Microbial consortia that form dipicolinic acid and methods for selecting microbes for co-formulation with carriers

Abstract

To provide compositions and methods that bring about improved microbial survival and / or improved co-formulation of microbes with carriers or seeds.SOLUTION: In some embodiments, the method comprises selecting one or more microbe species with expanded survivability or survival time alone and / or in co-formulation with one or more carriers or seeds. In some embodiments, the method comprises: selecting microbes for co-formulation with a carrier or seed, including identifying microbes comprising one or more dipicolinic acid (DPA) synthetase genes, microbes expressing one or more DPA synthetase proteins, and / or microbes producing a detectable amount of DPA; and selecting microbes for co-formulation with carriers.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This claims the benefit of U.S. Provisional Application No. 62 / 681,469, filed June 6, 2018, which is incorporated by reference in its entirety.

[0002] Field The present disclosure relates to methods for identifying microorganisms that produce dipicolinic acid and microorganisms with improved viability, methods for co-formulating the microorganisms with carriers, and compositions comprising the microorganisms and / or the co-formulations. [Background technology]

[0003] Microbial-based plant biostimulants provide a sustainable agricultural practice that protects the health of biological systems. Furthermore, supplementing plant and soil microbiomes with beneficial microorganisms has the potential to promote plant growth and plant fitness, increase productivity, improve soil fertility, and reduce chemical inputs, resulting in more sustainable agricultural practices. In current agricultural practices, microbial biostimulants can be co-applied and / or co-formulated with a number of wet or dry carriers. Summary of the Invention [Means for solving the problem]

[0004] Microbial inoculants may be sensitive to the chemical nature of the carrier(s) used. In addition, storage conditions and storage time before application may also affect microorganisms. These factors may negatively affect their viability and ultimately limit their effectiveness in the field. Disclosed herein are compositions and methods that result in improved survival of microorganisms and / or improved co-formulation of microorganisms with carriers or seeds. In some embodiments, the method includes selecting one or more microorganisms that have extended viability or survival time, either alone and / or co-formulated with one or more carriers or seeds.

[0005] In some embodiments, disclosed herein are microorganisms that produce dipicolinic acid (DPA) and compositions comprising such microorganisms. In one example, the composition includes Bacillus amyloliquefaciens, Bacillus firmus, Bacillus flexus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus koreensis, Bacillus drentensis, Bacillus subtilis, Clostridium bifermentans, Clostridium beijerinckii, Clostridium pasteurianum, Lactobacillus paracasei, Fontibacillus sp.(panacisegetis), Oceanobacillus oncorhynchi, Paenibacillus lautus, Paenibacillus azoreducens, Paenibacillus chibensis, Paenibacillus cookii, Paenibacillus sp.(chitinolyticus), Paenibacillus sp.(P1XP2), Pseudomonas sp. and Streptomyces griseus were listed. In one embodiment, the composition comprises cells of a microbial species deposited with the American Type Culture Collection (ATCC, Manassas, VA) on May 16, 2019 and assigned accession number PTA-125924. In other embodiments, the disclosed microbial consortia or compositions comprise, consist essentially of, or consist of two or more (e.g., five or more, ten or more, fifteen or more, twenty or more, or all) microorganisms having a 16S rDNA sequence having at least 95% identity (e.g., at least 96%, 97%, 98%, 99% identity, or a higher percentage identity) to SEQ ID NOs: 3-25.

[0006] Also disclosed are compositions comprising the microorganism or consortium (e.g., DFC consortium) of the present disclosure and one or more carriers (e.g., dry or liquid carriers) or one or more seeds. In some examples, the carriers include liquid or dry fertilizers, soil-derived materials, organic materials, inert materials, dust-suppressing chemicals, or mixtures of two or more thereof.

[0007] In some embodiments, the method includes selecting a microorganism for co-formulation with a carrier or seed, including identifying a microorganism that contains one or more dipicolinic acid (DPA) synthase genes, expresses one or more DPA synthase proteins, and / or produces a detectable amount of DPA; and selecting a microorganism for co-formulation with a carrier. In some embodiments, the method also includes co-formulating the selected microorganism with a carrier or seed. In some examples, the selected microorganism includes one or more of those included in Table 25 or 26, including, but not limited to, all of those listed in Table 26.

[0008] In some examples, the method includes detecting one or more DPA synthase genes (e.g., DPA synthase subunit A (DpaA) gene and / or DPA synthase subunit B (DpaB) gene) or one or more DpaA and / or DpaB proteins in a microorganism. The DpaA gene includes a nucleic acid encoding a DpaA protein having at least 20% (e.g., at least 60%) sequence identity to any one of the amino acid sequences of FIG. 1 (e.g., SEQ ID NOs: 26-41). The DpaB gene includes a nucleic acid encoding a DpaB protein having at least 20% (e.g., at least 60%) sequence identity to any one of the amino acid sequences of FIG. 2 (e.g., SEQ ID NOs: 42-56). The DpaA protein includes a DpaA protein having at least 20% (e.g., at least 60%) sequence identity to any one of the amino acid sequences of FIG. 1 (e.g., SEQ ID NOs: 26-41). The DpaB protein includes a DpaB protein having at least 20% (e.g., at least 60%) sequence identity to any one of the amino acid sequences of FIG. 2 (e.g., SEQ ID NOs: 42-56). In a further example, the method includes detecting one or more Isf genes or proteins in the microorganism. The Isf gene includes a nucleic acid encoding an Isf protein having at least 20% (e.g., at least 60%) sequence identity to any one of the amino acid sequences of FIG. 3 (e.g., SEQ ID NOs: 57-67). The Isf protein includes an Isf protein having at least 20% (e.g., at least 60%) sequence identity to any one of the amino acid sequences of FIG. 3 (e.g., SEQ ID NOs: 57-67). In another example, the method includes detecting DPA in the microorganism or in a medium containing the microorganism, for example, using a fluorescent assay.

[0009] In some embodiments, the method includes co-formulating one or more selected microorganisms with a carrier by contacting the selected microorganisms (including but not limited to the microbial consortia disclosed herein) in liquid or dry form with one or more liquid or dry carriers. In some examples, the carrier includes liquid or dry fertilizer, soil-derived material, organic material, inert material, dust-suppressing chemicals, or a mixture of two or more thereof. In other examples, the method includes treating a seed with one or more selected microorganisms (including but not limited to the microbial consortia disclosed herein). In some examples, the method further includes co-formulating one or more selected microorganisms with a carrier or seed and one or more microorganisms that do not contain one or more DPA synthase genes, do not express one or more DPA synthase proteins, and / or do not produce detectable amounts of DPA.

[0010] In certain embodiments, for example, the following items are provided: (Item 1) Bacillus amyloliquefaciens, Bacillus firmus, Bacillus flexus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus koreensis, Bacillus drentensis, Bacillus subtilis, Clostridium bifermentans, Clostridium beijerinckii, Clostridium pasteurianum, Lactobacillus paracasei, Fontibacillus sp.(panacisegetis), Oceanobacillus oncorhynchi, Paenibacillus lautus, Paenibacillus azoreducens, Paenibacillus chibensis, Paenibacillus cookii, Paenibacillus A composition comprising cells of a microbial species comprising or consisting of each of: Paenibacillus sp. (chitinolyticus), Paenibacillus sp. (P1XP2), Pseudomonas sp. and Streptomyces griseus. (Item 2) A composition comprising cells of a microbial species comprising or consisting of a microorganism having a 16S rDNA nucleic acid sequence having at least 99% sequence identity to each of SEQ ID NOs: 3-25. (Item 3) The composition according to item 2, comprising cells of a microbial species comprising or consisting of a microorganism having each of the 16S rDNA nucleic acid sequences of SEQ ID NOs: 3 to 25. (Item 4) Composition comprising American Type Culture Collection accession number PTA-125924. (Item 5) 5. The composition of any one of the preceding claims, further comprising one or more of chitin, chitosan, glucosamine, amino acids, and liquid fertilizer. (Item 6) 6. A method comprising contacting soil, a plant, a plant part, or a seed with a composition according to any one of items 1 to 5. (Item 7) 7. The method of claim 6, further comprising contacting the soil, plant, plant part, or seed with one or more of chitin, chitosan, glucosamine, and amino acids. (Item 8) 8. The method of claim 6 or 7, further comprising contacting the soil, plant, plant part, or seed with a liquid fertilizer. (Item 9) 9. The method of any one of items 6 to 8, further comprising contacting the soil, plant, plant part, or seed with one or more pesticides, one or more fungicides, one or more herbicides, one or more insecticides, one or more plant hormones, one or more plant inducers, or a combination of two or more thereof. (Item 10) 10. The method of any one of items 6 to 9, further comprising activating the microbial species in the composition prior to contacting the soil, plant, plant part, or seed with the composition. (Item 11) A composition comprising the composition according to any one of items 1 to 4 and a carrier or a seed. (Item 12) Item 12. The composition according to item 11, wherein the carrier comprises urea, potash, ammonium phosphate, ammonium nitrate, clay, peat, coal, mineral soil, charcoal, sawdust, wheat / soybean / oat bran, compost, coco coir, perlite, vermiculite, bentonite, Azomite®, kaolin, silicate, pumice, talc, liquid fertilizer or liquid dust control chemical. (Item 13) Item 12. The composition according to item 11, wherein the seeds comprise corn seeds, sunflower seeds, rapeseed seeds, wheat seeds, cucumber seeds, tomato seeds, rice seeds, and / or cotton seeds. (Item 14) 14. The composition according to any one of items 11 to 13, further comprising one or more insecticides and / or fungicides. (Item 15) 1. A method for selecting a microorganism for co-formulation with a carrier or seed, comprising: Identifying microorganisms that contain one or more dipicolinic acid (DPA) synthase genes, that express one or more DPA synthase proteins, and / or that produce DPA; and Selecting the microorganism for co-formulation with a carrier or seed. A method comprising: (Item 16) 16. The method of claim 15, further comprising co-forming the selected microorganism with the carrier or the seed. (Item 17) Item 17. The method of item 15 or 16, wherein the one or more DPA synthase genes include a DPA synthase subunit A (DpaA) gene and / or a DPA synthase subunit B (DpaB) gene. (Item 18) Item 18. The method according to Item 17, wherein the DpaA gene encodes a DpaA protein having at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 26 to 41, or the DpaB gene encodes a DpaB protein having at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 42 to 56. (Item 19) Item 19. The method according to Item 18, wherein the DpaA gene encodes a DpaA protein having at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 26 to 41, or the DpaB gene encodes a DpaB protein having at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 42 to 56. (Item 20) 20. The method according to item 19, wherein the DpaA gene encodes a DpaA protein comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 26 to 41, or the DpaB gene encodes a DpaB protein comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 42 to 56. (Item 21) 17. The method of claim 15 or 16, wherein the one or more DPA proteins comprise a DPA synthase subunit A (DpaA) protein and / or a DPA synthase subunit B (DpaB) protein. (Item 22) 22. The method according to item 21, wherein the DpaA protein comprises at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 26 to 41, or the DpaB protein comprises at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 42 to 56. (Item 23) 23. The method according to item 22, wherein the DpaA protein comprises at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 26 to 41, or the DpaB protein comprises at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 42 to 56. (Item 24) 24. The method according to item 23, wherein the DpaA protein comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 26 to 41, or the DpaB protein comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 42 to 56. (Item 25) 25. The method of any one of items 15 to 24, wherein identifying a microorganism containing one or more dipicolinic acid (DPA) synthase genes comprises detecting a nucleic acid or nucleic acid sequence encoding the one or more DPA synthase genes. (Item 26) 26. The method of claim 25, wherein the nucleic acid is DNA, mRNA, or cDNA. (Item 27) 27. The method of claim 25 or 26, wherein detecting the nucleic acid comprises one or more of nucleic acid sequencing, nucleic acid amplification, nucleic acid hybridization, and microarray analysis. (Item 28) 28. The method of any one of items 15 to 27, wherein identifying a microorganism expressing one or more DPA synthase proteins comprises immunoassay or mass spectrometry. (Item 29) 29. The method of claim 28, wherein the immunoassay comprises Western blotting, ELISA, flow cytometry, or immunohistochemistry. (Item 30) 17. The method of claim 15 or 16, wherein identifying a microorganism that produces DPA comprises detecting DPA in the microorganism or in a medium containing the microorganism. (Item 31) 31. The method of claim 30, wherein detecting DPA comprises a fluorescent assay. (Item 32) 32. The method of claim 31, wherein the fluorescence assay comprises a terbium-DPA fluorescence assay. (Item 33) Item 15. The method of item 15, wherein identifying a microorganism that produces DPA comprises identifying a microorganism that contains one or more EtfA or Isf genes and / or expresses one or more EtfA or Isf proteins. (Item 34) 34. The method according to item 33, wherein the Isf protein comprises at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 57 to 66. (Item 35) 35. The method according to item 34, wherein the Isf protein comprises at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 57 to 66. (Item 36) 36. The method according to item 35, wherein the Isf protein comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 57 to 66. (Item 37) 37. The method of any one of items 15 to 36, wherein the selected microorganism has increased viability when co-formulated with a carrier or seed, compared to a microorganism that does not contain one or more dipicolinic acid (DPA) synthase genes, does not express one or more DPA synthase proteins, and / or does not produce DPA. (Item 38) 38. The method of any one of items 16 to 37, wherein co-forming the selected microorganism with the carrier or seed comprises contacting one or more selected microorganisms with the carrier or seed. (Item 39) 39. The method of claim 38, wherein the one or more selected microorganisms are in a liquid medium. (Item 40) 40. The method of claim 39, wherein the one or more selected microorganisms are in solid or dried form. (Item 41) 41. The method according to any one of items 38 to 40, further comprising contacting the carrier or seed with one or more microorganisms that do not contain one or more DPA synthase genes, do not express one or more DPA proteins, and / or do not produce DPA. (Item 42) 42. The method of any one of items 16 to 41, wherein the support comprises a dry or solid support. (Item 43) 43. The method of claim 42, wherein the dry or solid carrier comprises dry fertilizer, soil-derived material, organic matter, inert material, or a mixture of two or more thereof. (Item 44) Item 44. The method of item 43, wherein the dry fertilizer comprises urea, potash, ammonium phosphate, and / or ammonium nitrate, the soil-derived material comprises clay, peat, coal, or mineral soil; the organic material comprises charcoal, sawdust, wheat / soybean / oat bran, compost, or coco coir; or the inert material comprises perlite, vermiculite, bentonite, Azomite®, kaolin, silicate, pumice, or talc. (Item 45) 43. The method of claim 42, wherein the liquid carrier comprises liquid fertilizer or liquid dust control chemical. (Item 46) 42. The method according to any one of items 16 to 41, wherein the seeds comprise corn seeds, sunflower seeds, rapeseed seeds, wheat seeds, cucumber seeds, tomato seeds, rice seeds, and / or cotton seeds. (Item 47) 47. The method of claim 46, wherein the treated seeds further comprise one or more insecticides and / or fungicides. The foregoing and other features of the present disclosure will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0011] [Figure 1A]FIG. 1 is an alignment of DpaA protein sequences from the indicated bacteria. Fourteen DpaA sequences (SEQ ID NOs: 26-39) from 13 strains were aligned using Clustal Omega (clustal.org / omega) with default settings. A minimum sequence identity threshold of 60% was then used to create a consensus sequence ("consensus 60"; SEQ ID NO: 40). PRK08306 (SEQ ID NO: 41) is the consensus sequence of the DpaA superfamily obtained from the NCBI CDD Conserved Domain Family database (ncbi.nih.gov / Structure / cdd / cddsrv.cgi). [Figure 1B] FIG. 1 is an alignment of DpaA protein sequences from the indicated bacteria. Fourteen DpaA sequences (SEQ ID NOs: 26-39) from 13 strains were aligned using Clustal Omega (clustal.org / omega) with default settings. A minimum sequence identity threshold of 60% was then used to create a consensus sequence ("consensus 60"; SEQ ID NO: 40). PRK08306 (SEQ ID NO: 41) is the consensus sequence of the DpaA superfamily obtained from the NCBI CDD Conserved Domain Family database (ncbi.nih.gov / Structure / cdd / cddsrv.cgi).

