriculturally acceptable carrier, additional agrochemicals, and fertilizers or combinations thereof. Suitable carriers and agrochemicals are described in Section XVI below.

[0277] VII. Enzymes and extended proteins used in conjunction with methods, plant seeds, or compositions

[0278] Phospholipases, lipases, xylanases, xylosidases, lactoneases, deacetylated chitosanases, proteases, glucans, extended proteins, phytases, acid phosphatases, pectinases, mannanases, and ACC deaminases suitable for use with the methods, seeds, and compositions described below.

[0279] A. Phospholipase

[0280] Enzymes can include phospholipases.

[0281] Phospholipases can be used for any of the plant growth stimulation or plant health promotion purposes described herein, but are particularly useful for stimulating plant growth, increasing nutrient uptake, and / or increasing root development and nodulation. Increased nodulation enhances the plant's ability to form a symbiotic relationship with nitrogen-fixing microorganisms in the soil, thereby increasing nitrogen uptake and improving growth rate. These effects also lead to reduced sensitivity to environmental stresses, such as drought.

[0282] Phospholipases are enzymes with specific activity against phospholipids, releasing free fatty acids from complex phospholipids. Phospholipases can be classified into five major classes: phospholipase A, phospholipase B, phospholipase C, phospholipase D, and phospholipase E. Each of these classes acts on a specific type of phospholipid.

[0283] If the enzyme contains phospholipase, then the phospholipase may contain phospholipase A, phospholipase B, phospholipase C, phospholipase D, phospholipase E, or any combination thereof.

[0284] For example, phospholipases can include phospholipase A, phospholipase C, phospholipase D, or any combination thereof.

[0285] When phospholipase contains phospholipase A, phospholipase A can contain phospholipase A1, phospholipase A2, or a combination thereof.

[0286] Phospholipase A2 may include group IIA phospholipase A2, group IIC phospholipase A2, group IID phospholipase A2, group IIE phospholipase A2, group IIF phospholipase A2, group III phospholipase A2, group IVA phospholipase A2, group IVB phospholipase A2, group IVC phospholipase A2, group IVD phospholipase A2, group IVE phospholipase A2, group VIF phospholipase A2, group V phospholipase A2, group VI phospholipase A2, group VII phospholipase A2, group X phospholipase A2, group XIIA phospholipase A2, group XIIB phospholipase A2, group XV phospholipase A2, group XVI phospholipase A2, or any combination thereof.

[0287] When phospholipase contains phospholipase B, phospholipase B can contain phospholipase B1.

[0288] When phospholipase includes phospholipase C, phospholipase C may include phospholipase Cβ1, phospholipase Cβ2, phospholipase Cβ3, phospholipase Cβ4, phospholipase Cδ1, phospholipase Cδ3, phospholipase Cδ4, phospholipase Cε1, phospholipase Cγ1, phospholipase Cγ2, phospholipase Cη1, phospholipase Cη2, phospholipase Cξ1, or any combination thereof.

[0289] When phospholipase includes phospholipase D, phospholipase D can include phospholipase D1, phospholipase D2, phospholipase D member 3, phospholipase D member 4, phospholipase D member 5, phospholipase D member 6, or any combination thereof.

[0290] Phospholipases may include 1-alkyl-2-acetylglycerol phospholipase, phosphatidylinositol deacylase, phosphoinositol phospholipase C, sphingomyelin phosphodiesterase, sphingomyelin phosphodiesterase D, alkylglycerol phosphoethanolamine phosphodiesterase, variant surface glycoprotein phospholipase C, glycosylphosphatidylinositol phospholipase D, N-acetylphosphatidylinositol hydrolyzing phospholipase D, phosphatidylinositol diacylglycerol lyase, glycosylphosphatidylinositol diacylglycerol lyase, protein 2 (PNPLA2) containing a potato glycoprotein-like phospholipase domain, protein 3 (PNPLA3) containing a potato glycoprotein-like phospholipase domain, or any combination thereof.

[0291] Phospholipases may include Streptomyces phospholipases (e.g., Streptomyces chromopyrospinata phospholipase D), Bacillus phospholipases (e.g., Bacillus cereus phospholipase, such as Bacillus cereus phosphatidylcholine-specific phospholipase C or Bacillus cereus phosphatidylinositol-specific phospholipase C or Bacillus thuringiensis phospholipase), Clostridium phospholipases (e.g., Clostridium perfringens phospholipase, such as Clostridium perfringens phospholipase C), or any combination thereof.

[0292] Phospholipases may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0293] Phospholipases may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0294] Phospholipases may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0295] Phospholipases may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0296] Phospholipases may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0297] Phospholipases may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0298] Phospholipases may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0299] Phospholipases may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0300] Phospholipases may contain amino acid sequences that are 100% identical to any one of SEQ ID NO. 13–19 and 115–117.

[0301] If the phospholipase includes Bacillus cereus phosphatidylcholine-specific phospholipase C (e.g., SEQ ID NO: 115), the method may also include applying mannanase (e.g., SEQ ID NO: 128) or xylose glucanase (e.g., SEQ ID NO: 125) to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0302] If the phospholipase includes Bacillus cereus phosphatidylcholine-specific phospholipase C (e.g., SEQ ID NO:115), the seeds can also be treated with mannanase (e.g., SEQ ID NO:128) or xylose glucanase (e.g., SEQ ID NO:125).

[0303] If the phospholipase comprises Bacillus cereus phosphatidylcholine-specific phospholipase C (e.g., SEQ ID NO: 115), the composition may also comprise mannanase (e.g., SEQ ID NO: 128) or xylose glucanase (e.g., SEQ ID NO: 125).

[0304] Bacillus cereus phosphatidylcholine-specific phospholipase C and mannanase can be present in synergistically effective amounts in the method, on the seed or in the composition.

[0305] Bacillus cereus phosphatidylcholine-specific phospholipase C and xylose glucanase can be present in synergistically effective amounts in the method, on the seed or in the composition.

[0306] B. Lipase

[0307] Enzymes can include lipases.

[0308] Lipases are enzymes with specific activity over lipids, cleaving the fatty acid chains of larger lipid molecules, such as triglycerides. Lipases can be used for any of the plant growth stimulation or plant health promotion purposes described herein, but are particularly well-suited for stimulating plant growth and enhancing nutrient uptake. These effects, in turn, lead to increased crop yield, improved seasonal vigor, and reduced plant sensitivity to early-season stress.

[0309] Lipases can include carboxyl ester lipase, diacylglycerol lipase α, diacylglycerol lipase β, lipase A, hepatic lipase, hormone-sensitive lipase, gastric lipase, endothelial lipase, member H lipase, lipase family member I, lipase family member J, lipase family member K, lipase family member M, lipase family member N, lipoprotein lipase, monoglyceride lipase, pancreatic lipase-related protein 2, pancreatic lipase-related protein 3, acylglycerol lipase, galactose lipase, lipoprotein lipase, or any combination thereof.

[0310] Lipases may include Bacillus subtilis lipase, Bacillus thuringiensis lipase, Bacillus cereus lipase, Bacillus clausti lipase, Burkholderia cepacia lipase, Burkholderia thermophila lipase, Pseudomonas spp. lipase, or any combination thereof.

[0311] Lipases may contain amino acid sequences that are at least 70% identical to SEQ ID NO:20, 21 and 118–120.

[0312] Lipases may contain amino acid sequences that are at least 75% identical to SEQ ID NO:20, 21 and 118–120.

[0313] Lipases may contain amino acid sequences that are at least 80% identical to SEQ ID NO:20, 21 and 118–120.

[0314] Lipases may contain amino acid sequences that are at least 85% identical to SEQ ID NO:20, 21 and 118–120.

[0315] Lipases may contain amino acid sequences that are at least 90% identical to SEQ ID NO:20, 21 and 118–120.

[0316] Lipases may contain amino acid sequences that are at least 95% identical to SEQ ID NO:20, 21 and 118–120.

[0317] Lipases may contain amino acid sequences that are at least 98% identical to SEQ ID NO:20, 21 and 118–120.

[0318] Lipases may contain amino acid sequences that are at least 99% identical to SEQ ID NO:20, 21 and 118–120.

[0319] Lipases may contain amino acid sequences that are 100% identical to SEQ ID NO:20, 21 and 118–120.

[0320] C. Xylanase

[0321] Enzymes can include xylanase.

[0322] Xylanase acts on xylan, a polysaccharide found in plants and soil, which is a common sugar. Xylanase can be used as a seed treatment, delivered to plant growth media (e.g., by furrow application or as a soil conditioner), or applied to plants as a foliar treatment to produce smaller sugar chains that can be absorbed by the plant or to feed the surrounding microbiome.

[0323] If the enzyme contains xylanase, then the xylanase may contain β-xylanase.

[0324] For example, β-xylanase may include glucuronide arabinoxylan end-1,4-β-xylanase, ex-1,4-β-xylanase, end-1,4-β-xylanase, or any combination thereof.

[0325] Xylanases may include xylanases from pyrolytic cellulosic bacteria (e.g., xylanases from extremely thermophilic cellulolytic bacteria), xylanases from Bacillus bacteria (e.g., xylanases from Bacillus subtilis or Bacillus stearothermophilus), xylanases from Neomycorrhizal fungi (e.g., xylanases from Neocomycorrhizal fungi), xylanases from thermophilic fungi (e.g., xylanases from Trichophyton spp.), or any combination thereof.

[0326] Xylanase may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0327] Xylanase may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0328] Xylanase may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0329] Xylanase may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0330] Xylanase may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0331] Xylanase may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0332] Xylanase may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0333] Xylanase may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0334] Xylanase may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO.22–25, 121 and 122.

[0335] D. Xylosidase

[0336] Enzymes can include xylosidases.

[0337] Xylosidases cleave individual xylose molecules from shorter fragments of xylan, a common polysaccharide found in plants and soil. Xylosidases can be used as seed treatments, delivered to plant growth media (e.g., by furrow application or as a soil conditioner), or applied to plants as foliar treatments to produce smaller sugar chains that can be passed through the plant or used to feed the surrounding microbiome.

[0338] For example, xylosidases can include xylosidases from extremely thermophilic cellulose-degrading bacteria, xylosidases from Bacillus pumilus, or combinations thereof.

[0339] Xylosidase may contain an amino acid sequence that has at least 70% identity with SEQ ID NO:26 or 123.

[0340] Xylosidase may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:26 or 123.

[0341] Xylosidase may contain an amino acid sequence that is at least 80% identical to SEQ ID NO:26 or 123.

[0342] Xylosidase may contain an amino acid sequence that has at least 85% identity with SEQ ID NO:26 or 123.

[0343] Xylosidase may contain an amino acid sequence that is at least 90% identical to SEQ ID NO:26 or 123.

[0344] Xylosidase may contain an amino acid sequence that is at least 95% identical to SEQ ID NO:26 or 123.

[0345] Xylosidase may contain an amino acid sequence that is at least 98% identical to SEQ ID NO:26 or 123.

[0346] Xylosidase may contain an amino acid sequence that is at least 99% identical to SEQ ID NO:26 or 123.

[0347] Xylosidase may contain an amino acid sequence that is 100% identical to SEQ ID NO:26 or 123.

[0348] E. lactonease

[0349] Enzymes can include lactones.

[0350] Lactoneases can be used for any of the plant growth stimulation or plant health promotion purposes described herein, but are particularly useful for reducing plant susceptibility to pathogens. Lactoneases are also described as acyl-homoserine lactoneases and are metalloenzymes produced by certain bacterial species. For example, lactoneases can be found in Bacteroidetes, Firmicutes, Actinobacteria, and other phyla within Pseudomonas and Bacillus species. Lactoneases target and inactivate acylated homoserine lactones. Lactoneases hydrolyze the ester bonds of small hormone-like molecules, commonly referred to as homoserine lactones. In the hydrolysis of these lactone bonds, lactoneases prevent these homoserine lactones from binding to their transcriptionally regulated targets, thereby interfering with quorum sensing. However, the lactoneases secreted by naturally occurring bacteria from soil or plant colonies are limited and inducible, thus requiring the provision of higher levels of lactoneases to the plant environment.

[0351] Free lactones or recombinant bacteria expressing lactones can be applied to plants (e.g., foliar application or as seed treatment) or to plant growth media to reduce lactone levels in the environment. Without being bound by any particular theory, it is believed that this reduction in lactone levels can, in turn, lead to a decrease in plant disease and a secondary increase in plant growth and development.

[0352] When expressed in recombinant microorganisms, adding a secretion signal to the lactonease will allow the microorganism to secrete the lactonease into the environment. Suitable secretion signals are further described in Section XII below.

[0353] If the enzyme contains a lactone, the lactone may include 1,4-lactone, 2-pyranone-4,6-dicarboxylate lactone, 3-oxoadipate enol-lactone, actinomycin lactone, deoxycitrate A-cyclic lactone, glucuronide lactone, L-rhamnose-1,4-lactone, citrate-D-cyclic lactone, steroid lactone, triacetate lactone, xylose-1,4-lactone, or any combination thereof.

[0354] Lactoneases may include Bacillus lactoneases (e.g., Bacillus thuringiensis lactonease, Bacillus pseudomycosis lactonease, or combinations thereof), Agrobacterium lactonease, Rhodococcus lactonease, Streptomyces lactonease, Arthrobacterium lactonease, Sphingosomal lactonease, Pseudomonas lactonease, Klebsiella lactonease, or any combination thereof.

[0355] Lactoneases contain AiiA.

[0356] Lactoneases are preferably specific to bacterial lactone homoserine signaling molecules.

[0357] Lactoneases may contain an amino acid sequence that has at least 70% identity with SEQ ID NO:27 or 28.

[0358] Lactoneases may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:27 or 28.

[0359] Lactoneases may contain an amino acid sequence that is at least 80% identical to SEQ ID NO:27 or 28.

[0360] Lactoneases may contain an amino acid sequence that has at least 85% identity with SEQ ID NO:27 or 28.

[0361] Lactoneases may contain an amino acid sequence that is at least 90% identical to SEQ ID NO:27 or 28.

[0362] Lactoneases may contain an amino acid sequence that is at least 95% identical to SEQ ID NO:27 or 28.

[0363] Lactoneases may contain an amino acid sequence that is at least 98% identical to SEQ ID NO:27 or 28.

[0364] Lactoneases may contain an amino acid sequence that is at least 99% identical to SEQ ID NO:27 or 28.

[0365] Lactoneases may contain an amino acid sequence that is 100% identical to SEQ ID NO:27 or 28.

[0366] F. Deacetylated chitosanase

[0367] Enzymes can include deacetylated chitosanases.

[0368] Deacetylated chitosanases can be used for any of the plant growth stimulation or plant health promotion purposes described herein, but are particularly useful for increasing nutrient uptake and enhancing plant growth. This, in turn, leads to increased crop yield, improved early-season vigor, and reduced susceptibility to early-season stress. Deacetylated chitosanases can also be used to protect plants from pathogens.

[0369] Deacetylated chitosanases may include exo-1,4-β-D-glucosidase, endo-1,4-β-d-glucosidase, or combinations thereof.

[0370] Deacetylated chitosanases may include Bacillus subtilis deacetylated chitosanases, Streptomyces deacetylated chitosanases, or any combination thereof.

[0371] Deacetylated chitosanase may contain an amino acid sequence that has at least 70% identity with SEQ ID NO:29 or 124.

[0372] The deacetylated chitosan polysaccharide enzyme may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:29 or 124.

[0373] Deacetylated chitosanase may contain an amino acid sequence that has at least 80% identity with SEQ ID NO:29 or 124.

[0374] The deacetylated chitosanase may contain an amino acid sequence that has at least 85% identity with SEQ ID NO:29 or 124.

[0375] Deacetylated chitosanase may contain an amino acid sequence that is at least 90% identical to SEQ ID NO:29 or 124.

[0376] The deacetylated chitosanase may contain an amino acid sequence that has at least 95% identity with SEQ ID NO:29 or 124.

[0377] The deacetylated chitosanase may contain an amino acid sequence that has at least 98% identity with SEQ ID NO:29 or 124.

[0378] The deacetylated chitosan polysaccharide enzyme may contain an amino acid sequence that has at least 99% identity with SEQ ID NO:29 or 124.

[0379] Deacetylated chitosanase may contain an amino acid sequence that is 100% identical to SEQ ID NO:29 or 124.

[0380] G. protease

[0381] Enzymes can include proteases.

[0382] Proteases can be used for any of the plant growth stimulation or plant health promotion purposes described herein, but are particularly useful for increasing nutrient uptake and stimulating plant growth. This, in turn, leads to increased crop yield, improved early-season vigor, and reduced susceptibility to early-season stress. Proteases can also be used to protect plants from pathogens.

[0383] Proteases may include subtilisin, acidic protease, alkaline protease, proteolytic enzyme, peptidase, endopeptidase, exopeptidase, thermophilic protease, papain, pepsin, trypsin, streptomycin, carboxylase, serine protease, glutamate protease, aspartic protease, cysteine ​​protease, threonine protease, asparagine protease, histidine protease, metalloproteinase, or any combination thereof.

[0384] For example, proteases may include cysteine ​​proteases, serine proteases, threonine proteases, aspartic proteases, asparagine proteases, metalloproteinases, glutamate proteases, or any combination thereof.

[0385] For example, proteases can include metalloproteinases, serine proteases, aspartic proteases, histidine proteases, or any combination thereof.

[0386] The protease is preferably not composed of methionine aminopeptidase.

[0387] The protease preferably does not contain methionine aminopeptidase.

[0388] Proteases may include Bacillus proteases (e.g., Bacillus subtilis protease), Aspergillus proteases, or combinations thereof.

[0389] The protease may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO.46–48 and 127.

[0390] The protease may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO.46–48 and 127.

[0391] The protease may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO.46–48 and 127.

[0392] The protease may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO.46–48 and 127.

[0393] The protease may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.46–48 and 127.

[0394] The protease may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO.46–48 and 127.

[0395] The protease may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO.46–48 and 127.

[0396] The protease may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO.46–48 and 127.

[0397] The protease may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO.46–48 and 127.

[0398] H. glucanase

[0399] Enzymes can include glucanase.

[0400] Glucanases can be used for any of the plant growth stimulation or plant health promotion purposes described herein, but are particularly useful for increasing nutrient uptake and stimulating plant growth. This, in turn, leads to increased crop yield, improved early-season vigor, and reduced sensitivity to early-season stress. Glucanases can also be used to protect plants from pathogens and reduce their sensitivity to environmental stresses.

[0401] Glucanases use water to break the chemical bonds between individual glucose molecules in the glucan chain, which is a long-chain polysaccharide. Glucans can be classified into two types: α-glucans, which are mainly composed of the α-chains of glucose molecules; and β-glucans, which are mainly composed of the β-chains of glucose molecules. Common α-glucans include glucan, glycogen, pullulan, and starch. α-glucans typically consist of combinations of α1,4; α1,6, and / or α1,3 glucans and branches. Glucanases specific to cleaving α-bonds are called α-glucanases. β-glucanases are specific to the β-bonds between glucans. Common β-glucans include cellulose, laminarin, lichen polysaccharides, and yeast glucans. β-glucans are commonly found between glucose molecules in the β-chains. Glucanases can be "exo- or "endo-" depending on the location of the polysaccharide cleavage. α-, β-, exo-, and endo-glucanases all effectively stimulate plant growth.

[0402] Glucanases can include endoglucanases, exoglucanases, or combinations thereof.

[0403] Glucanases include α-glucanase, β-glucanase, or combinations thereof.

[0404] If the glucanase includes α-glucanase, then the α-glucanase may include amylase, α-1,4-glucanase, α-1,6-glucanase, or any combination thereof.

[0405] If the glucanase includes β-glucanase, then the β-glucanase may include end-β-glucanase, ex-β-glucanase, or a combination thereof.

[0406] β-glucanase may include β-1,3-glucanase, β1,3 / 1,4-glucanase, β-1,4-glucanase, β-1,6-glucanase, or any combination thereof.

[0407] For example, β-glucanase may include β-1,3-glucanase, β-1,4-glucanase, or a combination thereof.

[0408] β-1,3-glucanase may include β-1,3-endoglucanase.

[0409] β-1,4-glucanase may include β-1,4-endoglucanase.

[0410] Glucanases may include cellulases, glycoside hydrolases, xyloglucans: xylose glucosyltransferases, cycloheptaneglucans, oligoglucans β-glucosidases, cyclohexosylglucases, xyloseglucans, cellulosic 1,4-β-cellobiosidases, glucan end-1,3-β-D-glucosidases, cyclomaltodextrinases, glucan 1,3-β-glucosidases, glucan end-1,3-α-glucosidases, end-1,3(4)-β-glucanases, ex-β-1,4-glucanases, laminarinases, glucan 1,4-β-glucosidases, glucan end-1,6-β-glucosidases, glucan 1,3-α-glucosidases, amyloprolases, laminarinases, or any combination thereof.

[0411] Glucanases can include non-cellulose-degrading glucans.

[0412] In any method, seed, or composition, the dextranase comprises a non-cellulose-degrading dextranase, which may comprise xylose-glucanase, sphagnanase, amylase, starch-glucanase, amylase, laminarinase, β-1,3-glucanase, β-1,6-glucanase, β-1,3 / 1,4-glucanase, α-1,4-glucanase, α-1,6-glucanase, or any combination thereof.

[0413] If the glucanase includes xylose glucanase, then the xylose glucanase may include xylose glucan-specific end-β-1,4-glucanase, xylose glucan-specific ex-β-1,4-glucanase, or a combination thereof.

[0414] Xylose glucanases can include Bacillus glucanases.

[0415] If the dextranase includes xylose-glucanase (e.g., SEQ ID NO:125), the method may also include applying mannanase (e.g., SEQ ID NO:128) to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0416] If the dextranase contains xylose-glucanase (e.g., SEQ ID NO:125), then mannanase (e.g., SEQ ID NO:128) can also be used to treat the seeds.

[0417] If the dextranase comprises xylose-glucanase (e.g., SEQ ID NO:125), the composition may also comprise mannanase (e.g., SEQ ID NO:128).

[0418] Xylose glucanase and mannanase can be present in synergistically effective amounts in the method, on seeds, or in the composition.

[0419] Glucanases can include cellulases.

[0420] Glucanases may include endocellulases, exocellulases, or combinations thereof.

[0421] Glucanases may include thermophilic bacteria glucanase, Trichoderma glucanase, Aspergillus glucanase, Bacillus-like glucanase, white snail glucanase, Bacillus glucanase, or any combination thereof.

[0422] For example, glucanases can include Bacillus circulans glucanase, Bacillus subtilis glucanase (e.g., Bacillus subtilis endoglucanase or Bacillus subtilis β-glucosidase), Bacillus thuringiensis glucanase (e.g., Bacillus thuringiensis endoglucanase or Bacillus thuringiensis β-glucosidase), Bacillus cereus glucanase (e.g., Bacillus cereus endoglucanase or Bacillus cereus β-glucosidase), and others. Trichoderma reesei glucanase (e.g., Trichoderma reesei cellulase or Trichoderma reesei β-1,4-endoglucanase), Bacillus clausti glucanase (e.g., Bacillus clausti endoglucanase or Bacillus clausti β-glucosidase), white jade snail glucanase (e.g., white jade snail β-1,3-endoglucanase), thermolytic cellulose-degrading bacteria glucanase (e.g., thermolytic cellulose-degrading bacteria β-1,4-endoglucanase), or any combination thereof.

[0423] The dextranase may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0424] The dextranase may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0425] The dextranase may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0426] The dextranase may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0427] The dextranase may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0428] The dextranase may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0429] The dextranase may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0430] The dextranase may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0431] The dextranase may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO. 30–45, 125 and 126.

[0432] When dextranase is applied in a formulation or in which seeds are coated with a seed coating formulation containing dextranase, the formulation may suitably contain additional agricultural chemicals and / or microbial inoculants. For example, the formulation may suitably contain fungicides, insecticides, nematicides, fertilizers, plant hormones, bacterial inoculants, fungal inoculants, or any combination thereof. Specific fungicides, insecticides, nematicides, fertilizers, plant hormones, bacterial inoculants, and fungal inoculants are described in Section XVI below.

[0433] I. Phytase

[0434] Enzymes can include phytase.

[0435] Plant enzymes act on phytic acid in the soil, a source of free phosphate used for plant growth. Phytase removes selected phosphates from phytic acid, and the released phosphates can be absorbed by nearby plants.

[0436] If the enzyme contains phytase, then the phytase can contain wheat phytase.

[0437] Phytase may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO. 132–134.

[0438] Phytase may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO. 132–134.

[0439] Phytase may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO. 132–134.

[0440] Phytase may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 132–134.

[0441] Phytase may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.132–134.

[0442] Phytase may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO. 132–134.

[0443] Phytase may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO. 132–134.

[0444] Phytase may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO. 132–134.

[0445] Phytase may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO. 132–134.

