MICROBIAL CONSORTIA PRODUCING DIPICOLINIC ACID AND METHODS FOR SELECTING MICROBES FOR CO-FORMULATION WITH CARRIERS
Methods for selecting a microbe for co-formulation with a carrier are provided. In some examples, the methods include identifying a microbe that comprises one or more dipicolinic acid (DPA) synthase genes, a microbe that expresses one or more DPA synthase proteins, and/or a microbe that produces DPA; and selecting the microbe for co-formulation with a carrier. The methods optionally include co-formulating the selected microbe with the carrier. In some examples, the methods include detecting one or more DPA synthase genes or one or more DpaA and/or DpaB proteins in a microbe. In other examples, the methods include detecting DPA in a microbe or medium containing a microbe, for example, utilizing a fluorescence assay. Microbial compositions including one or more microbes that comprise one or more DPA synthase genes, express one or more DPA synthase proteins and/or produce DPA are also provided.
This claims the benefit of U.S. Provisional Application No. 62/681,469, filed Jun. 6, 2018, which is incorporated herein by reference in its entirety.
FIELDThis disclosure relates to microbes producing dipicolinic acid and methods of identifying microbes with improved viability, methods of co-formulating microbes with carriers, and compositions including the microbes and/or co-formulations.
BACKGROUNDMicrobe-based plant biostimulants offer sustainable agriculture practices that protect the health of the ecosystem. Moreover, supplementation of the plant and soil microbiome with beneficial microorganisms has potential in promoting plant growth and plant fitness, increasing productivity, improving soil fertility, and reducing chemical inputs, resulting in more sustainable agricultural practices. In current agricultural practices, microbial biostimulants can be co-applied and/or co-formulated with numerous wet or dry carriers.
SUMMARYMicrobial inoculants can be susceptible to the chemistry of the carrier(s) used. Moreover, storage conditions and length of storage before application can also affect microbes. These factors can negatively impact their viability and ultimately limit their efficacy in the field. Disclosed herein are compositions and methods that result in improved microbe survival and/or improved co-formulation of microbes with carriers or seeds. In some embodiments, the methods include selecting one or more microbes with extended viability or survival either alone and/or in co-formulation with one or more carriers or seeds.
In some embodiments, disclosed herein are microbes that produce dipicolinic acid (DPA) and compositions including such microbes. 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 (in some examples, referred to herein as the “DFC” consortium). In one embodiment, the composition includes cells of microbial species deposited with the American Type Culture Collection (ATCC, Manassas, Va.) on May 16, 2019 and assigned deposit number PTA-125924. In other embodiments the disclosed microbial consortia or compositions include, consist essentially of, or consist of two or more (such as 5 or more, 10 or more, 15 or more, 20 or more, or all) microbes having 16S rDNA sequences with at least 95% identity (such as at least 96%, 97%, 98%, 99% identity, or more) with SEQ ID NOs: 3-25.
Also disclosed are compositions including the disclosed microbes or consortia (for example, the DFC consortium) and one or more carriers (such as a dry carrier or a liquid carrier) or one or more seeds. In some examples, the carrier includes a liquid or dry fertilizer, a soil-derived substance, an organic substance, an inert material, a dust control chemical, or a mixture of two or more thereof.
In some embodiments, the methods include selecting a microbe for co-formulation with a carrier or seed, including identifying a microbe that comprises one or more dipicolinic acid (DPA) synthase genes, a microbe that expresses one or more DPA synthase proteins, and/or a microbe that produces detectable amounts of DPA; and selecting the microbe for co-formulation with a carrier. In some embodiments, the methods also include co-formulating the selected microbe with the carrier or seed. In some examples, the selected microbes include one or more of those included in Tables 25 or 26, including, but not limited to all of those listed in Table 26.
In some examples, the methods include detecting one or more DPA synthase genes (such as a DPA synthase subunit A (DpaA) gene and/or or a DPA synthase subunit B (DpaB) gene) or one or more DpaA and/or DpaB proteins in a microbe. DpaA genes include nucleic acids that encode a DpaA protein with at least 20% (for example, at least 60%) sequence identity to any one of the amino acid sequences in
In some embodiments, the method includes co-formulating one or more selected microbes with a carrier by contacting the selected microbes (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 a liquid or dry fertilizer, a soil-derived substance, an organic substance, an inert material, a dust control chemical, or a mixture of two or more thereof. In other examples, the methods include treating seeds with the one or more selected microbes (including, but not limited to the microbial consortia disclosed herein). In some examples, the methods further include co-formulating the one or more selected microbes and one or more microbes that do not comprise one or more DPA synthase genes, do not express one or more DPA synthase proteins, and/or a microbe that does not produce detectable amounts of DPA with the carrier or seed.
The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
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 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NO: 1 is a consensus 16S rDNA nucleic acid sequence from Streptomyces pratensis.
SEQ ID NO: 2 is a consensus 16S rDNA nucleic acid sequence from Streptomyces venezuelae.
SEQ ID NO: 3 is a 16S rDNA nucleic acid sequence from Bacillus firmus.
SEQ ID NO: 4 is a consensus 16S rDNA nucleic acid sequence from Paenibacillus azoreducens.
SEQ ID NO: 5 is a 16S rDNA nucleic acid sequence from Bacillus amyloliquefaciens.
SEQ ID NO: 6 is a 16S rDNA nucleic acid sequence from Bacillus flexus.
SEQ ID NO: 7 is a 16S rDNA nucleic acid sequence from Bacillus licheniformis.