[0012] [Diagram 2]FIG. 2 is an alignment of DpaB protein sequences from the indicated bacteria. Thirteen DpaB sequences (SEQ ID NOs: 42-54) from 13 strains were aligned using Clustal Omega (clustal.org / omega) with default settings. A minimum sequence identity threshold of 60% was then used to create a consensus sequence ("consensus 60"; SEQ ID NO: 55). PRK08305 (SEQ ID NO: 56) is the consensus sequence of the DpaB superfamily obtained from the NCBI CDD Conserved Domain Family database (ncbi.nih.gov / Structure / cdd / cddsrv.cgi).

[0013] [Diagram 3] FIG. 3 shows an alignment of 10 Isf protein sequences (SEQ ID NOs: 57-66) from five bacterial species and the consensus sequence (SEQ ID NO: 67).

[0014] [Figure 4] FIG. 4 is a graph summarizing bacterial survival in combination with the indicated carriers.

[0015] [Diagram 5] FIG. 5 is a graph showing cucumber shoot dry weight at 32 days on plants treated with perlite or perlite impregnated with microbial consortium (AMC1).

[0016] [Figure 6] FIG. 6 is a graph showing cucumber shoot dry weight at 32 days for plants treated with perlite, perlite impregnated with a DFC microbial consortium, bentonite, and bentonite impregnated with a DFC microbial consortium.

[0017] [Figure 7]FIG. 7 is a graph showing cucumber shoot dry weight at 32 days for untreated plants, plants treated with a liquid DFC consortium, and plants treated with perlite impregnated with a DFC microbial consortium.

[0018] [Figure 8] FIG. 8 is a graph showing survival of bacteria from the DFC consortium in combination with corn and soybean seeds along with insecticide / fungicide treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Sequence Listing Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotides and amino acids as defined in 37 CFR § 1.822. In at least some instances, only a single strand of each nucleic acid sequence is shown, but any reference to any of the depicted strands is understood to include the complementary strand.

[0020] SEQ ID NO:1 is the 16S rDNA consensus nucleic acid sequence from Streptomyces pratensis.

[0021] SEQ ID NO:2 is the 16S rDNA consensus nucleic acid sequence from Streptomyces venezuelae.

[0022] SEQ ID NO:3 is the 16S rDNA nucleic acid sequence from Bacillus firmus.

[0023] SEQ ID NO:4 is the 16S rDNA consensus nucleic acid sequence from Paenibacillus azoreducens.

[0024] SEQ ID NO:5 is the 16S from Bacillus amyloliquefaciens rDNA nucleic acid sequence.

[0025] SEQ ID NO:6 is the 16S rDNA nucleic acid sequence from Bacillus flexus.

[0026] SEQ ID NO:7 is the 16S rDNA nucleic acid sequence from Bacillus licheniformis.

[0027] SEQ ID NO:8 is the 16S rDNA nucleic acid sequence from Bacillus megaterium.

[0028] SEQ ID NO:9 is the 16S rDNA nucleic acid sequence from Bacillus pumilus.

[0029] SEQ ID NO:10 is the 16S rDNA nucleic acid sequence from Bacillus koreensis.

[0030] SEQ ID NO:11 is the 16S rDNA nucleic acid sequence from Bacillus drentensis.

[0031] SEQ ID NO:12 is the 16S rDNA nucleic acid sequence from Bacillus subtilis.

[0032] SEQ ID NO:13 is the 16S rDNA nucleic acid sequence from Clostridium bifermentans.

[0033] SEQ ID NO:14 is the 16S rDNA nucleic acid sequence from Clostridium beijerinckii.

[0034] SEQ ID NO:15 is the 16S rDNA nucleic acid sequence from Clostridium pasteurianum.

[0035] SEQ ID NO:16 is the 16S rDNA nucleic acid sequence from Lactobacillus paracasei.

[0036] SEQ ID NO:17 is a partial 16S rDNA nucleic acid sequence from Fontibacillus sp. (panacisegetis).

[0037] SEQ ID NO:18 is the 16S rDNA nucleic acid sequence from Oceanobacillus oncorhynchi.

[0038] SEQ ID NO:19 is the 16S rDNA nucleic acid sequence from Paenibacillus lautus.

[0039] SEQ ID NO:20 is the 16S rDNA nucleic acid sequence from Paenibacillus chibensis.

[0040] SEQ ID NO:21 is the 16S rDNA nucleic acid sequence from Paenibacillus cookii.

[0041] SEQ ID NO:22 is the 16S rDNA nucleic acid sequence from Paenibacillus sp. (chitinolyticus).

[0042] SEQ ID NO:23 is a partial 16S rDNA nucleic acid sequence from Paenibacillus sp. (P1XP2).

[0043] SEQ ID NO:24 is the 16S rDNA nucleic acid sequence from Pseudomonas sp.

[0044] SEQ ID NO:25 is the 16S rDNA nucleic acid sequence from Streptomyces griseus.

[0045] SEQ ID NOs: 26 to 39 are the amino acid sequences of DpaA.

[0046] SEQ ID NOs: 40 to 41 are the DpaA consensus amino acid sequences.

[0047] SEQ ID NOs: 42 to 54 are the amino acid sequences of DpaB.

[0048] SEQ ID NOs:55 to 56 are the consensus amino acid sequences of DpaB.

[0049] SEQ ID NOs:57 to 66 are Isf amino acid sequences.

[0050] SEQ ID NO:67 is the Isf consensus amino acid sequence.

[0051] Detailed Description Non-spore-forming microorganisms are often more susceptible to adverse factors that arise during treatment and field application than spore-forming microorganisms. The selection process to identify spore-forming microorganisms from a large inventory of microorganisms can be tedious and time-consuming. This usually involves wet-lab testing for viability (e.g., in co-formulation with a selected carrier) with limited assurance of viability and extended shelf life of the final product.

[0052] Disclosed herein are new strategies and methods for selecting microorganisms with a high degree of reliability in shelf life extension as a single biostimulant formulation and / or in co-formulation with wet or dry carriers or seeds, thus significantly accelerating the lead time of new products to be tested in field trials.As described herein, spore-forming bacteria with identified DPA genes or proteins and / or that produce DPA outperform strains with no identified DPA genes and no detectable DPA production in terms of viability in co-formulation with carriers over time.Finally, a consortium of microorganisms comprising DPA-producing strains is described.

[0053] I. Terminology Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Krebs et al., Lewin's Genes XI, published by Jones and Bartlett Learning, 2012 (ISBN 1449659853);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 2011 (ISBN 8126531789);and George P. Redei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003 (ISBN: 0-471-26821-6).

[0054] The following explanations of terms and methods are provided to more fully describe the present disclosure and to guide those skilled in the art to practice the present disclosure. The singular forms "a", "an" and "the" refer to one or more than one, unless the context clearly indicates otherwise. For example, the term "comprising a cell" includes a single or multiple cells and is considered equivalent to the phrase "comprising at least one cell". As used herein, "comprises" means "includes". Thus, "comprising A or B" means "including A, B, or A and B" without excluding additional elements. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0055] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the techniques of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0056] In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0057] Carrier: A material (also referred to herein as an "agro-carrier") that can be used as a delivery vehicle (e.g., in a co-formulation or as an inoculant) for a microorganism, such as a microorganism or microbial consortium described herein. The carrier may be liquid or solid (dry). Exemplary carriers include liquid or dry fertilizers, soil-derived materials (e.g., charcoal, clay, peat), organic materials (e.g., sawdust, wheat / soybean / oat bran, compost) and inert materials (e.g., perlite, vermiculite, bentonite, Azomite®, kaolin, silicates, talc). In some cases, seeds may also be referred to as carriers.

[0058] Contacting: placing in direct physical association, including in either solid and / or liquid form. For example, contacting can occur between one or more microorganisms (e.g., microorganisms in a microbial consortium) and a carrier or a seed. Contacting can also occur between one or more microorganisms, a microorganism / carrier co-formulation, or a microorganism / seed co-formulation and soil, a plant, and / or a plant part (e.g., a leaf, stem, seedling, root, and / or seed).

[0059] Culturing: The deliberate growth of one or more organisms or cells in the presence of assimilable sources of carbon, nitrogen, and inorganic salts. In examples, such growth may occur in solid or semi-solid nutrient media, or in liquid media in which nutrients are dissolved or suspended. In further examples, culturing may occur on a surface or in submerged cultures. Nutrient media may be composed of complex nutrients or may be chemically defined.

[0060] Dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA): a compound with the following structure [ka] In most microorganisms, DPA is produced by converting dihydrodipicolinate to DPA by the enzyme dipicolinate synthase. DPA synthase has two subunits, subunit A (DpaA or spoVFA) and subunit B (DpaB or spoVFB). Exemplary DpaA and DpaB amino acid sequences are provided herein (Figures 1 and 2).

[0061] Some bacteria (e.g., some Clostridium) are able to synthesize DPA despite the lack of identifiable DpaA and DpaB genes. Without being bound by theory, it is proposed that these bacteria utilize a structurally related protein, namely, the electron transfer flavoprotein (etfA), which is a flavin mononucleotide (FMN) oxidoreductase. EtfA is thought to catalyze the final step of the biosynthetic pathway by converting dihydrodipicolinate to dipicolinic acid (Orsburn et al., Mol. Microbiol. 75:178-186, 2010). Alternatively, some bacteria can utilize an iron-sulfur flavoprotein (Isf) in the production of DPA.

[0062] Heterologous: Derived from a different genetic source or species. For example, a nucleic acid that is heterologous to a cell is derived from an organism or species other than the cell in which it is expressed. Methods for introducing heterologous nucleic acid into bacterial cells include transformation of the nucleic acid, including, for example, electroporation, lipofection, and particle gun acceleration.

[0063] In another example of the use of the term heterologous, a nucleic acid operably linked to a heterologous promoter is derived from an organism, species, or gene other than the organism, species, or gene to which the promoter belongs. In another example of the use of the term heterologous, a nucleic acid encoding a polypeptide or portion thereof is operably linked to a heterologous nucleic acid encoding a second polypeptide or portion thereof, for example, to form a non-naturally occurring fusion protein.

[0064] Isolated: An "isolated" biological component (e.g., a nucleic acid, a protein, or an organism) is substantially separated or purified from other biological components (e.g., other cells, cell debris, or other proteins or nucleic acids). An "isolated" biological component includes those components that have been purified by standard purification methods. The term also encompasses recombinant nucleic acids, proteins, or microorganisms, and chemically synthesized nucleic acids or peptides. The term "isolated" (or "enriched" or "purified") does not require extreme purity and can include microorganisms or molecules that are at least 50% isolated, e.g., at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.