[0446] Phytase may comprise a mixture of phytases containing SEQ ID NO. 132, 133 and 134.

[0447] J. acid phosphatase

[0448] Enzymes can include acid phosphatases.

[0449] Acid phosphatases act on insoluble and less soluble forms of phosphate in the soil and release them for absorption by plants.

[0450] If an enzyme contains acid phosphatase, then acid phosphatase can include wheat acid phosphatase.

[0451] Acid phosphatases may contain an amino acid sequence that is at least 70% identical to SEQ ID NO:130 or 131.

[0452] Acid phosphatases may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:130 or 131.

[0453] Acid phosphatases may contain an amino acid sequence that is at least 80% identical to SEQ ID NO:130 or 131.

[0454] Acid phosphatases may contain an amino acid sequence that has at least 85% identity with SEQ ID NO:130 or 131.

[0455] Acid phosphatases may contain an amino acid sequence that is at least 90% identical to SEQ ID NO:130 or 131.

[0456] Acid phosphatases may contain an amino acid sequence that is at least 95% identical to SEQ ID NO:130 or 131.

[0457] Acid phosphatases may contain an amino acid sequence that is at least 98% identical to SEQ ID NO:130 or 131.

[0458] Acid phosphatases may contain an amino acid sequence that is at least 99% identical to SEQ ID NO:130 or 131.

[0459] Acid phosphatases may contain an amino acid sequence that is 100% identical to SEQ ID NO:130 or 131.

[0460] Acid phosphatase may comprise a mixture of acid phosphatases containing SEQ ID NO. 130 and 131.

[0461] In any method described herein involving the use of acid phosphatase, the method may further include applying a second enzyme to a plant growth medium, a plant, a plant seed, or an area surrounding a plant seed.

[0462] In the plant seeds treated or coated with acid phosphatase as described in this article, the seeds may be further treated or coated with a second enzyme.

[0463] Any composition described herein that contains acid phosphatase also contains a second enzyme.

[0464] The second enzyme may comprise lipase, phospholipase, glucanase, xylanase, pectinase, mannanase, sphagnanase, or any combination thereof. Lipase, phospholipase, glucanase, xylanase, pectinase, mannanase, or sphagnanase may comprise any one of the lipase, phospholipase, glucanase, xylanase, pectinase, mannanase, or sphagnanase described herein.

[0465] K. pectinase

[0466] Enzymes can include pectinase.

[0467] Pectinase acts on pectin and related polysaccharides to release a small amount of sugar. The plant then absorbs this small amount of sugar as a carbon source and can also feed the native microorganisms surrounding the plant.

[0468] If the enzyme contains pectinase, then the pectinase can contain pectinase.

[0469] For example, pectinase can include Aspergillus niger pectinase.

[0470] Pectinase may contain an amino acid sequence that has at least 70% identity with SEQ ID NO:129.

[0471] Pectinase may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:129.

[0472] Pectinase may contain an amino acid sequence that has at least 80% identity with SEQ ID NO:129.

[0473] Pectinase may contain an amino acid sequence that has at least 85% identity with SEQ ID NO:129.

[0474] Pectinase may contain an amino acid sequence that is at least 90% identical to SEQ ID NO:129.

[0475] Pectinase may contain an amino acid sequence that has at least 95% identity with SEQ ID NO:129.

[0476] Pectinase may contain an amino acid sequence that has at least 98% identity with SEQ ID NO:129.

[0477] Pectinase may contain an amino acid sequence that is at least 99% identical to SEQ ID NO:129.

[0478] Pectinase may contain an amino acid sequence that is 100% identical to SEQ ID NO:129.

[0479] L. mannanase

[0480] Enzymes can include mannanase.

[0481] Mannanase acts on glucomannan and related polysaccharides to release small amounts of sugar. Plants absorb these small amounts of sugar as a carbon source and can also feed the native microorganisms around the plant.

[0482] If the enzyme contains mannanase, then the mannanase can contain Bacillus mannanase.

[0483] Mannanase may contain an amino sequence that has at least 70% identity with SEQ ID NO:128.

[0484] Mannanase may contain an amino sequence that has at least 75% identity with SEQ ID NO:128.

[0485] Mannanase may contain an amino sequence that has at least 80% identity with SEQ ID NO:128.

[0486] Mannanase may contain an amino sequence that has at least 85% identity with SEQ ID NO:128.

[0487] Mannanase may contain an amino sequence that has at least 90% identity with SEQ ID NO:128.

[0488] Mannanase may contain an amino sequence that has at least 95% identity with SEQ ID NO:128.

[0489] Mannanase may contain an amino sequence that has at least 98% identity with SEQ ID NO:128.

[0490] Mannanase may contain an amino sequence that is at least 99% identical to SEQ ID NO:128.

[0491] Mannanase may contain an amino sequence that is 100% identical to SEQ ID NO:128.

[0492] M.ACC deaminase

[0493] Enzymes may include ACC deaminase.

[0494] ACC deaminase may include any of the enzymes described in Part II above.

[0495] ACC deaminase may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO.7–12, 113 and 114.

[0496] ACC deaminase may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0497] ACC deaminase may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0498] ACC deaminase may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0499] ACC deaminase may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0500] ACC deaminase may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO.7–12, 113 and 114.

[0501] ACC deaminase may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0502] ACC deaminase may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0503] ACC deaminase may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO. 7–12, 113 and 114.

[0504] N. extended protein

[0505] Expansion proteins contribute to the expansion of plant walls during plant growth. Therefore, expansion proteins are applicable to any method used to stimulate plant growth as described in this article.

[0506] The extended protein may contain an amino acid sequence that is at least 70% identical to SEQ ID NO:74.

[0507] The extended protein may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:74.

[0508] The extended protein may contain an amino acid sequence that is at least 80% identical to SEQ ID NO:74.

[0509] The extended protein may contain an amino acid sequence that has at least 85% identity with SEQ ID NO:74.

[0510] The extended protein may contain an amino acid sequence that is at least 90% identical to SEQ ID NO:74.

[0511] The extended protein may contain an amino acid sequence that is at least 95% identical to SEQ ID NO:74.

[0512] The extended protein may contain an amino acid sequence that is at least 98% identical to SEQ ID NO:74.

[0513] The extended protein may contain an amino acid sequence that is at least 99% identical to SEQ ID NO:74.

[0514] The extended protein may contain an amino acid sequence that is 100% identical to SEQ ID NO:74.

[0515] VIII. Use of fertilizers and / or biostimulants in relation to the methods, seeds, and compositions described herein

[0516] In any of the methods described herein, the method may also include applying fertilizers, biostimulants, or combinations thereof to plant growth media, plants, plant seeds, or the area surrounding plants or plant seeds.

[0517] For any of the plant seeds described herein, the plant seeds may also be treated or coated with fertilizers, biostimulants, or combinations thereof.

[0518] For any method, seed, or composition described herein, the fertilizer may contain nitrogen, phosphates (e.g., monoammonium phosphate, diammonium phosphate, orthophosphate, polyphosphate, or any combination thereof), potassium (e.g., potassium acetate), zinc, iron, selenium, boron, copper, or any combination thereof.

[0519] For example, fertilizers can contain 12% ammonia nitrogen and 58% available phosphate.

[0520] Other fertilizers described in Section XVI below may be used.

[0521] Biostimulants may include gibberellic acid, indole-3-butyric acid, kinetin, auxin, auxin homologs or derivatives, or any combination thereof.

[0522] In any method or seed involving the use of fertilizers and / or biostimulants, the enzyme suitably comprises acid phosphatase, phospholipase, mannanase, dextranase, or any combination thereof. Acid phosphatase, phospholipase, mannanase, or dextranase may comprise any one of the acid phosphatase, phospholipase, mannanase, or dextranase described herein.

[0523] IX. Enzyme preparations

[0524] In any of the methods, seeds, or compositions described herein involving the use of free enzymes and / or extended proteins, the enzymes or extended proteins may comprise crude cell per-extracts containing enzymes or extended proteins, partially purified enzymes or extended proteins, or substantially purified enzymes or extended proteins.

[0525] In any of the methods, seeds, or compositions described herein involving the use of free enzymes and / or extended proteins, the enzymes or extended proteins preferably do not contain enzymes or extended proteins that bind to the outer spore wall of members of the Bacillus cereus family.

[0526] In any of the methods, seeds, or compositions described herein that involve the use of free enzymes and / or extended proteins, the enzymes or extended proteins preferably do not bind to the outer spore wall of intact Bacillus cereus family members.

[0527] X. Immobilization of enzymes and / or extended proteins

[0528] In any method, seed, or composition described herein that includes the use of free enzymes and / or extended proteins, the enzymes or extended proteins may comprise enzymes or extended proteins immobilized on a matrix or support.

[0529] The matrix or support may contain charcoal, biochar, nano-carbon, agarose, alginate, cellulose, cellulose derivatives, silica, plastic, stainless steel, glass, polystyrene, ceramic, dolomite, clay, diatomaceous earth, talc, polymer, gum, water-dispersible material, or any combination thereof.

[0530] Compared to the release rate of the same immobilized enzyme or extended protein under the same conditions, the immobilization of the enzyme or extended protein on a matrix or support preferably results in a slower release of the enzyme or extended protein into the environment or onto the plant or plant seed.

[0531] XI. Methods for preparing free enzymes

[0532] Free enzymes can be prepared using many standard biochemical and molecular biological methods commonly known in the art. For example, a gene encoding the enzyme can be amplified from chromosomal DNA using polymerase chain reaction (PCR) and cloned into a suitable vector (e.g., a plasmid vector). The vector appropriately contains multiple cloning sites into which DNA molecules encoding fusion proteins can be readily inserted. The vector also appropriately contains selection markers, such as antibiotic resistance genes, to facilitate the identification and isolation of transformed, transfected, or vector-paired bacteria. When the vector is a plasmid, the plasmid also appropriately contains an origin of replication. Alternatively, the DNA encoding the enzyme protein can be integrated into the chromosomal DNA of the microbial host.

[0533] The host can then be cultured and the enzyme harvested from the culture. The enzyme can be partially or substantially purified using cell extracts or standard biochemical techniques.

[0534] The host can then be cultured and the enzyme harvested from the culture. Crude cell extracts can be used, or the enzyme can be partially or substantially purified using standard biochemical techniques. Suitable hosts for large-scale enzyme production include, but are not limited to, Bacillus species (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus megaterium, Bacillus thuringiensis, Clostridium, Bacillus cereus, or Bacillus mycosis fungoides), Escherichia coli, Aspergillus niger, Aspergillus oryzae, Streptomyces species, Klebsiella species, Mucor species, Rhizopus species, Mucor species, Kluyveromyces species, Candida species, Penicillium chrysogenum, Trichoderma species, Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yersinia lipolytica, Schizosacchari sacchari, and Candida przewalskii.

[0535] Enzymes can be collected from whole fermented broths or partially or substantially purified from fermented batch cultures.

[0536] Alternatively, enzymes can be produced by screening microorganisms and selecting those expressing high levels of the enzyme. This can be accomplished through initial selection, enrichment, and / or screening in a nutrient medium containing the enzyme substrate as a nutrient source for the microorganism. Further selection is typically performed using differential nutrient media with indicators to demonstrate the enzyme levels and activities produced by the identified microorganisms. These microorganisms can be mutated, and isolates producing increased levels of these enzymes can be screened. These microorganisms can be used in both high-volume and continuous fermentation methods to produce and secrete sufficient quantities of the enzyme. Optimizing the fermentation process and conditions can generally increase the output of the microorganism.

[0537] Eukaryotic cell lines can also be used to produce enzymes at high levels. Many of these cell lines can be engineered to secrete high levels of enzymes, offering the advantages of varying levels of key post-translational modifications and reduced host enzyme production problems. These can also be scaled up to larger cell culture production scales, with the enzymes purified and processed as described above. Examples of suitable eukaryotic cell lines for enzyme production include, but are not limited to: insect cells derived from insects, such as silkworms, hepialus moths, fall armyworms, white armyworms, or Drosophila melanogaster; and vertebrate cell lines derived from vertebrates, such as mice, rats, hamsters, humans, or dogs.

[0538] Other potential sources of enzymes include cell-free protein expression vectors, including those derived from animal, bacterial, fungal, and plant sources.

[0539] Genetically modified organisms such as plants, rabbits, mice, chickens, or frogs can also be used to produce recombinant enzymes. For example, plants can be modified to overexpress enzymes, and the enzymes can then be harvested from the plants and purified or used as crude extracts. This production system allows for low-cost expression of enzymes and provides a source of material for delivery to plants. These methods have the added advantage of being easy to scale up and requiring minimal investment.

[0540] In each of these production systems, the yield and quality of the desired enzyme can be increased through genetic modification and formulation methods. For example, genetic engineering may involve creating high-level expression cassettes and production systems, removing protease and degradation genes from the production microorganisms, optimizing the enzyme's thermostability and long-term storage stability, and enhancing the enzyme's or the production microorganism's ability to secrete mature enzymes into the culture medium for easy collection and use. Additionally, expression strains can be used to induce point mutations, which can lead to an increased ability to produce sufficient or increased enzyme levels. In some cases, the production microorganisms can also be used and delivered to plant seeds, the vicinity of the plant, plant roots, or nearby areas to obtain the desired effect in situ on the plant.

[0541] Other sources of enzymes include extraction from animal, plant, insect, seaweed, or other biological extracts. Common sources of enzymes produced and / or purified on an industrial scale in this manner include internal tissues of pigs and cattle, such as the abomasum, liver, mucosa, and pancreas, as well as plant sources such as papaya. Another example is the purification of glucanase from barley.

[0542] Many commercially available enzymes come from tissues with high levels of their target enzymes and can be used as is or in purified form for agricultural applications.

[0543] XII. Signal peptide

[0544] Any signal peptide can be used to modify any enzyme described herein, causing the enzyme to be secreted from the host microorganism that expresses it. The type of signal peptide used depends primarily on the characteristics of the host microorganism, as the secretion mechanisms of different microorganisms vary in their ability to recognize specific signal peptides. Exemplary signal peptide sequences, along with bacterial species in nature where signal peptides are found, are provided in Table 16 below. The signal peptide will cause the secretion of its associated protein in the bacterial genus where it is found, as well as closely related genera. For example, a signal sequence from Bacillus thuringiensis will cause the secretion of a protein in bacteria of the genus Bacillus, as well as in bacteria of the genera Bacillus-like and Lysinobacter.

[0545] For ease of reference, the amino acid sequence of an exemplary signal peptide is described, which can be added to any enzyme or extended protein described herein to induce its secretion. Microorganisms expressing this are shown in Table 16 below. Any signal peptide listed in Table 16 below can be added to the N-terminus of any enzyme or extended protein described herein to induce its secretion.

[0546] Table 16. Amino acid sequences of signal peptides

[0547]

[0548]

[0549] For example, the signal peptide may contain an amino acid sequence that is at least 70% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0550] For example, the signal peptide may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0551] For example, the signal peptide may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0552] For example, the signal peptide may contain an amino acid sequence that has at least 85% identity with any one of SEQ ID NO.49–73, 135 and 137–147.

[0553] For example, the signal peptide may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0554] For example, the signal peptide may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0555] For example, the signal peptide may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0556] For example, the signal peptide may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO.49–73, 135, and 137–147.

[0557] For example, the signal peptide may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO.49–73, 135 and 137–147.

[0558] Signal peptides applicable to Bacillus, Bacillus-like bacteria, or Lysinobacteria are shown in SEQ ID NO. 49–51, 54, 56–73, 135, 139, 140, and 142.

[0559] Therefore, for example, the signal peptide may contain an amino acid sequence that has at least 70% identity with any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0560] The signal peptide may contain an amino acid sequence that is at least 75% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0561] The signal peptide may contain an amino acid sequence that is at least 80% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0562] The signal peptide may contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0563] The signal peptide may contain an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0564] The signal peptide may contain an amino acid sequence that is at least 95% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0565] The signal peptide may contain an amino acid sequence that is at least 98% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0566] The signal peptide may contain an amino acid sequence that is at least 99% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0567] The signal peptide may contain an amino acid sequence that is 100% identical to any one of SEQ ID NO.49–51, 54, 56–73, 135, 139, 140 and 142.

[0568] Therefore, for example, when the signal peptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO. 49–51, 54, 56–73, 135, 139, 140, and 142, the microorganism that appropriately expresses the enzyme or extended protein comprises Bacillus, Bacillus-like bacteria, Lysinobacter, Pseudomonas, or any combination thereof.

[0569] For example, microorganisms may include Bacillus mycosisviridae, Bacillus pseudomycosisviridae, Bacillus cereus, Bacillus sturdius, Bacillus thuringiensis, Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus circumflex, Bacillus curvatureis, Bacillus nissiellus, Bacillus pumilus, Bacillus licheniformis, Bacillus longisineus, Bacillus spheroidis, Bacillus fusiformis, or any combination thereof.

[0570] The microorganisms preferably include Bacillus thuringiensis, Bacillus cereus, Bacillus pseudomycosis, Bacillus mycosis, Bacillus longisinus, Bacillus lysinus, Bacillus fusiformis, Bacillus spheroidis, or any combination thereof.

[0571] The signal peptide is preferably located at the amino terminus of the enzyme or extended protein.

[0572] XIII. Recombinant Microorganisms

[0573] Recombinant vitamins, preparations and compositions containing recombinant microorganisms, methods for using recombinant microorganisms, and seeds treated with recombinant microorganisms are as described above.

[0574] In any recombinant microorganism, formulation, composition, method, or seed described herein, the enzyme or extended protein may be expressed under the control of a constitutive promoter.

[0575] In any recombinant microorganism, formulation, composition, method, or seed described herein, the enzyme or extended protein may be expressed under the control of an inducible promoter.

[0576] In any recombinant microorganism, formulation, composition, method, or seed described herein, the recombinant microorganism may comprise Bacillus, Bacillus-like bacteria, Lysinobacillus, Aspergillus, Gastromycosis, Pseudomonas, Arthrobacter, Paracoccus, Rhizobium, Rhizobium, Azotobacter, Enterobacter, Escherichia, or any combination thereof.

[0577] If the recombinant microorganism contains a recombinant spore-forming microorganism, then the recombinant spore-forming microorganism may contain Bacillus, Bacillus-like bacteria, Lysinobacillus, Aspergillus, Gynotrophomonas fungi, or any combination thereof.

[0578] For any recombinant microorganism, formulation, composition, method or seed described herein, the recombinant microorganism suitably comprises Bacillus, Bacillus-like bacteria, Lysinobacter, or any combination thereof.

[0579] For example, recombinant microorganisms may include Bacillus mycosisviridae, Bacillus pseudomycosisviridae, Bacillus cereus, Bacillus thuringiensis, Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus circumflex, Bacillus curvatureis, Bacillus nissiellus, Bacillus pumilus, Bacillus licheniformis, Bacillus spheroidis, Bacillus fusiformis, or any combination thereof.

[0580] Recombinant microorganisms suitably include Bacillus thuringiensis, Bacillus cereus, Bacillus pseudomycosis, Bacillus longisinus, Bacillus spheroidis, Bacillus fusiformis, or combinations thereof.

[0581] For any recombinant microorganism, formulation, composition, method or seed described herein, the recombinant microorganism may comprise a bacterial strain that promotes plant growth, a bacterial endoparasitic strain, or a strain that promotes both plant growth and endoparasitism.

[0582] The strain can produce insecticidal toxins (e.g., Cry toxin), fungicidal compounds (e.g., β-1,3-glucanase, deacetylated chitosanase, lysozyme, or combinations thereof), nematicidal compounds (e.g., Cry toxin), fungicidal compounds, resistance to one or more antibiotics, contain one or more freely replicable plasmids, bind to plant roots, colonize plant roots, form biofilms, dissolve nutrients, secrete organic acids, or combinations thereof.

[0583] For example, strains may include:

[0584] (a) Bacillus aryepedodes CAP53 (NRRL No. B-50819),

[0585] (b) Bacillus aryabdominis CAP56 (NRRL No. B-50817),

[0586] (c) Bacillus curvatureis BT054 (NRRL No. B-50816),

[0587] (d) Paracoccus kohlii NC35 (NRRL No. B-50820),

[0588] (e) Bacillus mycosis fungoides BT155 (NRRL No. B-50921),

[0589] (f) Enterobacter cloacae CAP12 (NRRL No. B-50822),

[0590] (g) Bacillus niger BOBA57 (NRRL No. NRRL B-50821),

[0591] (h) Bacillus mycosis fungoides EE118 (NRRL No. B-50918),

[0592] (i) Bacillus subtilis EE148 (NRRL No. B-50927),

[0593] (j) Fecal alkaloid-producing bacteria EE107 (NRRL No. B-50920),

[0594] (k) Bacillus mycosis fungoides EE141 (NRRL NO. B-50916),

[0595] (l) Bacillus mycoides BT46-3 (NRRL No. B-50922),

[0596] (m) Member of the Bacillus cereus family EE128 (NRRL No. B-50917),

[0597] (n) Bacillus musei BT23 (NRRL No. B-50923),

[0598] (o) Member of the Bacillus cereus family EE349 (NRRL No. B-50928),

[0599] (p) Bacillus subtilis EE218 (NRRL No. B-50926),

[0600] (q) Bacillus megaterium EE281 (NRRL No. B-50925),

[0601] (r) Member of the Bacillus cereus family EE-B00377 (NRRL B-67119);

[0602] (s) Bacillus pseudomycosis EE-B00366 (NRRL B-67120),

[0603] (t) Bacillus mycoides EE-B00363 (NRRL B-67121),

[0604] (u) Bacillus pumilus EE-B00143 (NRRL B-67123),

[0605] (v) Bacillus thuringiensis EE-B00184 (NRRL B-67122),

[0606] (w) Bacillus mycosis fungoides EE116 (NRRL No. B-50919),

[0607] (x) Member of the Bacillus cereus family EE417 (NRRL No. B-50974),

[0608] (y) Bacillus subtilis EE442 (NRRL No. B-50975),

[0609] (z) Bacillus subtilis EE443 (NRRL No. B-50976),

[0610] (aa) Member of the Bacillus cereus family EE444 (NRRL No. B-50977),

[0611] (bb) Bacillus subtilis EE405 (NRRL No. B-50978),

[0612] (cc) Bacillus cereus family member EE439 (NRRL No. B-50979),

[0613] (dd) Bacillus megaterium EE385 (NRRL No. B-50980),

[0614] (ee) Bacillus cereus family member EE387 (NRRL No. B-50981),

[0615] (ff) Bacillus circulans EE388 (NRRL No. B-50982),

[0616] (gg) Bacillus thuringiensis EE319 (NRRL No. B-50983),

[0617] (hh) Member of the Bacillus cereus family EE377 (NRRL No. B-67119),

[0618] (ii) Bacillus mycosis fungoides EE363 (NRRL No. B-67121),

[0619] (jj) Pseudomycium mycosis EE366 (NRRL No. B-67120);

[0620] (kk) Bacillus thuringiensis BT013A (NRRL No. B-50924);

[0621] Or a combination thereof.

[0622] These strains have been deposited individually with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), 1815 North University Street, Peoria, Illinois 61604 USA, and identified with the NRRL accession numbers provided in parentheses. Strains (a)–(d), (f), and (g) were deposited on March 11, 2013. Strains (e), (h)–(q), (w), and (kk) were deposited on March 10, 2014. Strains (x)–(ff) were deposited on September 10, 2014. Strain (gg) was deposited on September 17, 2014. Strains (r)–(v), (hh), (ii), and (jj) were deposited on August 19, 2015. Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7.

[0623] The isolation and characterization of these strains are described in the examples below. The partial 16S ribosomal RNA sequences of each strain are provided in the sequence listing and summarized together with their SEQ ID NO in Table 17.

[0624] Table 17. Partial 16S ribosomal RNA sequences

[0625]

[0626]

[0627] Endoparasitic microorganisms can be used for enzyme expression. Although many rhizosphere microorganisms have symbiotic relationships with plants, only a small fraction of these microorganisms are able to be ingested by the plant and grow as endoparasites. Several strains of Bacillus cereus and several strains of non-Bacillus cereus have been isolated from maize seedlings and found to have the ability to grow as endoparasites in plants. Other endoparasitic microorganisms may also be useful, including but not limited to bacterial endophytes from the following genera: Fibriophyta, Corynebacterium, Brachybacterium breve, Pseudomonas, Bacillus subtilis, Enterobacter, Bacillus, Klebsiella, Mycobacterium, Lysinobacter, Pantothecinia clumps, Actinomyces, Saccharomyces, Alcaligenes, and Microbacteria. Endophytic fungi can also be used, including endophytes from the following genera: Neotyphodium, Gliocadium, Acremonium lolii, ergot, ascomycetes, Idriella, Xylariaceous, ascomycetes, deuteromycetes, Aspergillus, pseudostem mold, Wardomyces, Fusarium, Dreschrella, discochaetes, curvularia, humic mold, multi-segmented mold, and Penicillium.