SEQ ID NO: 8 is a 16S rDNA nucleic acid sequence from Bacillus megaterium.
SEQ ID NO: 9 is a 16S rDNA nucleic acid sequence from Bacillus pumilus.
SEQ ID NO: 10 is a 16S rDNA nucleic acid sequence from Bacillus koreensis.
SEQ ID NO: 11 is a 16S rDNA nucleic acid sequence from Bacillus drentensis.
SEQ ID NO: 12 is a 16S rDNA nucleic acid sequence from Bacillus subtilis.
SEQ ID NO: 13 is a 16S rDNA nucleic acid sequence from Clostridium bifermentans.
SEQ ID NO: 14 is a 16S rDNA nucleic acid sequence from Clostridium beijerinckii.
SEQ ID NO: 15 is a 16S rDNA nucleic acid sequence from Clostridium pasteurianum.
SEQ ID NO: 16 is a 16S rDNA nucleic acid sequence from Lactobacillus paracasei.
SEQ ID NO: 17 is a partial 16S rDNA nucleic acid sequence from Fontibacillus sp. (panacisegetis).
SEQ ID NO: 18 is a 16S rDNA nucleic acid sequence from Oceanobacillus oncorhynchi.
SEQ ID NO: 19 is a 16S rDNA nucleic acid sequence from Paenibacillus lautus.
SEQ ID NO: 20 is a 16S rDNA nucleic acid sequence from Paenibacillus chibensis.
SEQ ID NO: 21 is a 16S rDNA nucleic acid sequence from Paenibacillus cookii.
SEQ ID NO: 22 is a 16S rDNA nucleic acid sequence from Paenibacillus sp. (chitinolyticus).
SEQ ID NO: 23 is a partial 16S rDNA nucleic acid sequence from Paenibacillus sp. (P1XP2).
SEQ ID NO: 24 is a 16S rDNA nucleic acid sequence from Pseudomonas sp.
SEQ ID NO: 25 is a 16S rDNA nucleic acid sequence from Streptomyces griseus.
SEQ ID NOs: 26-39 are DpaA amino acid sequences.
SEQ ID NO: 40-41 are DpaA consensus amino acid sequences.
SEQ ID NOs: 42-54 are DpaB amino acid sequences.
SEQ ID NO: 55-56 are DpaB consensus amino acid sequences.
SEQ ID NOs: 57-66 are Isf amino acid sequences.
SEQ ID NO: 67 is an Isf consensus amino acid sequence.
DETAILED DESCRIPTIONMicrobes that do not form spores are often more susceptible to deleterious factors occurring during processing and field application than microbes that form spores. The selection process for identifying microbes that form spores from large microbial inventories can be tedious and time consuming. This usually involves wet-lab testing for survivability (for example, in co-formulations with carriers of choice), with limited guarantee of survivability and final product extended shelf-life.
Disclosed herein is a novel strategy and method for selecting microbes with high confidence of extended shelf-life as either standalone biostimulant formulations and/or in co-formulation with wet or dry carriers or seeds, thus significantly accelerating lead time to new product testing in field trials. As described herein, spore forming bacteria with identified DPA genes or proteins and/or producing DPA outperform strains with no identified DPA genes and no detectable DPA production in terms of survivability in co-formulation with carriers over time. Finally, a consortium of microbes that includes DPA-producing strains is described.
I. TermsUnless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may 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. Rédei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003 (ISBN: 0-471-26821-6).
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill 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 dictates otherwise. For example, the term “comprising a cell” includes single or plural 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.
Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:
Carrier: A substance that can be used as a delivery vehicle (for example, in co-formulation or as inoculant) for microbes, such as the microbes or microbial consortia described herein (also referred to herein as “agro-carriers”). The carrier may be liquid or solid (dry). Exemplary carriers include liquid or dry fertilizers, soil-derived substances (for example, charcoal, clays, turf) organic substances (for example, sawdust, wheat/soy/oat bran, composts,) and inert materials (for example, perlite, vermiculite, bentonite, Azomite®, kaolin, silicates, talc). In some examples, seeds may also be referred to as carriers.
Contacting: Placement in direct physical association, including in either solid and/or liquid form. For example, contacting can occur with one or more microbes (such as the microbes in a microbial consortium) and a carrier or a seed. Contacting can also occur with one or more microbes, microbe/carrier co-formulation, or microbe/seed co-formulation and soil, plants, and/or plant parts (such as foliage, stem, seedling, roots, and/or seeds).
Culturing: Intentional growth of one or more organisms or cells in the presence of assimilable sources of carbon, nitrogen and mineral salts. In an example, such growth can take place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. In a further example, the culturing may take place on a surface or by submerged culture. The nutritive medium can be composed of complex nutrients or can be chemically defined.
Dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA): A compound with the structure
In most microbes, DPA is produced by conversion of 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 (
Some bacteria (e.g., some Clostridium) are able to synthesize DPA, despite lacking identifiable DpaA and DpaB genes. Without being bound by theory, these bacteria are proposed to utilize a structurally related protein, electron transfer flavoprotein (etfA), which is a flavin mononucleotide (FMN) oxidoreductase. EtfA is thought to catalyze the final step in the biosynthesis pathway by converting dihydrodipicolinate to dipicolinic acid (Orsburn et al., Mol. Microbiol. 75:178-186, 2010). Alternatively, some bacteria may utilize and iron-sulfur flavoprotein (Isf) in production of DPA.