[0065] Microorganism: A microorganism, including but not limited to bacteria, archaea, fungi, and algae (e.g., microalgae). In some examples, a microorganism is a unicellular organism (e.g., bacteria, blue-green algae, some fungi, or some algae). In other examples, the term microorganism includes multicellular organisms, such as certain fungi or algae (e.g., multicellular filamentous fungi or multicellular algae).

[0066] Microbial composition: a composition (which may be solid, liquid, or at least partially both) that includes at least one type (or species) of microbial cells (or at least one type of microbial cell population). In some examples, the microbial composition includes one or more types (or species) of microbial cells (or one or more microbial populations) in a liquid (e.g., a storage, culture, or fermentation medium or liquid fertilizer), e.g., as a suspension in the liquid. In other examples, the microbial composition includes one or more types (or species) of microbial cells (or one or more microbial populations) on or embedded in a solid or gelatinous medium (including, but not limited to, a culture plate) or a slurry or paste. In other examples, the microbial composition includes one or more types (or species) of microbial cells (or one or more microbial populations) associated with dry materials or seeds, e.g., on or impregnated in the dry materials or seeds.

[0067] Microbial consortium: A mixture, community, or collection of cells of two or more microbial species that are in physical contact with each other in some cases. Microorganisms in a consortium may affect each other by direct physical contact or by biochemical interactions, or both. For example, microorganisms in a consortium may exchange nutrients, metabolites, or gases with each other. Thus, in some cases, at least some of the microorganisms in a consortium are metabolically interdependent. Such interdependent interactions may change in character and extent over time and with changing culture conditions.

[0068] Transduced and transformed: A virus or vector "transduces" a cell when it transfers a nucleic acid into the cell. A cell is "transformed" by transducing a nucleic acid into the cell, by integrating the nucleic acid into the cell genome, or by episomal replication, when the DNA is replicated by the cell. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including bacterial conjugation, transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun technology.

[0069] Vector: A nucleic acid molecule that can be introduced into a host cell, thereby generating a transformed or transduced host cell. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain a nucleic acid sequence that allows replication in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes, a cloning site for the introduction of heterologous nucleic acid, a promoter (e.g., for expression of an operably linked nucleic acid), and / or other genetic elements known in the art. Vectors include plasmid vectors, including plasmids for expression in cells of gram-negative and / or gram-positive bacteria. Exemplary vectors include those for use in E. coli.

[0070] Viability: The ability of a cell (e.g., a microbial cell) to grow or reproduce under conditions suitable for growth or reproduction. In some instances, "survival" or "viability" refers to the viability of a cell (e.g., a microbial cell) after a period of storage in liquid or dry state, alone, in a mixture with other microbial cells, and / or when co-formulated with a carrier or seed.

[0071] II. Methods for identifying viable microorganisms in coformulations Disclosed herein is a method for identifying microorganisms that can survive or remain viable when co-formulated with liquid or solid carriers.Microorganisms can be individually co-formulated with carriers or seeds (e.g., a single strain or species of microorganism is co-formulated with carriers or seeds) or can be part of a consortium or mixture of microorganisms (e.g., two or more strains or species of microorganisms) that are co-formulated with carriers or seeds.In other embodiments, the method includes identifying microorganisms that can survive or remain viable for extended periods in consortia (either as a single consortium or co-formulated with carriers or seeds).

[0072] In some examples, microorganisms identified by the methods disclosed herein, such as those that contain one or more DPA synthase genes, express one or more DPA proteins, and / or produce detectable amounts of DPA, have improved viability (alone or in a co-formulation) over microorganisms that do not contain one or more DPA synthase genes, do not express one or more DPA synthesis proteins, and / or do not produce detectable amounts of DPA. In some examples, microorganisms identified by the methods disclosed herein have at least 10% increased viability (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1.5-fold, at least 2-fold, at least 5-fold, or more increased viability) compared to microorganisms that do not contain one or more DPA synthase genes, do not express one or more DPA synthesis proteins, and / or do not produce detectable amounts of DPA. Increased viability can include a greater number of viable cells after a period of time and / or viability for a longer period of time.

[0073] In some embodiments, the method disclosed herein comprises identifying a microorganism that contains one or more genes encoding DPA synthase in its genome, expresses one or more DPA synthase proteins, and / or produces detectable amounts of DPA. Such a microorganism is identified as a microorganism that can survive or remain viable when individually or co-formulated with a liquid or solid carrier (e.g., compared to one or more microorganisms that do not contain a gene encoding DPA synthase, do not express one or more DPA synthase proteins, and / or do not produce detectable amounts of DPA). The identified microorganism can be further selected for downstream use, for example, for co-formulation with a liquid or solid carrier or seed. In some examples, the microorganisms are viable or remain viable (individually or when co-formulated with a carrier or seed) for at least 1 day, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, at least 1 year, at least 2 years, or longer (e.g., at least 1-28 days, at least 5-21 days, at least 2-6 weeks, at least 4-8 weeks, at least 2-6 months, at least 3-9 months, at least 4-10 months, at least 6 months to 1 year, at least 1-2 years, or longer).

[0074] The method of determining the viability or survival of a microorganism includes detecting the growth of the microorganism in culture. In some examples, a preparation containing microbial cells (in liquid or dry state, or in co-formulation with carriers or seeds) is inoculated into a liquid medium, incubated under suitable conditions for microbial growth, and the presence and / or amount of the microorganism is measured after a defined period of time. In other examples, a preparation containing microbial cells (in liquid or dry state, or in co-formulation with carriers or seeds) is streaked onto a plate containing a solid or semi-solid medium, incubated under suitable conditions for microbial growth, and the presence and / or amount of the microorganism (e.g., the presence, size, and / or number of colonies) is measured. In some examples, the microorganism is identified, for example, using PCR methods. An exemplary method for determining the viability and identity of microbial cells is provided in Example 1.

[0075] In further embodiments, the method includes selecting one or more microorganisms that express one or more DPA synthase proteins, including one or more DPA synthase genes, and / or produce detectable amounts of DPA, and optionally co-formulating the one or more selected microorganisms with one or more carriers or seeds. In some examples, the method includes preparing a co-formulation of one or more selected microorganisms with one or more carriers or seeds. The method includes contacting one or more microorganisms with one or more carriers or seeds, for example, in solid (dry) or liquid form. In some examples, the carrier(s) or seed(s) are contacted with a mixture of microorganisms. The mixture includes microorganisms that express DPA synthase or produce DPA (e.g., microorganisms selected or produced using the methods described herein, including, but not limited to, DFC), and may include one or more microorganisms that do not express DPA synthase or produce DPA. In some examples, the carrier is added to about 10 mL of 10 ... 3 ~10 9 or more cells (e.g., approximately 1 x 10 per mL) 3 Approximately 5 x 10 cells per mL 3Approximately 1 x 10 cells per mL 4 Approximately 5 x 10 cells per mL 4 Approximately 1 x 10 cells per mL 5 Approximately 5 x 10 cells per mL 5 Approximately 1 x 10 cells per mL 6 Approximately 5 x 10 cells per mL 6 Approximately 1 x 10 cells per mL 7 Approximately 5 x 10 cells per mL 7 Approximately 1 x 10 cells per mL 8 Approximately 5 x 10 cells per mL 8 Approximately 1 x 10 cells per mL 9 Approximately 5 x 10 cells per mL 9 The microorganism is contacted with a liquid containing each of the microorganisms (cells or more).

[0076] In some embodiments, a liquid containing one or more of the selected microorganisms (and optionally one or more additional microorganisms) is placed in contact with one or more dry carriers or seeds. In some examples, the liquid containing the microorganisms is a fresh or frozen bacterial culture or a mixture of fresh or frozen bacterial cultures. In other examples, the liquid containing the microorganisms is a liquid to which freeze-dried microorganisms have been added. The liquid containing one or more microorganisms is soaked into the dry carrier or seed. In some examples, an amount of liquid containing one or more microorganisms is used such that the dry carrier or seed is saturated, e.g., resulting in a relatively uniform distribution of the microorganisms throughout the carrier or seed. However, a non-saturating amount of liquid may be used. In non-limiting examples, the amount is about 35 μL / g to 6 mL / g. In some examples, the carrier or seed impregnated with the microorganisms is dried (e.g., at room temperature or about 30-35° C.) and stored at ambient temperature (e.g., in a closed or sealed container).

[0077] In other embodiments, a liquid containing one or more of the selected microorganisms (and optionally one or more additional microorganisms) is mixed with one or more liquid carriers. In some examples, the liquid containing the microorganism is a fresh or frozen bacterial culture or a mixture of fresh or frozen bacterial cultures. In other examples, the liquid containing the microorganism is a liquid to which freeze-dried microorganisms have been added. The liquid containing the microorganism can be mixed with the liquid carrier in any selected amount, for example, 0.1% to 90% (v / v), for example, 0.5 to 1%, 1 to 5%, 2 to 10%, 3 to 6%, 4 to 8%, 5 to 15%, 8 to 20%, 10 to 25%, 20 to 40%, 30 to 50%, 40 to 60%, 50 to 75%, or 70 to 90% (v / v). In some examples, the microorganisms are mixed with the liquid carrier at about 0.1%, about 0.2%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% (v / v). In one non-limiting example, the mixture of microorganisms is added to the liquid carrier at 0.5% (v / v) or a ratio of 1:180. In another non-limiting example, the mixture of microorganisms is added to a concentrated liquid carrier (e.g., a 10-fold concentrated liquid carrier) at 90% (v / v), resulting in a 1-fold concentration of the liquid carrier. The amount of microbial cells in the mixture can be adjusted to achieve a desired final concentration of microbial cells, depending on the dilution factor used. The mixture of microorganisms is stored at ambient temperature (e.g., in a closed or sealed container).

[0078] In some embodiments, dry preparations of microorganisms (e.g., freeze-dried microorganisms) are used in co-formulation with dry carriers or seeds. In some examples, freeze-dried microorganisms are mixed with dry carriers or seeds (e.g., about 40 mg of microorganisms per kg of carriers or seeds to about 1 g of microorganisms per kg of carriers or seeds). In some examples of this embodiment, freeze-dried microorganisms are added to a liquid and then contacted with dry carriers or seeds. In other examples, freeze-dried microorganisms are added to a liquid and then contacted with the above-mentioned dry carriers or seeds.

[0079] A. Detection of DPA synthase nucleic acid In some embodiments, the method includes identifying the presence of one or more DPA synthase nucleic acid molecules (e.g., DNA, cDNA, or mRNA) in a microorganism or population of microorganisms. In some examples, the method includes detecting one or more DPA synthase genes (e.g., DpaA and / or DpaB) in the genome of the microorganism. In some examples, the microorganism includes both the DpaA gene and the DpaB gene. Exemplary DPA synthase genes include DpaA (GenBank Accession Nos. NC_000964.3, 1744367-1745260, which are incorporated herein by reference as they exist in GenBank on June 3, 2018) and DpaB (GenBank Accession Nos. NC_000964.3, 1745236-1745865, which are incorporated herein by reference as they exist in GenBank on June 3, 2018) of B. subtilis. In some examples, the DpaA gene encodes a protein having at least 20% sequence identity (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more) to the protein shown in FIG. 1 or to a protein shown in FIG. 1 (e.g., SEQ ID NOs: 26-39). In some examples, the DpaB gene encodes a protein having at least 20% sequence identity (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more) to the protein shown in FIG. 2 or the proteins shown in FIG. 2 (e.g., SEQ ID NOs: 42-54).In some examples, the DpaA or DpaB protein has one or more conserved regions identified in the "consensus 60" sequence in Figures 1 and 2, respectively (SEQ ID NOs: 40 and 55, respectively).

[0080] In other embodiments, the method includes identifying the presence of one or more nucleic acids (e.g., DNA, mRNA, or cDNA) involved in an alternative pathway for DPA synthesis. In some examples, the method includes identifying the presence or expression of one or more nucleic acids encoding an electron transfer flavoprotein (e.g., EtfA) or an iron-sulfur flavoprotein (e.g., Isf). Electron transfer flavoproteins are heterodimers consisting of alpha and beta subunits and are part of the adenine nucleotide alpha hydrolase superfamily. Exemplary bacterial EtfA nucleic acid sequences include GenBank Accession Nos. CP000312.1 (2508382-2509389), NC_004578.1 (2407768-2408697), NC_009089.1 (977905-978927), NC_019382.1 (1357738-1356809, complement), NC_003030.1 (2833696-2833268, complement), NC_002971.4 (1062557-1061613, complement), and NC_003063.2 (650447-651376), each of which is incorporated by reference herein as present in GenBank on Jun. 3, 2018. Exemplary bacterial EtfA amino acid sequences include ABG86939, NP_792007.1, YP_001087282.1, YP_006967336.1, NP_349315.1, NP_820116.1, and NP_357016.2, each of which is incorporated by reference herein as present in GenBank on Jun. 3, 2018. Exemplary Isf nucleic acid and protein sequences include GenBank Accession Nos. CP016318 (3060700-3061308) and ARE63607 (each of which is incorporated by reference herein as present in GenBank on Jun. 3, 2018), and those depicted in FIG. 3 (e.g., SEQ ID NOs: 57-66), respectively.

[0081] In some examples, DPA synthase nucleic acid (or EtfA or Isf nucleic acid) can be identified by sequence analysis of microorganisms (e.g., genome sequencing and / or sequencing using oligonucleotides specific for DPA synthase). In some examples, sequence analysis is performed using sequences present in one or more databases, including GenBank (ncbi.nlm.nih.gov / nucleotide / ), ENSEMBL (ensembl.org / index.html), IMG (img.jgi.doe.gov), MicrobesOnline (microbesonline.org), SEED (theseed.org), or GOLD (gold.jgi-psf.gov). An exemplary method for identifying DPA synthase genes is provided in Example 1 below. Similar methods can be used to identify EtfA or Isf genes.