[0628] Many microorganisms can colonize, live near, on, or become endophytic within plants. These microorganisms provide useful delivery mechanisms for target enzymes to plants, seeds, the vicinity of plants, or plant growth media. Microorganisms that can colonize roots or become endophytic can be screened, recombinantly modified to express or overexpress commercially produced enzymes, and applied to seeds, plants, or the vicinity of plants to enable the strain to produce target enzymes in situ (within or near the plant). These microorganisms can also be enhanced through point mutations or genetic modification to express higher or novel target enzymes to benefit plants. Point mutations can be screened by mutating the host microorganism and selecting mutants with higher enzyme expression levels using enzyme assays or selective media that identify strains that highly express the enzyme. Common strains of beneficial enzyme producers and colonizers / endophytic species include: *Bacillus sphaeroides*, *Bacillus tsutsuga*, *Bacillus lactis*, *Bacillus amyloliquefaciens*, *Bacillus cereus*, *Bacillus coagulans*, *Bacillus endoparasiticus*, *Haemophilus endogenosus*, *Kustella*, *Bacillus lactis*, *Bacillus thuringiensis*, *Bacillus jellyfish*, *Bacillus laterosporus*, *Bacillus sclerotiorum*, *Bacillus licheniformis*, *Bacillus megaterium*, *Bacillus metiens*, *Bacillus natto*, *Bacillus niger*, *Bacillus beetle*, *Bacillus pumilus*, *Bacillus sicca*, *Bacillus spheroides*, *Bacillus subtilis*, *Bacillus thuringiensis*, *Bacillus unifagellatu*, other species of the genus *Bacillus* or combinations thereof, and those listed in the categories of *Bacillus Genus* in Bergey's Manual of Systematic Bacteriology, 1st edition (1986), which are incorporated herein by reference in their entirety. Other possible strains may include, but are not limited to: *Fibromonas*, *Corynebacterium*, *Bacillus*, *Pseudomonas*, *Bacillus*, *Enterobacter*, *Bacillus*, *Klebsiella*, *Arthrobacter*, *Lysinobacter*, *Pantotheca cumulus*, *Actinomyces*, *Saccharomyces*, *Rhizobium*, *Rhizobium*, *Candida*, *Streptomyces*, *Alcaligenes*, Chromobacteriales, *Rhizobium*, *Rhizobium*, *Rhodospiralles*, Rhizobiumales, Rhizobaceae, and Microbacteria.

[0629] For any method or seed described herein, the recombinant microorganism may comprise any mixture of two or more of the recombinant microorganisms described herein.

[0630] For any recombinant microorganism, formulation, method, or seed described herein, the recombinant microorganism can be inactivated. Inactivation produces microorganisms that cannot replicate. Inactivation of microorganisms may be advantageous, for example, because it allows for the delivery of microorganisms to plants or plant growth media while reducing or eliminating the potentially harmful effects of live microorganisms on plants or the environment. Recombinant microorganisms can be inactivated by any physical or chemical means, such as by heat treatment, gamma irradiation, X-ray irradiation, UV-A irradiation, UV-B irradiation, or treatment with solvents such as glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform, or phenol, or any combination thereof.

[0631] XIV. Methods for preparing recombinant microorganisms

[0632] Recombinant microorganisms can be prepared using standard molecular biology methods known in the art. For example, the gene encoding the enzyme can be amplified by polymerase chain reaction (PCR). If a signal sequence is used, the gene encoding the enzyme can be ligated into DNA encoding the signal sequence. The gene can then be cloned into any suitable vector, such as a plasmid vector. This vector suitably contains a multiple cloning site that facilitates insertion into a DNA molecule encoding a fusion protein. The vector also suitably contains selection markers, such as antibiotic resistance genes, to facilitate identification and isolation of bacteria transformed, transfected, or paired with the vector. If the vector is a plasmid, the plasmid also suitably contains an origin of replication. Alternatively, the DNA encoding the enzyme or extended protein can be integrated into the chromosomal DNA of the microbial host.

[0633] XV. Effects on plants

[0634] In any of the methods described herein, plants grown in the presence of enzymes, expansive proteins, or microorganisms may exhibit increased growth compared to plants grown under the same conditions in the absence of enzymes, expansive proteins, or microorganisms.

[0635] For any of the seeds described herein, plants grown from seeds treated with free enzymes, expansive proteins, or microorganisms can exhibit increased growth compared to plants grown from seeds never treated with free enzymes, expansive proteins, or microorganisms under the same conditions.

[0636] For any of the methods or seeds described herein, seeds that have been treated with enzymes or microorganisms may exhibit an increased germination rate compared to seeds that have not been treated with enzymes or microorganisms under the same conditions.

[0637] In any of the methods described herein, plants grown in the presence of enzymes, expansive proteins, or microorganisms may exhibit increased nutrient uptake compared to plants grown under the same conditions in the absence of enzymes, expansive proteins, or microorganisms.

[0638] For any of the seeds described herein, plants grown from seeds treated with free enzymes, expansive proteins, or microorganisms can show increased nutrient uptake compared to plants grown from seeds never treated with free enzymes, expansive proteins, or microorganisms under the same conditions.

[0639] In any of the methods described herein, plants grown in the presence of enzymes or microorganisms may exhibit reduced susceptibility to pathogens compared to plants grown under the same conditions in the absence of enzymes or microorganisms.

[0640] For any of the seeds described herein, plants grown from seeds treated with free enzymes or microorganisms may exhibit reduced susceptibility to pathogens compared to plants grown from seeds never treated with free enzymes or microorganisms under the same conditions.

[0641] In any of the methods described herein, plants grown in the presence of enzymes or microorganisms may exhibit reduced susceptibility to environmental stress compared to plants grown under the same conditions in the absence of enzymes or microorganisms.

[0642] For any of the seeds described herein, plants grown from seeds treated with free enzymes or microorganisms may exhibit reduced susceptibility to environmental stress compared to plants grown from seeds never treated with free enzymes or microorganisms under the same conditions.

[0643] For example, plants can exhibit reduced sensitivity to drought, floods, heat, cold, salt, heavy metals, low pH, high pH, ​​or any combination thereof.

[0644] In any of the methods described herein, plants grown in the presence of enzymes, expansive proteins, or microorganisms may exhibit increased nutrient content compared to plants grown under the same conditions in the absence of enzymes, expansive proteins, or microorganisms.

[0645] For any of the seeds described herein, seeds treated with free enzymes, expansive proteins, or microorganisms, or microorganisms or plants grown from seeds treated with free enzymes, expansive proteins, or microorganisms, may exhibit increased nutrient content compared to seeds grown under the same conditions without treatment with free enzymes, expansive proteins, or microorganisms, or plants grown from seeds never treated with free enzymes, expansive proteins, or microorganisms.

[0646] For example, nutrients can include polysaccharides, proteins, phytic acid, phosphates, phospholipids, or any combination thereof.

[0647] In any of the methods described herein, plants grown in the presence of enzymes or microorganisms may exhibit increased root nodules compared to plants grown under the same conditions in the absence of enzymes or microorganisms.

[0648] For any of the seeds described herein, plants grown from seeds treated with free enzymes or microorganisms can exhibit increased root nodules compared to plants grown from seeds never treated with free enzymes or microorganisms under the same conditions.

[0649] In any of the methods described herein, plants grown in the presence of enzymes or microorganisms can exhibit slower fruit ripening than plants grown under the same conditions in the absence of enzymes or microorganisms.

[0650] For any of the seeds described herein, plants grown from seeds treated with free enzymes or microorganisms can exhibit slower fruit ripening than plants grown from seeds never treated with free enzymes or microorganisms under the same conditions.

[0651] In any of the methods described herein, plants grown in the presence of enzymes, expansive proteins, or microorganisms can exhibit higher crop yields than plants grown under the same conditions in the absence of enzymes, expansive proteins, or microorganisms.

[0652] For any of the seeds described in this article, plants grown from seeds treated with free enzymes or microorganisms can exhibit higher crop yields than plants grown from seeds never treated with free enzymes or microorganisms under the same conditions.

[0653] In any of the methods described herein, plants grown in the presence of enzymes or microorganisms may exhibit altered leaf wilting compared to plants grown under the same conditions in the absence of enzymes or microorganisms.

[0654] For any of the seeds described herein, plants grown from seeds treated with free enzymes or microorganisms may exhibit altered leaf wilting compared to plants grown from seeds never treated with free enzymes or microorganisms under the same conditions.

[0655] Slower leaf wilting can lead to later, higher levels of photosynthesis during the season, resulting in more photosynthetic products, fuller and larger grains, and / or increased yield.

[0656] XVI. Formulations, compositions and co-applications of agrochemicals

[0657] In any of the methods described herein, the method may include administering the enzyme, extended protein, or microorganism in a formulation comprising an agriculturally acceptable carrier.

[0658] For any of the seeds described herein, the seeds may be coated with a formulation containing free enzymes, extended proteins, or recombinant microorganisms and agriculturally acceptable carriers.

[0659] Any composition described herein may contain an agriculturally acceptable carrier.

[0660] Agriculturally acceptable carriers may include dispersants, surfactants, additives, water, thickeners, anti-sticking agents, residue decomposition products, compost, granules, diatomaceous earth, oils, colorants, stabilizers, preservatives, polymers, coatings, or combinations thereof.

[0661] Additives may include oils, gums, resins, clays, polyethylene glycols, terpenes, viscous organics, fatty acid esters, sulfated alcohols, alkyl sulfonates, petroleum sulfonates, ethanol sulfates, sodium alkylbutane, sodium thiobutanoate, polyesters, phenylacetonitrile derivatives, protein materials (e.g., dairy products, flour, soy flour, blood, albumin, gelatin, alfalfa powder, yeast extract, or any combination thereof) or any combination thereof.

[0662] Thickeners may contain long-chain alkyl sulfonates of polyethylene glycol, polyoxyethylene oleate, or any combination thereof.

[0663] Surfactants may include heavy petroleum, heavy petroleum distillates, polyol fatty acid esters, polyethoxylated fatty acid esters, arylalkyl polyethylene glycol, alkylamine acetates, alkylaryl sulfonates, polyols, alkyl phosphate esters, or any combination thereof.

[0664] Surfactants can include nonionic surfactants.

[0665] Anti-sticking agents may contain sodium salts (such as sodium monomethylnaphthalene sulfonate, sodium dimethylnaphthalene sulfonate, sodium sulfite, sodium sulfate, or any combination thereof), calcium carbonate, diatomaceous earth, or any combination thereof.

[0666] Agriculturally acceptable carriers may include gold dust, charcoal, sugar mill carbonized pressing mud, rice husks, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, starch, talc, polyvinylpyrrolidone, or any combination thereof.

[0667] The formulation or composition may comprise a seed-coating formulation or composition, a liquid formulation or composition applied to a plant or a plant growth medium, or a solid formulation or composition applied to a plant or a plant growth medium.

[0668] Seed coating formulations or compositions may comprise an aqueous or oil-based solution applied to the seed or a powdered or granular formulation applied to the seed.

[0669] Liquid formulations or compositions applied to plants or to plant growth media may contain concentrated formulations or compositions readily available.

[0670] Solid formulations or compositions applied to plants or to plant growth media may contain granular formulations or compositions or powdered reagents.

[0671] Formulations or compositions may also contain agrochemicals.

[0672] Alternatively, any method described herein may also involve applying agricultural chemicals to a plant growth medium, a plant, a plant seed, or the area surrounding a plant or plant seed.

[0673] Any of the plant seeds described in this article may also be treated or coated with agricultural chemicals.

[0674] Agricultural chemicals may include fertilizers, micronutrient fertilizers, insecticides, nematicides, herbicides, plant growth modifiers, fungicides, insecticides, molluscicides, algaecides, bacterial inoculants, fungal inoculants, plant hormones, or any combination thereof.

[0675] Bacterial inoculum may contain plant growth-promoting bacterial strains, endophytic bacterial strains, or plant growth-promoting endophytic bacterial strains.

[0676] Plant-promoting bacterial strains can produce insecticidal toxins (e.g., Cryotoxin), fungicidal compounds (e.g., β-1,3-glucanase, deacetylated chitosanase, lysozyme, or combinations thereof), nematicidal compounds (e.g., Cryotoxin), fungicidal compounds, resistance to one or more antibiotics, contain one or more freely replicable plasmids, bind to plant roots, colonize plant roots, form biofilms, dissolve nutrients, secrete organic acids, or combinations thereof.

[0677] Bacterial strains that promote plant growth may include *Bacillus aryepasis* CAP53 (NRRL No. B-50819), *Bacillus aryepasis* CAP56 (NRRL No. B-50817), *Bacillus curvularia* BT054 (NRRL No. B-50816), *Paragonimococcus kohlii* NC35 (NRRL No. B-50820), *Bacillus mycoides* BT155 (NRRL No. B-50921), *Enterobacter cloacae* CAP12 (NRRL No. B-50822), *Bacillus niger* BOBA57 (NRRL No. NRRL B-50821), *Bacillus mycoides* EE118 (NRRL No. B-50918), *Bacillus subtilis* EE148 (NRRL No. B-50927), *Bacillus faecalis* EE107 (NRRL No. B-50920), and *Bacillus mycoides* EE141 (NRRL No. B-50920). Bacillus cereus (NRRL No. B-50916), Bacillus mycosis BT46-3 (NRRL No. B-50922), Bacillus cereus EE128 (NRRL No. B-50917), Bacillus maseiformis BT23 (NRRL No. B-50923), Bacillus cereus EE349 (NRRL No. B-50928), Bacillus subtilis EE218 (NRRL No. B-50926), Bacillus megaterium EE281 (NRRL No. B-50925), Bacillus cereus EE-B00377 (NRRL B-67119); Pseudomonas mycosis EE-B00366 (NRRL B-67120), Bacillus mycosis EE-B00363 (NRRL B-67121), Bacillus pumilus EE-B00143 (NRRL No. B-50916), Bacillus mycosis BT46-3 (NRRL No. B-50922), Bacillus cereus EE128 (NRRL No. B-50917), Bacillus maseiformis BT23 (NRRL No. B-50923), Bacillus cereus EE349 (NRRL No. B-50928), Bacillus subtilis EE218 (NRRL No. B-50926), Bacillus megaterium EE281 (NRRL No. B-50925), Bacillus cereus EE-B00377 (NRRL B-67119); Pseudomonas mycosis EE-B00366 (NRRL B-67120), Bacillus mycosis EE B-67123), or Bacillus thuringiensis EE-B00184 (NRRL B-67122), Bacillus mycosis fungoides EE116 (NRRL No. B-50919), Bacillus cereus family member EE417 (NRRL No. B-50974), Bacillus subtilis EE442 (NRRL No. B-50975), Bacillus subtilis EE443 (NRRL No. B-50976), Bacillus cereus family member EE444 (NRRL No. B-50977), Bacillus subtilis EE405 (NRRL No. B-50978), Bacillus cereus family member EE439 (NRRL No. B-50979), Bacillus megaterium EE385 (NRRL No. B-50980), Bacillus cereus family member EE387 (NRRL B-67123), or Bacillus thuringiensis EE-B00184 (NRRL B-67122), Bacillus mycosis fungoides EE116 (NRRL No. B-50919), Bacillus cereus family member EE417 (NRRL No. B-50974), Bacillus subtilis EE442 (NRRL No. B-50975), Bacillus subtilis EE443 (NRRL No. B-50978), Bacillus cereus family member EE439 (NRRL No. B-50979), Bacillus megaterium EE385 (NRRL No. B-50980), Bacillus cereus family member EE387 (NRRL B-67123), or Bacillus cereus family member EE387 (NR Bacillus circulans No. B-50981), Bacillus circulans EE388 (NRRL No. B-50982), Bacillus thuringiensis EE319 (NRRL No. B-50981).Bacillus cereus (B-50983), Bacillus cereus family members EE377 (NRRL No. B-67119), Bacillus mycosis fungoides EE363 (NRRL No. B-67121), Bacillus pseudomycosis fungoides EE366 (NRRL No. B-67120), Bacillus thuringiensis BT013A (NRRL No. B-50924), or any combination thereof.

[0678] Agricultural chemicals can include fertilizers.

[0679] Fertilizers can include liquid fertilizers or dry fertilizers.

[0680] Agricultural chemicals may include micronutrient fertilizer materials, which include boric acid, borates, boron glaze, copper sulfate, copper glaze, copper chelate, sodium tetraborate decahydrate, ferric sulfate, ferric oxide, ferric ammonium sulfate, iron glaze, iron chelate, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese glaze, sodium molybdate, molybdic acid, zinc sulfate, zinc oxide, zinc carbonate, zinc glaze, zinc phosphate, zinc chelate, or any combination thereof.

[0681] Agricultural chemicals may include insecticides, which include organophosphates, amino acid formates, pyrethroids, acaricides, alkyl phthalates, boric acid, borates, fluorides, sulfur, halogenated aromatic substituted ureas, hydrocarbon esters, biologically based insecticides, or any combination thereof.

[0682] Agricultural chemicals may include herbicides, which contain chlorophenoxy compounds, nitrophenol compounds, nitrocresol compounds, dipyridyl compounds, acetamide, aliphatic acids, acylaniline, benzamide, benzoic acid, benzoic acid derivatives, anisic acid, anisic acid derivatives, benzyl nitrile, benzothiadiazinone dioxide, thiocarbamate, carbamate, phenylcarbamate, chloropyridyl compounds, chlorohexenone derivatives, dinitroaminobenzene derivatives, fluorodinitrotoluidine compounds, isoxazolidinone, nicotinic acid, isopropylamine, isopropylamine derivatives, dioxazolinone, phosphate esters, phthalates, pyridinecarboxylic acid compounds, triazine, triazole, uracil, urea derivatives, herbicides, sodium chlorate, or any combination thereof.

[0683] Agricultural chemicals may contain fungicides, said fungicides comprising substituted benzene, thiocarbamate, ethylene dithiocarbamate, thiophthalamide, copper compounds, organomercury compounds, organotin compounds, cadmium compounds, thiophanate-methyl, benomyl, cyclohexylamide, doxycycline, terbufos, iprodione, metalaxyl, thiamine, cymoxanil, or any combination thereof.

[0684] Agricultural chemicals may contain fungal inoculants, including fungal inoculants from the families Cynophoraceae, Hypophyceae, Megasporidiaceae, Anophelesaceae, Cynosporidiaceae, Endotrophicoceraceae, Pittosporaceae, Polysporidiaceae, Cynophoraceae, Protosporidiaceae, Lysimachiae, Amphicoeliaceae, Megasporidiaceae, Bryophyceae, Paramecaceae, Basidiomycetes, Ascomycetes, Zygomycetes, or any combination thereof.

[0685] Agricultural chemicals may contain bacterial inoculants, including Rhizobium spp., Rhizobium brevichorum spp., Rhizobium intermedium spp., Azotobium spp., Heterogeneous Rhizobium spp., Rhizobium spp. of soybean, Crophyllella spp., Azotobacter spp., Pseudomonas spp., Azospirillum spp., Bacillus spp., Streptomyces spp., Bacillus-like bacteria spp., Paracoccus spp., Enterobacter spp., Mycobacterium spp., Trichoderma spp., Glycotric acid spp., Gastrodia spp., Klebsiella spp., or any combination thereof.

[0686] Agricultural chemicals may contain an effective amount of rhizobia. Rhizobia may include bacteria of the genus *Rhizobium* (e.g., *Rhizobium japonicum*), bacteria of the genus *Rhizobium* (e.g., *Rhizobium beanense*, *Rhizobium peaense*, or combinations thereof), or combinations thereof.

[0687] Agricultural chemicals may include fungicides, and fungicides include: thiamethoxam, aminophosphonic acid, potassium aminophosphonate, methamidophos, thiamethoxam, azaconazole, pyrimethanil, benzylpyridinium chloride, benomyl, benzyl ethyl acetate, dipropamidophos, chlorpyrifos, biphenyl, bifenthrin, blastomycin-S, cyazofamid, furazolidone, sulfadiazine, thiophanate-methyl, calcium polysulfide, carbadoxam, captan, methyl benzimidazole, carvone, quinaloxam, cymoxanil, chlorothalonil, ethoxysulfuron, clozapine, thiophanate-methyl, cymoxanil, cyproconazole, cyproconazole, pyraclostrobin, pyraclostrobin, imazalil, diclofenac, benzyltriazole, pyridaben, chlorpyrifos, acetamiprid, acetamiprid, and dimethomorph. Methimil, dimethomorph, azoxystrobin, tebuconazole, tebuconazole-M, acaricide, diphenylamine, pyrimethanil, phosmet, dicyandioxonone, dodecyl morpholine, doxycycline, hydrazine, isoprothiolane, epoxiconazole, eticonazole, etoxazole, tebuconazole, oxadixyl, imidacloprid, lebifen, cypermethrin, methoxyfenozide, seed dressing ester, seed dressing agent, benzyl sulfadiazine, butyl morpholine, triphenyltin acetate, toxic tin, ferrous sulfate, pyraclostrobin, fluazinam, fluazinam, fluchloroquine, furazolidone, flusilazole, sulfadiazine, fluamide, fenazol, captan, aluminum tris(ethyl phosphonate), sodium ethyl acetate, tetrachlorophthalide, malathion, furazolidone, furabi, furazolidone, furazolidone-cis, methamidophos, biguanide octylamine, hexachlorobenzene, hexaconazole Hymexazol, tebuconazole, imazalil, Pefloxacin, alkylbenzene sulfonate, biguanide octylamine acetate, cymoxanil, isoprothiolane (IBP), iprodione, irulin, isoprothiolane, isoamyl ketone, kasugamycin, phenoxypyr, copper preparations such as: copper hydroxide, copper naphthenate, copper oxychloride, copper sulfate, copper oxide, quinoline copper and Bordeaux mixture, mancozeb, mancozeb, mancozeb, mancozeb, pyraclostrobin, pyraclostrobin, cymoxanil, tebuconazole, sulfadiazine, thiamethoxam, fenvalerate, methamidophos, nickel dimethyl dithiocarbamate, phthalimide, flufenoxuron, mefenoxuron, oxychloride, glufosinate, oxadiazon, oxychloride, paclobutrazol, isoprothiolane, penadazole, sheath blight, chlorpyrifos, etc. Varmus, Powder-resistant fungicide, Polyoxin, Polyoxin, Allylfenoxam, Prochloraz, Iprodione, Cymoxanil, Sodium Propyleneoxide, Propiconazole, Propineb, Thioconazole, Phosphate, Cantharidin, Dimethomorph, Quinolones, Clopidogrel, Quinconazole, Pentachloronitrobenzene (PCNB), Sulfur and sulfur preparations, Tebuconazole, Phosphatic acid, Tetrachloronitrobenzene, Tetracycline, Flufenoxam, Thiabendazole, Thiamethoxam, Thiofenylbenzamide, Thiofenylbenzamide, Methyl thiophanate, Para-methyl, Triadimefon, Triadimefon, Butyltriazole, Imidazin, Salicylic acid, Tricyclazole, Dimethyltridecylmorpholine, Ibuprofen, Powder-resistant fungicide, Azoxystrobin, Uniconazole, Enteromycin A, Vafozoline, Voriconazole, Cyanobacterium, Zinc ethyl thiophanate, and Dagger. G, OK-8705, OK-8801, a-(1,1-Dimethylethyl)-(3-(2-phenoxyethyl)-1H-1,2,4-triazol-1-ethanol, α-(2,4-dichlorophenyl)-[3-fluoro-3-propyl-1H-1,2,4-triazol-1-ethanol, α-(2,4-dichlorophenyl)-[3-methoxy-α-methyl-1H-1,2,4-triazol-1-ethanol, α-(5-methyl-1,3-dioxane-5-yl)-[3-[[4-(trifluoromethyl)-phenyl]-methylene]-1H-1,2,4-triazol-1-ethanol, (5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(1H-1,2,4-triazol-1-yl)-3-octanone, (E)-α-(methyl) (Oxy-imino)-N-methyl-2-phenoxy-phenylacetamide, carbamate {2-methyl-1-[[[1-(4-methylphenyl)-ethyl]-amino]-carbonyl]-propyl}1-isopropyl ester, 1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-acetone-O-(phenylmethyl)-oxime, 1-(2-methyl-1-naphthyl)-1H-pyrrole-2,5-dione, 1-(3,5-dichlorophenyl)-3-(2-propenyl)-2,5-pyrrolidinedione, 1-[(diiodomethyl)-sulfonyl]-4-methyl-benzene, 1-[[2-(2,4-dichlorophenyl)-1,3-dioxolane-2-yl]-methyl]-1H-imidazolium, 1 -[[2-(4-chlorophenyl)-3-phenylepoxyethylene]-methyl]-1H-1,2,4-triazole, 1-[1-[2-[(2,4-dichlorophenyl)-methoxy]-phenyl]-vinyl]-1H-imidazol, 1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidone, 2',6'-dibromo-2-methyl-4'-trifluoromethoxy-4'-trifluoro-methyl-1,3-thiazolyl-carboxamide, 2,2-dichloro-N-[1-(4-chlorophenyl)-ethyl]-1-ethyl-3-methyl-cyclopropanecarboxamide, 2,6-dichloro-5-(methylthio)-4-pyrimidinyl-thiocyanate, 2,6-dichloro-N-(4-trifluoromethylbenzyl)-benzamide, 2,6-Dichloro-N-[[4-(trifluoromethyl)-phenyl]-methyl]-benzamide, 2-(2,3,3-triiodo-2-propenyl)-2H-tetrazole, 2-[(1-methylethyl)-sulfonyl]-5-(trichloromethyl)-1,3,4-thiadiazole, 2-[[6-deoxy-4-O-(4-O-methyl-(3-D-glucyranopy ...6-Dimethylphenyl)-N-(isothiocyanate methyl)-acetamide, 2-phenylphenol (OPP), 3,4-dichloro-1-[4-(difluoromethoxy)-phenyl]-pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[(1-methyl-2-propynyl)-oxy]-methyl]-benzamide, 3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carboxynitrile), 3-[2-(4-chlorophenyl)-5-ethoxy-3-isooxazolyl]-pyridine, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazolium-1-sulfonamide, 4-methyl-tetrazolo[1,5-a]quinazolin-5(4H)-one, 8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4,5]decane-2-methylamine, 8-hydroxyquinoline sulfate, 9H-xanthon-2-[(phenylamino)-carbonyl]-9-carboxylhydrazine, bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)-oxy]-2,5-thiophene dicarboxylate, cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-cycloheptanol, cis-4-[3-[4-(1,1-dimethylpropyl)-phenyl-2-methylpropyl]-2,6-dimethyl-morpholine hydrochloride, [(4-chlorophenyl)-azo]-cyanoethyl acetate, potassium bicarbonate, methane Tetrathiol sodium salt, methyl 1-(2,3-dihydro-2,2-dimethyl-inden-1-yl)-1H-imidazolium-5-carboxylate, N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alanine methyl ester, N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alanine methyl ester, N-(2,3-dichloro-4-hydroxyphenyl)-1-methyl-cyclohexanecarboxamide, N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-furanyl)-acetamide, N-(2,6-dimethylphenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)-acetamide, N-(2-chloro-4-nitro)-acetamide, N-(4-cyclohexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamide, N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamide, N-(5-chloro-2-methylphenyl)-2-methoxy-N-(2-oxo-3-oxazolyl)-acetamide, N-(6-methoxy)-3-pyridyl)-cyclopropanecarboxamide, N-[2,2,2-trichloro-1-[(chloroacetyl)-amino]-ethyl]-benzamide, N-[3-chloro-4,5-bis(2-propynyloxy)-phenyl]-N'-methoxy-formimide, N-formyl-N-hydroxy-DL-alanine sodium salt, 0,O-Diethyl[2-(dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate, O-methylS-phenylphenylpropylphosphoramidothioate, S-1,2,3-benzothiadiazole-7-methylthioate and spiro[2H]-1-benzopyran-2,1'(3'H)-isobenzofuran]-3'-one, N-trichloromethyl)thio-4-cyclohexane-1,2-dicarboximide, tetramethylthioperoxydicarbonamide, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine methyl ester, 4-(2,2-difluoro-1,3-benzo[m-dioxapenten-4-yl]-1-H-pyrrole-3-carboxynitrile or any combination thereof.