Heterologous: Originating from a different genetic sources or species. For example, a nucleic acid that is heterologous to a cell originates from an organism or species other than the cell in which it is expressed. Methods for introducing a heterologous nucleic acid into bacterial cells include for example transformation with a nucleic acid, including electroporation, lipofection, and particle gun acceleration.
In another example of use of the term heterologous, a nucleic acid operably linked to a heterologous promoter is from an organism, species, or gene other than that of the promoter. In other examples 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.
Isolated: An “isolated” biological component (such as a nucleic acid, protein or organism) has been substantially separated or purified away from other biological components (such as other cells, cell debris, or other proteins or nucleic acids). Biological components that have been “isolated” include those components purified by standard purification methods. The term also embraces recombinant nucleic acids, proteins, or microbes, as well as chemically synthesized nucleic acids or peptides. The term “isolated” (or “enriched” or “purified”) does not require absolute purity, and can include microbes or molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.
Microbe: A microorganism, including but not limited to bacteria, archaebacteria, fungi, and algae (such as microalgae). In some examples, microbes are single-cellular organisms (for example, bacteria, cyanobacteria, some fungi, or some algae). In other examples, the term microbes includes multi-cellular organisms, such as certain fungi or algae (for example, multicellular filamentous fungi or multicellular algae).
Microbial composition: A composition (which can be solid, liquid, or at least partially both) that includes cells of at least one type (or species) of microbe (or a population of cells of at least one type of microbe). In some examples, a microbial composition comprises cells of one or more types (species) of microbes (or one or more populations of microbes) in a liquid (such as a storage, culture, or fermentation medium or a liquid fertilizer), for example, as a suspension in the liquid. In other examples, a microbial composition includes cells of one or more types (species) of microbes (or one or more populations of microbes) on the surface of or embedded in a solid or gelatinous medium (including but not limited to a culture plate), or a slurry or paste. In other examples, a microbial composition includes cells of one or more types (or species) of microbes (or one or more populations of microbes) in association with a dry material or seed, such as on the surface of or impregnated in a dry material or seed.
Microbial consortium: A mixture, association, or assemblage of cells of two or more microbial species, which in some instances are in physical contact with one another. The microbes in a consortium may affect one another by direct physical contact or through biochemical interactions, or both. For example, microbes in a consortium may exchange nutrients, metabolites, or gases with one another. Thus, in some examples, at least some of the microbes in a consortium are metabolically interdependent. Such interdependent interactions may change in character and extent through time and with changing culture conditions.
Transduced and Transformed: A virus or vector “transduces” a cell when it transfers nucleic acid into the cell. A cell is “transformed” by a nucleic acid transduced into the cell when the DNA becomes replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including bacterial conjugation, transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
Vector: A nucleic acid molecule that can be introduced into a host cell, thereby producing a transformed or transduced host cell. Recombinant DNA vectors are vectors including recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes, a cloning site for introduction of heterologous nucleic acids, a promoter (for example 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 gram negative and/or gram positive bacterial cells. Exemplary vectors include those for use in E. coli.
Viability: Ability of a cell (such as a microbial cell) to grow or reproduce under appropriate conditions for growth or reproduction. In some examples, “survival” or “survivability” refers to the viability of a cell (such as a microbial) cell after a period of storage in a liquid or dry state, alone, in a mixture with other microbial cells, and/or when co-formulated with a carrier or seed.
II. Methods of Identifying Microbes with Viability in Co-Formulations
Disclosed herein are methods of identifying microbes that remain viable or survive when co-formulated with a liquid or solid carrier. The microbes may be individually co-formulated with a carrier or seed (e.g., a single strain or species of microbes is co-formulated with a carrier or seed) or may be part of a consortium or mixture of microbes (e.g., two or more strains or species of microbes) that is co-formulated with a carrier or seed. In other embodiments, the methods include identifying microbes that remain viable or survive for an extended period of time in a consortium (as a standalone consortium or co-formulated with a carrier or seed).
In some examples, microbes identified with the methods disclosed herein, for example, microbes that include 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) than microbes that do not include one or more DPA synthase genes, do not express one or more DPA synthase proteins, and/or do not produce detectable amounts of DPA. In some examples, the microbes identified with the methods disclosed herein have at least 10% increased viability (for example, 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 a microbe that does not include one or more DPA synthase genes, does not express one or more DPA synthase proteins, and/or does not produce detectable amounts of DPA. Increased viability may include a greater number of viable cells after a set period of time and/or a viability for a longer period of time.
In some embodiments, the methods disclosed herein include identifying microbes that include in their genome one or more genes encoding a DPA synthase, express one or more DPA synthase proteins, and/or produce detectable amounts of DPA. Such microbes are identified as microbes that can remain viable or survive individually or when co-formulated with a liquid or solid carrier (for example, compared to one or more microbes that do not include genes encoding a DPA synthase, do not express one or more DPA synthase proteins, and/or do not produce detectable amounts of DPA). The identified microbes may further be selected for downstream use, such as for co-formulation with a liquid or solid carrier or seed. In some examples, the microbes remain viable or survive (either 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 more (for example, 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 more).