[0082] In some examples, nucleic acid from a microorganism or a population of microorganisms is isolated, amplified, or both, before detection. In some examples, amplification and expression detection are performed simultaneously or nearly simultaneously. In some examples, expression of nucleic acid can be detected by PCR (e.g., PCR, real-time PCR, RT-PCR or quantitative RT-PCR). For example, nucleic acid can be isolated and amplified by using a commercially available kit. In some examples, nucleic acid can be incubated with primers that allow amplification of DpaA and / or DpaB (or EtfA or Isf) nucleic acid under conditions sufficient to allow amplification of such products. The resulting amplicon can be detected.

[0083] In another example, the nucleic acid from a microorganism or a population of microorganisms is incubated with a probe that can bind to DpaA and / or DpaB (or EtfA or Isf) nucleic acid molecules (e.g., cDNA, genomic DNA, or RNA (e.g., mRNA)) under very stringent conditions. The resulting hybridization can then be detected. In another example, a microorganism or a population of microorganisms is screened by applying isolated nucleic acid molecules obtained from the microorganism(s) to an array. In one example, the array comprises oligonucleotides complementary to DpaA and / or DpaB (or EtfA or Isf) nucleic acids. In one example, the microbial nucleic acid molecules are incubated with an array comprising oligonucleotides complementary to DpaA and / or DpaB (or EtfA or Isf) for a sufficient time to allow hybridization between the isolated nucleic acid molecules and the oligonucleotide probes, thereby forming an isolated nucleic acid molecule:oligonucleotide complex. The isolated nucleic acid molecule:oligonucleotide complex is then analyzed to determine whether the nucleic acid is present in the sample.

[0084] B. Detection of DPA synthase protein As an alternative or in addition to detecting the DPA synthase nucleic acid, the protein can be detected using methods such as immunoassays (e.g., Western blot, immunohistochemistry, flow cytometry, or ELISA) or mass spectrometry. In some examples, the DpaA protein includes a protein having at least 20% sequence identity (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more) with the proteins shown in FIG. 1 (e.g., SEQ ID NOs: 26-39). In some examples, the DpaB protein includes a protein having at least 20% sequence identity (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more) with the protein shown in Figure 2 (e.g., SEQ ID NOs: 42-54). In some examples, the DpaA or DpaB protein has one or more conserved regions identified in the "consensus 60" sequence of Figures 1 and 2, respectively (SEQ ID NOs: 40 and 55, respectively). In other examples, Isf proteins include proteins having at least 20% sequence identity (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more) to the proteins shown in FIG. 3 (e.g., SEQ ID NOs:57-66).

[0085] In some examples, the proteins are purified prior to detection. In one example, DpaA and / or DpaB (or Isf) proteins can be detected by incubating a microbial sample with an antibody that specifically binds DpaA and / or DpaB (or Isf). The antibody ("primary antibody") can include a detectable label. For example, the primary antibody can be directly labeled, or the sample can then be incubated with a labeled (e.g., fluorescently labeled) secondary antibody. The label can then be detected, for example, by microscopy, ELISA, flow cytometry, or spectrophotometry. In another example, the sample is analyzed by Western blot to detect expression of DpaA and / or DpaB proteins. Antibodies against DpaA, DpaB, or Isf can be generated by one skilled in the art, for example, using the amino acid sequences of Figures 1-3.

[0086] Suitable labels for antibodies or secondary antibodies include various enzymes, conjugated groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable conjugated group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and a non-limiting exemplary radioactive label is, 125 I, 131 I, 35 S or 3 Examples include H.

[0087] C. DPA detection In some embodiments, the method includes identifying a microorganism or population of microorganisms that produces DPA (e.g., detectable levels of DPA). In some examples, the method includes detecting at least 1 nM of DPA (e.g., at least 2 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM of DPA, or more). In one example, the method includes detecting DPA using a terbium-DPA fluorescence assay (see, e.g., Rosen, Anal. Chem. 69:1082-1085, 1997; Pellegrino et al., Anal. Chem. 70:1755-1760, 1998; Ammann et al., Int. J. Microbiol. 2011:435281, 2011). Briefly, DPA is contacted with terbium(III) to form a complex that has increased fluorescence relative to terbium(III), allowing for detection and / or quantification of DPA in a sample. An exemplary terbium-DPA assay is described in Example 1.

[0088] III. Microorganisms and Coformulations Disclosed herein is a microorganism that comprises one or more DPA synthase genes in its genome, expresses one or more DPA synthase proteins, and / or produces DPA.In some examples, the microorganism comprises one or more DPA synthase genes in its genome, expresses one or more DPA synthase proteins, and / or is modified to produce DPA.Also disclosed is the co-formulation of the microorganism with one or more carriers or seeds.

[0089] A. Microorganisms Microorganisms having one or more DPA synthase genes, expressing one or more DPA synthase proteins, and / or producing DPA include, but are not limited to, Bacillus amyloliquefaciens, Bacillus flexus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis, Bacillus sp. (closely related to B. kochii, B. pocheonensis, and Bacillus sp. (strain R-27341)), Clostridium beijerinckii, Oceanobacillus oncorhynchi, Paenibacillus chibensis, Paenibacillus cookii, Paenibacillus lautus, Virgibacillus halophilus, Paenibacillus azoreducens and Bacillus firmus. In some examples, these bacteria include those described in PCT Publication No. WO2018 / 045004, which is incorporated herein by reference in its entirety. Additional microorganisms include those listed in Tables 25 and 26. In some examples, these microorganisms also have sporulation ability, but the sporulation ability and the presence of a distinguishable DPA synthase gene or DPA production are not completely consistent (see, for example, Table 6).

[0090] In additional embodiments, compositions are disclosed that include microorganisms that have one or more DPA synthase genes, express one or more DPA synthase proteins, and / or produce DPA, such as those referred to herein as Dry Formulation Consortiums (DFCs). Microorganisms in DFCs include, but are not limited to, Bacillus amyloliquefaciens, Bacillus firmus, Bacillus flexus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus koreensis, Bacillus drentensis, Bacillus subtilis, Clostridium bifermentans, Clostridium beijerinckii, Clostridium pasteurianum, Lactobacillus paracasei, Fontibacillus sp. (panacisegetis), Oceanobacillus oncorhynchi, Paenibacillus lautus, Paenibacillus azoreducens, Paenibacillus chibensis, Paenibacillus cookii, Paenibacillus sp. (chitinolyticus), Paenibacillus sp. (P1XP2), Pseudomonas sp. and Streptomyces griseus. In one embodiment, the composition comprises cells of a microbial species deposited with the American Type Culture Collection (ATCC, Manassas, VA) on May 16, 2019 and assigned accession number PTA-125924.

[0091] Those skilled in the art will recognize that it is not always possible to identify microorganisms, especially at the species or strain level. In some examples, the microorganisms in the compositions described herein have been analyzed by 16S rDNA sequencing and whole genome sequencing, followed by comparison of sequences in public databases. However, limited information in sequence databases (including little or no information for some species or strains and / or changes in nomenclature over time) may make it difficult to ultimately identify species or strains. Thus, in some embodiments, the microbial species contained in the compositions of the present disclosure are identified by sequence identity to the 16S rDNA sequences (SEQ ID NOs: 3-25) provided herein. In some examples, a microbial consortium or composition of the present disclosure comprises, consists essentially of, or consists of two or more (e.g., five or more, ten or more, fifteen or more, twenty or more, or all) of microorganisms having a 16S rDNA sequence with at least 95% identity (e.g., at least 96%, 97%, 98%, 99%, or more) to SEQ ID NOs:3-25.

[0092] Microorganisms that have one or more DPA synthase genes, express one or more DPA synthase proteins, and / or produce detectable amounts of DPA also include microorganisms that do not naturally have one or more DPA synthase genes, do not express one or more DPA synthase proteins, and / or do not produce DPA, but are modified to do so. In some examples, microorganisms that do not naturally have one or more DPA synthase genes, do not express one or more DPA synthase proteins, and / or do not produce DPA are modified to express one or more heterologous DPA synthase genes, such as DpaA and / or DpaB or one or more Isf genes. Exemplary DpaA and DpaB genes and proteins and Isf genes and proteins are described in Section II and Figures 1-3 (including SEQ ID NOs: 26-67).

[0093] Bacteria that can be engineered to express one or more heterologous DPA synthase genes include, but are not limited to, Azotobacter (e.g., Azotobacter vinelandii), Clostridium (e.g., Clostridium pasteurianum), Streptomyces (e.g., Streptomyces griseus, Streptomyces venezuelae, Streptomyces pratensis), Sporolactobacillus spp. (e.g., Sporolactobacillus dextrus), Sporosarcina spp. (e.g., Sporosarcina halophila), Desulfotomaculum spp. (e.g., Desulfotomaculum guttoideum) , Nocardiopsis spp. (e.g., Nocardiopsis sinuspersici), Promicromonospora spp. (e.g., Promicromonospora enterophila, Promicromonospora soli), Brevibacillus spp. (e.g., Brevibacillus centrosporus), Rummeliibacillus spp. (e.g., Rummeliibacillus pycnus), Lysinibacillus spp., Terribacillus spp. (e.g., Terribacillus shanxiensis), Micromonospora spp. (e.g., Micromonospora fulva, Micromonospora palomenea), Saccharopolyspora spp. (e.g., Saccharopolyspora spinose, Saccharopolyspora indica) and Fontibacillus spp. (e.g., Fontibacillus panacisegetis). In some examples, these bacteria include those described in PCT Publication No. WO2018 / 045004, which is incorporated by reference in its entirety.

[0094] In some examples, the heterologous DpaA and / or DpaB genes are placed under the control of a promoter. In some examples, the promoter is a constitutive promoter, while in other examples, the promoter is inducible (e.g., an inducible T7 promoter). In additional examples, the promoter is an arabinose-inducible promoter (e.g., pBAD system), a lac promoter (IPTG / lactose direct induction), a trc promoter (IPTG / lactose direct induction), a tetracycline-inducible promoter, or a pho promoter (phosphate starvation inducible). The heterologous DpaA and / or DpaB genes can be included in a vector, for example, operably linked to a promoter. Similar methods can be used for EtfA or Isf genes.

[0095] Multiple genes (e.g., two or more DPA synthases and / or Isf genes) can be expressed simultaneously in bacteria. To ensure proper and equal production of multiple enzymes from a single pathway, each nucleic acid encoding a heterologous gene is optionally placed under the control of a single type of promoter, such as an inducible T7 promoter. One example is the Duet™ vector (Novozymes), which is designed with compatible replicons and drug resistance genes for efficient propagation and maintenance of four plasmids in a single cell. This allows for the co-expression of up to eight different proteins. In another example, the vector is a pET vector, such as a pET21 or pET28 vector. pET and pET-based vectors are commercially available, for example, from Novagen (San Diego, CA), or Clontech (Mountain View, CA).

[0096] In one example, the vector is pET21a or pET28a. In some examples, the pET vector contains a resistance marker (e.g., ampicillin or kanamycin resistance) and a T7 promoter. Multiple cloning sites are engineered so that more than one gene (e.g., 2, 3, 4, or more) can be expressed from a single vector. In some examples, genes are expressed as multicistronic products (e.g., bicistronic, tricistronic, etc. products) that contain a single mRNA and multiple polypeptides generated. In other examples, genes are expressed as multiple monocistronic products that contain individual mRNAs and polypeptides generated for each gene. The appropriate vector can be selected depending on the gene(s) to be expressed and the host cell to be transformed.

[0097] In some cases, the plasmid is introduced extrachromosomally and replicated within the host microorganism, while in other cases, introduction of the plasmid is followed by a double homologous recombination event, which inserts one or more genes into the genome.

[0098] Transformation of bacterial cells with recombinant DNA may be performed. When the host is bacteria, competent cells capable of DNA uptake can be prepared from cells harvested after exponential growth phase, and then treated by CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl may be used. Bacteria can also be transformed by electroporation, conjugation, or transduction.

[0099] B. Carriers and Seed Treatment Disclosed herein are methods for co-formulating one or more microorganisms with one or more carriers, and compositions comprising one or more microorganisms and one or more carriers. Carriers may be liquid or solid (dry). Carriers include liquid or dry fertilizers (e.g., fertilizers containing urea, potash, ammonium phosphate, and / or ammonium nitrate), soil-derived materials (e.g., clay, peat, coal, mineral soil), organic materials (e.g., charcoal, sawdust, wheat / soybean / oat bran, compost, coco coir), and / or inert materials (e.g., perlite, vermiculite, bentonite, Azomite®, kaolin, silicate, pumice, talc). Exemplary carriers include Azomite®, perlite, biochar, dry fertilizers (e.g., urea, MOP, or MAP), liquid fertilizers (e.g., UAN), and dust suppression chemicals (e.g., those available from ArrMaz, FL, USA). Additional exemplary supports include montmorillonite, attapulgite, hydrous aluminosilicate (Agsorb Products Group, IL, USA), akadama (Eastern Leaf Inc, CA, USA), Seramis Clay granules (Greens hydroponics, UK), Aquasmart™ Pro (Aquasmart, TX, USA), Pyro-Gro (Green Air products, OR, USA), crushed lava rock, clay pebbles.