[0688] Agricultural chemicals may contain Bacillus spp. inoculants, including Bacillus spp., Bacillus tumefaciens, Lactobacillus albus (Lactobacillus lactis), Bacillus amyloliquefaciens, Bacillus cereus, Bacillus coagulans, Bacillus endoparasiticus, Bacillus congenitalis, Kustalbacterium, Lactobacillus spp., Lactobacillus lactis, Lactobacillus, Lactobacillus laterosporus, Lactobacillus licheniformis, Bacillus megaterium, Bacillus cereus, Bacillus alfalfa, Bacillus natto, Bacillus niger, Bacillus papilloma, Bacillus pumilus, Bacillus sicca, Bacillus spheroidosa, species of Bacillus spp., Bacillus subtilis, Bacillus thuringiensis, Bacillus monotypicus, or any combination thereof.

[0689] Agricultural chemicals may include herbicides, and these herbicides may include 2,4-D, 2,4-DB, acetochlor, trifluralin, metolachlor, atrazine, atrazine, chlorpyrifos, fluroxypyr, thiamethoxam, dimethoate, bentazon, bensulfuron-methyl, bromobenzonitrile, butachlor, oxychlorfenapyr, chlorsulfuron-methyl, clethodim, isoxaflutole, dichloropyridine, chlorpyrifos-methyl, cyclohexane, DCPA, betaine, and dicamba. , Dichlorvos, dichlorophenoxypropionic acid, bis(chlorothalonil), diflubenzuron, dimethyl phenoxychloride, diquat, diuron, DSMA, EPTC, butyraz, ethoxysulfuron, oxazolidinone, dihydropyridine-P, fluazolidinone, fluthiamethoxam, pyrazosulfuron, flumethrin, flumetsulam, propyzoxystrobin, fluroxypyr, fluroxypyr, cyhalofop-P-methyl, formamidosulfuron, glufosinate, glyphosate, chlorpyrifos, cycloazine Acetate, imazalil, methoxypromethazine, methyl imazalil, metribuzin, imazalil, isoxaflutole, isoxazolidinone, lactoferrin, linuron, MCPA, MCPB, mesotrione, pretilachlor, metsulfuron-methyl, quizalofop-p-ethyl, methylsulfuron, quizalofop-p-ethyl, MSMA, fenpropathrin, ciprofloxacin, nicosulfuron, daburine, ammoniacalsulfuron, oxadiazon, ethoxyflufenoxam, paraquat, nonanoic acid, pendimethalin, betaine, toxic Atrazine, flupyrfluthrin, amflurazole, pyrazosulfuron, fenvalerate, propargite, flusulfuron, phenylbutazone calcium, pyrimisulfuron, quinclorac, quizalofop-P-ethyl, pendimethalin, thiamethoxam, cyclosulfuron, simazine, metsulfuron-methyl, sulfonylurea, buprofen, terbuprofen, thiamethoxam, broadleaf chlorpyrifos, quizalofop-P-ethyl, styrax, fensulfuron-methyl, fensulfuron-methyl, fensulfuron-methyl, fensulfuron-methyl, flusulfuron-methyl, or any combination thereof.

[0690] Agricultural chemicals can include fertilizers, and fertilizers include ammonium sulfate, ammonium nitrate, ammonium nitrate sulfate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, ammonia water, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcite limestone, calcium oxide, calcium nitrate, dolomite limestone, quicklime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrate, and magnesian limestone. Magnesium oxide, urea, urea-formaldehyde, urea-ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylene diurea, K2SO4–2MgSO4, potassium magnesium sulfate, potassium halite, sulfur magnesium sulfate, Epsom salt, elemental sulfur, mud ash, ground oyster shells, fish meal, oil cake, fish fertilizer, blood meal, phosphate rock, superphosphate, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, humic acid or any combination thereof.

[0691] Agricultural chemicals may contain plant hormones, and plant hormones may include gibberellins, auxins, kinetin, or any combination thereof.

[0692] Enzymes can be formulated in many ways. Common goals for formulated enzyme products include extending shelf life, protecting the product from microorganisms, and enhancing enzyme activity. Enzyme products can be lyophilized to extend the shelf life of most enzymes by freeze-drying, spray-drying, or removing the liquid side of the enzyme product. Liquid and lyophilized products are often swelled with additives such as buffers, stabilizers, antimicrobial agents, and volumetric additives. Enzymes can often be encapsulated or granulated to make the final product safer and easier to use. Granular products can have an enhanced shelf life and exhibit minimal enzyme activity exposed on the outer surface of the particles. Enzymes can also be attached to organic or inorganic platforms such as plastic beads, dolomite, clay, charcoal, biochar, nanoparticles, alginate, and silica beads, which help bind them and make them easier to use. Typically, enzymes are immobilized on a matrix to allow for longer activity and shelf life of the enzyme product. Common matrices include carbon, carbon nanotubes, agarose, alginate, cellulose and cellulose materials, silica, plastics, stainless steel, glass, polystyrene, and ceramics.

[0693] Many enzyme formulations can be used to extend the enzyme activity or shelf life of a product. These include, but are not limited to, preservatives, biocides, stabilizers, color enhancers, odor reducers, surfactants, detergents, buffers, cofactors, ions, and other modifications to the formulation to enhance enzyme performance.

[0694] XVII. Plant Growth Medium

[0695] In any of the methods described herein involving the application of plant growth media, the plant growth media may comprise soil, water, aqueous solution, sand, gravel, polysaccharides, mulch, compost, peat moss, wheat straw, logs, clay, soybean flour, yeast extract, or combinations thereof.

[0696] Plant growth media may contain fertilizers or consist mainly of fertilizers.

[0697] In addition, substrates for enzymes can be added to plant growth media.

[0698] The substrate may include tryptophan, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate (e.g., adenosine-3-triphosphate), polyphosphate, protein powder, trimetaphosphate, cellulose, methylcellulose, chitosan, deacetylated chitosan, cellulose derivatives, phosphate, fat, wax, phospholipid, phytic acid, or any combination thereof.

[0699] XVIII. Plants

[0700] In any of the above methods involving plants, the plant is a dicotyledonous plant, a monocotyledonous plant, or a gymnosperm.

[0701] Similarly, for any seed mentioned herein, the seed can be a seed of a dicotyledonous plant, a monocotyledonous plant, or a gymnosperm.

[0702] For example, if the plant is a dicotyledonous plant or the seed is a dicotyledonous seed, then the dicotyledonous plant can be selected from beans, peas, tomatoes, peppers, squash, alfalfa, almonds, fennel, apples, apricots, arracha, artichokes, avocados, Bambara peanuts, beets, bergamot, black pepper, black thorn, blackberries, blueberries, bitter oranges, bok choy, Brazil nuts, breadfruit, broccoli, broad beans, Brussels sprouts, buckwheat, cabbage, flaxseeds, Chinese cabbage, cocoa, cantaloupe, caraway, chard, carob, carrots, cashews, cassava, castor beans, cauliflower, celery, cherries, chestnuts, chickpeas, chicory, peppers, chrysanthemums, cinnamon, citron, cloves, clover, coffee, cola nuts, rapeseed, corn, cotton, cottonseed. Cowpea, Capsella, Cranberry, Watercress, Cucumber, Gooseberry, Custard Apple, Drumstick Tree, Earth Pea, Eggplant, Lettuce, Fennel, Fenugreek, Fig, Hazelnut, Flax, Geranium, Gooseberry, Gourd, Grape, Grapefruit, Guava, Hemp, Cannabis, Henna, Jumping Point, Horse Bean, Horseradish, Indigo, Jasmine, Jerusalem Artichoke, Jute, Kale, Kapok, Hemp, Turnip, Kumquat, Lavender, Lemon, Lentil, Lespedeza, Lettuce, Lime, Licorice, Lychee, Loquat, Lupin, Macadamia Nut, Maces, Mandarin, Forage Beetroot, Mango, Goji Berry, Melon, Mint, Mulberry, Mustard, Nectarine, Niger Seed, Nutmeg, Okra, Olive, Opium, Orange, Papaya, Parsnip, Pea, Peach, Peanut, Pear, Hickory Persimmon, pigeon pea, pistachio, plantain, plum, pomegranate, pomelo, poppy seeds, potato, sweet potato, prune, pumpkin, freckle, papaya, cinchona, quinoa, radish, ramie, rapeseed, raspberry, lily, rhubarb, rose, rubber, turnip, safflower, yew, Brahman ginseng, hematologice evergreen, Samoyed, Allium, sesame, shea butter, soybean, spinach, pumpkin, strawberry, sugar beet, sugarcane, sunflower, cabbage, bell pepper, orange, tea, Ethiopian thrush, tobacco, tomato, clover, paulownia, radish, Brahma, milkvetch, walnut, watermelon, yerba mate, shiitake mushroom, shepherd's purse, garden watercress, Sichuan pepper, watercress, pennycress, star anise, laurel, cassia Jamun, Dill, Tamarind, Mint, Oregano, Rosemary, Sage, Soursop, Borage, Calophyll, Bitter Melon, Kukui Nuts, Tamarind Chestnut, Basil, Blueberry, Hibiscus, Passion Fruit, Star Apple, Sinensis, Cactus, St. John's Wort, Pearl Green, Hawthorn, Coriander, Curry Plant, Kiwi, Thyme, Zucchini, Ulluco, Yak Beef, Watercress, Spinach, Yellow Mombin, Star Fruit, Amaranth, Mustard, Japanese Chili, Yellow Plum, Mashua, Chinese Toon, New Zealand Spinach, Gazebo Spinach, Ugu, Tansy, Chicory, Jocote, Malaysian Apple, Paracress, Bitter Lettuce, Chinese Potato, Parsley, Hedge Mustard, Campion, AgateCassodtree, Thistle, Burnett, Star Kiwi, Salt Thistle, European Seaweed, Maroon, Silver Lace Fern, Kale, Primrose, Cowslip, Purslane, Knotgrass, Terebinth, Tree Lettuce, Wild Areca, West African Pepper, Yerba Santa Claus, Tarragon, Parsley, Mountain Radish, Land Watercress, Burnet Saxifrage, Honeyherb, Coltsfoot, Perilla, Water Pepper, Perilla, Bitter Bean, OCA, Kampong, Chinese Celery, Lemon Basil, Thai Basil, Water Mimosa, Root Parsley, Cabbage Tree, Moringa, Mauka, Ostrich Fern, Paddy Grass, Yellow Saw Lettuce, Angelica pubescens, Pepper Grass, Maca, Gourd, Hyacinth Bean, Water Spinach, Cat's Ear, Fishwort, Okinawa Spinach, Lotus Sweet Juice, Brave Soldier, Culantro, Arugula, Artichoke, Caigua, Mitsuba, Chipilin, Samphire, Mampat, Ebolo, Ivy Gourd, White Artichoke, Sea Mustard, Amaranth, Huauzontle, Ethiopian Mustard, Magenta Spreen, good king henry, epazole, lamb's quarters, centella-plumed cockscomb, capers, rapini, napa cabbage, mizuna, Chinese appetizer, kerosene, mustard greens, Malabar spinach, beetroot, marshmallow, climbing fence, Chinese jute, paprika, annatto seeds, spearmint, savory, marjoram, fennel, chamomile, lemon balm, allspice, blueberry, custard apple, cloudberry, Budapest, dragon fruit, durian, elderberry, feijoa, jackfruit, Hainan rose apple, jujube, ground cherry, purple mangosteen, rambutan, red currant, black currant, salmonberry, mandarin orange, ugli fruit, azuki bean, black bean, black-eyed pea, borlotti bean, common bean, mung bean, kidney bean, lima bean, mung bean, navy bean, pinto bean, flax bean, mangetout, snappea, broccoflower, calabrese, nettle, sweet pepper, raddichio, white radish, white radish, skirret, tatsoi, broccoli, black radish, burdock root, broad bean, broccoli raab, lentil, lupin, malva nut, velvet bean, winged bean, yam bean, mulga, iron grass, umbrella shrub, tjuntjula, wakalpulka, witchetti bush, wiry wattle, chia, beech nut, tung tree, medicinal watermelon, honey fruit, Maya nut, mongongo, ogbono nut, paradise nut, and cempedak.

[0703] If the plant is a monocotyledonous plant or the seed is a monocotyledonous seed, then the monocotyledonous plant can be selected from corn, wheat, oats, rice, barley, millet, banana, onion, garlic, asparagus, ryegrass, millet, crabgrass, raishan, nipa grass, turmeric, saffron, galangal, leek, cardamom, date, pineapple, scallion, chives, onion, water chestnut, ramp, Job's tears, bamboo, barnyard grass, clean rootless duckweed, sword-leaved elephant ear, Tahitian spinach, abaca, areca nut, foxtail millet, areca nut, broom millet, broom sorghum, lemongrass, coconut. Seed, cocoyam, corn, taro, sorghum, durum wheat, edo, fique, formio, ginger, orchard grass, Spanish grass, Sudan grass, Guinea corn, Manila hemp, agave fiber, hybrid corn, sorghum, lemongrass, agave, reed millet, finger millet, foxtail millet, Japanese millet, yellow millet, New Zealand flax, oats, oil palm, palm scallop, sago palm, red cap, sisal, sorghum, blended wheat, sweet corn, sweet sorghum, taro, Ethiopian thrush, cat's tail grass, triticale, vanilla, wheat and yam.

[0704] If the plant is a gymnosperm or the seed is a gymnosperm seed, then the gymnosperm can be selected from the following families: Araucariaceae, Cycas Bowenaceae, Cephalotaxaceae, Cupressaceae, Cycas, Ephedraceae, Ginkgoaceae, Gnetaceae, Pinaceae, Podocarpusaceae, Taxaceae, Taxaceae, Welwitschiaceae, and Zemiaceae.

[0705] The plants and plant seeds described herein may include genetically modified plants or plant seeds, such as genetically modified cereals (wheat, rice), corn, soybeans, potatoes, cotton, tobacco, rapeseed, and fruit plants (apples, pears, citrus fruits, and grapes). Preferred genetically modified plants include corn, soybeans, potatoes, cotton, tobacco, and oilseeds.

[0706] Suitable transgenic plants and seeds are characterized by toxin formation, particularly from Bacillus thuringiensis genetic material (e.g., genes CryIA(a), CryIA(b), CryIA(c), CryIIA, CryIIIA, CryIIIB2, Cry9c, Cry2Ab, Cry3Bb, CryIF, or combinations thereof). The formation of plant toxins increases the plant's resistance to insects, spiders, nematodes, gastropods, and snails (hereinafter referred to as "Bt plants"). Bt plants, for example, are commercially available under the trade name YIELD. (e.g., corn, cotton, soybeans) (e.g., corn) (e.g., corn) (cotton), (cotton) and (Potato) varieties of corn, cotton, soybean, and potato. Herbicide-resistant plants include those under the following trade names: Roundup (Glyphosate resistance, such as in corn, cotton, and soybeans) (e.g., corn), Liberty (Glufosinate resistance, for example, in oilseeds) (with imidazolinone resistance) and (Sulfonylurea resistance, for example, corn).

[0707] Plant seeds as described herein can be genetically modified (e.g., any seed that results in a transgenic plant or plant part expressing herbicide tolerance, tolerance to environmental factors such as water stress, drought, viruses, and nitrogen production, or resistance to bacterial, fungal, or insect toxins). Suitable transgenic seeds include rapeseed crops, vegetables, fruits, trees, fiber crops, oil crops, tuber crops, coffee, flowers, legumes, cereals, and other monocotyledonous and dicotyledonous plants. Preferably, genetically modified seeds include peanuts, tobacco, grass, wheat, barley, rye, sorghum, rice, rapeseed, sugar beets, sunflowers, tomatoes, peppers, beans, lettuce, potatoes, and carrots. Most preferably, genetically modified seeds include cotton, soybeans, and corn (sweet, field, seed, or popcorn).

[0708] Particularly useful transgenic plants that can be treated according to the present invention are plants containing transformation events or combinations of transformation events, listed in the examples from databases of various national or regional regulatory agencies (see examples http: / / gmoinfo.jrc.it / gmp_browse.aspx and http: / / www.agbios.com / dbase.php). The invention has been described in detail, and it will be apparent that modifications and variations can be made without departing from the scope of the invention as defined by the appended claims.

[0709] Example

[0710] The following non-limiting embodiments are provided to further illustrate the invention.

[0711] Example 1: Free endoglucanase on corn, greenhouse

[0712] β-1,4-endoglucanase from *Thermolyticus fibrosis* (SEQ ID NO: 30; commercially available from Sigma-Aldrich, St. Louis MO, trade name E2164) was diluted to concentrations ranging from 12.5 to 1600 mU / mL in citrate dilution buffer. Endoglucanase activity (U units or ISUs) was determined by the amount of enzyme that degraded 1 μmol / min / mL of substrate under ideal temperature and conditions. For each treatment group, 18 seeds of commercially available hybrid BECK'S 6626RR maize containing glyphosate resistance were placed in 50 mL conical tubes without seed treatment. Vortex each conical tube and add 18 μL of enzyme solution to each tube to a final concentration of 0, 12.5 μU, 25 μU, 50 μU, 100 μU, 200 μU, 400 μU, 800 μU, or 1600 μU / endonuclease for the seeds. Vortex the conical tube again for 20 seconds to obtain a uniform coating on each seed. Allow the seeds to dry for 5 minutes, then plant them at a depth of 2.54 cm into a 39.7 cm layer of commercial topsoil. 3 Two seeds were placed in each pot. After planting, 50 mL of room temperature water was added to each pot to allow germination. The pots were kept in an artificially lit plant growing room with a 13 / 11 hour light / day cycle and a daytime / nighttime temperature range of 21°C / 15°C. The plants were watered as needed and randomized over a 3-day cycle to avoid any cool spots in the room. At the end of 14 days, the height of the maize plants for each treatment was measured and calibrated against the height of the control plants from seeds coated only with water.

[0713] The experiment was repeated three times, and the average of the multiple experiments was taken. Table 18 shows that the main effect of endoglucanase as a seed treatment on BECK'S 6626RR (a glyphosate-resistant maize hybrid) was within the enzyme activity range of 100-1600 μU / seed. At these values, it had a significant and reproducible effect on maize growth. Values ​​below 50 μU / seed had a much lower effect on the growth rate of this hybrid maize. These enzyme treatments were very effective as individual treatments on the crop.

[0714] Table 18. Height effect of β-1,4-endoglucanase treatment as a seed treatment

[0715]

[0716] Example 2: Free endoglucanase on corn, greenhouse

[0717] β-1,4-endoglucanase from *Thermolyticus fibrosis* (SEQ ID NO: 30; commercially available from Sigma-Aldrich, St. Louis MO, trade name E2164) was diluted to concentrations ranging from 50 to 1200 mU / mL in citrate dilution buffer. Endoglucanase activity (U) was determined by the amount of enzyme that degraded 1 μmol / min / mL of substrate under ideal temperature and conditions. Eighteen seeds of commercially available hybrid BECK'S 5140HR maize containing the HERCULEX corn borer (insect protection trait) and glyphosate resistance trait were placed in 50 mL conical tubes without seed treatment. Vortex each conical tube and add 18 μL of enzyme solution to each tube to a final concentration of 0, 50 μU, 100 μU, 200 μU, 400 μU, 600 μU, 800 μU, or 1200 μU / seed enzyme. Vortex the conical tube again for 20 seconds to obtain a uniform coating on each seed. Allow the seeds to dry for 5 minutes, then plant them at a depth of 2.54 cm into a 39.7 cm layer of commercial topsoil. 3 Two seeds were placed in each pot. After planting, 50 mL of room temperature water was added to each pot to allow germination. The pots were kept in an artificially lit plant growing room with a 13 / 11 hour light / day cycle and a daytime / nighttime temperature range of 21°C / 15°C. The plants were watered as needed and randomized over a 3-day cycle to avoid any cool spots in the room. At the end of 14 days, the height of the maize plants for each treatment was measured and calibrated against the height of the control plants that were seed-coated with water only. Each experiment was repeated three times.

[0718] As shown in Table 19, the main effect of endoglucanase as a seed treatment on BECK'S 5140HR was within the enzyme activity range of 600-1200 μU / seed. At these values, it had a significant and reproducible effect on maize growth. Values ​​below 400 μU / seed had a lower impact on the growth rate of this hybrid maize. These enzyme treatments were very effective as individual treatments on the crop.