Methods of determining viability or survival of a microbe include detecting growth of the microbe in culture. In some examples, a preparation containing microbial cells (in a liquid or dry state, or in co-formulation with a carrier or seed) is inoculated in a liquid medium, incubated under conditions suitable for microbial growth, and presence and/or amount of microbes after a defined period of time are measured. In other examples a preparation containing microbial cells (in a liquid or dry state, or in co-formulation with a carrier or seed) is streaked on a plate containing solid or semi-solid medium, incubated under conditions suitable for microbial growth, and presence and/or amount of microbes (such as presence, size, and/or number of colonies) are measured. In some examples, the microbes are identified, for example, using PCR methods. Exemplary methods for determining microbial cell viability and identity are provided in Example 1.
In further embodiments, the methods include selecting one or more microbes that include one or more DPA synthase genes, express one or more DPA synthase proteins, and/or produce detectable amounts of DPA and optionally co-formulating the one or more selected microbes with one or more carriers or seeds. In some examples, the methods include preparing a co-formulation of one or more of the selected microbes with one or more carriers or seed. The methods include contacting the one or more microbes with the one or more carriers or seeds, for example, in a solid (dry) or liquid form. In some example, the carrier(s) or seed(s) are contacted with a mixture of microbes. The mixture includes microbes that express DPA synthase or produce DPA (such as microbes selected or produced using the methods described herein, including, but not limited to DFC) and may also include one or more microbes that do not express DPA synthase or produce DPA. In some examples, the carrier is contacted with a liquid that includes about 103-109 cells/mL or more (e.g., about 1×103 cells/mL, about 5×103 cells/mL, about 1×104 cells/mL, about 5×104 cells/mL, about 1×105 cells/mL, about 5×105 cells/mL, about 1×106 cells/mL, about 5×106 cells/mL, about 1×107 cells/mL, about 5×107 cells/mL, about 1×108 cells/mL, about 5×108 cells/mL, about 1×109 cells/mL, about 5×109 cells/mL, or more) of each microbe.
In some embodiments, a liquid including one or more of the selected microbes (and optionally one or more additional microbes) is placed in contact with one or more dry carriers or seeds. In some examples, the liquid including the microbes is a fresh or frozen bacterial culture or a mixture of fresh or frozen bacterial cultures. In other examples, the liquid including the microbes is a liquid to which freeze-dried microbes have been added. The liquid including the one or more microbes is allowed to soak into the dry carrier or seed. In some examples, an amount of liquid including the one or more microbes is used so that the dry carrier or seed is saturated, for example to provide relatively even distribution of the microbes throughout the carrier or seed. However, non-saturating amounts of liquid may also be used. In non-limiting examples, the amount is about 35 μL/g to 6 mL/g. In some examples, the microbe-impregnated carrier or seed is dried (such as at room temperature or at about 30-35° C.) and stored at ambient temperature (for example, in a closed or air-tight container).
In other embodiments, a liquid including one or more of the selected microbes (and optionally one or more additional microbes) is mixed with one or more liquid carriers. In some examples, the liquid including the microbes is a fresh or frozen bacterial culture or a mixture of fresh or frozen bacterial cultures. In other examples, the liquid including the microbes is a liquid to which freeze-dried microbes have been added. The liquid including the microbes can be mixed with the liquid carrier at any selected amount, for example, from 0.1%-90% (v/v), such as 0.5-1%, 1-5%, 2-10%, 3-6%, 4-8%, 5-15%, 8-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-75%, or 70-90% (v/v). In some examples, the microbes 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 microbes 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 microbes is added to a concentrated liquid carrier (such as a 10× concentrated liquid carrier) at 90% (v/v) to produce a 1× 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 that will be used. The mixture of microbes is stored at ambient temperature (for example, in a closed or air-tight container).
In some embodiments, a dry preparation of microbes (such as freeze-dried microbes) is used in the co-formulation with a dry carrier or seed. In some examples, freeze-dried microbes are mixed with a dry carrier or seed (such as about 40 mg microbes/kg carrier or seed to about 1 g microbes/kg carrier or seed). In some examples, of this embodiment, the freeze-dried microbes are added to a liquid that is then contacted with the dry carrier or seed. In other examples, the freeze-dried microbes are added to a liquid and then contacted with the dry carrier or seed as described above.
A. Detecting DPA Synthase Nucleic Acids
In some embodiments, the methods include identifying presence of one or more DPA synthase nucleic acid molecules (such as DNA, cDNA, or mRNA) in a microbe or population of microbes. In some examples, the methods include detecting one or more DPA synthase genes (such as DpaA and/or DpaB) in the genome of a microbe. In some examples, a microbe includes both DpaA and DpaB genes. Exemplary DPA synthase genes include B. subtilis DpaA (GenBank Accession No. NC_000964.3, 1744367-1745260, incorporated herein by reference as present in GenBank on Jun. 3, 2018) and DpaB (GenBank Accession No. NC_000964.3, 1745236-1745865, incorporated herein by reference as present in GenBank on Jun. 3, 2018). In some examples, a DpaA gene encodes a protein shown in
In other embodiments, the methods include identifying presence of one or more nucleic acids (such as DNA, mRNA, or cDNA) that are involved in an alternative pathway for DPA synthesis. In some examples, the method includes identifying presence or expression of one or more nucleic acids encoding an electron transfer flavoprotein (such as EtfA) or an iron-sulfur flavoprotein (such as Isf). The electron transfer flavoprotein is a heterodimer consisting of an alpha and a beta subunit, 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 herein by reference 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 herein by reference as present in GenBank on Jun. 3, 2018). Exemplary Isf nucleic acid and protein sequences include GenBank Accession Nos. CP016318 (3060700-3061308) and ARE63607, respectively (incorporated herein by reference as present in GenBank on Jun. 3, 2018) and those shown in
In some examples, DPA synthase nucleic acids (or EtfA or Isf nucleic acids) can be identified by sequence analysis of a microbe (for example, whole genome sequencing and/or sequencing using DPA synthase-specific oligonucleotides). In some examples, the 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). Exemplary methods for identifying DPA synthase genes are provided in Example 1, below. Similar methods can be used for identifying EtfA or Isf genes.