[0100] In some embodiments, the co-formulation with the carrier comprises a consortium of 22 microorganisms described in WO2018 / 045004 (herein incorporated by reference in its entirety; referred to as AMC1) and one or more of the carriers described herein. In other embodiments, the co-formulation with the carrier comprises a consortium of 23 microorganisms disclosed herein (e.g., the microorganisms listed in Table 26 or ATCC deposit PTA-125924) and one or more carriers. The co-formulation also comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or more) microorganisms and one or more of the carriers described herein. In some examples, the co-formulation comprises at least one microorganism that contains or expresses one or more DPA synthases and / or Isf gene(s) (or produces DPA) and a carrier. In other examples, the co-formulation comprises at least one microorganism that contains or expresses DPA synthase gene(s) or produces DPA and at least one microorganism that does not contain or express DPA synthase gene(s) or does not produce DPA, together with a carrier. The method of co-formulating a carrier and one or more microorganisms comprises contacting one or more carriers with one or more microorganisms. In some examples, the one or more microorganisms are in liquid form (e.g., in liquid medium) or in solid or dry form.

[0101] Also disclosed herein are methods for treating seeds with one or more microorganisms (e.g., co-formulating one or more microorganisms with one or more seeds) and compositions comprising one or more microorganisms and one or more seeds. In such embodiments, the seed is the "carrier" for the microorganism. In some embodiments, the seed treatment comprises a consortium of 22 microorganisms described in WO2018 / 045004 (incorporated herein by reference in its entirety) and one or more seeds. In other embodiments, the seed treatment comprises a consortium of 23 microorganisms disclosed herein (e.g., the microorganisms listed in Table 26 or ATCC deposit PTA-125924) and one or more seeds. In other examples, the co-formulation comprises at least one microorganism and a seed that comprises or expresses (or produces DPA) one or more DPA synthases and / or Isf gene(s). In other examples, the co-formulation includes at least one microorganism that contains or expresses a DPA synthase gene(s) or produces DPA and at least one microorganism that does not contain or express a DPA synthase gene(s) or does not produce DPA, and a seed. Exemplary seeds that can be treated with one or more microorganisms include, but are not limited to, corn seeds, sunflower seeds, canola seeds, wheat seeds, cucumber seeds, tomato seeds, rice seeds, and cotton seeds.

[0102] In some examples, the microorganism-treated seeds are prepared by applying the microorganism directly to the seed (e.g., contacting the seed with one or more microorganisms). In other examples, the microorganism-treated seeds are prepared by applying the microorganism as an overcoat to a seed previously treated with an insecticide and / or fungicide (e.g., contacting the insecticide and / or fungicide-treated seed with one or more microorganisms). In yet further examples, the microorganism-treated seeds are prepared by mixing the microorganism with an insecticide and / or fungicide (e.g., an insecticide / fungicide slurry) and applying the mixture to the seed (e.g., contacting the seed with a mixture of an insecticide and / or fungicide and one or more microorganisms). Exemplary insecticides and fungicides that can be used in combination with the microorganisms include, but are not limited to, metalaxyl, trifloxystrobin, ipiconazole, clothianidin, thiamethoxam, fludioxonil, mefenoxam, azoxystrobin, thiabendazole, pyraclostrobin, imidacloprid, fluxapyroxad, and / or cedexane. In some examples, the one or more microorganisms applied to the seeds are in liquid form (e.g., in a liquid medium) or in solid or dry form. Methods for preparing treated seeds include, but are not limited to, those described in Seed Treatment: Oregon Pesticide Applicator Training Manual (Paulsrud et al., Urbana, Illinois, 2001) and Example 13. Some of the things that are being considered are:

[0103] IV. How to use The microbial compositions of the present disclosure can be used alone or in a co-formulation with one or more liquid or dry carriers to treat soil, plants, or plant parts (e.g., roots, stems, leaves, seeds, or seedlings). In other examples, the microbial compositions of the present disclosure can be used in the form of treated seeds.

[0104] In some examples, treatment with the disclosed compositions and / or carriers or seeds treated with the disclosed compositions improves plant growth, improves stress tolerance, and / or increases crop yield. In some embodiments, the method includes contacting soil, plants (e.g., plant leaves, stems, roots, seedlings, or other plant parts), or seeds with the microbial composition or co-formulation disclosed herein. In other embodiments, the method includes sowing seeds treated with the disclosed compositions. The method may also include growing the treated plants, plant parts, or seeds and / or cultivating the plants, plant parts, or seeds in the treated soil.

[0105] In some examples, the amount of composition(s) is calculated as a co-formulation of one or more microorganisms and carrier or seed to be applied (e.g., per acre or hectare), and the composition is diluted in water (or in some examples, liquid fertilizer) to a sufficient amount to spray or irrigate (if the composition is a liquid) the area to be treated. The composition can be applied at the time of sowing at a rate of 0.5 to 2 liters per acre (e.g., 0.5 L per acre, 1 L per acre, 1.5 L per acre, or 2 L per acre). The composition can also be applied to the soil (e.g., near the roots of the plant) or to the plant in the same or different amounts one or more times during growth. In other examples, the composition can be mixed with a diluted herbicide, insecticide, pesticide, or plant growth regulator chemical. If the composition to be applied is a solid (e.g., a dry formulation), the solid can be applied directly to the soil, plant, or plant part, or can be suspended or dissolved in water (or other liquid) before use.

[0106] In some examples, treatment of soil, seeds, plants, or plant parts with a composition of the present disclosure increases plant growth (e.g., overall plant size, leaf mass, root number, root diameter, root length, tiller production, fruit production, pollen production, and / or seed production) by at least about 5% (e.g., at least about 10%, at least about 30%, at least about 50%, at least about 75%, at least about 100%, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, or more). In other examples, the methods of the present disclosure increase crop production by about 10-75% (e.g., about 20-60% or about 30-50%) compared to an untreated crop. Other measures of crop performance include fruit quality, yield, starch or solids content, sugar content or Brix, shelf life of fruit or harvestable produce, saleable yield or target size production, fruit or produce quality, resistance to grass tillering and turf foot traffic, pollination and fruit set, flowering, flower number, flower longevity, flowering quality, rooting and root mass, crop resistance to lodging, resistance to and recovery after abiotic stresses of heat, drought, cold, adaptability to poor soils, photosynthesis and greening levels, and plant health. To determine production efficiency, controls include the same agricultural practices without the addition of microorganisms, run in parallel.

[0107] The disclosed methods and compositions and / or co-formulations can be used in conjunction with any crop (e.g., direct crop treatment or pre- or post-sowing soil treatment). Exemplary crops include, but are not limited to, alfalfa, almonds, bananas, barley, broccoli, cabbage, hemp, oilseed rape, carrots, citrus and fruit tree crops, corn, cotton, cucumber, flowers and ornamentals, garlic, grapes, hops, horticultural plants, leeks, melons, oil palm, onions, peanuts and legumes, pineapple, poplar, pine and wood-bearing trees, potatoes, raspberries, rice, sesame, sorghum, soybeans, pumpkins, strawberries, sugarcane, sunflowers, tomatoes, turf and pasture grasses, watermelon, wheat, and eucalyptus. EXAMPLES

[0108] The following examples are provided to illustrate certain particular features and / or embodiments, and should not be construed as limiting the disclosure to the particular features or embodiments described.

[0109] Example 1 material and method Microorganism isolation and identification: All microorganisms were from the Agrinos Microorganism Collection (AMC) and were previously described in WO2018 / 045004, except for the four additional microorganisms described herein, which were isolated as described below.

[0110] The bacteria Streptomyces pratensis and Streptomyces venezuelae into bulk soil (N38° 38' 49.402”, W121° 40" 5.775"). Briefly, soil samples were suspended in sterile phosphate-buffered saline-TWEEN® 80 solution before being serially diluted and plated on several types of semi-solid media. After incubation at 30°C for up to 3 days, S. pratensis and S. venezuelae were isolated from Azotobacter agar containing mannitol (HIMEDIA no. M372) plates. These strains were repeatedly streaked onto semi-solid MP medium (see below) until isogenic.

[0111] Bacillus firmus was isolated from a sample of HYT® A (Agrinos AS) premixed with fertilizer (UAN32) in a ratio of 1:180. After 3 weeks of incubation at room temperature, aliquots of the mixture were plated on several types of semi-solid media and incubated at 30°C for up to 3 days. Colonies of B. firmus were picked from Pikovskaya agar plates (HIMEDIA number M520). The strain was repeatedly streaked on semi-solid MP medium (see below) until it was isogenic.

[0112] Paenibacillus azoreducens was isolated from a sample of HYT® A (Agrinos AS). P. azoreducens was isolated as a colony growing on 1-10 mM ammonium sulfate agar medium (0.585 g / L NaCl, 0.075 g / L KCl, 0.147 g / L CaCl2, 0.049 g / L MgSO4, 1.32-0.132 g / L (NH4)2SO4, 0.054 g / L KH2PO4 in HEPES buffer (pH 7.5). This strain was repeatedly streaked on semi-solid MP medium (see below) until an isogenic type was obtained.

[0113] Taxonomic classification of the newly described microorganisms: For all four newly described strains, whole genome sequencing of biologically pure isolates was performed as described below: De novo genome assembly was performed by the Hierarchical Genome Assembly Process (HGAP, Pacific Biosciences, Menlo Park, CA USA).

[0114] Taxonomic identification was initially performed using 16S ribosomal RNA (rRNA) sequences. 16S rRNA sequences were first identified within the de novo genome assemblies using RNAmmer (cbs.dtu.dk / services / RNAmmer / ). Then, NCBI BLASTn, Ribosomal Database Project (RDP) Naive Bayesian Classifier (Wang et al. Appl. Environ. Microbiol. 73:5261-5267, 2007), and Greengene 16S sequences were classified using pairwise alignment by de novo phylogenetic tree construction and rank mapping (DeSantis et al. Appl. Environ. Microbiol. 72:5069-502, 2006). Species were then assigned using the consensus of the three methods.

[0115] Based on the above, the following microorganisms were identified: - Streptomyces pratensis. Taxonomic classification of the whole genome was also performed using 16S sequences. Protein coding sequences were identified using Prodigal (Hyatt et al. BMC Bioinformatics, 11:119, 2010). To find a set of corresponding sequences matching the genome, a set of 49 conserved Clusters of Orthologous Groups (COG) families (Tatsuov et al. Science 278:631-637, 1997) was utilized. Sequences from the genomes were inserted into a reference alignment, nearest neighbors were extracted and enriched, and the sequences were matched using FastTree2 (an approximate maximum likelihood method; Price et al. PLoS One 5:e9490, A tree was derived from them using the NCBI RefSeq (2010). This strict classification method selected Streptomyces pratensis as the most appropriate reference species. The reference genome for Streptomyces pratensis ATCC 33331 was downloaded from NCBI RefSeq and aligned against the resulting whole genome sequence using MUMmer (mummer.sourceforge.net / ). The alignment revealed extensive overall agreement, confirming that the two sequences are very closely related on a genome-wide scale. The consensus 16S sequence is provided as SEQ ID NO:1.

[0116] - Streptomyces venezuelae. The results of all analyses strongly supported the identification of this isolate as S. venezuelae. The consensus 16S sequence is provided as SEQ ID NO:2.

[0117] - Bacillus firmus. The results of all analyses strongly supported the identification of this isolate as B. firmus. The consensus 16S sequence is provided as SEQ ID NO:3.

[0118] - Paenibacillus azoreducens. The results of all analyses strongly supported the identification of this isolate as P. azoreducens. The consensus 16S sequence is provided as SEQ ID NO:4.

[0119] Identification of microbial metabolic activity potential: All potential microbial metabolic activities were evaluated using laboratory assays as described in WO2018 / 045004, which is incorporated herein by reference in its entirety. To determine whether the microorganisms reduce sulfur-containing compounds to sulfides during metabolic processes, bioMerieux's API® Identification products were used according to the manufacturer's recommendations (bioMerieux, Inc., Durham, NC USA). The results of key metabolic activities profiling the newly identified microorganisms are shown in Table 1.

[0120] [Table 1]

[0121] Assessment of dipicolinic acid (DPA) production in bacteria: DPA production in bacterial strains of interest was assessed using terbium-DPA fluorescence assay, essentially as described by Rosen (Anal. Chem. 69:1082-1085, 1997); Pellegrino et al. (Anal. Chem. 70:1755-1760, 1998); and Ammann et al. (Int. J. Microbiol. 2011:435281, 2011). The assay was carried out according to Say. Briefly, each isogenic strain was grown on agar medium (see Table 2) either aerobically or anaerobically. Aerobic strains were grown at 30°C for up to 3 days, while anaerobic strains were grown in a BD GasPak EZ container system (Becton, Dickinson and Company, Franklin Lakes, NJ USA) with Pack-Anaero anaerobic gas generation sachets (Mitsubishi Gas Chemical Co., Tokyo, Japan) at 35°C for up to 3 days. For the DPA assay, approximately 5 μL of bacteria were taken from colonies growing on the plate and resuspended in 10 mL of sodium acetate buffer (0.2 M, pH 5). The suspension was autoclaved at 121°C, 15 psig for 15 min and cooled at room temperature for approximately 30 min. Equal volumes of the autoclaved suspension and 30 μM terbium(III) chloride hexahydrate (Sigma-Aldrich, Saint Louis, MO USA) solution were then mixed. Fluorescence was then measured (excitation at 272 nm, emission at 545 nm) using a Cytation 5 Imaging Reader (BioTek, Winooski, VT USA).