[0719] Table 19. Height effect of β-1,4-endoglucanase treatment as a seed treatment

[0720]

[0721] Example 3: Glucanase and phospholipase on corn, in the field

[0722] The β-1,4-endoglucanase from *Thermolyticus celluloseis* (SEQ ID NO: 30; available from Sigma-Aldrich, St. Louis MO, trade name E2164), the β-1,3-d-glucanase from *Trichoderma reesei* (SEQ ID NO: 126; available from Sigma-Aldrich, St. Louis MO, trade name 67138), the β-1,4-endoglucanase "cellulase" from *Trichoderma reesei* (SEQ ID NO: 36; available from Worthington Biochemical Corp., Lakewood, NJ, trade name ATCC26921), and the exo-β-1,3-glucanase from *Aspergillus oryzae* (SEQ ID NO: 30) were compared with those from *Thermolyticus celluloseis* (SEQ ID NO: 30; available from Sigma-Aldrich, St. Louis MO, trade name E2164), and those from *Trichoderma reesei* (SEQ ID NO: 30; available from Worthington Biochemical Corp., Lakewood, NJ, trade name ATCC26921), and those from *Aspergillus oryzae* (SEQ ID NO: 30). NO:41; available commercially from Megazyme, Chicago, IL, under the trade name E-EXG5AO) was diluted in citrate dilution buffer to concentrations of 12.5 to 1600 mU / mL (for thermophilic bacteria β-1,4-endoglucanase and Trichoderma β-1,4-endoglucanase) or 252 mU / mL (for snail β-1,3-d-glucanase). This group contains several cellulase (celluloglucanase) and non-celluloglucanase activities, including β-1,4-endoglucanase and β-1,3-d-glucanase activities, respectively. Enzyme activity (U) was determined by the amount of enzyme that degraded 1 μmol / min / mL of substrate under ideal temperature and conditions. Phosphatidylinositol-specific phospholipase C (SEQ ID NO: 116; available from Sigma-Aldrich, St. Louis MO, trade name P5524), phosphatidylcholine-specific phospholipase C (SEQ ID NO: 115; available from Sigma-Aldrich, St. Louis MO, trade name P6621), Clostridium perfringens phospholipase C (SEQ ID NO: 18; available from Sigma-Aldrich, St. Louis MO, trade name P7633), and Streptomyces bruncii phospholipase D (SEQ ID NO: 116) were used in this study. NO:19; available commercially from Sigma-Aldrich, St. Louis, MO under the trade name P0065) is diluted in citrate dilution buffer to a final concentration of 2.5 U / mL (for Bacillus phosphatidylcholine phospholipase C, Clostridium phospholipase C, and Streptomyces phospholipase D) or 100 U / mL (for Bacillus phosphatidylinositol-specific phospholipase C). Each of these phospholipases has specific activity against phospholipids and against different phospholipid cleavage sites.Commercially available hybrid BECK'S 6175YE maize seeds were used without seed treatment. These seeds contain HERCULEX (protective traits against rootworms and corn borers), MON810 (containing corn borer resistance traits), glufosinate resistance, and glyphosate resistance traits. Seeds were placed in a batch processor at 400 seeds per treatment. 400 μL of solution was added to each batch, resulting in a final enzyme concentration of 600 μU / seed (Thermolyticus β-1,4-endoglucanase and Trichoderma β-1,4-endoglucanase), 252 μU / seed (Snail β-1,3-d-glucanase), 100 μU / seed (phosphatidylinositol-specific phospholipase C), or 2.5 μU / seed (Bacillus phosphatidylcholine-specific phospholipase C and phospholipase C and D coated seeds). Each batch was mixed for 20 seconds to obtain a uniform coating on each seed. In addition, these seeds were coated with commercially available EVERGOL Energy / PONCHO Seed Treatment (available from Bayer CropScience) (“Substrate”). Each experiment was repeated three times. The seeds were allowed to dry for three weeks and then planted in natural soil in 9.14 m rows spaced 10.16 cm apart at a depth of 3.81 cm. The height of the plants was measured two weeks after planting and calibrated against the height of control plants, which were seeds coated only with water containing the substrate. The results are shown in Table 16 below.

[0723] Table 20. Height effect of glucanase and phospholipase treatments as seed treatments

[0724]

[0725]

[0726] β-1,3-exoglucanase (Aspergillus oryzae; SEQ ID NO:41; available from Megazyme, Chicago, IL, trade name E-EXG5AO), phosphatidylinositol-specific phosphatase C (Bacillus cereus; SEQ ID NO:116; available from Sigma-Aldrich, St. Louis MO, trade name P6621), phosphatidylcholine-specific phosphatase C (Bacillus cereus; SEQ ID NO:115; available from Sigma-Aldrich, St. Louis, MO, trade name P5542), and phosphatase D (Streptomyces brownii; SEQ ID NO:41) were used. NO:19 (available commercially from Sigma-Aldrich, St. Louis, MO, under the trade name P0065) was diluted in water to 182 mU / mL (for β-1,3-exoglucanase), 100 U / mL (for phosphatidylinositol-specific phospholipase C), or 2.5 U / mL (for phosphatidylcholine phospholipase C and streptomycin phospholipase D). The enzymes were applied as seed treatment to maize (BECK'S 5828YH) (which contains the HERCULEX trait (protective traits against rootworms and corn borers), glufosinate resistance, and glyphosate resistance), planted, and allowed to grow to harvest using the same method described above. Seed treatment was performed based on a substrate containing prothioconazole, pendimethalin, metalaxyl, and thiamethoxam (“substrate”) and as described in the preceding section of this example. The yields of the treated crops (quantified in bushels per acre (Bu / Ac) or metric tons per hectare (MT / ha)) were compared and calibrated with those of crops grown from water-treated seeds. Each treatment was performed independently at least four times. Compared with control maize plants that did not receive seed treatment, maize seed treatments using these free enzymes resulted in increased maize yields. β-1,3-exoglucanase increased crop yield by approximately 4%, phosphatidylinositol-specific phospholipase C increased crop yield by approximately 3%, and phospholipase D increased crop yield by approximately 2%. The average weight per ear was also increased for maize plants grown from seeds treated with these three free enzymes. The results are shown in Table 21 below.

[0727] Table 21. Glucanase and phospholipase applied as seed treatments to increase maize yield

[0728]

[0729]

[0730] Of the phospholipases and dextranases tested in this experiment, β-1,3-exoglucanase, Bacillus cereus phosphatidylinositol-specific phospholipase C, and Streptomyces phospholipase D exhibited the best plant responses. These enzyme treatments are effective for various hybridization and trait packaging.

[0731] Example 4: Glucanase on corn, in the field

[0732] β-1,4-endoglucanase from *Thermolyticus fibrosis* (SEQ ID NO: 30; commercially available from Sigma-Aldrich, St. Louis MO, trade name E2164) was diluted in citrate dilution buffer to concentrations ranging from 200 mU / mL to 450 mU / mL. Endoglucanase activity (U) was determined by the amount of enzyme that degraded 1 μmol / min / mL of substrate under ideal temperature and conditions. Without seed treatment, 150 seeds of commercially available hybrid BECK'S 6175YE maize (containing HERCULEX (protective traits against rootworms and corn borers), MON810 (resistance to corn borers), glufosinate resistance, and glyphosate resistance) were placed in 50 mL conical tubes, 50 seeds per tube. 50 μL of enzyme was added to each tube, along with 250 μL of a slurry containing prothioconazole, pendimethalin, metalaxyl, and thiamethoxam (EVERGOL Energy / PONCHO Seed Treatment) (“substrate”). This resulted in final enzyme concentrations of 200 μU / seed and 450 μU / seed. The tubes were vortexed for 20 seconds to obtain a uniform coating on each seed. The seeds were allowed to dry for 3 weeks and then planted in natural soil in 9.14 m rows spaced 10.16 cm apart at a depth of 3.81 cm. Plant height was measured 2 weeks after planting, and the results were calibrated against the height of control plants, which were seeds coated only with water containing the substrate (prothioconazole, pendimethalin, metalaxyl, and thiamethoxam).

[0733] The experiment was repeated three times, and the average of the multiple experiments was taken. The data in Table 22 below show that, two weeks after planting, both concentrations of β-1,4-endoglucanase increased maize growth rates. These concentrations demonstrated significant and reproducible effects on maize growth. These enzyme treatments are based on their effectiveness as a comprehensive approach for both crop and substrate treatments for various hybridization and trait packaging.

[0734] Table 22. Height effect of endoglucanase treatment on crop seeds

[0735]

[0736] Example 5: Phospholipases on corn, greenhouse, high range

[0737] Phosphatidylcholine-specific phospholipase C from *Bacillus cereus* (SEQ ID NO: 115; available from Sigma-Aldrich, St. Louis MO, trade name P6621), phospholipase C from *Clostridium perfringens* (SEQ ID NO: 18; available from Sigma-Aldrich, St. Louis MO, trade name P7633), and phospholipase D from *Streptomyces brunneoincarnata* (SEQ ID NO: 19; available from Sigma-Aldrich, St. Louis MO, trade name P0065) were diluted to concentrations ranging from 100 U / mL to 450 U / mL in 100 mMtris buffer, pH 7.0. For each treatment group, 18 seeds of commercially available hybrid BECK'S 6626RR maize containing glyphosate resistance were placed in 50 mL conical tubes without seed treatment. Vortex each conical tube and add 18 μL of enzyme solution to each tube, resulting in a final phospholipase concentration of 100 mU / mL, 200 mU / mL, or 450 mU / mL / seed. Vortex again for 20 seconds to obtain a uniform coating on each seed. Allow the seeds to dry for 5 minutes, then plant them at a depth of 2.54 cm in 42.24 indole soil containing commercial topsoil. 3 (692.19cm 3 Two seeds were placed in each pot. After planting, 50 mL of room temperature water was added to each pot to allow germination. The pots were kept in an artificially lit plant growing chamber with a 13 / 11 hour light / day cycle and a daytime / nighttime temperature range of 21°C / 15°C. The plants were watered as needed and rotated every 3 days to avoid any cool spots in the chamber. At the end of 14 days, the height of the maize plants for each treatment was measured, and the height was calibrated against the height of the control plants with water-coated seeds only. Triple copies were used for the experiment.

[0738] Table 23 clearly shows that the effects of phospholipase C and D are optimal at or below 100 mU / seed. At these values, they have significant and reproducible effects on maize growth. Values ​​equal to or higher than 200 mU / seed are detrimental to maize growth. This is true for both phospholipase C and D.

[0739] Table 23. Height effect of phospholipase treatment on crop seed treatment

[0740]

[0741] Example 6: 2015 soybean yield, endoglucanase and phospholipase

[0742] The β-1,4-endoglucanase of *Thermolyticus celluloseis* (SEQ ID NO: 30; available from Sigma-Aldrich, St. Louis MO, trade name E2164), the β-1,3-d-glucanase of *Solanum lyratum* (SEQ ID NO: 126; available from Sigma-Aldrich, St. Louis MO, trade name 67138), and the β-1,4-endoglucanase "cellulase" of *Trichoderma reesei* (SEQ ID NO: 36; available from Worthington Biochemical Corp., Lakewood, NJ) were diluted in water to concentrations of 1600 mU / mL (for both β-1,4-endoglucanases) or 252 mU / mL (for β-1,3-d-glucanase). This group contains several cellulose-degrading and non-cellulose-degrading glucanase activities, including β-1,4-endoglucanase and β-1,3-d-glucanase activities. Phosphatidylcholine-specific phospholipase C from *Bacillus cereus* (SEQ ID NO: 115; commercially available from Sigma-Aldrich, St. Louis MO, trade name P6621), phospholipase C from *Clostridium perfringens* (SEQ ID NO: 18; commercially available from Sigma-Aldrich, St. Louis MO, trade name P7633), and phosphatidylinositol-specific phospholipase C from *Bacillus cereus* (SEQ ID NO: 116; commercially available from Sigma-Aldrich, St. Louis MO, trade name P5524) were diluted in water to a final concentration of 2.5 U / mL (for *Bacillus* phosphatidylcholine phospholipase C and *Clostridium* phospholipase C) or 100 U / mL (for *Bacillus* phosphatidylinositol-specific phospholipase C). Each of these phospholipases has specific activities targeting phospholipids and different cleavage sites of phospholipids. 720 seeds of commercially available hybrid BECK'S 294NR soybean (containing nematode resistance traits (SCN-SB) and glyphosate resistance traits (ROUNDUP READY 1)) were placed in lacquered cans with metalaxyl and thiamethoxam substrate seed treatment packages (“substrate”) and coated with the commercially available seed treatment (substrate). Each batch was mixed, and 720 μL of the solution was added to each batch to obtain the final enzyme concentrations listed in Table 24 below. The seeds were allowed to dry for 3 weeks and then planted in natural soil in 9.14 m rows spaced 6.35 cm apart and at a depth of 3.81 cm. The plants were harvested and the yield was measured at harvest. Each treatment was repeated 4 times and planted in the field 4 times. The results are shown as a weight percentage relative to the control (substrate) treatment in Table 24 below.

[0743] Table 24. Production increase as a control percentage

[0744]

[0745] As can be observed in Table 24, all three glucanases resulted in a significant increase in the yield of soybeans, as well as PC-PLC and PI-PLC from Bacillus cereus.

[0746] Example 7: 2015 maize yield, endoglucanase

[0747] β-1,4-endoglucanase from *Thermolyticus fibrosis* (SEQ ID NO: 30; commercially available from Sigma-Aldrich, St. Louis MO, under the trade name E2164) was diluted in citrate dilution buffer to concentrations of 250 and 600 mU / mL. Commercially available hybrid BECK'S 5828YH maize seeds were placed in seed processors with seed treatment packages (“substrates”) containing prothioconazole, pendimethalin, metalaxyl, and thiamethoxam (EVERGOL Energy / PONCHO), 250 seeds per package. Each batch was mixed, and 250 μL of the solution was added to each tube, resulting in a final enzyme concentration of 200 or 600 μU / seed for the endoglucanase-coated seeds. Each batch was mixed again for 20 seconds to obtain a uniform coating on each seed. The seeds were allowed to dry for 3 weeks and then planted in natural soil in 9.14 m rows spaced 6.35 cm apart and at a depth of 3.81 cm. Harvest the plants and measure the yield at harvest. Each treatment was repeated four times. The results are shown in Table 25 below as a percentage of harvest weight relative to the harvest weight of the control treatment (calibrated).

[0748] Table 25. Production increase as a control percentage

[0749]

[0750] As can be observed in Table 25, both rates of thermophilic bacteria β-1,4-endoglucanase lead to increased maize yield.

[0751] Example 8: Isolation and Identification of Bacterial Strains that Promote Plant Growth

[0752] Rhizosphere soil samples were collected from the healthiest and most resistant potato, yellow zucchini, tomato, and bean plants, diluted with sterile water, and spread on nutrient agar plates. Bacterial isolates with high growth rates and the ability to subculture and reproduce were selected for further studies. The selected strains were grown in a basal medium (KH₂PO₄ 3 g, Na₂HPO₄ 6 g, NH₄Cl 1 g, NaCl 0.50 g, MgSO₄ 7H₂O 0.15 g, CaCl₂ 2H₂O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures of the selected strains were centrifuged (30 °C), the medium was decanted, and resuspended in an equal volume of distilled water. Ten bulb lettuce seeds from each treatment were planted at a depth of 1 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. Seeds were inoculated into 4cm pots, with 0.5μl of bacteria resuspended in water mixed with 10ml of H2O. 10ml of H2O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm 3 Seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–75°F (18–24°C) with 11 hours of light daily and 5 ml of water every 3 days. After one week, plant height, leaf diameter, and overall plant health were collected. Preliminary screening of rhizosphere isolates yielded over 200 different bacterial and fungal species from the rhizosphere of four plant species. Some bacterial species are described in Table 26. Identified strains are indicated by appropriate bacterial identification. Other strains are indicated by an unknown identification number. Inoculants with results close to the control (+ / - 2%) are not included in the table.

[0753] Table 26

[0754] bacterial inoculum Average height (cm) contrast SEM Unvaccinated 1.8 Comparison .07 Paracoccus kongeri NC35 2 111.1％ .05 Bacillus aryepoda CAP53 3.65 202.8％ .45 Bacillus curvatureii BT054 2.45 136.1％ .11 Bacillus mycoides strain BT155 2.17 120.4％ .21 Bacillus aryepoda CAP56 2.1 116.7％ .20 Bacillus niger BOBA57 2.8 155.6％ .03 Enterobacter cloacae CAP12 2.4 133.3％ .41 Unknown 8 1.77 77.8％ .65 Unknown122 1.9 105.6％ .11 Unknown 15 1.4 77.8％ .41 Unknown 39 1.8 100.0％ .20 Unknown 401 2 111.1％ .21 Unknown 402 1.53 85.2％ .27 Unknown 41 1.45 80.6％ .31 Unknown 42 1.4 77.8％ .15 Unknown 44 2.2 133.3％ .08 Unknown 51 1.83 102.9％ .21

[0755] The bacterial strains that had the greatest impact on overall plant health and plant height in the initial lettuce trial were further identified. The bacterial strains were cultured overnight at 37°C in Luria Bertani broth, and the overnight culture was centrifuged. The culture medium was decanted, and chromosomal DNA was isolated from the remaining bacterial pellet using the Qiagen Bacterial Chromosomal DNA Isolation Kit. PCR amplification of the 16S rRNA coding region of the chromosomal DNA was performed using primers E338F 5'-ACT CCT ACG GGA GGC AGC AGT-3' (SEQ ID NO: 108), E1099R A 5'-GGG TTG CGC TCG TTG C-3' (SEQ ID NO: 109), and E1099R B 5'-GGG TTG CGCTCG TTA C-3' (SEQ ID NO: 110). The PCR amplicons were purified using the Promega PCR Purification Kit, diluted, and sent to the University of Missouri DNACore for DNA sequencing. DNA sequences were compared with bacterial isolates, genera, and species in the NCBI BLAST database for identification by direct comparison with known strains. Table 26 lists the most frequently identified species. In many cases, the 16S rRNA DNA sequence was only sufficient to characterize the genus of the selected bacterial strain. In the absence of direct identification, additional biochemical analyses were performed using standard methods in the art to differentiate at the species and strain levels, and are listed in Table 27.

[0756] Table 27

[0757]

[0758] Example 9: Isolation and identification of bacterial strains that additionally promote plant growth

[0759] Soil samples were collected from fields near Gas, Kansas, diluted with sterile water, and spread on nutrient agar plates. Bacterial isolates with high growth rates and the ability to subculture and reproduce were selected for further studies. The selected strains were grown in basal medium (3 g KH₂PO₄, 6 g Na₂HPO₄, 1 g NH₄Cl, 0.50 g NaCl, 0.15 g MgSO₄·7H₂O, 0.013 g CaCl₂·2H₂O, and 1 g glucose per L dry weight). Overnight cultures of the selected strains were centrifuged (30°C), the medium was decanted, and the isolates were resuspended in an equal volume of distilled water. Maize seeds were coated with either a commercial seed polymer mixed with water alone (1.6 μl total of each seed) or a commercial seed polymer containing the selected bacterial strains (1.6 μl total of each seed). The coated seeds were planted in 3-inch (7.62 cm) diameter pots of loam topsoil (Columbia, MO) that had been sieved to remove large debris. Plants were grown at temperatures between 18–24°C (65–75°F) with 11 hours of light per day and 50 ml of water at planting time and every 3 days. After two weeks, plant height, leaf diameter, and overall plant health were collected. For germination determination and 3-day root length assessment, seeds were coated as described above and evenly distributed on each paper towel (10 seeds per towel). The paper towels were moistened with 10 ml of water, rolled up, placed in small plastic bags, and incubated at 30°C or on a germination heat pad at 27–30°C (80–85°F). Root measurements were recorded after 3 days. Preliminary screening of rhizosphere isolates resulted in the acquisition of over 100 different bacterial and fungal species from the rhizosphere. Some bacterial species are described in Table 28. Identified strains were indicated by appropriate bacterial identification.

[0760] Table 28

[0761]

[0762] Table 28 describes the bacterial strains that had the greatest impact on plant health. The bacterial strains were cultured overnight at 37°C in Luria Bertani broth, and the overnight culture was centrifuged. The culture medium was decanted, and the remaining bacterial pellet was used to isolate chromosomal DNA using the Qiagen Bacterial Chromosomal DNA Isolation Kit. PCR amplification of the 16S rRNA coding region of the chromosomal DNA was performed using primers E338F5'-ACT CCT ACG GGA GGC AGC AGT-3' (SEQ ID NO: 108), E1099R A5'-GGG TTG CGC TCGTTG C-3' (SEQ ID NO: 109), and E1099R B 5'-GGG TTG CGC TCG TTA C-3' (SEQ ID NO: 110). The PCR amplicons were purified using the Promega PCR Purification Kit, diluted, and sent to the University of Missouri DNA Core for DNA sequencing. DNA sequences were compared with bacterial isolates, genera, and species in the NCBI BLAST database for identification by direct comparison with known strains. Table 28 lists the most frequently identified species. In many cases, the 16S rRNA DNA sequence was only sufficient to characterize the genus of the selected bacterial strain. In the absence of direct identification, additional biochemical analyses were performed using standard methods in the art to differentiate strains at the species and strain levels, and the differentiated strains are listed in Table 29.

[0763] Table 29

[0764]

[0765] wk = weak growth or low growth

[0766] Example 10: Testing of plant growth-promoting bacterial strains on alfalfa

[0767] The selected strains were grown in a basic culture medium (3g KH₂PO₄, 6g Na₂HPO₄, 1g NH₄Cl, 0.50g NaCl, 0.15g MgSO₄·7H₂O, 0.013g CaCl₂·2H₂O, and 1g glucose, per L dry weight). The overnight culture of the selected strains (30°C) was centrifuged, the culture medium was decanted, and the bacteria were resuspended in an equal volume of distilled water. Ten ZEBA-coated alfalfa seeds from each treatment were planted at a depth of 0.6 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. ZEBA is a superabsorbent corn starch-based polymer used as a moisture-retaining seed coating. Seeds were inoculated with 0.5 μl of the resuspended bacteria mixed in water with 10 ml of H₂O. 10 ml of H₂O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm 3 The seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–75°F (18–24°C) with 11 hours of light per day and 5 ml of water every 3 days. Alfalfa was allowed to grow for one week to analyze germination and initial growth under the stated conditions. Table 30 lists the identified strains and final height data, as indicated by appropriate bacterial identification.

[0768] Table 30

[0769] bacterial inoculum Average height (cm) Compare SEM Unvaccinated 4.82 – .008 Bacillus aryepoda CAP56 4.85 101.20％ .016 Bacillus niger BOBA57 4.86 101.70％ .021 Enterobacter cloacae CAP12 5.6 116.23％ .020

[0770] Example 11: Testing of plant growth-promoting bacterial strains on cucumbers

[0771] The selected strains were grown in basal medium (KH₂PO₄ 3g, Na₂HPO₄ 6g, NH₄Cl 1g, NaCl 0.50g, MgSO₄·7H₂O 0.15g, CaCl₂·2H₂O 0.013g, and glucose 1g, per L dry weight). The overnight culture of the selected strains (30°C) was centrifuged, the medium was decanted, and the culture was resuspended in an equal volume of distilled water. Ten cucumber seeds from each treatment were planted at a depth of 1 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. For seed inoculation, 0.5 μl of the resuspended bacteria was mixed with 10 ml of H₂O in water. 10 ml of H₂O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm 3 The seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–75°F (18–24°C) with 11 hours of light per day and 5 ml of water every 3 days. Cucumbers were allowed to grow for 2 weeks to analyze emergence and initial growth under the stated conditions. Table 31 lists the identified strains and final height data, as indicated by their appropriate bacterial identification.

[0772] Table 31

[0773]

[0774]

[0775] Example 12: Testing of plant growth-promoting bacterial strains on yellow pumpkins

[0776] The selected strains were grown in basal medium (KH₂PO₄ 3g, Na₂HPO₄ 6g, NH₄Cl 1g, NaCl 0.50g, MgSO₄·7H₂O 0.15g, CaCl₂·2H₂O 0.013g, and glucose 1g, per L dry weight). The overnight culture of the selected strains (30°C) was centrifuged, the medium was decanted, and the culture was resuspended in an equal volume of distilled water. Ten cucumber seeds from each treatment were planted at a depth of 1 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. For seed inoculation, 0.5 μl of the resuspended bacteria was mixed with 10 ml of H₂O in water. 10 ml of H₂O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm 3 The seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–75°F (18–24°C) with 11 hours of light per day and 5 ml of water every 3 days. Cucumbers were allowed to grow for 2 weeks to analyze emergence and initial growth under the stated conditions. Table 31 lists the identified strains and final height data, as indicated by their appropriate bacterial identification.

[0777] Table 32

[0778]

[0779] Example 13: Testing of plant growth-promoting bacterial strains on ryegrass

[0780] The selected strains were grown in basal medium (KH₂PO₄ 3g, Na₂HPO₄ 6g, NH₄Cl 1g, NaCl 0.50g, MgSO₄·7H₂O 0.15g, CaCl₂·2H₂O 0.013g, and glucose 1g, per L dry weight). The overnight culture of the selected strains (30°C) was centrifuged, the medium was decanted, and the culture was resuspended in an equal volume of distilled water. Thirty ryegrass seeds from each treatment were planted in 0.3 cm deep topsoil (Columbia, MO) that had been sieved to remove large debris. Seed inoculation was performed with 0.5 μl of the resuspended bacteria mixed in 10 ml of H₂O. 10 ml of H₂O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm3 The seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–75°F (18–24°C) with 11 hours of light per day and 5 ml of water every 3 days. The ryegrass was allowed to grow for 1.5 weeks to analyze germination and initial growth under the stated conditions. Table 33 lists the identified strains and height data, as indicated by appropriate bacterial identification.