In some examples, nucleic acids from a microbe or population of microbes are isolated, amplified, or both, prior to detection. In some examples, amplification and detection of expression occur simultaneously or nearly simultaneously. In some examples, nucleic acid expression can be detected by PCR (for example, PCR, real-time PCR, RT-PCR or quantitative RT-PCR). For example, nucleic acids can be isolated and amplified by employing commercially available kits. In an example, the nucleic acids can be incubated with primers that permit the amplification of DpaA and/or DpaB (or EtfA or Isf) nucleic acids, under conditions sufficient to permit amplification of such products. The resulting amplicons can be detected.
In another example, nucleic acids from a microbe or population of microbes are incubated with probes that can bind to DpaA and/or DpaB (or EtfA or Isf) nucleic acid molecules (such as cDNA, genomic DNA, or RNA (such as mRNA)) under high stringency conditions. The resulting hybridization can then be detected. In other examples, a microbe or population of microbes is screened by applying isolated nucleic acid molecules obtained from the microbe(s) to an array. In one example, the array includes oligonucleotides complementary to DpaA and/or DpaB (or EtfA or Isf) nucleic acids. In an example, the microbial nucleic acid molecules are incubated with an array including oligonucleotides complementary to DpaA and/or DpaB (or EtfA or Isf) for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes. The isolated nucleic acid molecule:oligonucleotide complexes are then analyzed to determine if the nucleic acids are present in the sample.
B. Detecting DPA Synthase Proteins
As an alternative, or in addition to detecting DPA synthase nucleic acids, proteins can be detected using methods such as immunoassays (such as Western blot, immunohistochemistry, flow cytometry, or ELISA) or mass spectrometry. In some examples, a DpaA protein includes a protein with at least 20% sequence identity (such as 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 a protein shown in
In some examples, proteins are purified before 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 to 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 be subsequently incubated with a secondary antibody that is labeled (for example with a fluorescent label). The label can then be detected, for example by microscopy, ELISA, flow cytometry, or spectrophotometry. In another example, the sample is analyzed by Western blotting for detecting expression of DpaA and/or DpaB proteins. Antibodies for DpaA, DpaB, or Isf can be generated by one of ordinary skill in the art, for example, using the amino acid sequences in
Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic 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 prosthetic 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 non-limiting exemplary radioactive labels include 125I, 131I, 35S or 3H.
C. Detecting DPA
In some embodiments, the methods include identifying a microbe or population of microbes that produces DPA (such as detectable levels of DPA). In some examples, the methods include detecting at least 1 nM DPA (such as 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 DPA, or more). In one example, the methods include 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, contacting DPA with terbium(III) forms a complex that has increased fluorescence compared to terbium(III), allowing detection and/or quantitation of DPA in a sample. An exemplary Terbium-DPA assay is described in Example 1.
III. Microbes and Co-FormulationsDisclosed herein are microbes that include one or more DPA synthase genes in their genome, express one or more DPA synthase proteins, and/or produce DPA. In some examples, the microbes are modified to include one or more DPA synthase genes in their genome, express one or more DPA synthase proteins, and/or produce DPA. Also disclosed are co-formulations of the microbes with one or more carriers or seeds.
A. Microbes
Microbes that possess one or more DPA synthase genes, express one or more DPA synthase proteins, and/or produce 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. WO 2018/045004 (incorporated herein by reference in its entirety). Additional microbes include those listed in Tables 25 and 26. In some examples, these microbes also have sporulation ability; however, sporulation ability and presence of identifiable DPA synthase genes or DPA production are not completely concordant (see, e.g., Table 6).
In additional embodiments, disclosed are compositions including microbes that possess one or more DPA synthase genes, express one or more DPA synthase proteins, and/or produce DPA, including those referred to herein as Dry Formulation Consortium (DFC). The microbes in DFC 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 includes cells of microbial species deposited with the American Type Culture Collection (ATCC, Manassas, Va.) on May 16, 2019 and assigned deposit number PTA-125924.
One of ordinary skill in the art will recognize that identification of microbes, particularly at the species or strain level, is not always possible. In some examples, the microbes in the compositions described herein were analyzed by 16S rDNA sequencing and whole genome sequencing followed by comparison to sequences in public databases. However, due to limitations of information in sequence databases (including little or no information for some species or strains and/or changes in nomenclature over time) it can be challenging to provide definitive species or strain identifications. Thus, in some embodiments, the microbial species included in the disclosed compositions are identified by their sequence identity to the 16S rDNA sequences provided herein (SEQ ID NOs: 3-25). In some examples, the disclosed microbial consortia or compositions include, consist essentially of, or consist of two or more (such as 5 or more, 10 or more, 15 or more, 20 or more, or all) of the microbes having 16S rDNA sequences with at least 95% identity (such as at least 96%, 97%, 98%, 99%, or more) to SEQ ID NOs: 3-25.