[0122] Culture of bacteria used in this study on semi-solid media: Isogenic bacterial strains stored at -80°C as master cell banks were grown on agar media (Table 2) until the formation of individual colonies was observed. Briefly, aerobic strains were grown at 30°C for up to 3 days, while anaerobic strains were grown at 35°C for up to 3 days in BD GasPak EZ container systems (Becton, Dickinson and Company, Franklin Lakes, NJ USA) with Pack-Anaero anaerobic gas-generating sachets (Mitsubishi Gas Chemical Company, Tokyo, Japan).

[0123] [Table 2]

[0124] Cultivation of individual bacterial strains used in this study in liquid media: Selected colonies from agar-grown strains were inoculated into the appropriate sterile liquid medium (Table 3) and incubated for up to 3 days. Aerobic strains were cultivated at 30 °C with shaking (125-175 rpm), while anaerobic strains were cultivated at 35 °C without stirring in sealed serum bottles or Hungate tubes under N2 gas. When required, microbial consortia were produced by mixing equal volumes of individually grown strains. Typical results (see below) showing the number of cells per mL per strain are summarized in Table 4. Microbial content was determined by Droplet Digital PCR (ddPCR) using Supermix For Probes (Bio-Rad Laboratories, Hercules, CA) as described in WO2018 / 045004.

[0125] [Table 3]

[0126] [Table 4]

[0127] Generation of co-cultured microbial consortia by fermentation: Both aerobic and / or anaerobic bacteria were cultivated in a medium containing 2% molasses supplemented with essential elements such as phosphate, sodium, potassium and chloride (in the form of commercial phosphate buffered saline) as well as amino acids, nitrogen and peptides / proteins in the form of food grade whey powder (0.1% w / v) and enzymatically produced non-GMO soy extract (0.25% w / v; Ferti-Nitro Plus Plant N; Ferti-Organic, Brownsville, TX USA). The concentration of sodium chloride ranged from 0 to 4% w / v. Strains from Table 4 above (referred to herein as AMC1) were cultured in a medium containing 2% molasses supplemented with essential elements such as phosphate, sodium, potassium and chloride (in the form of commercial phosphate buffered saline) as well as amino acids, nitrogen and peptides / proteins in the form of food grade whey powder (0.1% w / v) and enzymatically produced non-GMO soy extract (0.25% w / v; Ferti-Nitro Plus Plant N; Ferti-Organic, Brownsville, TX USA). The concentration of sodium chloride ranged from 0 to 4% w / v. The OD for each strain was 1.0. 600 The final inoculum amount ranged between 6.67E-05 and 6.67E-04, and the pH was maintained between pH 5.5 and 6.9 using ammonium hydroxide and phosphoric acid as base and acid solutions, respectively. The temperature was controlled between 28°C and 35°C. The anaerobic fermentation was continuously sparged with N2 gas to maintain an anaerobic environment, while sparged air was used in the aerobic fermentation as the oxygen source for the microorganisms during the fermentation period (typically up to 3 days). In some experiments, after fermentation, batches containing the various strains were pooled to generate one complete 22-strain bacterial mixture. Typical results showing the number of cells per mL per strain (see below) are summarized in Table 5. The microbial content in the fermentation broth was analyzed using Supermix as described in WO2018 / 045004. was determined by Droplet Digital PCR (ddPCR) using For Probes (Bio-Rad Laboratories, Hercules, CA).

[0128] [Table 5]

[0129] Generation of freeze-dried microbial consortia: In some experiments, the generated microbial consortia were freeze-dried prior to experiments in co-formulation with agricultural carriers. Consortia were generated by either pooling equal volumes of individually cultured bacteria or using co-cultured fermentation broth (above). Freeze-drying was performed essentially as described in WO2018 / 045004. Briefly, freeze-dried microbial preparations were generated by mixing liquid microbial consortia with mannitol / cryoprotectant solution (OPS Diagnostics Lebanon, NJ, USA) as recommended by the manufacturer and aliquoting the microbial suspension into freeze-drying vials (OPS Diagnostics, Lebanon, NJ, USA). After 60 min at -80°C, the mixture was placed in a FreeZone6 freeze-drying system (Labconco, Kansas City, MO) and vacuum was applied to sublimate the water in the samples. Samples were stored at 4°C until required. In some experiments, cryoprotectant solutions were prepared by adding the above chemicals to the microbial cultures to achieve final concentrations of 0.75 g / L Tryptic Soy Broth (Becton, Dickinson and Company, USA), 10 g / L sucrose (Sigma Aldrich, USA) and 5 g / L skim milk (Carnation, Nestle SA, CH).

[0130] Co-formulation of microorganisms with agricultural carriers: Liquid microbial consortia generated as above (produced by co-culture or grown individually and then pooled) or individual bacterial strains were impregnated onto agricultural carriers such as perlite, Azomite® (Azomite, UT, USA), pumice, monoammonium phosphate fertilizer (MAP; Mosaic, MN, USA), potassium chloride fertilizer (MOP; Mosaic, MN, USA), and biochar (Cool Terra®; Cool Planet Energy system, CO, USA). Depending on the water retention properties of the carrier, various volumes of microbial consortia were used to saturate the carriers from as low as 35 μL per gram to as high as 6 mL per gram. The microorganism / agricultural carrier mixture was then dried overnight at 30°C-35°C before being stored in airtight containers for further microbial viability studies and plant assays for shelf life.

[0131] Urea and Ammonium Nitrate (UAN) in Water 32 In case of co-formulation with liquid pesticides such as EDTA (TGI, CA, USA) or fertilizer dust control (DUSTROL; ArrMaz FL, USA), the liquid microbial consortia generated as detailed above were mixed in various ratios (described in the Examples below) prior to storage and microbial survivability analysis. All operations were performed under sanitary conditions to minimize contamination.

[0132] In some cases, freeze-dried bacterial consortia were used to minimize the influence of the culture broth on the chemistry of the carrier, details of which are provided in the Examples where appropriate.

[0133] Analysis of bacterial genomes for DPA synthase production Microbial genomic DNA extraction: Bacterial cells of various species were grown and recovered from optimized liquid broth and culture conditions. PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc, Carlsbad, CA USA) was used for small-scale genomic DNA extraction. For large-scale genomic DNA extraction, GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO USA) or Qiagen Genomic DNA Buffer were used. Set and Genomic-tip 500 / G (Qiagen, Hilden, Germany) were used according to the manufacturer's recommended method. The resulting genomic DNA was then precipitated with an equal volume of isopropanol, washed with 70% ethanol, air-dried, and resuspended in TE buffer.

[0134] Whole genome sequencing (WGS): Whole genome sequencing of biologically pure isolates was performed using the PacBio RSII system (Pacific Biosciences, Menlo Park, CA USA) following the manufacturer's recommended methods for sequence library preparation and sequencing. An average of 73,000 reads of 24 kb length were generated from the microbial isolates, followed by sequencing using the Hierarchical Genome Assembly Process (HGAP, Pacific A de novo genome assembly was performed by Microbial Pathway Biosciences, Menlo Park, CA USA).

[0135] Identification of DPA synthase coding sequences in selected bacterial strains: Initial bioinformatics analysis included selected members of the class Bacilli; Bacillus (Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus sp., and Bacillus flexus), and Lactobacillus (Lactobacillus delbrueckii). First, using the previously acquired whole genome sequences, a bacterial pan-genome analysis was performed using BPGA-Version-1.3 (iicb.res.in / bpga / index.html) to assess genome diversity and determine the core (conserved), accessory (not necessarily), and unique (strain-specific) gene pools. A search was then performed for any dipicolinic acid synthase subunit A (DpaA) in the accessory gene set. This was followed by a full search of the strains in Table 1 for DpaA and DpaB using custom Bash scripts (Free Software Foundation, 2007).

[0136] Briefly, protein-coding genes were annotated in two steps using Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm (Hyatt et al., BMC Bioinformatics 11:119, 2010). ) was used to identify the coordinates of candidate genes, but no putative gene products were described. These candidate genes were then compared to large databases in a hierarchical manner, starting with smaller reliable databases, moving to intermediate size but domain-specific databases, and finally to curated models of protein families. By default, 10 -6 An e-value threshold of was used for the following set of databases:

[0137] 1. All bacterial proteins in UniProt with evidence of actual proteins or transcripts and not fragments. Use BLAST+ to search.

[0138] 2. All proteins from RefSeq sequenced bacterial genomes for a particular genus. Use BLAST+ for the search.

[0139] 3. A set of hidden Markov model profile databases, including Pfam (Punta et al., 2012) and TIGRFAMs (Haft et al., 2013), which are part of the HMMER This is performed using hmmscan from the 3.1 package (Eddy, 2011).

[0140] 4. If no match is found, label it as a "hypothetical protein."

[0141] These data were then tabulated along with sporulation ability and viability of dry preparations to establish a correlation between DPA synthase and viability (see Table 6). Alignment of the DpaA and DpaB genes was then performed using Clustal Omega (www.ebi.ac.uk / Tools / msa / clustalo / ) and amino acid composition and frequency statistics calculated using Seaview4 (Gouy et al. Mol. Biol. Evol. 27:221-224, 2010). The DpaA and DpaB amino acid alignment was then used to generate constructs using the packages Biostrings and seqinr (Pages et al., 2016; Charif and Lobry, Structural Approaches to Sequence Evolution, pp. 207-232, 2007; R Core Team, 2016). The census sequence and the probability matrix in R were calculated.

[0142] [Table 6]

[0143] Bacteria in the consortia viability assay Preparation of liquid co-formulation samples: For each shelf-life time point, 1 mL of the co-formulation (pesticide / bacterial consortium) was diluted at 10 mL in sterile peptone water. -1 From 10 -5 Serial dilutions were made.

[0144] Preparation of dried formulation samples: At each shelf-life time point, 0.03–1 g of dried formulation (agricultural carrier / bacterial consortium) was suspended in up to 3 mL of culture broth, such as peptone water, or other suitable medium, and incubated at room temperature for up to 1 h. In some cases, gentle sonication (35 khz) was used to release the bacteria from the dried matrix using a water bath sonicator (VWR ultrasonic cleaner). The suspension was then diluted in sterile peptone water or other type of culture broth for 10 min. -1 From 10 -5 In other cases, the dry material was added directly to liquid medium for growth.

[0145] Viability Assay: Bacterial strains receiving treatment(s) were given an opportunity to grow. To maximize the growth potential of each strain in the consortium, several different agar media were used, such as chA (semi-dry chitin, 5 g / L; K2HPO4, 0.7 g / L; KH2PO4, 0.3 g / L; MgSO4.5H2O, 0.5 g / L; FeSO4.7H2O, 0.01 g / L; ZnSO4, 0.001 g / L; MnCl2, 0.001 g / L and agar, 15 g / L), RCM, NA, MP, YPD, RhX, AMAS, and / or BHIS. In some cases, each serial dilution generated above was plated and spread in duplicate on one or more agar media. In other cases, the dry material generated above was suspended in one or more liquid media in sextuplicate. For plate cultures, plates were incubated in a static incubator, one set at 30° C. aerobic conditions and the other at 35° C. anaerobic conditions for 3 days. For liquid cultures, tubes were incubated at 30° C. aerobically with shaking or at 35° C. anaerobically statically for 7 days. Bacteria that survived the treatment(s) were grown by forming colonies or growing in liquid medium, then sampled for this growth and identified using droplet digital PCR (ddPCR).

[0146] Genomic DNA was extracted from harvested cells using a DNeasy PowerLyzer PowerSoil kit (Qiagen, Inc., Germantown, MD USA) according to the manufacturer's recommendations. DNA was then quantified using a Quantas Fluorometer and QuantiFluor dsDNA (Promega Corporation, Madison, WI USA) and processed for identification and quantification using strain-specific probes in conjunction with ddPCR (Dreo et al., 2011). Anal. Bioanal. Chem. 406:6513-6528, 2014;Yin, et al., Journal of Microbiological Methods 65:21-31, 2006). Briefly, DNA samples, primers, and probes (designed using unique sequences from 16S genes and / or unique coding gene sequences identified from WGS genome assemblies as previously described in WO2018 / 045004, incorporated herein by reference) were combined with Bio-Rad's ddPCR Supermix for Probes according to the manufacturer's recommendations to prepare ddPCR reactions. Droplets were then generated using either a QX200™ Droplet Generator or an AutoDG™ Instrument according to the manufacturer's recommendations. Polymerase chain reaction (PCR) was performed using an Eppendorf Mastercycler® nexus gradient using the temperature cycle conditions recommended by Bio-Rad's ddPCR Supermix for Probes. Following the PCR protocol, reactions were read out using a QX200 droplet reader.Finally, enrichments were analyzed using QuantaSoft™ software.

[0147] In cases where the viability of a single strain was identified following co-formulation with a liquid or dry agricultural carrier, a single plating was performed using the agar medium most compatible with the given strain as listed in Table 2.

[0148] Example 2 Identification of DPA synthase coding sequences in selected bacterial strains Dipicolinate synthase subunit A (DpaA): Amino acid alignment of DpaA from bacterial strains revealed diverse amino acid alignments with only 28.4% conserved amino acids. Results are summarized in Tables 7-9 and Figure 1. Oceanobacillus oncorhynchi was revealed to be more diverse than most other strains. By calculating percent identity with the denominator defined as the aligned position, O. oncorhynchi had a percent identity of 58% with the consensus sequence using BLOSUM62. Furthermore, these amino acid changes appeared to be in conserved regions for most of the analyzed strains. Amino acid alignment of DpaA also revealed that Virgibacillus halophilus possesses two copies of the gene (likely due to gene duplication). The first copy was highly diverse (51% identity with the consensus sequence, same method as above) and the second copy appeared to be truncated by 29 amino acids in addition to significant amino acid changes. Furthermore, all other instances of DpaA were located contiguous to DpaB as part of an operon, whereas in V. halophilus the two genes are separated by 539 other genes.