[0781] Table 33

[0782] bacterial inoculum Average height (cm) Compare SEM Unvaccinated 1.61 – .023 Bacillus aryepoda CAP53 2.01 124.70％ .012 Bacillus curvatureii BT054 2.21 137.30％ .034 Bacillus mycoides BT155 2.29 142.20％ .049 Bacillus aryepoda CAP56 2.19 136.00％ .009 Bacillus niger BOBA57 2.29 142.40％ .045 Enterobacter cloacae CAP12 1.98 122.50％ .015

[0783] Example 14: Testing of plant growth-promoting bacterial strains on corn

[0784] The selected strains were grown in basal medium (KH₂PO₄ 3g, Na₂HPO₄ 6g, NH₄Cl 1g, NaCl 0.50g, MgSO₄·7H₂O 0.15g, CaCl₂·2H₂O 0.013g, and glucose 1g, per L dry weight). The overnight culture of the selected strains (30°C) was centrifuged, the medium was decanted, and the culture was resuspended in an equal volume of distilled water. Ten maize seeds from each treatment were planted at a depth of 2.5 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. Seeds were inoculated with 0.5 μl of the resuspended bacteria mixed in 10 ml of H₂O. 10 ml of H₂O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm 3 The seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–75°F (18–24°C) with 11 hours of light per day and 5 ml of water every 3 days. The maize was allowed to grow for 2 weeks to analyze emergence and initial growth under the stated conditions. Table 34 lists the identified strains and final height data, as indicated by their appropriate bacterial identification.

[0785] Table 34

[0786]

[0787]

[0788] Example 15: Testing of plant growth-promoting bacterial strains on soybeans

[0789] The selected strains were grown in basal medium (KH₂PO₄ 3g, Na₂HPO₄ 6g, NH₄Cl 1g, NaCl 0.50g, MgSO₄·7H₂O 0.15g, CaCl₂·2H₂O 0.013g, and glucose 1g per L dry weight, or yeast-mannitol medium for short rhizobia or rhizobia). Overnight cultures of the selected strains were centrifuged (30°C), the medium was decanted, and the culture was resuspended in an equal volume of distilled water. Ten soybean seeds from each treatment were planted at a depth of 2.5 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. Seeds were inoculated with 0.5 μl of the resuspended bacteria mixed in 10 ml of H₂O. When testing two bacterial strains, 0.5 μl of each resuspended bacterial strain was mixed in 10 ml of H₂O. 10 ml of H₂O was sufficient to transport the bacteria to a depth of 3 inches. 3 (49.16cm 3 Soybeans were placed in soil saturated to allow for proper seed germination. Plants were grown at temperatures between 65-75°F (18-24°C) with 11 hours of light per day and 5 ml of water every 3 days. Soybeans were allowed to grow for 2 weeks to analyze emergence and initial growth under the stated conditions. Table 35 lists the identified strains and final height data, as indicated by their appropriate bacterial identification. Co-inoculation of the bacterial strains of this invention with a species of *Rhizobium brevicornum* or a species of *Rhizobium* resulted in increased plant growth compared to inoculum alone.

[0790] Table 35

[0791]

[0792] Example 16: Members of the Bacillus cereus family with plant growth-promoting properties

[0793] Bacillus strains BT155, EE118, EE141, BT46-3, EE349 (a member of the Bacillus cereus family), BT013A, and EE281 were grown in Luria Bertani broth at 37°C. Overnight cultures were vortexed to settle the cultures, the medium was poured off, and the cultures were resuspended in an equal volume of distilled water. Twenty maize seeds from each treatment were planted at a depth of 2.5 cm in topsoil (Columbia, MO) that had been sieved to remove large debris. Seeds were inoculated with 0.5 μl of the resuspended bacteria mixed in 50 ml of H2O. 50 ml of H2O was sufficient to transport the bacteria to a depth of 29 inches. 3 (475.22cm 3The seeds were placed in soil saturated to allow for proper germination. Plants were grown at temperatures between 65–72°F, with 13 hours of light per day and 5 ml of water every 3 days. Seedlings were allowed to grow for 2 weeks to analyze emergence and initial growth under the conditions described. Table 36 lists the identified strains and final height data, as indicated by their appropriate bacterial identification.

[0794] Table 36

[0795] bacterial inoculum Average height, cm, corn percentage SEM, <![CDATA[H2O control]]> 11.41 100％ .123 Bacillus mycosis fungoides EE118 12.43 108.9％ .207 Bacillus mycosis fungoides EE141 12.84 112.5％ .231 Bacillus mycoides BT46-3 11.81 103.5％ .089 Bacillus thuringiensis BT013A 12.05 105.6％ .148 Bacillus cereus family member EE128 13.12 114.9％ .159 Bacillus mycoides BT155 12.85 112.6％ .163 Bacillus megaterium EE281 11.99 105.1％ .098

[0796] Under the conditions described, all plant growth-promoting bacteria tested had a beneficial effect on maize height at two weeks. The Bacillus cereus family member strain EE128 showed the greatest effect in this experiment, increasing maize height by more than 14%.

[0797] Example 17: Isolation, identification and characterization of endophytic Bacillus cereus family bacterial strains

[0798] Member 349 of the Bacillus cereus family discussed in the preceding examples was found to possess endogenetic growth capabilities. Several other Bacillus cereus family members with endogenetic growth capabilities were also identified: Bacillus cereus family member EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus mycosis fungoides EE-B00363, Bacillus pseudomycosis fungoides EE-B00366, and Bacillus cereus family member EE-B00377.

[0799] To obtain these additional members of the Bacillus cereus family, commercial hybrid maize seeds were planted in potting soil and allowed to grow. The maize seeds were coated with a fungicide and a bio-inoculum. The plants were grown under artificial light for 14 hours, and plant growth was measured over the 14-day period. The plants were watered every three days during the experiment. After 14 days, the plants were extracted from the soil and washed to remove excess debris. The plants were then inverted, exposed to 5% bleach for 10 minutes, washed in water, exposed to hydrogen peroxide (10%) for 10 minutes, washed again in water, and the stems were separated using a sterile razor blade. The separated halves of the stems were placed face down on nutrient agar plates for two hours. After two hours, the stems were removed, and the agar plates were incubated at 30°C for 48 hours. After 48 hours, the colony morphology of the plates was examined, and colonies of Bacillus cereus family members found inside the plants were picked and transferred to nutrient agar. These were then grown overnight in brain heart infusion broth at 30°C and centrifuged at 10000×g for 5 minutes. The supernatant was removed, and the precipitate was frozen overnight at -20°C. Chromosomal DNA was then extracted from each clone, and the identity of each colony was verified by PCR using 16S rRNA primers. The amplicon was then sent for DNA sequencing and identification. The 16S rRNA sequences of these strains are provided in Table 17 above.

[0800] Example 18: Isolation, identification, and characterization of additional endophytic bacterial strains (non-Bacillus cereus family members)

[0801] Endophytic bacterial strains were isolated from maize seedlings: *Bacillus megaterium* EE385, one species of *Bacillus* EE387, *Bacillus circulans* EE388, *Bacillus subtilis* EE405, *Bacillus lysinophilus* EE442, certain species of *Bacillus lysinophilus* EE443, and *Bacillus pumilus* EE-B00143. Two-week-old maize seedlings were first sterilized. They were extracted from the soil and washed to remove excess debris. The plants were then inverted and exposed to 5% bleach for 10 minutes, washed in water, exposed to hydrogen peroxide (10%) for 10 minutes, and washed again in water. The stems were then separated using a sterile razor blade. The separated halves of the stems were placed face down on nutrient agar plates for two hours. After two hours, the stems of the plants were removed from the plates, and the plates were then incubated at 30°C for 48 hours. Endophytic *Bacillus* colonies were selected for further analysis. These strains were grown overnight in brain heart infusion broth at 30°C, and DNA was extracted from the cultures using the Qiagen Chromosome DNA Kit. The 16S rRNA gene was obtained by PCR amplification and then sent for DNA sequencing. A BLAST search of the obtained sequence was performed using the NCBI database to determine the species identity of Bacillus. The 16S rRNA sequence is provided in Table 17 above.

[0802] 1-Aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) was applied as a foliar spray to corn plants. Two amino acids of the D-cysteine ​​desulfurase from Bacillus thuringiensis strain IS5056 (SEQ ID NO: 113) were mutated, resulting in a moderate increase in ACC deaminase (1-aminocyclopropane-1-carboxylic acid ester) activity. Besides its D-cysteine ​​desulfurase activity, the native D-cysteine ​​desulfurase from Bacillus thuringiensis strain IS5056 possesses sufficient inherent ACC deaminase activity. However, for the purposes of this embodiment and Example 20, the native D-cysteine ​​desulfurase from Bacillus thuringiensis strain IS5056 (SEQ ID NO: 113) will be referred to as "D-cysteine ​​desulfurase". Since the mutant form of the enzyme (SEQ ID NO: 114) has increased ACC deaminase activity, for the purposes of this embodiment, the mutant enzyme will be referred to as "ACC deaminase". The mutated sequences are provided as SEQ ID NO:112 (nucleic acid) and SEQ ID NO:114 (protein). In Table 2, the two amino acid substitutions shown are indicated in bold and underlined. Using standard PCR mutagenesis techniques in the art, threonine at position 290 of SEQ ID NO:113 was substituted with a glutamic acid residue, and serine at position 317 of SEQ ID NO:113 was substituted with a leucine residue. The genes encoding d-cysteine ​​deaminase (SEQ ID NO:111) and ACC deaminase (SEQ ID NO:112) were then cloned into a Gram-positive pBC vector (a miniaturized version of the naturally occurring plasmid pBC16) under the control of the BclA spore formation promoter. The vector was then transformed into Bacillus thuringiensis. After spore formation in a basal medium releasing cell contents (including enzymes), all cells were removed by filtration, and the remaining active enzyme fraction was applied to the plants. ACC deaminase activity was quantified using the standard dinitrophenol hydrazine assay (Li et al., A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase, Lett Appl. Microbiol. 53(2):178–85 (2011).

[0803] Wild-type enzyme (SEQ ID NO:113) and enzyme with two point mutations (SEQ ID NO:114) were provided as free enzymes and delivered foliarly to 2-week-old maize (BECK'S 5828YH, V2-V3 developmental stage) and 4-week-old soybean plants (BECK'S 297NR, V2-V3 developmental stage). BECK'S 5828YH maize contained HERCULEX (rootworm resistance and corn borer resistance traits), glufosinate resistance, and glyphosate resistance traits. BECK'S 297NR soybean contained nematode resistance (SCN-SB) and glyphosate resistance traits.

[0804] Foliar application of D-cysteine ​​desulfurase and ACC deaminase was performed individually on 16 replicate plants in each experiment and compared with a separate surfactant control. The activities of the foliar-applied D-cysteine ​​desulfurase (SEQ ID NO: 113) and ACC deaminase (SEQ ID NO: 114) enzymes were normalized to the same protein content and applied at a consistent rate as a foliar spray containing 0.1% nonionic surfactant (NIS) (ALLIGARE SURFACE, Alligare LLC) to deliver the enzymes to maize and soybean plants at a rate of 10 ml / plant. The ACC deaminase activity is described herein as 1 mU equal to 1 nmol product / mg protein / hour at 30°C. The initial activity of D-cysteine ​​desulfurase used in this assay was 500 mU / ml, and the activity of ACC deaminase was 2,124 mU / ml. After dilution to 10 ml per plant, the enzyme delivery rate is 2.5% by volume, with a final concentration of 12.5 mU / ml (D-cysteine ​​desulfurase) and 53.1 mU / ml (ACC deaminase) per plant. ALLIGARE SURFACE surfactant is a blend of alkyl polyoxyethylene, ethylene glycol derivatives, humectants, and formulation aids.

[0805] Two weeks after foliar application, roots were harvested from corn or soybean plants, rinsed with water, and gently blotted dry to remove any excess water. The fresh root weight (g) was then determined. The fresh root weight of each treatment was calibrated against control plants treated only with a carrier containing only basal medium and 0.1% nonionic surfactant. The results are shown in Tables 37 and 38 below.

[0806] As shown in Table 37, foliar application of ACC deaminase in maize resulted in a significant (approximately 12%) increase in fresh root mass compared to plants treated with nonionic surfactant alone (*p = 0.015). In contrast, the mean fresh root mass of maize plants treated with D-cysteine ​​desulfurase was comparable to that of the control plants treated with surfactant alone.

[0807] In soybean plants treated with D-cysteine ​​desulfurase (Table 38), root weight increased slightly two weeks after foliar application. In contrast, soybean plants treated with ACC deaminase showed an average increase of 12% in root weight compared to the control.

[0808] This study simultaneously observed monocotyledonous maize and dicotyledonous soybean, demonstrating that foliar application of ACC deaminase (and to a lesser extent, D-cysteine ​​desulfurase) directly resulted in increased root mass in foliar-treated plants compared to the control.

[0809] Table 37. Average root mass of maize plants treated with ACC deaminase by foliar application compared to control plants

[0810]

[0811] Table 38. Average root mass of soybean plants treated with ACC deaminase by foliar application compared to control plants

[0812]

[0813]

[0814] ACC deaminase (SEQ ID NO: 114) was also applied as a furrow (soil application) treatment around hybrid rice seeds, which also resulted in increased plant growth. As described above, ACC deaminase (SEQ ID NO: 114) was produced and purified at the aforementioned initial concentration and delivered at a rate of 8 fl oz / Ac (584.2 ml / ha) per 2.5 gallons of water / Ac (23.4 liters / ha). After dilution in water, a final activity of 6.25 mU / ml was produced for D-cysteine ​​desulfurase and 52.1 mU / ml for ACC deaminase. The product was applied directly to the top of the seeds at a rate of 1 ml / seed and dried in the soil before the seeds were covered with loose soil. The results are shown in Table 39 below. For the furrow treatment using rice hybrids, an increase in mean height of approximately 131% was observed in both trials (36 plants each) calibrated against the control. This study shows that exogenous application of free ACC deaminase in furrows directly affects plant growth and vitality by increasing plant height.

[0815] Table 39. Growth-promoting properties of ACC deaminase when applied as a furrow treatment

[0816]

[0817] Example 20: ACC deaminase free enzyme delays fruit ripening

[0818] 1-Aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) degrades 1-aminocyclopropane-1-carboxylic acid ester (ACC), a natural precursor of ethylene (C2H4), which stimulates and regulates fruit ripening. Trace levels of ethylene play a role throughout the plant's life cycle by stimulating or regulating fruit ripening, flower opening, and fruit and leaf abscission or breakage. Ethylene is an important natural plant hormone used in agriculture to promote fruit ripening (Lin et al., Recent advances inethylene research, Journal of Experimental Botany 60:3311–3336 (2009)). Ethylene-induced ripening is characterized by accelerated discoloration (pigment accumulation) accompanied by softening of the peel or pericarp and the fleshy region inside the exocarp. To determine whether the application of free ACC deaminase or D-cysteine ​​sulfhydrylase to fruit could delay fruit ripening, both enzymes were applied to unripe mango fruit.

[0819] ACC deaminase and D-cysteine ​​thiolase were characterized and possessed the activities described in Example 19 above. The ACC deaminase (SEQ ID NO: 114) with two amino acid mutations described in Example 19 and the native D-cysteine ​​dethiolase (SEQ ID NO: 113) were expressed and provided as free enzymes using the method described in Example 19. As described in Example 19 above, the native D-cysteine ​​dethiolase (SEQ ID NO: 113) possessed both D-cysteine ​​dethiolase and ACC deaminase activities.

[0820] Immature mango fruits (commercially available variety, Keitt) were treated with either ACC deaminase or D-cysteine ​​desulfurase and compared to mango fruits treated with water (control) or a separate filtrate control (no enzyme expressed by the strain). Four fruits were used for each treatment group. The outer layer of the mango fruit was completely wetted with 1 mL of free enzyme (equivalent to a final protein concentration of 10 μg / mL in the filtrate). For this assay, the estimated ACC deaminase activity applied to the fruit with D-cysteine ​​desulfurase was 500 mU / mL, and the estimated ACC deaminase activity was 2124 mU / mL. Both control treatments (filtrate or water only) were also applied to the mango fruits using 1 mL volume. The mango fruits were then placed in sealed plastic bags overnight. The next day, excess liquid was removed with paper towels, and the fruit was blotted dry. The dried mango fruits were then placed in sealed brown bags (separate bags were used for different treatments) to enhance the ripening reaction over 4 days. The maturation reaction was scored on a scale of 1-5, with 1 being the least mature (firm, green or no color change) and 5 being the most mature (softened, color changed from green to yellow / pink). Different degrees of maturity (2-4) existed between these low and high scores. The maturation reactions of both softening and color change were then combined to produce a "fully matured reaction" on a scale of 1-10, which was used to determine the effectiveness of the treatment.

[0821] Data are provided in Table 40 below and represent the mean score of fruit in each treatment group. Treatment of mango fruit with ACC deaminase and D-cysteine ​​desulfurase as free enzymes resulted in delayed ripening compared to the water or filtrate control treatment alone after 4 days. Free enzyme treatment with either ACC deaminase or D-cysteine ​​desulfurase produced similar effects in the overall ripening response based on softening and color change when applied to mangoes. These results suggest that both types of enzymes can be used as fruit washing / rinsing treatments to delay fruit ripening and can be applied to other economically important fruits to prevent other stresses from accelerating ripening or causing fruit loss.

[0822] Table 40. ACC deaminase and D-cysteine ​​desulfatase free enzymes cause delayed ripening in mango trees.

[0823]

[0824]

[0825] Example 21: Glucanase and phospholipase on soybean seeds, in the field

[0826] β-1,4-endoglucanase (Thermolyticus; SEQ ID NO:30), β-1,3-D-glucanase (Snail spp.; SEQ ID NO:126), phosphatidylinositol-specific phospholipase C (Bacillus cereus; SEQ ID NO:116), and phosphatidylcholine-specific phospholipase C (Bacillus cereus; SEQ ID NO:115) were applied as free enzymes to soybean seeds (BECK'S 294NR). The free enzymes were diluted in water to the concentrations listed in Table 41 below (μU / seed or mU / seed). The unit (U) of endoglucanase or phospholipase activity was determined by the amount of enzyme required to break down 1 μmol / min / mL substrate (1U = 1 μmol substrate / min) under ideal temperature and conditions. The amount of enzyme solution required for the final activity of each seed treatment (1 μL / seed) was mixed with the seed treatments of metalaxyl and thiamethoxam. Dry the seeds completely before planting them in the field, using standard practices for planting depth and row spacing (1.5 to 2 inches (3.8 to 5 cm) deep to ensure normal root development and an average of 150,000 plants per acre (370,658 plants per hectare), with row widths of 30 inches (76.2 cm) and seed spacing of approximately 7 to 8 seeds per foot (26 seeds per meter)). Apply fertilizer as recommended by soil testing. Use herbicides for weed control and supplement with fertilizer as needed.

[0827] Three replicate trials were conducted, each consisting of 600 seeds. Soybean yield was measured approximately six months after sowing and reported in Table 41 below as absolute change in bushels per acre (Bu / Ac) or metric tons per hectare (MT / ha) relative to the control (water only) and the percentage yield calibrated against the control. Application of endoglucanase or phospholipase (β-1,4-endoglucanase (Acidobacterium pyridostigmae), β-1,3-D-glucanase (Solanum lyratum), phosphatidylinositol-specific phospholipase C (Bacillus cereus), and phosphatidylcholine-specific phospholipase C (Bacillus cereus)) as seed treatment resulted in increased yield compared to the control (water-treated) seeds. Of the enzymes tested, phosphatidylcholine-specific phospholipase C (Bacillus cereus) provided the largest yield increase compared to the control, resulting in an increase of more than 8 Bu / Ac (more than 0.5 MT / ha) or a 145% increase in yield compared to untreated control seeds (see Table 41).

[0828] Table 41. Application of glucanase and phospholipase as seed treatments to increase soybean yield

[0829]

[0830]

[0831] Example 22: Free phospholipase on maize seeds, greenhouse

[0832] Phosphatidylcholine-specific phospholipase C (PLC) from Bacillus cereus (SEQ ID NO: 115) was diluted in water to concentrations ranging from 20 mU / seed to 800 mU / seed (as listed in Table 42 below). The unit of PLC enzyme activity was determined by the amount of enzyme required to decompose 1 μmol / min / mL of substrate (1 U = 1 μmol substrate / min) under ideal temperature and conditions.

[0833] Two replicate experiments consisted of 18 seeds, with each commercial hybrid BECK'S 5828YH maize seed placed in a 50 mL conical tube. Each conical tube was vortexed, and 18 μL of enzyme solution was added to each tube to achieve the final enzyme concentration of 20, 50, 100, 200, 400, 600, or 800 mU of PLC activity per seed. The conical tube was vortexed again for 20 seconds to obtain a uniform coating on each seed. The seeds were dried for 5 minutes and then planted at a depth of 2.54 cm to a depth of 39.7 cm containing topsoil. 3 Two seeds per pot. After planting, add 50 mL of room temperature water to each pot to allow germination. Store the pots in a growth chamber with artificial lighting, receiving approximately 300 μmol m... -2 s -1 Light levels, continuous 13 / 11 lighting / daytime cycle and 21°C daytime / 15°C nighttime temperature range.

[0834] Mean plant height was measured in two replicates using 18 plants per trial in each treatment group. Differences in plant height after seed treatment with the PLC enzyme were calibrated against control plants that received only water treatment. Table 42 shows the changes in plant height as a percentage of the calibrated mean plant height against the control, and reports the standard deviation (STDEV) for both trials. As can be seen from Table 42, when compared and calibrated against control plants treated with water (non-enzyme), PLC enzyme activity ranging from 50 mU / seed to 600 mU / seed resulted in a significant increase in maize plant height (cm).

[0835] Table 42. Phospholipase C (PLC) as a seed treatment applied to maize to promote growth.

[0836]

[0837]

[0838] In the second experiment, the potency of phospholipase D required to achieve optimal growth was determined. Phospholipase D (PLD) (SEQ ID NO: 117) from oat acidophilus was diluted in water to concentrations ranging from 20 mU / seed to 800 mU / seed. Units of PLD enzyme activity were determined by the amount of enzyme required to break down 1 μmol / min / mL substrate (1 U = 1 μmol substrate / min) under ideal temperature and conditions. Each enzyme activity level was tested in duplicate using 18 plants per experiment. For each treatment group in both experiments, 18 seeds of a commercial maize hybrid (BECK'S 5828 YH) were placed in 50 mL conical tubes. Each conical tube was vortexed and 18 μL of enzyme solution was added to each tube to achieve a final enzyme concentration of 20, 50, 100, 200, 400, 600, or 800 mU per PLD seed. Using 1 μl volumes, PLD potency ranging from 20 mU / seed to 800 mU / seed was applied to maize seeds to determine the optimal PLD seed treatment for promoting growth. The conical tube was vortexed again for 20 seconds to obtain a uniform coating on each seed. The seeds were dried for 5 minutes and then spread at a depth of 2.54 cm on a 39.7 cm bed containing topsoil. 3 Two seeds were placed in each pot. After planting, 50 mL of room temperature water was added to each pot to allow germination. The pots were then kept in a growing chamber with artificial light, receiving approximately 300 μmol of water. -2 s -1 Light levels, with a continuous 13 / 11 lighting / day cycle and a day / night temperature range of 21°C.

[0839] Mean plant height (in cm) in two replicate trials, with 18 plants per treatment per trial. Plant heights produced from PLD-treated seeds are compared to the control and expressed as a percentage of the mean plant height calibrated against the non-enzyme-treated (water) control plants, and reported in Table 43 below with the standard deviation (STDEV) of the two trials.

[0840] As shown in Table 43, phospholipase D applied to maize seeds had a positive effect on plant growth at each tested enzyme activity level. In each case, plants treated with PLD as seeds had increased height compared to control plants.

[0841] Table 43. Phospholipase D (PLD) as a seed treatment applied to maize to promote growth.

[0842]

[0843]

[0844] Example 23: Free phospholipase and xylose glucanase in corn and soybean, foliar application, greenhouse

[0845] Free xylose glucanase (SEQ ID NO: 125; Bacillus spp.) and phospholipase D (SEQ ID NO: 117; Acidobacterium oatum) were applied as foliar treatments using spray bottles, delivering 10 ml / plant, and 0.1% nonionic surfactant (ALLIGARE SURFACE) to 2-week-old hybrid maize (BECK'S 5828YH). The average plant height was calibrated against control plants, which received foliar application of water plus surfactant alone. Both xylose glucanase and phospholipase D treatments applied as foliar sprays to maize plants resulted in increased plant height compared to the control plants (Table 44). The greatest increases in plant growth were observed with xylose glucanase applied at a foliar application rate of 600 μU / ml and phospholipase D applied at a foliar application rate of 200 μU / ml, resulting in increases of 106.5% and 111.1%, respectively, compared to the control plants.

[0846] Table 44. Foliar treatment of maize using xylose glucanase and phospholipase D as free enzymes to promote maize plant growth.