Microbes that possess one or more DPA synthase genes, express one or more DPA synthase proteins, and/or produce detectable amounts of DPA also include microbes that do not naturally have one or more DPA synthase genes, express one or more DPA synthase proteins, and/or produce DPA, but are modified to do so. In some examples, a microbe that does not naturally have one or more DPA synthase genes, express one or more DPA synthase proteins, and/or produce DPA is 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
Bacteria that may be modified to express one or more heterologous DPA synthase genes include, but are not limited to, Azotobacter (such as Azotobacter vinelandii), Clostridium (such as Clostridium pasteurianum), Streptomyces (such as 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 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. WO 2018/045004 (incorporated herein by reference in its entirety).
In some examples, the heterologous DpaA and/or DpaB gene is placed under control of a promoter. In some examples, the promoter is a constitutive promoter, while in other examples, the promoter is inducible (for example, an inducible T7 promoter). In additional examples, the promoter is an arabinose-inducible promoter (for example, the pBAD system), a lac promoter (direct IPTG/lactose induction), a trc promoter (direct IPTG/lactose induction), a tetracycline-inducible promoter, or a pho promoter (phosphate deprivation induced). The heterologous DpaA and/or DpaB gene may be included in a vector, for example operatively linked to a promoter. Similar methods can be used for EtfA or Isf genes.
Multiple genes (such as two or more DPA synthase and/or Isf genes) can be expressed simultaneously in bacteria. To ensure adequate and coordinate production of multiple enzymes from a single pathway, each nucleic acid encoding a heterologous gene is optionally placed under control of a single type of promoter, such as the inducible T7 promoter. One example is the Duet™ vectors (Novozymes), which are designed with compatible replicons and drug resistance genes for effective propagation and maintenance of four plasmids in a single cell. This allows for the coexpression of up to eight different proteins. In other examples, 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, Calif.), or Clontech (Mountain View, Calif.).
In one example, the vector is pET21a or pET28a. In some examples, the pET vector includes a resistance marker (e.g. ampicillin or kanamycin resistance) and a T7 promoter. The multiple cloning site has been manipulated such that more than one gene (such as 2, 3, 4, or more) can be expressed from a single vector. In some examples, the genes are expressed as a multicistronic product (for example, a bi-cistronic, tri-cistronic, etc. product), with a single mRNA and multiple polypeptides produced. In other examples, the genes are expressed as multiple monocistronic products, with an individual mRNA and polypeptide produced for each gene. Appropriate vectors can be selected depending on the gene(s) to be expressed and the host cell being transformed.
In some examples, a plasmid is introduced extrachromosomally and replicated within the host microbe. In other examples, after introduction of the plasmid, a double homologous recombination event occurs and the one or more genes are inserted into the genome.
Transformation of a bacterial cell with recombinant DNA can be carried out. Where the host is bacterial, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Bacteria can also be transformed by electroporation, conjugation, or transduction.
B. Carriers and Seed Treatments
Disclosed herein are methods for co-formulating one or more microbes with one or more carriers and compositions including one or more microbes and one or more carriers. The carrier may be liquid or solid (dry). Carriers include liquid or dry fertilizers (such as fertilizers including urea, potash, ammonium phosphate, and/or ammonium nitrate), soil-derived substances (for example, clay, peat, coal, inorganic soil) organic substances (for example, charcoal, sawdust, wheat/soy/oat bran, compost, coco coir), and/or inert materials (for example, perlite, vermiculite, bentonite, Azomite®, kaolin, silicates, pumice, talc). Exemplary carriers include Azomite®, perlite, biochar, dry fertilizers (such as urea, MOP, or MAP), liquid fertilizer (such as UAN), and dust control chemicals (such as those available from ArrMaz, FL, USA). Additional exemplary carriers 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, clay pebbles).
In some embodiments, co-formulations with carriers include the consortium of 22 microbes described in WO 2018/045004 (incorporated herein by reference in its entirety; referred to herein as AMC1) and one or more of the carriers described herein. In other embodiments, co-formulations with carriers include the consortium of 23 microbes disclosed herein (e.g., the microbes listed in Table 26 or ATCC deposit PTA-125924) and one or more carriers. Co-formulations also include one or more (such as 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) microbes and one or more of the carriers described herein. In some examples, a co-formulation includes at least one microbe that includes or expresses one or more DPA synthase and/or Isf gene(s) (or produces DPA) and a carrier. In other examples, a co-formulation includes at least one microbe that includes or expresses DPA synthase gene(s) or produces DPA and at least one microbe that does not include or express DPA synthase gene(s) or does not produce DPA with a carrier. Methods of co-formulating a carrier and one or more microbes include contacting the one or more carriers with the one or more microbes. In some examples, the one or more microbes are in liquid form (e.g., are in a liquid medium) or are in a solid or dry form.
Also disclosed herein are methods for treating seeds with one or more microbes (e.g., co-formulating one or more microbes with one or more seeds) and compositions including one or more microbes and one or more seeds. In such embodiments, the seeds are the “carrier” for the microbes. In some embodiments, seed treatments include the consortium of 22 microbes described in WO 2018/045004 (incorporated herein by reference in its entirety) and one or more seeds. In other embodiments, seed treatments include the consortium of 23 microbes disclosed herein (e.g., the microbes listed in Table 26 or ATCC deposit PTA-125924) and one or more seeds. In other examples, a co-formulation includes at least one microbe that includes or expresses one or more DPA synthase and/or Isf gene(s) (or produces DPA) and a seed. In other examples, a co-formulation includes at least one microbe that includes or expresses DPA synthase gene(s) or produces DPA and at least one microbe that does not include or express DPA synthase gene(s) or does not produce DPA and a seed. Exemplary seeds that can be treated with the one or more microbes include, but are not limited to, corn seeds, sunflower seeds, canola seeds, wheat seeds, cucumber seeds, tomato seeds, rice seeds, and cotton seeds.