[0149] [Table 7]

[0150] [Table 8]

[0151] [Table 9]

[0152] Dipicolinate synthase subunit B (DpaB): Amino acid alignment of DpaB from bacterial strains revealed that the conserved amino acids were 45.5%, making the amino acid alignment more conserved than that of DpaA. The results are summarized in Tables 10-12 and Figure 2. Neither duplication nor major truncations were detected. As mentioned above, all copies of DpaB were located contiguously with DpaA as part of an operon, except in V. halophilus, where DpaB was 539 genes upstream from DpaA.

[0153] [Table 10]

[0154] [Table 11]

[0155] [Table 12]

[0156] Correlation between DPA production (in the above assays) and the presence of DPA genes: As described in Table 6, we detected Clostridium beijerinckii as a DPA-producing strain by the terbium-DPA fluorescence assay, but neither DpaA nor DpaB was identifiable in its genome. We found that C. beijerinckii possesses an iron-sulfur flavoprotein (Isf) that is structurally related to EtfA, previously implicated in DPA production (Orsburn et al., Mol. Microbiol. 75:178-186, 2010). Six copies of the iron-sulfur flavoprotein were expressed in C. beije These sequences were then detected in the genome of four different bacteria: Archaeoglobus fulgidus, Methanocaldococcus The Isf protein was aligned with Isf sequences from Clostridium pasteurianum, Methanosarcina thermophila, and Peptoclostridium difficile (FIG. 3). The Isf protein is not present in Clostridium pasteurianum, and DPA production was not detected in C. pasteurianum. Further support for the role of Isf in DPA production is provided in Example 12.

[0157] Example 3 Survival of selected single strains on carriers An evaluation of bacterial viability on Azomite® impregnated with individual strains was performed. Four strains were selected based on their ability to produce DPA in lab assays and / or with respect to the results of DPA gene identification. The impregnated materials were dried and stored for 5 days before evaluating bacterial viability. The results are shown in Table 13. 100% (2 / 2) of the strains with detectable DPA production remained viable for 5 days. For strains with undetectable DPA production, 50% (1 / 2 strains) remained viable for 5 days.

[0158] [Table 13]

[0159] In a second experiment, the number of strains was expanded. Evaluation of bacterial viability in Azomite® after impregnation was performed over a one-month period. Briefly, Azomite® was impregnated with individual isogenic strains, dried, and samples were taken at 3, 7, 14, 21, and 28 days for bacterial viability analysis. The results are shown in Table 14 and show that 100% (4 / 4) of the bacteria with identified DPA genes and producing DPA remained viable for one month. For strains with unidentified DPA genes and undetectable DPA production, 40% (2 / 5 strains) remained viable for one month.

[0160] [Table 14]

[0161] A second agricultural carrier (perlite) was then tested. Microbial viability evaluation was performed over a one-month period as previously described for Azomite®. The results are shown in Table 15, and show that 100% (4 / 4) of the bacteria with the identified DPA gene and producing DPA remained viable for one month. For strains without a detectable DPA gene and with undetectable DPA production, 60% (3 / 5 strains) remained viable for one month.

[0162] [Table 15]

[0163] Example 4 Survival of microbial consortia on supports In the following experiments, 22 species of bacteria were grown individually and then mixed to produce a consortium as described above. Dry Azomite® in pelleted form was impregnated with 0.23-0.4 mL of the consortium (to preserve the integrity of the pellet), dried, and stored for 2 weeks before analyzing which bacteria survived using an assay to assay the viability of microorganisms from the consortium (Example 1). Two independent tests were performed and the results are summarized in Table 16. We observed that 83% and 92% of the bacterial strains in which the DPA gene was identified and / or which produce DPA remained viable 2 weeks after impregnation on Azomite®. The strain that did not seem to show consistent viability in the two tests was O. oncorhynchi. As mentioned above, the DPA synthase subunit A of O. oncorhynchi appears to be more diverse than most other investigated strains, with a percent identity of 58% with the consensus sequence using BLOSUM62. For strains in which the DPA gene was not identified and DPA production was not detectable, the results varied between 60% and 70% of the bacteria remaining viable within the same period. The reproducibility between the two tests was low and could be attributed to the various metabolic states of this set of microorganisms at the time of impregnation.

[0164] [Table 16-1] [Table 16-2]

[0165] As in the Azomite® experiment above, the 22 species of bacteria were grown individually and then mixed to produce a consortium. After impregnating 2-6 mL of the consortium on perlite, drying, and storing for 2 weeks, an assay to assay the viability of microorganisms from the consortium was used to analyze which bacteria survived (Example 1). Two independent tests were performed and the results are summarized in Table 17. We observed that 58% and 100% of the bacterial strains with identified DPA genes and / or producing DPA remained viable 2 weeks after impregnation on perlite. For strains with no identified DPA genes and no detectable DPA production, the results varied between 60% and 80% of the bacteria remaining viable within the same period. Compared to the Azomite® experiment, the bacteria with identified DPA genes and / or producing DPA showed good reproducibility even when different impregnation substrates were used. They also outperformed those in which the DPA gene was not identified or DPA production was undetectable in our assay.

[0166] [Table 17]

[0167] Example 4 Survival of microbial consortia co-formulated with liquid anti-dust agents: In the following experiments, bacterial strains of interest were grown on semi-solid agar plates using the media described above (see Table 2). Anti-dust chemical / bacteria co-formulations were made by suspending a 1 μL loopful scraped from the agar plate in 200 μL of sterile peptone water and then mixing with the anti-dust chemical at a ratio of 5% v / v. The viability of each strain in the co-formulation was then determined by inoculating the bacteria / pesticide mixture into fresh media (Table 3) and scoring signs of growth.

[0168] Viability of bacteria co-formulated with MDC-200: Evaluation of bacterial viability after co-formulation with dust control agent MDC-200 (ArrMaz, FL, USA) was performed over a one-month period. The results are shown in Table 18. We observed that 92% (11 / 12) of bacteria with identified DPA genes and / or producing DPA remained viable for one month. For strains with no identified DPA genes and undetectable DPA production, 0% (0 / 10 strains) remained viable for one month.

[0169] Viability of bacteria co-formulated with DUSTROL® 3275: Evaluation of bacterial viability after co-formulation with DUSTROL® 3275 (ArrMaz, FL, USA) was performed over a one-month period. The results are shown in Table 18. We observed that 100% (12 / 12) of bacteria with identified DPA genes and / or producing DPA remained viable for one month. For strains with no identified DPA genes and undetectable DPA production, 0% (0 / 10 strains) remained viable for one month.

[0170] Viability of bacteria co-formulated with DUSTROL® 3133: Evaluation of bacterial viability after co-formulation with DUSTROL® 3133 (ArrMaz, FL, USA) was performed over a one-month period. The results are shown in Table 18. We observed that 100% (12 / 12) of bacteria with identified DPA genes and / or producing DPA remained viable for one month. For strains with no identified DPA genes and undetectable DPA production, 0% (0 / 10 strains) remained viable for one month.

[0171] Viability of bacteria co-formulated with DUSTROL® 3139: Evaluation of bacterial viability after co-formulation with DUSTROL® 3139 (ArrMaz, FL, USA) was performed over a one-month period. The results are shown in Table 18. We observed that 92% (11 / 12) of bacteria with identified DPA genes and / or producing DPA remained viable for one month. For strains with no identified DPA genes and undetectable DPA production, 0% (0 / 10 strains) remained viable for one month.

[0172] Viability of bacteria co-formulated with DUSTROL® 3001: Evaluation of bacterial viability after co-formulation with DUSTROL® 3001 (ArrMaz, FL, USA) was performed over a one-month period. The results are shown in Table 18. We observed that 33% (4 / 12) of bacteria with identified DPA genes and / or producing DPA remained viable for one month. 0% (0 / 10 strains) of strains with no identified DPA genes and undetectable DPA production remained viable for one month. The reduced survival in this experiment may be due to incompatibility of the microorganisms (whether they produce DPA or not) with one or more components of DUSTROL® 3001.

[0173] Viability of bacteria co-formulated with DUSTROL® 3010: Evaluation of bacterial viability after co-formulation with DUSTROL® 3010 (ArrMaz, FL, USA) was performed over a one-month period. The results are shown in Table 18. We observed that 75% (9 / 12) of bacteria with identified DPA genes and / or producing DPA remained viable for one month. For strains with no identified DPA genes and undetectable DPA production, 20% (2 / 10 strains) remained viable for one month.

[0174] [Table 18-1] [Table 18-2]

[0175] Example 5 Survival of microbial consortia co-formulated with liquid UAN fertilizer Assessment of bacterial viability after co-formulation with liquid UAN fertilizer was performed at various time points over a one-month period. Briefly, each isogenic strain was grown in liquid medium (as described above) either aerobically or anaerobically. The cultures were then mixed in equal proportions, and the mixture was then combined with UAN32 at a ratio of 1:180 (microorganism:UAN). Microbial viability was assessed at days 0, 7, 14, and 28 and is summarized in Table 19. At day 0 (approximately 1 hour after co-formulation), 83% (10 / 12) of bacterial strains with identified DPA genes and / or producing DPA remained viable. For bacteria with no detectable DPA production, only 20% (2 / 10 strains) remained viable at day 0. At day 7, 67% (8 / 12) of bacterial strains with identified DPA genes and / or producing DPA remained viable. There was no change in viability for this set of strains after day 7. For bacteria with no detectable DPA production, only 10% (1 / 10 strains) remained viable at the 7 day time point.

[0176] [Table 19-1] [Table 19-2]

[0177] Example 6 Survival of microbial consortia coformulated with biochar In this experiment, the 22 species of bacteria shown in Table 4 were grown individually and then mixed to generate a consortium as described above. One gram of biochar (Cool Terra®; Cool Planet Energy system, CO, USA) was impregnated with approximately 0.5 mL of the consortium, dried, and stored for 7 days, after which the previously described viability assay was used to analyze which bacteria survived. The results are summarized in Table 20. 66.7% (7 / 12 strains) of bacteria with identified DPA genes and / or producing DPA remained viable after 7 days of impregnation on biochar. For strains with no identified DPA genes and no detectable DPA production, 30% (3 / 10 strains) remained viable within the same time frame.

[0178] [Table 20]

[0179] Example 7 Survival of microbial consortia co-formulated with dry fertilizer granules In this experiment, the 22 species of bacteria shown in Table 4 were grown individually and then mixed to generate consortia as described above and impregnated into MOP(0-0-60) dry fertilizer at a rate of 35 μL / g. The fertilizer was then stored at room temperature and periodically sampled to assess microbial viability as previously described. The results are summarized in Table 21. 66.7% (8 / 12 strains) of bacteria with identified DPA genes and / or producing DPA remained viable for up to 7 days. For strains with no identified DPA genes and no detectable DPA production, 30% (3 / 10 strains) remained viable within the same time frame.

[0180] [Table 21-1] [Table 21-2]

[0181] The 22 species of bacteria shown in Table 4 were also grown individually and then mixed to form consortia as described above and impregnated into MAP (11-52-0) at a rate of 35 μL / g. The fertilizer was then stored at room temperature and periodically sampled to assess microbial viability as previously described. The results are summarized in Table 22. In this case, 58.3% (7 / 12 strains) of bacteria with identified DPA genes and / or producing DPA remained viable for up to 7 days. For strains with no identified DPA genes and no detectable DPA production, 40% (4 / 10 strains) remained viable within the same time frame.

[0182] [Table 22]

[0183] Example 9 Survival of bacteria from the consortium compared with DPA expression / production In this experiment, the 22 bacterial species shown in Table 4 were grown individually and then mixed to generate consortia as described above. The consortia were then stored at room temperature and sampled periodically to assess microbial viability as previously described. The results are summarized in Table 23.

[0184] At 2 weeks, 83% (10 / 12 strains) of bacteria with identified DPA genes and / or producing DPA remained viable. For strains without identified DPA genes and no detectable DPA production, 60% (6 / 10 strains) remained viable within the same time frame. After 7.5 weeks, 58% (7 / 12 strains) of bacteria with identified DPA genes and / or producing DPA remained viable. For strains without detected DPA genes and no detectable DPA production, 50% (5 / 10 strains) remained viable within this period.

[0185] [Table 23-1] [Table 23-2]

[0186] Example 10 Summary of Microbial Survival In all examples tested, spore-forming bacteria with identified DPA genes and / or DPA production outperformed strains with no identified DPA genes and no detectable DPA production in terms of viability over time in co-formulation with agricultural carriers, either as liquid or dry formulations (Table 24 and Figure 4). Thus, in the rational design of microbial consortia with selected plant / soil beneficial traits for co-formulation with agricultural carriers (wet and / or dry) or as seed treatments, the selection of spore-forming and DPA-producing microorganisms can help ensure that desired microbial functions (e.g., but not limited to, nitrogen metabolism, sulfur metabolism, salt tolerance, mineral salt solubility, cellulose decomposition, chitin decomposition, plant hormone production, iron metabolism, dephosphorylation of organic matter) are retained in the surviving microorganisms. In some examples, this can ensure sufficient redundancy in microbial functions present in the rationally designed consortium to meet the needs of the target crop, soil, and application practice as well as geographic and regulatory challenges.