[0847]

[0848] In another experiment, phospholipase D (PLD) from the genus *Acidobacterium* was applied as a seed treatment to soybean seeds (BECK'S 297NR), using 1 μL volume equivalent to providing 600 mU / seed and 800 mU / seed of final activity (these activities were selected for testing in soybean based on the titers described for maize in Example 22 above). Using PLD activities of 600 mU / seed and 800 mU / seed as seed treatments for soybean seeds had a positive effect on plant growth rate.

[0849] Treated seeds were planted in a greenhouse and allowed to grow. When the plants reached developmental stages V2-V3, their total biomass, root biomass, and nodule count were measured. Stages V2-V3 are the earliest developmental stages for nodule formation. Nodulation begins in soybean seedlings once root hairs are present on the taproot or branch roots. Nitrogen fixation begins approximately 2-3 weeks after initial rhizobium infection. Soybean plants fully develop their first trifoliate leaf at stages V1-V2 and are measured at the estimated peak nitrogen fixation. Effective nodulation of soybean roots results in higher yields per acre and higher quality seed, protein, and oil production.

[0850] Two independent experiments were conducted (18 replicates per treatment group per experiment). Data from plants in the PLD treatment were calibrated against control plants grown from water-treated control seeds.

[0851] Compared with plants grown from water-treated control seeds that had not received PLD free enzymes, the application of PLD at a seed treatment of 800 mU / soybean seed resulted in a significant increase in total biomass and root biomass (Table 45).

[0852] PLD treatment also increased the nodule count on plant roots. Both seed treatments with 600 mU or 800 mU PLD activity resulted in increased nodule formation compared to the untreated control, with the 800 mU treatment nearly doubling the number of nodules on soybean plant roots.

[0853] Table 45. Biomass effect of phospholipase D treatment as a soybean seed treatment

[0854]

[0855] Example 24: Free enzymes on corn, in the field

[0856] Free xyloglucanase, xylanase, deacetylated chitosanase, bryosinase, xylosidase, protease, and lipase were diluted in water to the activity levels listed in Table 46 below. Hybrid maize (BECK'S 5828 YH) seeds were treated with 1 μL of free enzyme solution / seed to obtain the activity per seed (1 U = 1 μmol substrate / min), as shown in Table 46 below. Seeds were completely dried and planted in 24' (7.3 m) rows with four replicates per treatment, with a seed spacing of 1.72 seeds / ft / row (5.64 seeds / m / row). Field seedbeds were prepared for each location using conventional or conservation tillage methods for maize cultivation. Herbicides were applied for weed control and supplemented with culture as needed. Each trial was repeated four times. Seed treatments included prothioconazole, pendimethalin, metalaxyl, and thiamethoxam.

[0857] After harvest, the absolute change in bushels per acre (Bu / Ac) or metric tons per ton for each free enzyme treatment was measured and calibrated against the yield of untreated control (water) plants (Table 46, below). The mean yield for control maize seeds was 162 Bu / Ac (10.17 MT / ha). Seed treatments with bryosinase, protease, or lipase resulted in the greatest increase in maize yield relative to the control plants. Treatment with bryosinase showed the greatest yield increase compared to the control plants, with an average increase of 22 Bu / Ac (1.39 MT / ha), equivalent to an increase of 114% when calibrated against maize control plants.

[0858] Table 46. Increased yield of free enzymes applied to maize

[0859]

[0860] In the second experiment, free enzymes (endoglucanase, exoglucanase, deacetylated chitosanase, protease, and phytase) were applied via foliar application to maize (BECK'S Hybrid 5140HR) at the V5-V8 developmental stage, exhibiting HERCULEX rootworm traits and glyphosate resistance, at four sites across the Midwest. To ensure uniform leaf coverage, all enzyme treatments and controls were additionally treated with a nonionic surfactant (ALLIGARE SURFACE) at a final concentration of 0.1%. Absolute changes in bushels per acre (Bu / Ac) (and equivalents in MT / ha) were reported on control plants, as well as yields calibrated against control plants (“water / surfactant control”) (Table 47). Results of foliar treatments using free enzymes were reported as absolute yields in Bu / Ac (or MT / ha) and absolute changes in yield (Bu / Ac or MT / ha) calibrated by four replicates against control plants (Table 47). Compared with the control plants (plants treated with water and surfactant only), the positive yields of enzyme treatment increased. Phytase used as a foliar treatment resulted in the greatest overall increase in yield (absolute yield change of ~24 Bu / Ac (~1.51 MT / ha) relative to the control).

[0861] Table 47. Increased yield of free enzymes when used as a foliar treatment on maize

[0862]

[0863] Example 25: Lipase on corn seeds, greenhouse

[0864] Experiments were conducted to determine whether lipase applied as a seed treatment in maize also promoted plant growth. Lipase (Pseudomonas fluorescens; SEQ ID NO: 119) was diluted in water to concentrations providing 3000 μU and 6000 μU of lipase activity per seed. Lipase was applied to maize seeds (BECK'S maize variety 5828YH) using 3000 μU / seed and 6000 μU / seed activities, with 1 μL of enzyme / seed used to achieve the reported activity for each seed. Seeds were dried for 5 minutes and then planted at a depth of 2.54 cm to a depth of 39.7 cm containing topsoil. 3 Two seeds were placed in each pot. After planting, 50 mL of room temperature water was added to each pot to allow germination. The pots were then kept in a growing chamber with artificial light, receiving approximately 300 μmol of water. -2 s -1 The light level was maintained at a continuous 13 / 11 light / day cycle and a day / night temperature range of 21°C. At the end of 2 weeks, when all plants had reached the V2 to V3 developmental stages, the height of the maize plants treated with lipase was measured and calibrated with the height of the control plants whose seeds were treated with water only.

[0865] The experiment was repeated twice, with 18 plants per treatment group (3 replicates per treatment group), and the values ​​from multiple experiments were averaged and reported in Table 48 along with the standard deviation (STDEV). Lipase was applied as a free enzyme, with activities of 3000 μU and 6000 μU per seed, resulting in average increases in plant height of approximately 106% and 103%, respectively.

[0866] Table 48. Lipase treatment as a height effect of maize seed treatment

[0867]

[0868] Example 26: Lipase or phospholipase on corn, greenhouse furrows

[0869] Lipase (Berberis cepacia) applied as a furrow treatment was used to determine whether applying lipase as a free enzyme to the area surrounding maize seeds resulted in an early positive growth benefit for maize plants. The lipase (Berberis cepacia, SEQ ID NO: 118) was diluted in water to the activity levels listed in Table 49 below. Maize seeds (BECK'S 6626RR) were planted at a depth of 2.54 cm to a depth of 39.7 cm containing topsoil. 3 Two seeds were placed in each pot. After planting but before covering the seeds, 1 μL of lipase with an activity ranging from 2 μU to 200 μU was applied to each furrow around the seed. Instead of treating the seed subset, β-1,4-endoglucanase (Thermolytic alginate; SEQ ID NO: 30) was applied to the area around the seed in the furrow at an activity of 1000 μU. The pots were kept in an artificially lit growth chamber and received approximately 300 μmol / m³ of lipase. -2 s -1 Light levels were maintained at a continuous 13 / 11 light / day cycle and a day / night temperature range of 21°C. Approximately two weeks later, when the plants reached the V2 to V3 developmental stage, their height was measured and calibrated against control plants that received only water. Plants treated with lipase were further compared to plants that received β-1,4-endoglucanase (a thermophilic bacterium).

[0870] The experiments were repeated twice (18 plants per treatment group per experiment). The mean plant height of the treatments versus the control was reported as standard deviation (STDEV) over both experiments (Table 49). Lipase applied at 20 μU per seed as a furrow treatment for maize resulted in the greatest increase in plant height compared to other lipase activities used as furrow treatments. β-1,4-endoglucanase, used as a free enzyme in furrow treatments, also resulted in a positive change in plant height and a growth-promoting effect was reported in maize plants. Lipase applied around the seed at 20 μU / area (volume per mL of water) was comparable to that of seeds treated with β-1,4-endoglucanase in the furrow treatment.

[0871] Table 49. Increased levels and effects of β-1,4-endoglucanase plant furrow treatment applied to the area surrounding maize seed plants to promote growth.

[0872]

[0873] In the second experiment, phosphatidylcholine-specific phospholipase C (SEQ ID NO: 115) from Bacillus cereus was applied to maize seeds (BECK'S 5828 YH) together with fertilizer (SF) containing 12% ammonia nitrogen and 58% available phosphate (derived from monoammonium phosphate) using the direct furrowing method described above. The enzyme was applied at a rate of 8 fl oz / Ac (584.2 ml / ha) or approximately 1200 mU to the area surrounding the seeds. This treatment resulted in a mean increase in plant height over three replicates, which was calibrated to 105% compared to the control treated with water and fertilizer alone. The results are shown in Table 50 below.

[0874] Table 50. Effects of furrow treatment on plant height in maize using free phospholipase C

[0875]

[0876] Example 27: Acid phosphatase on pumpkin and corn, in furrows

[0877] The effects of acid phosphatases (alone or in combination with lipases, β-xylanases, pectinases, mannanases, bryophyllases, or xylanases) on plant growth were tested. The free enzyme containing acid phosphatase (wheat, a mixture of two different isotypes, sequences provided by SEQ ID NO. 130 and 131, commercially available from Sigma-Aldrich, St. Louis MO, product number P3627) was used alone or in combination with lipase (Pseudomonas fluorescens, SEQ ID NO: 119), β-xylanase (Neocomyces parva, SEQ ID NO: 122), pectinase (Aspergillus, SEQ ID NO: 129), mannanase (a species of Bacillus, SEQ ID NO: 128), bryotanase (Bacillus subtilis, SEQ ID NO: 43), or xylanase (Trichophyton thermophilum, SEQ ID NO: 121) at the activity levels listed in Table 51, in the same manner as described in Example 26, and applied directly to the area around the pumpkin seed (continental hybrid pumpkin, commercially available from Park Seed, product 05298). Enzyme treatment was administered to pumpkin seeds containing a seed treatment (Thiram) and fertilizer (SF) containing 12% ammonia nitrogen and 58% available phosphate. Fermentation enzyme and fertilizer were applied separately using the application rates listed in Table 51 below in units of activity per ml volume, delivered in 1 ml per seed to the soil surrounding the seed. Plant height was determined in two trials, with 18 plants measured in each trial. Data are reported in Table 51 below, providing the percentage change in plant height of pumpkin seeds treated with free furrow enzymes compared to control seeds (fertilizer alone). Free acid phosphatase treatment alone showed an average increase in plant height of 49.6% compared to control plants. Pumpkin seeds treated with free enzyme furrows, containing acid phosphatase in combination with enzymes such as lipase, β-xylanase, pectinase, mannanase, bryophyllase, or xylanase, showed increased plant height compared to pumpkins treated with water and fertilizer. Compared to combinations of acid phosphatase with other enzymes (lipase, β-xylanase, pectinase, mannanase, bryophyllase, or xylanase), furrow treatment with acid phosphatase alone resulted in the greatest average percentage increase in overall growth, as indicated by an increase in plant height.

[0878] Table 51. Changes in plant height in pumpkins treated with application of acid phosphatase and free enzyme.

[0879]

[0880] The effects of acid phosphatases (alone or in combination with lipases, β-xylanases, pectinases, mannanases, bryophyllases, or xylanases) on plant growth were tested. The free enzyme containing acid phosphatase (wheat, a mixture of two different isotypes, sequences provided by SEQ ID NO. 130 and 131, commercially available from Sigma-Aldrich, St. Louis MO, product number P3627) was used alone or in combination with lipase (Pseudomonas fluorescens, SEQ ID NO: 119), β-xylanase (Neocomyces parva, SEQ ID NO: 122), pectinase (Aspergillus, SEQ ID NO: 129), mannanase (a species of Bacillus, SEQ ID NO: 128), bryotanase (Bacillus subtilis, SEQ ID NO: 43), or xylanase (Trichophyton thermophilum, SEQ ID NO: 121) at the activity levels listed in Table 51, in the same manner as described in Example 26, and applied directly to the area around the pumpkin seed (continental hybrid pumpkin, commercially available from Park Seed, product 05298). Enzyme treatment was administered to pumpkin seeds containing a seed treatment (Thiram) and fertilizer (SF) containing 12% ammonia nitrogen and 58% available phosphate. Fermentation enzyme and fertilizer were applied separately using the application rates listed in Table 51 below in units of activity per ml volume, delivered in 1 ml per seed to the soil surrounding the seed. Plant height was determined in two trials, with 18 plants measured in each trial. Data are reported in Table 51 below, providing the percentage change in plant height of pumpkin seeds treated with free furrow enzymes compared to control seeds (fertilizer alone). Free acid phosphatase treatment alone showed an average increase in plant height of 49.6% compared to control plants. Pumpkin seeds treated with free enzyme furrows, containing acid phosphatase in combination with enzymes such as lipase, β-xylanase, pectinase, mannanase, bryophyllase, or xylanase, showed increased plant height compared to pumpkins treated with water and fertilizer. Compared to combinations of acid phosphatase with other enzymes (lipase, β-xylanase, pectinase, mannanase, bryophyllase, or xylanase), furrow treatment with acid phosphatase alone resulted in the greatest average percentage increase in overall growth, as indicated by an increase in plant height.

[0881] Table 52. Changes in plant height in maize treated with furrowing agents using a combination of acid phosphatase, phospholipase C, and β-1,4-endoglucanase with biostimulants.

[0882]

[0883] Free enzymes containing acid phosphatase (wheat, a mixture of two different isotypes having the sequences provided herein by SEQ ID NO. 130 and 131) or phosphatidylcholine-specific phospholipase C (Bacillus cereus; SEQ ID NO: 115) were applied to the area surrounding hybrid maize seeds (BECK'S 5828 YH) using direct furrow application. The furrow treatment with the enzyme was combined with treatment with the hormone biostimulant (CYTOPLEX, commercially available from Miller Chemical & Fertilzier, LLC) containing marine plant extracts, activators, gibberellic acid, and indole-3-butyric acid. The furrow treatment was applied at application rates of 2, 4, and 8 fl oz / seed area (59.14, 118.29, and 236.59 ml / seed area, respectively). Plant height was determined for two trials, with 18 plants measured in each trial. Data are reported in Table 53 below as the percentage change in plant height of maize seeds treated with either acid phosphatase or phospholipase C compared to control seeds (biostimulant alone). Treatment with free acid phosphatase at rates of 2, 4, and 8 Fl. oz (59.14, 118.29, and 236.59 ml) per applied seed region (approximately 150 mU / ml, 300 mU / ml, and 600 mU / ml per seed region) increased plant height compared to control plants, with application at 4 Fl. oz (118.29 ml) resulting in an 8.3% increase relative to the control at a rate of 300 mU / ml. Furrow treatment of maize grown with phospholipase C at rates of 2 and 4 Fl. oz (59.14 and 118.29 ml) per subregion (approximately 150 and 300 mU per subregion, respectively) resulted in increased plant height compared to control maize grown with biostimulant alone. For plant growth, a preferred application rate of 4 fl oz (118.29 ml) resulted in an 11.4% increase in plant height compared to the biostimulant-only control. The biostimulant-only control resulted in slower maize plant growth compared to the water-only treatment.

[0884] Table 53. Changes in plant height in maize treated with furrowing using a combination of acid phosphatase or phospholipase C and biostimulants.

[0885]

[0886]

[0887] Example 28: Protease or xylosidase on corn, furrows

[0888] Protease A (Cetorhizium anisopliae; SEQ ID NO: 127) and xylosidase (Bacillus pumilus; SEQ ID NO: 123) were applied as furrowing free enzyme treatments to maize, and their effects on plant height and growth were examined. For protease A and xylosidase, a similar method was used for lipase furrowing treatments with maize as described in Example 26. After planting maize but before covering the seeds with loose soil, the furrowing treatment (1 ml per seed) was applied to the area surrounding the maize seeds (BECK'S 5828 YH). The furrowing treatments using protease A and xylosidase were delivered in 1 μL volumes, representing 428 μU / seed area activity for protease and 714 μU / seed area activity for xylosidase (per ml). When calibrated against control plants (water treatment only), both protease A and xylosidase resulted in increased plant height. The results are shown in Table 54 below.

[0889] Table 54. Plant height of maize treated with furrows using protease A or xylosidase.

[0890]

[0891] Example 29: Xylanase or xylosidase on corn and soybean seeds, greenhouse

[0892] Free enzymes were applied as seed treatments to maize and soybean. Maize (BECK'S 5828 NR) and soybean (BECK'S 297 NR) seeds were treated with xylanase derived from *Trichophyton thermophilum* (SEQ ID NO: 121) or *Neocomonas parva* (SEQ ID NO: 122) and xylanase derived from *Bacillus pumilus* (SEQ ID NO: 123), using 2 μL volumes of the activity equivalent to 600 μU per seed for xylanase (*Trichophyton thermophilum*; *Neocomonas parva*) and 714 μU per seed for xylosidase (*Bacillus pumilus*). Two separate groups of maize and soybean seeds were treated with β-1,4-endoglucanase (*Thermolyticus cytosolicus*; SEQ ID NO: 30) at 1000 μU activity / seed. Seeds were then engraved and planted in commercial topsoil as described in Example 1. At the end of 14 days, the mean percentage change in plant height compared to the water control was determined for two replicate maize trials and one soybean trial (12 plants in each trial). The change in mean plant height (cm) was compared with the control plants and maize and soybean plants grown from seeds treated with β-1,4-endoglucanase (thermosinophil), which resulted in increased plant growth when applied as seed treatment for maize and soybean. The mean percentage change in plant height calibrated against the water control treatment is reported in Table 55 below, with the standard deviation (STDEV) of the means from the two trials conducted in maize and soybean.

[0893] Compared to control plants, xylanase (*Trichophyton thermophilum*; *Neocoma parvifolium*) applied as a seed treatment at an activity of 600 μU / seed to maize and soybean seeds resulted in increased plant height. Xylanase (*Trichophyton thermophilum*) treatment applied to maize seeds resulted in an average increase of 9% in maize plant height and an average increase of 12% in soybean. β-xylanase (*Neocoma parvifolium*) treatment applied to maize seeds resulted in an average increase of 4% in maize plant height. Xylosidase (*Bacillus pumilus*) applied as a seed treatment at 714 μU / seed to maize and soybean seeds resulted in increases of approximately 9–11% in both maize and soybean plants compared to control plants. The positive effects of xylanase and xylosidase treatments as free enzyme treatments on plant height in maize and soybean seeds were comparable to or better than those of β-1,4-endoglucanase (*Thermophyton*). Plant height was measured after two weeks and will be calibrated for plants that received only fertilizer treatment.

[0894] Table 55. Corn and soybean treated with endoglucanase, endoglucanase and xylosidase as seed treatments

[0895]

[0896] Example 30: Free enzymes and activity increments for seed treatment and furrow treatment on corn and soybeans

[0897] The following enzymes were diluted with water: bryophyllase (Bacillus subtilis, product E-LICHN, purchased from Megazyme; SEQ ID NO:43), xylose glucanase (Bacillus species, product E-XEGP, purchased from Megazyme; SEQ ID NO:125), β-xylanase (Bacillus stearothermophilus, product E-XYNBS, purchased from Megazyme; SEQ ID NO:25), mannanase (Bacillus species, product E-BMABS, purchased from Megazyme; SEQ ID NO:128), lipase (Burkholderia stearothermophilus, product 534641, purchased from Sigma-Aldrich; SEQ ID NO:120), pectinase (Aspergillus japonicus, product P3026, purchased from Sigma-Aldrich; SEQ ID NO:129), and β-1,4-endoglucanase (Thermolytic acid-degrading bacteria, product E2164, purchased from Sigma-Aldrich; SEQ ID NO:129). NO:30) to obtain the activity levels listed in Table 56 below. Aliquots (1 μL) of these formulations were used to treat the seeds in the experiments described in this example and Example 31.

[0898] Table 56. Titration of enzymes used to determine optimal activity as seed treatments for corn and soybean seeds to promote plant growth.

[0899] enzymes Source of organisms Increasing utilization rate (μU activity) Water control – 0μU Polysaccharide enzyme Bacillus subtilis 400μU Polysaccharide enzyme Bacillus subtilis 500μU Polysaccharide enzyme Bacillus subtilis 600μU Polysaccharide enzyme Bacillus subtilis 700μU Polysaccharide enzyme Bacillus subtilis 800μU Polysaccharide enzyme Bacillus subtilis 900μU Xylo-glucanase Bacillus species 500μU Xylo-glucanase Bacillus species 600μU Xylo-glucanase Bacillus species 1500μU Xylo-glucanase Bacillus species 3000μU Xylo-glucanase Bacillus species 4000μU β-xylanase Thermophilic liposporidum 50μU β-xylanase Thermophilic liposporidum 300μU β-xylanase Thermophilic liposporidum 500μU β-xylanase Thermophilic liposporidum 1500μU β-xylanase Thermophilic liposporidum 3000μU β-xylanase Thermophilic liposporidum 5000μU Mannanase Bacillus species 60μU Mannanase Bacillus species 300μU Mannanase Bacillus species 600μU Mannanase Bacillus species 1200μU Mannanase Bacillus species 3000μU Mannanase Bacillus species 6000μU Lipase Thermophilic Burkholderia 2μU Lipase Thermophilic Burkholderia 5μU Lipase Thermophilic Burkholderia 10μU Lipase Thermophilic Burkholderia 20μU Lipase Thermophilic Burkholderia 50μU Lipase Thermophilic Burkholderia 200μU pectinase Aspergillus japonicum 60μU pectinase Aspergillus japonicum 300μU pectinase Aspergillus japonicum 600μU pectinase Aspergillus japonicum 1200μU pectinase Aspergillus japonicum 3000μU pectinase Aspergillus japonicum 6000μU β-1,4-endoglucanase Fiber-degrading heat-loving bacteria 1000μU

[0900] The potency of six free enzymes (bryospinase, xyloglucanase, xylanase, mannanase, lipase, and pectinase) was tested to determine the optimal activity when used as seed treatments to promote the growth of maize (BECK'S 5828 YH) and soybean (BECK'S 297NR). The potency activities determined to be most suitable for use as seed treatments of the six enzymes are listed in Table 57 below (listed as free enzyme activity for each seed). Experiments were conducted under the same environmental conditions in the controlled growth environment described in Example 29. The percentage change in mean plant height for the six enzymes used as seed treatments applied to maize or soybean seeds was determined (Table 57 below). The mean plant height for each of the six enzymes was calibrated against the mean plant height of plants grown from seeds that received water-controlled treatment and recorded as a percentage change (Table 57). Furthermore, the treatment of maize seeds with the free enzyme was compared with treatment with the free enzyme β-1,4-endoglucanase, as this enzyme has been shown to promote growth when used as a seed treatment agent on maize plants (see Examples 1-4, 7, 26 and 29 above).

[0901] Compared to control plants grown from non-enzyme-treated seeds, all six free enzymes (bryosinase, xyloglucanase, β-xylanase, mannanase, lipase, and pectinase) increased plant height in maize and soybean when used as seed treatments for their optimized activity levels. The results are shown in Table 57 below. After calibration with control plants, application of β-1,4-endoglucanase to maize seeds resulted in an increase in maize plant height. When considering maize and soybean plant varieties, mannanase resulted in the largest increase in plant height after calibration with control plants (107% increase in maize and 110% increase in soybean).

[0902] Table 57. High Efficacy of Free Enzymes Applied to Plant Seed Treatments in Maize and Soybean Plants

[0903]

[0904] The potency of four identical enzymes (bryosinase, xylose-glucanase, mannanase, and pectinase, listed in Table 56 above) was determined to determine their optimal activity for promoting plant growth when applied as a furrow treatment to maize (BECK'S 5828YH). The potency of each enzyme was optimized for growth potential (Table 58), and each enzyme was applied directly to the area around the seed with 1 ml of water and covered with soil before planting. Two weeks after planting, plant height was measured and calibrated against the height of plants that did not receive enzyme treatment but only water control. The experiment was repeated in three trials with 18 plants per trial, and measurements were averaged across trials to produce the percentage change in average maize plant height (compared to control). Data for the four free enzymes: bryosinase, xylose-glucanase, mannanase, and pectinase are reported in Table 58. All maize plants showed increased height when the free enzymes bryosinase, xylose-glucanase, and pectinase were applied as a furrow treatment around the maize seed, compared to the water-only control.

[0905] Table 58. Height effect of free enzymes applied as furrow treatment around maize seeds.

[0906]

[0907] The potency of the same six free enzymes (bryosinase, xyloglucanase, xylanase, mannanase, lipase, and pectinase listed in Table 56) was determined to determine the optimal activities for seed treatment in soybean (BECK'S 297 NR). The activity (μU / seed) of each enzyme is reported in Table 59 below. Three trials were conducted, each with 18 plants, and changes in total biomass, shoot biomass, root biomass, and nodulation were measured. The experiments were conducted under the same environmental conditions in the controlled growth environment described in Example 6 above. In some trials, additional seed groups were treated with β-1,4-endoglucanase (1000 μU / seed). Changes in total biomass, shoot biomass, root biomass, and nodulation are reported in Table 59 below as percentage changes (%) calibrated to soybean seeds not treated with the free enzymes (water treatment control).