In some examples, microbe-treated seeds are prepared by applying microbes directly to seeds (e.g., contacting seed with one or more microbes). In other examples, microbe-treated seeds are prepared by applying the microbes as an overcoat to seeds that have been previously treated with an insecticide and/or fungicide (e.g., contacting insecticide and/or fungicide treated seed with one or more microbes). In yet further examples, microbe-treated seeds are prepared by mixing the microbes with an insecticide and/or fungicide (such as an insecticide/fungicide slurry) and applying the mixture to the seeds (e.g., contacting seed with a mixture of insecticide and/or fungicide and one or more microbes). Exemplary insecticides and fungicides that can be used in combination with the microbes include, but are not limited to, metalaxyl, trifloxystrobin, ipconazole, clothianidin, thiamethoxam, fludioxonil, mefenoxam, azoxystrobin, thiabendazole, pyraclostrobin, imidacloprid, fluxapyroxad, and/or sedexane. In some examples, the one or more microbes applied to the seed are in liquid form (e.g., are in a liquid medium) or are in a solid or dry form. Methods of preparing treated seeds include, but are not limited to those described in Seed Treatment: Oregon Pesticide Applicator Training Manual (Paulsrud et al., Urbana, Ill., 2001) and Example 13.
IV. Methods of UseThe disclosed microbial compositions, alone or in co-formulation with one or more liquid or dry carriers, can be used to treat soil, plants, or plant parts (such as roots, stems, foliage, seeds, or seedlings). In other examples, the disclosed microbial compositions can be used in the form of treated seeds.
In some examples, treatment with the disclosed compositions and/or carriers or seeds treated with the disclosed compositions improve plant growth, improve stress tolerance, and/or increase crop yield. In some embodiments the methods include contacting soil, plants (such as plant foliage, stems, roots, seedlings, or other plant parts), or seeds with a microbial composition or co-formulation disclosed herein. In other embodiments, the methods include planting seeds treated with the disclosed compositions. The methods may also include growing the treated plants, plant parts, or seeds and/or cultivating plants, plant parts, or seeds in the treated soil.
In some examples, the amount of the composition(s), alone or as a co-formulation of one or more microbes and carriers or seeds to be applied (for example, per acre or hectare) is calculated and the composition is diluted in water (or in some examples, liquid fertilizer) to an amount sufficient to spray or irrigate the area to be treated (if the composition is a liquid). The composition can be applied at the time of seed planting at a rate of 0.5-2 liters per acre (such as 0.5 L/acre, 1 L/acre, 1.5 L/acre, or 2 L/acre). The composition can also be applied to the soil (e.g., near the plant roots) or plant one or more times during growth, in the same or a different amount. In other examples, the composition can be mixed with diluted herbicides, insecticides, pesticides, or plant growth regulating chemicals. If the composition to be applied is a solid (such as a dry formulation), the solid can be applied directly to the soil, plants, or plant parts or can be suspended or dissolved in water (or other liquid) prior to use.
In some examples, treatment of soil, seeds, plants, or plant parts with a disclosed composition increases plant growth (such as overall plant size, amount of foliage, root number, root diameter, root length, production of tillers, fruit production, pollen production, and/or seed production) by at least about 5% (for example, 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 disclosed methods result in increased crop production by about 10-75% (such as about 20-60% or about 30-50%) compared to untreated crops. Other measures of crop performance include quality of fruit, yield, starch or solids content, sugar content or brix, shelf-life of fruit or harvestable product, production of marketable yield or target size, quality of fruit or product, grass tillering and resistance to foot traffic in turf, pollination and fruit set, bloom, flower number, flower lifespan, bloom quality, rooting and root mass, crop resistance to lodging, abiotic stress tolerance to heat, drought, cold and recovery after stress, adaptability to poor soils, level of photosynthesis and greening, and plant health. To determine efficacy of products, controls include the same agronomic practices without addition of microbes, performed in parallel.
The disclosed methods and compositions and/or co-formulations can be used in connection with any crop (for example, for direct crop treatment or for soil treatment prior to or after planting). Exemplary crops include, but are not limited to alfalfa, almond, banana, barley, broccoli, cabbage, cannabis, canola, carrots, citrus and orchard tree crops, corn, cotton, cucumber, flowers and ornamentals, garlic, grapes, hops, horticultural plants, leek, melon, oil palm, onion, peanuts and legumes, pineapple, poplar, pine and wood-bearing trees, potato, raspberry, rice, sesame, sorghum, soybean, squash, strawberry, sugarcane, sunflower, tomato, turf and forage grasses, watermelon, wheat, and eucalyptus.
EXAMPLESThe following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
Example 1 Materials and MethodsIsolation and Identification of Microbes: All microbes were derived from Agrinos microbial collection (AMC) and previously described in WO 2018/045004, except for four additional microbes described herein. These additional microbes were isolated as described below.
The bacteria Streptomyces pratensis and Streptomyces venezuelae were isolated from bulk soil (N38° 38′ 49.402″, W121° 40″ 5.775″). Briefly, the soil sample was suspended in a sterile phosphate buffered saline-TWEEN® 80 solution before serial dilution and plating onto several types semi-solid media. S. pratensis and S. venezuelae were isolated from Azotobacter medium agar with mannitol (HIMEDIA #M372) plates after incubation for up to 3 days at 30° C. The strains were repeatedly streaked onto semi-solid media MP (see below) until isogenic.