[0187] [Table 24]

[0188] Example 11 Evaluation of plant growth promoting activity Cucumber seeds purchased from Seed Kingdom (Lubbock, TX) were pregerminated for 4 days at 22–24°C in rolled germination paper (Anchor Paper, Saint Paul, MN) impregnated with a dilute mixture of liquid fertilizer (25 ppm NPK in water). The potting medium (Sunshine Mix) was pretreated with Hoagland's solution (Hoagland, Calif. Agric. Exp. Stn. Bull. 347:36-39, 1938) and modified to contain P, 30.97 ppm; K, 39.1 ppm; Ca, 40.0 ppm; Mg, 14.59 ppm; S, 20.143 ppm; Fe, 1.010 ppm; Cu, 0.019 ppm; Co, 0.012 ppm; B, 2.44 ppm; Mn, 0.494 ppm; Mo, 0.001 ppm, and Zn, 0.056 ppm. A ratio of 1 L of potting medium per pound was used. To each pot, 1 mL of 10% w / v urea solution was added before transplanting pre-germinated cucumber seedlings of similar length. For each treatment (including the control), 17–18 plants were randomized in flats with defined growth conditions, controlling temperature (16–24 °C) and 12 h photoperiod. Control pots contained 2 g of untreated perlite. Experimental pots were treated with perlite impregnated with 2 mL of the 22 species of bacteria listed in Table 4. The impregnated perlite was stored dry for 2 weeks before use in this assay. Flats were watered with modified Hoagland's solution three times a week. After 28 to 32 days, shoots were allowed to dry and weights were recorded for each plant. Data were analyzed with one-way ANOVA (analysis of variance) and samples were compared within experiments using post hoc Tukey's test. Shoot dry weight increased in plants treated with perlite impregnated with microorganisms, but did not reach statistical significance (Figure 5). As described primarily in Example 4 (Table 17), microorganisms for which DPA genes were identified and / or for which DPA production was detectable survived on impregnated perlite, a subset of the starting consortium.

[0189] Example 12 DPA Generation Consortium The in-house microbial collection was screened to identify suitable strains for use in designing additional DPA-producing consortia. Strains from genera known to form spores were revived from bacterial glycerol stocks onto appropriate media and grown for up to 3 days. Single colonies were then selected and passaged at least three times to assess purity. Strains were tested for DPA production using a terbium-DPA fluorescence assay as described in Example 1. Strains shown to produce DPA were selected for whole genome sequencing. Whole genome sequencing of biologically pure isolates was performed using an Illumina NovaSeq 6000 sequencing system (Illumina, Inc., San Diego, CA USA) following the manufacturer's recommended methods for sequence library preparation and sequencing. An average of 5,133,928 reads, averaging 87 bp in length, were generated from the microbial isolates. De novo genome assembly was performed using SPAdes 3.12.0 (Nurk et al., J. Comput. Biol. 20:714-737, 2013). The assembled contigs were then Annotated with PROKKA (Seemann, Bioinformatics 30(14):2068-2069, 2014 ). The annotated genomes were then assessed for the presence of dipicolinate synthase subunit A (DpaA) and dipicolinate synthase subunit B (DpaB) and iron-sulfur flavoprotein (Isf, Table 25). In most cases, strains that produced DPA possessed both the DpaA and DpaB genes and lacked the Isf gene. All strains that produced DPA and lacked DpaA and DpaB possessed one or more copies of the Isf gene. In three cases, strains possessed all three (DpaA, DpaB, and Isf) genes. Thus, detection of DPA production by the terbium-DPA fluorescence assay correlated perfectly with the presence of the DpaA, DpaB, and / or Isf genes.

[0190] [Table 25-1] [Table 25-2] [Table 25-3]

[0191] For strains that tested positive for DPA production, potential microbial metabolic activities (e.g., but not limited to, nitrogen metabolism, sulfur metabolism, salt tolerance, mineral salt solubility, cellulose degradation, chitin degradation, plant hormone production, iron metabolism, dephosphorylation of organic matter) were evaluated using laboratory assays as described in WO2018 / 045004 and Example 1. Strains with the greatest number of metabolic activities were selected for co-fermentation in a microbial consortium with five strains lacking DPA production and selected for their metabolic activities. (See Example 1.) Those strains that proved suitable for co-fermentation in a consortium (see Table 26), also referred to herein as the dry formulation consortium (DFC), were selected for co-formulation with carriers such as bentonite, perlite and / or urea.

[0192] All consortia included in this example were fermented as follows: both aerobic and anaerobic bacterial strains were cultured in media containing 2-10% sugar source. Amino acids, nitrogen and peptides were provided in the form of one or more of the following: food grade whey powder (0.1-0.5% w / v), yeast extract (0.1-0.5% w / v), enzymatically produced non-GMO soy extract (0.1-0.5% w / v; Ferti-Nitro Plus Plant N; Ferti-Organic, Brownsville, TX USA), spirulina (0.1-0.5% w / v), and / or peptones (0.1-0.5% w / v). When required, additional vitamins and micronutrients were provided by kelp extract (0.1-0.5% w / v), purified vitamin B (Sigma), and / or Wolfe's trace metals solution. When necessary, additional salts were added as phosphate buffered saline and / or sodium chloride additions (0-4% w / v). Strains from AMC1 (described above) were inoculated into 2 L DASGIP bioreactors (Eppendorf North America Hauppauge, NY) with a working volume of 1.5 liters. The pH during fermentation was maintained between 5.0 and 7.0. Aeration conditions during fermentation were adjusted to achieve the target oxygen transfer rate (k L a) was estimated using k L a (per hour) ranged from 0 to 110. Temperature was controlled between 28°C and 35°C.

[0193] [Table 26-1] [Table 26-2]

[0194] To generate the co-formulations, the DFC fermentation broth was applied to bentonite or perlite and then allowed to dry for approximately 24 hours. In some cases, the fermentation broth was concentrated by centrifugation prior to application. The co-formulations were then tested for viability. Additionally, certain co-formulations were tested in plant growth chamber studies to evaluate beneficial plant traits.

[0195] Viability of DFC and AMC1 with Carriers: In the preparations for field testing that included DFC liquor, AMC1 liquor, DFC-impregnated perlite, AMC1-impregnated perlite, DFC-impregnated bentonite, and AMC1-impregnated bentonite, DFC and AMC1 were co-fermented and impregnated at 2 mL / g or 1 mL / g, respectively, into the perlite or bentonite carrier. The impregnated perlite and bentonite were stored dry for 1 to 3 weeks, at which point viability assays were performed to determine the number of strains viable at the time of application. Additionally, the liquors were stored for 1 week, at which point viability assays were performed to determine the number of strains viable at the time of application. In the liquors, 16 of the 23 DFC strains were viable after 1 week versus 14 of the 22 AMC1 strains (Table 27).

[0196] [Table 27-1] [Table 27-2]

[0197] On impregnated perlite, 17 of 23 DFC strains versus 14 of 22 AMC1 strains remained viable after up to 3 weeks (Table 28).

[0198] [Table 28-1] [Table 28-2]

[0199] On impregnated bentonite, 17 of the 23 DFC strains versus 14 of the 22 AMC1 strains were viable after up to 3 weeks (Table 29), demonstrating how DPA-producing strains maintain improved viability when stored in liquid and when dried on a carrier such as perlite or bentonite.

[0200] [Table 29-1] [Table 29-2]

[0201] After 3 months of aging, 15 strains were shown to be viable on perlite impregnated with DFC, and only 11 strains were shown to be viable on perlite impregnated with AMC1, only one of which did not produce DPA (Tables 30 and 31).

[0202] [Table 30-1] [Table 30-2]

[0203] After the same 3 months of aging, bentonite impregnated with DFC showed 19 strains to be viable, whereas perlite impregnated with AMC1 showed viability for 13 strains, only 3 of which did not produce DPA (Tables 30 and 31).

[0204] [Table 31-1] [Table 31-2]

[0205] Evaluation of DFC plant beneficial activity: Cucumber seeds purchased from Seed Kingdom (Lubbock, TX) were pre-germinated in rolled germination paper (Anchor Paper, Saint Paul, MN) impregnated with a dilute mixture of liquid fertilizer (25 ppm NPK in water) for 4 days at 22° C. At the time of seed preparation, potting medium (Sunshine Mix) was prepared with 67.60 kg per hectare of tricalcium phosphate (TCP) and a pretreatment of modified Hoagland solution (Hoagland, Calif. Agric. Exp. Stn. Bull. 347:36-39, 1938). The Hoagland solution, NK+, was modified to contain N, 56.03 ppm; P, 0 ppm; K, 39.1 ppm; Ca, 40.0 ppm; Mg, 14.59 ppm; S, 20.143 ppm; Fe, 1.010 ppm; Cu, 0.019 ppm; Co, 0.012 ppm; B, 2.44 ppm; Mn, 0.494 ppm; Mo, 0.001 ppm, and Zn, 0.056 ppm, and was applied at a rate of 1 L per pound of potting medium. Control treatments also contained 10 g of untreated perlite or 20 g of untreated bentonite per pound of potting medium. In comparison, the experimental pots had the same amount of perlite or bentonite as the control pots, but these carriers were impregnated with 2 mL / g or 1 mL / g of DFC, respectively. The impregnated perlite and bentonite were stored dry for 2 weeks prior to use in this assay, at which point viability assays were performed to determine the number of strains from the DFC that were viable at the time of application. The majority of strains (15–18 of the 23 viable strains) survived the coformulation and aging process.

[0206] Following pre-germination of cucumber seeds, seedlings of similar length were selected and one was transplanted into each pot. For each treatment (including the control), 18 plants were randomized in flats with defined growth conditions, controlled temperature (16-24 °C) and 12 h photoperiod. The flats were watered for the first time 3 days after transplanting with modified Hoagland solution (PK+ containing N, 0 ppm; P, 14.49 ppm; K, 19.55 ppm; Ca, 20.0 ppm; Mg, 14.59 ppm; S, 20.143 ppm; Fe, 1.010 ppm; Cu, 0.019 ppm; Co, 0.012 ppm; B, 2.44 ppm; Mn, 0.494 ppm; Mo, 0.001 ppm and Zn, 0.056 ppm). The flats were then watered three times a week with NK+ Hoagland solution. After 32 days, shoots were harvested and the dry weight was recorded for each plant. Data were analyzed with one-way ANOVA and post-hoc Tukey's test was used to compare samples within experiments. These experiments were performed twice on two separate occasions.

[0207] Results from the first trial showed a significant increase in shoot weight for both DFC-impregnated perlite and DFC-impregnated bentonite when compared to the control (Figure 6). Results from the second trial also showed a significant increase in shoot weight for both DFC liquid treatment and DFC-impregnated perlite, with DFC-impregnated perlite performing better that the DFC liquid treatment, but these were not significantly different (Figure 7). Thus, co-formulation with a carrier did not affect efficacy when compared to the new liquid product.

[0208] Example 13 Microbial seed treatment Microbial seed treatments were applied to corn and soybean seeds using a batch treater SGS (SGS North America, Brookings, SD USA). 1 kg of seeds were treated for each treatment. For separate seed treatments, the microbes were applied directly to untreated seeds, applied as an overcoat to seeds pretreated with an insecticide / fungicide package, or mixed with an insecticide / fungicide slurry before application to the seeds. For corn, the insecticide / fungicide treatments consisted of either an Acceleron mix containing metalaxyl, trifloxystrobin, ipiconazole, and clothianidin, or a CruiserMaxx mix containing Cruiser (thiamethoxam), fludioxonil, mefenoxam, azoxystrobin, and thiabendazole. In soybean, insecticide / fungicide treatments consisted of either the Acceleron mix, containing metalaxyl, pyraclostrobin, imidacloprid, and fluxapyroxad, or the CrusierMaxx mix, containing Cruiser (thiamethoxam), mefenoxam, fludioxonil, and cedexan.

[0209] Viability was tested within 48 hours and three weeks after seed treatment (as in Example 1). The vast majority of strains (19-20 of 23 viable strains) survived the initial co-formulation process. After three weeks, only a slight decrease in viability was observed (17-18 of 23 viable strains; Figure 8). This indicated that selecting DPA-producing strains is not only effective for dry fertilizers such as bentonite and perlite, but also for seed treatment. Additionally, seeds were tested for germination to determine the effect of seed treatment on germination potential. A cold vigor test was performed in which four replicates of 100 seeds were tested for germination. Each replicate of 100 seeds was tested for germination. The seeds were sown on moistened crepe cellulose paper and chilled overnight at 10°C. The seeds were then covered with 1 inch of non-sterile sand moistened to 70% water holding capacity and returned to 10°C for 7 days without light. The seeds were then shifted to 25°C for 4 days. The seedlings that germinated from the sand were evaluated. Results were reported as a percentage representing the number of seedlings classified as normal according to AOSA rules. A score of 82% or higher is considered the minimum acceptable for marketing a corn seed lot according to Iowa State Seed Lab and SGS. All treatments had a germination percentage of at least 83.8% or higher (Table 32). This indicates that in addition to performing well in the form of a seed treatment, the DPA-producing strains did not negatively affect the germination of the seeds.

[0210] [Table 32]

[0211] In view of the many possible embodiments to which the principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are examples only and should not be construed as limiting the scope of the invention, which is rather defined by the following claims, and the inventors therefore claim as their invention all that comes within the scope and spirit of these claims.

Claims

[Claim 1] The invention described in the specification.

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