[0908] Table 59. Free enzymes applied as seed treatment to promote plant growth

[0909]

[0910]

[0911] Example 31: Free enzymes used as seed treatment to increase zucchini yield

[0912] The bryophyllase, xyloglucanase, xylanase, lipase (free enzyme), and β-1,4-endoglucanase (Thermolyticus celluloseis, SEQ ID NO: 30) described in Example 30 above were applied as seed treatments at the optimal rate. The titer of the enzymes, with activity reported in μU / seed (Table 60), was determined by titer assay series and applied to zucchini seeds (Spineless Beauty, commercially available from Park Seed). The total yield of seeds treated with the free enzymes of bryophyllase, xyloglucanase, xylanase, lipase, and β-1,4-endoglucanase is reported in Table 60 as the total weight of harvested zucchini calibrated against the control, and is the average of two harvests completed in August (Columbia, Missouri). Treatment of zucchini seeds with free enzymes of biosyntheticase (700 μU / seed), xylanase (3000 μU / seed), and lipase (50 μU / seed) all showed positive yield increases compared to the control. The increase in total harvestable yield of zucchini plants with free enzyme seed treatments of biosyntheticase, xylanase, and lipase showed a similar overall yield advantage to that of β-1,4-endoglucanase (1000 μU).

[0913] Table 60. Zucchini yield after treatment with free enzymes in zucchini seeds.

[0914] deal with Total production as a percentage of the control Polysaccharidase 700 μU 113％ Xyloglucanase 3000 μU 89％ β-xylanase 300 μU 118％ lipase 50μU 130％ β-1,4-Endoglucanase 1000 μU 132％

[0915] Example 32: Synergistic effect of multiple enzymes on maize, furrows

[0916] Mannanase (Bacillus spp.; SEQ ID NO: 128), xyloglucanase (Bacillus spp., SEQ ID NO: 125), phosphatidylcholine-specific phospholipase C (Bacillus cereus, SEQ ID NO: 115), and xylosidase (Bacillus pumilus; SEQ ID NO: 123) were applied as furrowing free enzyme treatments to maize (BECK'S 5828YH), and the effects on plant height and growth were assessed. Enzyme treatments, including combinations of enzymes, are described in Table 61. For all free enzymes, a method similar to that described in Example 26 for lipase furrowing treatment with maize was used. Briefly, after planting maize but before covering the seeds with loose soil, the furrowing treatment was applied to the area around the maize seeds. Each treatment was applied at a volume of 1 ml per seed, comprising the enzyme and a fertilizer containing ortho-polyphosphate and potassium acetate. Furrow treatments were administered using each enzyme at ratios of 300 mU / seed region (mannanase and phosphatidylcholine-specific phospholipase C), 500 mU / seed region (xyloglucanase), and 714 mU / seed region (xylosidase). The enzyme was delivered to the seeds in a 1 ml volume for each subregion, containing both the enzyme and fertilizer. Fifty-four seeds were used per treatment, divided into three replicates of 18 plants each. Plant height was measured approximately two weeks later, and calibration was performed against control plants treated only with fertilizer.

[0917] The results are shown in Table 61 below. Neither mannanase nor xylose glucanase alone resulted in a significant increase in height. Both phospholipase C and xylose glucanase alone resulted in an increase in plant height. Surprisingly, the combination of phospholipase C and either mannanase or xylose glucanase resulted in a synergistic increase in plant height compared to either treatment alone. The combination of mannanase and xylose glucanase was also more effective than either enzyme alone.

[0918] Table 61. Plant height of maize treated with furrows using free mannanase, xylose glucanase, xylosidase, phospholipase C or a combination thereof.

[0919]

[0920]

[0921] Example 33: The cumulative effect of multiple enzymes on pumpkin, furrows

[0922] Mannanase (Bacillus; SEQ ID NO: 128), bryotanase (Bacillus subtilis, SEQ ID NO: 43), acid phosphatase (wheat, a mixture of two different isotypes having sequences SEQ ID NO: 130 and 131 provided herein), pectinase (Aspergillus japonicus, SEQ ID NO: 129), β-xylanase (Neocallismastixpatriciarum, SEQ ID NO: 122), and β-xylanase (Bacillus thermophilus, SEQ ID NO: 25) were applied as furrow treatments to Ambassador hybrid squash (commercially available from Park Seed, product 05298), and the effects on plant height and growth were examined. For all free enzymes, a method similar to that described in Example 26 for lipase furrow treatment with maize was used. In short, the furrow treatment was applied to the area around the squash seeds after planting but before covering the seeds with loose soil. Each treatment was applied in a 1 ml volume for each seed, comprising the enzyme and fertilizer containing monoammonium phosphate. Furrow treatments using each enzyme were delivered at ratios of 300 mU / seed region (mannanase), 600 mU / seed region (bryophyllase), 30 mU / seed region (pectinase), 35 μU / seed region (acid phosphatase), and 1500 mU / seed region (β-xylanase). The enzyme was delivered to the seed in a 1 ml volume for each seed region, containing both the enzyme and fertilizer. Two weeks later, plant height was measured and calibrated against plants that received only the fertilizer treatment.

[0923] The results are shown in Table 62 below. Acid phosphatase alone led to an increase in height relative to the fertilizer-only control, and this effect was slightly better when bryotanase was applied in combination with acid phosphatase. Large increases were observed when the fertilizer / acid phosphatase combination was further enhanced with any of the xylanases, pectinase, or mannanase. These non-cellulose-degrading carbohydrate hydrolases combined with acid phosphatase significantly increased plant height as a soil transport mechanism.

[0924] Table 62. Plant height of pumpkins treated with furrows using free mannanase, xylanase, acid phosphatase, pectinase, bryotanase or combinations thereof.

[0925]

[0926] Implementation

[0927] For further examples, other non-limiting embodiments of this disclosure are described below.

[0928] Implementation 1 is an enzyme comprising an amino acid sequence encoding an enzyme having 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity and a signal peptide that causes the enzyme to be secreted when expressed in a microorganism.

[0929] Embodiment 2 is the enzyme of Embodiment 1, wherein the enzyme having ACC deaminase activity comprises an enzyme derived from Bacillus bacteria.

[0930] Embodiment 3 is an enzyme having ACC deaminase activity, wherein the amino acid sequence of the enzyme contains at least one amino acid substitution relative to the sequence of wild-type D-cysteine ​​desulfurase or ACC deaminase from Bacillus spp., and wherein the amino acid substitution results in an increase in ACC deaminase activity compared to the ACC deaminase activity of wild-type D-cysteine ​​desulfurase or ACC deaminase under the same conditions.

[0931] Implementation 4 is the enzyme of Implementation 3, wherein the enzyme further comprises a signal peptide that causes the enzyme to be secreted when expressed in a microorganism.

[0932] Implementation 5 is an enzyme of any one of Implementation 1, 2 and 4, wherein the microorganisms include Bacillus, Pseudomonas, Rhizobium, Bacillus-like bacteria, Lysinobacter, Paracoccus, Intermediate Rhizobium, Short Rhizobium, Azotobacter, Arthrobacter, Azotobacter, Azospirobacter, Pink pigment facultative methyltrophic bacteria, mycorrhizal fungi, Gastromycosis fungi, Trichoderma fungi, Kruvorella fungi, Glycotric fungi or any combination thereof.

[0933] Implementation 6 is the enzyme of Implementation 5, wherein the microorganisms include Bacillus, Lysinobacillus, Pseudomonas, Bacillus-like bacteria, or any combination thereof.

[0934] Embodiment 7 is an enzyme from any one of Embodiments 1–6, wherein the enzyme comprises Bacillus thuringiensis enzyme or Bacillus pseudomycota enzyme.

[0935] Embodiment 8 is the enzyme of Embodiment 7, wherein the enzyme comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with any one of SEQ ID NO. 7–9 and 113, and has ACC deaminase activity.

[0936] Embodiment 9 is an enzyme of any one of Embodiments 3–8, wherein the amino acid sequence of the enzyme contains two amino acid substitutions relative to the sequence of wild-type D-cysteine ​​desulfurase or ACC deaminase, wherein the amino acid substitutions result in an increase in ACC deaminase activity compared to the ACC deaminase activity of the wild-type enzyme under the same conditions.

[0937] Embodiment 10 is an enzyme of any one of Embodiments 3–9, wherein the amino acid sequence of the enzyme comprises:

[0938] The threonine residue at position 290 of SEQ ID NO:7 is replaced by a glutamic acid residue, and the serine residue at position 317 of SEQ ID NO:7 is replaced by a leucine residue;

[0939] The threonine residue at position 290 of SEQ ID NO:8 is replaced by a glutamic acid residue, and the serine residue at position 317 of SEQ ID NO:8 is replaced by a leucine residue;

[0940] The threonine residue at position 290 of SEQ ID NO:9 is replaced by a glutamic acid residue, and the serine residue at position 317 of SEQ ID NO:9 is replaced by a leucine residue; or

[0941] The threonine residue at position 290 of SEQ ID NO:113 is replaced by a glutamic acid residue, and the serine residue at position 317 of SEQ ID NO:223 is replaced by a leucine residue.

[0942] Embodiment 11 is an enzyme of any one of Embodiments 3–10, wherein the enzyme comprises or is composed of SEQ ID NO: 10, 11, 12 or 114.

[0943] Embodiment 12 is an enzyme of Embodiment 1 or 2, wherein the enzyme comprises an amino acid sequence that is 100% identical to any one of SEQ ID NO. 7–9 and 113.

[0944] Implementation method 13 is a recombinant microorganism expressing any one of the enzymes in implementation methods 1–12.

[0945] Implementation method 14 is a recombinant microorganism of implementation method 13, wherein the expression of enzymes is increased compared with the enzyme expression level of wild-type microorganisms of the same species under the same conditions.

[0946] Embodiment 15 is a formulation comprising an enzyme of any one of Embodiments 1–12 or a recombinant microorganism of Embodiment 13 or 14 and an agriculturally acceptable carrier.

[0947] Embodiment 16 refers to plant seeds treated with any of the enzymes in Embodiments 1–12, the recombinant microorganisms in Embodiments 13 or 14, or the preparation in Embodiment 15.

[0948] Embodiment 17 is a method for stimulating plant growth and / or promoting plant health, comprising applying an enzyme of any one of Embodiments 1–12, a recombinant microorganism of Embodiment 13 or 14, or a preparation of Embodiment 15 to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or a plant seed.

[0949] Implementation 18 is the method of Implementation 17, wherein the method includes applying an enzyme from any of Implementations 1–12 to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed.

[0950] Implementation 19 is a method of Implementation 17 or 18, wherein the method includes applying a free enzyme to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or a plant seed.

[0951] Embodiment 20 is a method of any one of Embodiments 17–19, wherein the method includes applying the recombinant microorganism of Embodiment 13 or 14 to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or a combination of seeds.

[0952] Implementation 21 is a method for stimulating plant growth and / or promoting plant health, comprising applying a free enzyme to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed, wherein the enzyme is selected from phospholipases, lipases, xylanases, xylosidases, lactones, mannanases, pectinases, deacetylated chitosanases, proteases, acid phosphatases, non-cellulose-degrading dextranases, ACC deaminases, and any combination thereof.

[0953] Implementation 22 is a method for stimulating plant growth and / or promoting plant health, comprising applying two or more free enzymes to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed, wherein the enzymes are independently selected from phospholipases, lipases, xylanases, xylosidases, lactones, deacetylated chitosanases, proteases, phytases, acid phosphatases, mannanases, pectinases, glucans, and ACC deaminases.

[0954] Implementation 23 is a method for stimulating plant growth and / or promoting plant health, comprising applying a free enzyme to a plant or plant seed, wherein the enzyme comprises a glucanase, and wherein applying the enzyme to the plant seed comprises: (a) applying the enzyme to the plant seed at the time of planting; or (b) coating the plant seed with the enzyme.

[0955] Implementation 24 is the method of Implementation 23, wherein the method includes coating plant seeds with a seed coating preparation containing an enzyme and an agriculturally acceptable carrier.

[0956] Implementation 25 is a method of implementation 23 or 24, wherein the method further includes applying an enzyme or extended protein to a plant growth medium or a region around a plant or plant seed.

[0957] Embodiment 26 is a method of Embodiment 25, wherein the method further includes applying the enzyme or extended protein to a plant growth medium.

[0958] Embodiment 27 is a method of Embodiment 26, wherein the method further includes applying the enzyme and the extended protein to a plant growth medium.

[0959] Implementation 28 is a method for stimulating plant growth and / or promoting plant health, comprising applying a free enzyme to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed, wherein the enzyme comprises a glucanase, and the method further comprising applying an extended protein to the plant growth medium, the plant, the plant seed, or a region surrounding a plant or plant seed.

[0960] Implementation 29 is a method of implementation 28, wherein applying the enzyme or expansive protein to plant seeds includes: (a) applying the enzyme or expansive protein to the plant seeds at the time of planting; or (b) coating the plant seeds with the enzyme or expansive protein.

[0961] Implementation method 30 is the method of implementation method 29, wherein the method includes coating plant seeds with a seed coating agent, the seed coating agent comprising:

[0962] Enzyme, extended protein, or both enzyme and extended protein; and

[0963] An agriculturally acceptable carrier.

[0964] Implementation 31 is a method for stimulating plant growth and / or promoting plant health, which includes applying a free enzyme to a plant or plant seed, wherein the enzyme comprises phytase.

[0965] Implementation 32 is a method for stimulating plant growth and / or promoting plant health, comprising applying fertilizer and free enzyme to a plant growth medium, an area around a plant or plant seed, or to a plant or plant seed, wherein the free enzyme comprises phytase.

[0966] Implementation method 33 is a method for stimulating plant growth and / or promoting plant health, comprising applying recombinant microorganisms to a plant growth medium, a plant, a plant seed, or a region surrounding a plant seed, wherein:

[0967] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0968] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, mannanase, pectinase, protease, phytase, acid phosphatase, and any combination thereof; and

[0969] Enzymes or extended proteins are expressed during the vegetative growth of recombinant microorganisms.

[0970] Implementation method 34 is a method for stimulating plant growth and / or promoting plant health, comprising applying recombinant microorganisms to a plant growth medium, a plant, a plant seed, or a region surrounding a plant or plant seed, wherein:

[0971] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0972] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, mannanase, pectinase, phytase, acid phosphatase, ACC deaminase, and any combination thereof; and

[0973] The enzyme or extended protein also contains a signal peptide that causes the enzyme or extended protein to be secreted.

[0974] Implementation method 35 is a method for stimulating plant growth and / or promoting plant health, which includes

[0975] Recombinant microorganisms are applied to plant growth media, plants, plant seeds, or the area surrounding plants or plant seeds, wherein:

[0976] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0977] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, phytase, mannanase, pectinase, acid phosphatase, and any combination thereof; and

[0978] The enzyme or extended protein does not bind to the outer spore wall of recombinant Bacillus cereus family members.

[0979] Implementation method 36 is a method for stimulating plant growth and / or promoting plant health, comprising:

[0980] Recombinant microorganisms are applied to plant growth media, plants, plant seeds, or the area surrounding plants or plant seeds, wherein:

[0981] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0982] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, phytase, mannanase, pectinase, acid phosphatase, and any combination thereof; and

[0983] Enzymes or extended proteins are not components of fusion proteins.

[0984] Implementation method 37 is plant seeds coated with recombinant microorganisms, wherein:

[0985] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0986] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, phytase, mannanase, pectinase, acid phosphatase, ACC deaminase, and any combination thereof; and

[0987] Enzymes or extended proteins are expressed during the vegetative growth of recombinant microorganisms.

[0988] Implementation method 38 is a plant seed coated with recombinant microorganisms, wherein:

[0989] Recombinant microorganisms express enzymes or extended proteins, wherein the expression level of the enzyme or extended protein is increased compared with the expression level of the enzyme or extended protein in the same species of wild-type microorganisms under the same conditions;

[0990] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, mannanase, pectinase, phytase, acid phosphatase, ACC deaminase, and any combination thereof; and

[0991] The enzyme or extended protein also contains a signal peptide that causes the enzyme or extended protein to be secreted.

[0992] Implementation method 39 is plant seeds coated with recombinant microorganisms, wherein:

[0993] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0994] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, mannanase, pectinase, phytase, acid phosphatase, ACC deaminase, and any combination thereof; and

[0995] The enzyme or extended protein does not bind to the outer spore wall of recombinant Bacillus cereus family members.

[0996] Implementation method 40 is a plant seed coated with recombinant microorganisms, wherein:

[0997] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[0998] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, deacetylated chitosanase, glucanase, protease, mannanase, pectinase, phytase, acid phosphatase, ACC deaminase, and any combination thereof; and

[0999] Enzymes or extended proteins are not components of fusion proteins.

[1000] Embodiment 41 is a composition comprising fertilizer and an enzyme or extended protein, wherein the enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof.

[1001] Embodiment 42 is the composition of Embodiment 41, wherein the enzyme comprises a free enzyme.

[1002] Implementation method 43 is a composition comprising fertilizer and recombinant microorganisms, wherein:

[1003] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[1004] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof; and

[1005] Enzymes or extended proteins are expressed during the vegetative growth of recombinant microorganisms.

[1006] Implementation method 44 is a composition comprising fertilizer and recombinant microorganisms, wherein:

[1007] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[1008] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof; and

[1009] The enzyme or extended protein also contains a signal peptide that causes the enzyme or extended protein to be secreted.

[1010] Embodiment 45 is a composition comprising fertilizer and recombinant microorganisms, wherein:

[1011] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of enzymes or extended proteins is increased compared with the expression level of enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[1012] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof; and

[1013] The enzyme or extended protein does not bind to the outer spore wall of recombinant Bacillus cereus family members.

[1014] Embodiment 46 is a composition comprising fertilizer and recombinant microorganisms, wherein:

[1015] Recombinant microorganisms express enzymes or extended proteins, wherein the expression of the enzymes or extended proteins is increased compared with the expression level of the enzymes or extended proteins in wild-type microorganisms of the same species under the same conditions;

[1016] The enzyme is selected from phospholipase, lipase, xylanase, xylosidase, lactonease, mannanase, pectinase, deacetylated chitosanase, protease, acid phosphatase, phytase, glucanase, ACC deaminase, and any combination thereof; and

[1017] Enzymes or extended proteins are not components of fusion proteins.

[1018] Embodiment 47 is a composition of any one of Embodiments 41–46, wherein the composition further comprises an agriculturally acceptable carrier, additional agrochemicals, or a combination thereof.

[1019] Embodiment 48 is a method of any one of Embodiments 34–36, a seed of any one of Embodiments 38–40, or a composition of any one of Embodiments 44–47, wherein the enzyme or extended protein is expressed during the vegetative growth of the recombinant microorganism.

[1020] Embodiment 49 is the method of Embodiment 33 or 48, the seed of Embodiment 37 or 48, or a composition of any one of Embodiments 43, 47, and 48, wherein the recombinant microorganism comprises a recombinant spore-forming microorganism.

[1021] Embodiment 50 is a method of any one of Embodiments 33, 35, 48 and 49, a seed of any one of Embodiments 37, 39, 48 and 49, or a composition of any one of Embodiments 43 and 45–49, wherein the enzyme or extended protein further comprises a signal peptide that causes the enzyme or extended protein to be secrete...

Claims

1. A composition comprising a fertilizer and a free enzyme, wherein the free enzyme comprises a phospholipase, and wherein: Phospholipases include phospholipase C or phospholipase D, and phospholipases are streptococcal phospholipases, bacillus phospholipases, clostridium phospholipases, or acidophilus phospholipases.

2. The composition of claim 1, wherein the composition further comprises a second free enzyme, wherein the second free enzyme is selected from xylose glucanase and mannanase.

3. The composition of claim 1, wherein the composition further comprises an agriculturally acceptable carrier, additional agrochemicals, or a combination thereof.

4. The composition of claim 1, wherein the phospholipase comprises Bacillus phospholipase.

5. The composition of claim 1, wherein the Streptomyces phospholipase comprises Streptomyces crotonii phospholipase, wherein the Bacillus phospholipase comprises Bacillus cereus phospholipase or Bacillus thuringiensis phospholipase, or wherein the Clostridium phospholipase comprises Clostridium perfringens phospholipase.

6. The composition of claim 5, wherein the *Streptomyces cereus* phospholipase comprises *Streptomyces cereus* phospholipase D, wherein the *Bacillus cereus* phospholipase comprises *Bacillus cereus* phosphatidylcholine-specific phospholipase C or *Bacillus cereus* phosphatidylinositol 1-specific phospholipase C, or wherein the *Clostridium perfringens* phospholipase comprises *Clostridium perfringens* phospholipase C.

7. The composition of claim 1, wherein the phospholipase comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO. 18, 19, and 115–117.

8. The composition of claim 7, wherein the phospholipase comprises the amino acid sequence shown in any one of SEQ ID NO. 18, 19 and 115–117.

9. The composition of claim 2, wherein the composition further comprises an agriculturally acceptable carrier, additional agrochemicals, or a combination thereof.

10. The composition of claim 2, wherein the phospholipase comprises Bacillus phospholipase.

11. The composition of claim 2, wherein the Streptomyces phospholipase comprises Streptomyces crotonii phospholipase, wherein the Bacillus phospholipase comprises Bacillus cereus phospholipase or Bacillus thuringiensis phospholipase, or wherein the Clostridium phospholipase comprises Clostridium perfringens phospholipase.

12. The composition of claim 11, wherein the *Streptomyces cereus* phospholipase comprises *Streptomyces cereus* phospholipase D, wherein the *Bacillus cereus* phospholipase comprises *Bacillus cereus* phosphatidylcholine-specific phospholipase C or *Bacillus cereus* phosphatidylinositol 1-specific phospholipase C, or wherein the *Clostridium perfringens* phospholipase comprises *Clostridium perfringens* phospholipase C.

13. The composition of claim 2, wherein the phospholipase comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO. 18, 19, and 115–117.

14. The composition of claim 2, wherein the phospholipase comprises Bacillus cereus phosphatidylcholine-specific phospholipase C, and wherein the second free enzyme is Bacillus mannanase or Bacillus xyloglucanase.

15. The composition of claim 14, wherein Phospholipase C and mannanase; or Phospholipase C and xylose glucanase It exists in an effective quantity due to synergistic effects.

16. The composition of claim 13, wherein the phospholipase comprises the amino acid sequence shown in any one of SEQ ID NO. 18, 19 or 115–117.

17. The composition of claim 2, wherein the second free enzyme is xylose glucanase.

18. The composition of claim 2, wherein the second free enzyme is mannanase.

19. The composition of claim 17, wherein the xylose glucanase comprises an amino acid sequence having at least 98%, at least 99%, or 100% identity with SEQ ID NO:

125.

20. The composition of claim 18, wherein the mannanase comprises an amino acid sequence having at least 98%, at least 99%, or 100% identity with SEQ ID NO:

128.

21. The composition of any one of claims 1-20, wherein the fertilizer comprises nitrogen, phosphate, potassium, zinc, iron, boron, copper or any combination thereof.

22. The composition of claim 21, wherein the fertilizer comprises 12% ammonia nitrogen and 58% available phosphate.

23. The composition of any one of claims 1-20, wherein the first free enzyme and / or the second free enzyme comprises a crude cell extract containing the enzyme.

24. The composition of any one of claims 1-20, wherein the first free enzyme and / or the second free enzyme comprises a partially purified enzyme.

25. The composition of any one of claims 1-20, wherein the first free enzyme and / or the second free enzyme comprises purified enzymes.

26. The composition of claim 3 or 9, wherein the agriculturally acceptable carrier comprises a dispersant, a surfactant, an additive, water, a thickener, an anti-sticking agent, compost, granules, a colorant, a stabilizer, a preservative, a polymer, a coating material, or a combination thereof.

27. The composition of claim 26, wherein the additive comprises oil.

28. The composition of claim 26, wherein the anti-sticking agent comprises diatomaceous earth.

29. The composition of any one of claims 3-20, wherein the composition comprises a seed coating composition; a liquid composition applied to a plant or a plant growth medium; or a solid composition applied to a plant or a plant growth medium.

30. The composition of claim 29, wherein the seed coating composition comprises an aqueous or oil-based solution applied to the seed or a powder or granular formulation applied to the seed.

31. The composition of claim 29, wherein the liquid composition applied to the plant or the plant growth medium comprises a concentrated composition or a ready-to-use composition.

32. The composition of claim 29, wherein the solid composition applied to the plant or the plant growth medium comprises a granular composition or a powdered reagent.

33. A method of stimulating plant growth and / or promoting plant health, comprising applying the composition of claim 1 or 2 to a plant, a plant seed, or an area surrounding the plant or plant seed.

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