Bacillus firmus was isolated from a sample of HYT® A (Agrinos AS) which had been previously mixed with fertilizer (UAN32) at a ratio of 1:180. After three weeks incubation at room temperature, aliquots of the mixture were plated on several types of semi-solid media and incubated for up to 3 days at 30° C. A B. firmus colony was collected from a Pikovskaya's medium agar plate (HIMEDIA #M520). The strain was repeatedly streaked onto semi-solid media MP (see below) until isogenic.
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 media (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). The strain was repeatedly streaked onto semi-solid media MP (see below) until isogenic.
Taxonomic classification of newly described microbes: For all four newly described strains, whole-genome sequencing of biologically pure isolates was performed as described below. De novo genome assembly was performed with Hierarchical Genome Assembly Process (HGAP, Pacific Biosciences, Menlo Park, Calif. USA).
Taxonomic identifications were primarily made using 16S ribosomal RNA (rRNA) sequences. 16S rRNA sequences were first identified within the de novo genome assembly using RNAmmer (cbs.dtu.dk/services/RNAmmer/). 16S sequences were then classified using pairwise alignment with NCBI BLASTn, the Ribosomal Database Project (RDP) Nave Bayesian Classifier (Wang et al. Appl. Environ. Microbiol. 73:5261-5267, 2007), and Greengenes de novo phylogenetic tree construction and rank mapping (DeSantis et al. Appl. Environ. Microbiol. 72:5069-502, 2006). Species assignments were then made using a consensus of the three methods.
Based on the above, microbial identifications were made as follows:
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- Streptomyces pratensis. Using 16S sequences, a whole genome taxonomic classification was also performed. Protein coding sequences were identified using Prodigal (Hyatt et al. BMC Bioinformatics, 11:119, 2010). This classification utilized a set of 49 conserved Clusters of Orthologous Groups (COG) families (Tatsuov et al. Science 278:631-637, 1997) to find the matching corresponding set of sequences for a specific genome. The sequences from the selected genome were then inserted into the reference alignments, the closest neighbors were extracted and concatenated, and a tree was rendered from them using FastTree2 (an approximate maximum likelihood method; Price et al. PLoS One 5:e9490, 2010). This rigorous 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 obtained whole genome sequence using MUMmer mummer.sourceforge.net/). The alignment revealed broad global agreement and confirmed that the two are very closely related on a genome-wide scale. A consensus 16S sequence is provided as SEQ ID NO: 1.
- Streptomyces venezuelae. The results of all analyses strongly supported the identification of this isolate as S. venezuelae. A consensus 16S sequence is provided as SEQ ID NO: 2.
- Bacillus firmus. The results of all analyses strongly supported the identification of this isolate as B. firmus. A consensus 16S sequence is provided as SEQ ID NO: 3.
- Paenibacillus azoreducens. The results of all analyses strongly supported the identification of this isolate as P. azoreducens. A consensus 16S sequence is provided as SEQ ID NO: 4.
Identification of Microbial Metabolic Activity Potential: All potential microbial metabolic activities were assessed using laboratory assays as described in WO 2018/045004, incorporated herein by reference in its entirety. In order to determine whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism, bioMérieux's API® identification products were used according to the manufacturer's recommendations (bioMérieux, Inc., Durham, N.C. USA). The results of the key metabolic activity profiling for newly identified microbes are shown in Table 1.
Evaluation of di picolinic acid (DPA) production in bacteria: Evaluation of DPA production in bacterial strains of interest was performed with a 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). Briefly, each isogenic strain was grown on agar media (see Table 2) either aerobically or anaerobically. Aerobic strains were grown at 30° C. for up to 3 days, while anaerobic strains were grown at 35° C. in BD GasPak EZ container systems (Becton, Dickinson and Company, Franklin Lakes, N.J. USA) with Pack-Anaero anaerobic gas generating sachets (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) for up to 3 days. For the DPA assay, approximately 5 μL of bacteria was taken from a colony growing on a plate and resuspended in 10 mL of sodium acetate buffer (0.2 M, pH 5). The suspension was autoclaved for 15 min at 121° C., 15 psig and cooled at room temperature for about 30 min. Equal volumes of the autoclaved suspension and a 30 μM Terbium(III) chloride hexahydrate (Sigma-Aldrich, Saint Louis, Mo. USA) solution were subsequently mixed. Fluorescence was then measured (272 nm excitation, 545 nm emission) using a the Cytation 5 Imaging Reader (BioTek, Winooski, Vt. USA).
Culturing 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 formation of distinct colonies was observed. Briefly, aerobic strains were grown at 30° C. for up to 3 days, while anaerobic strains were grown at 35° C. in BD GasPak EZ container systems (Becton, Dickinson and Company, Franklin Lakes, N.J. USA) with Pack-Anaero anaerobic gas generating sachets (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) for up to 3 days.
Culturing of individual bacteria strains used in this study in liquid media: Selected colonies from agar-grown strains were inoculated in appropriate sterilized liquid media (Table 3) and incubated for up to 3 days. Aerobic strains were cultured at 30° C. with shaking (125-175 rpm) while anaerobic strains were cultured under N2 gas in sealed serum bottles or Hungate tubes at 35° C. with no agitation. When needed, microbial consortia were produced by mixing equal volumes of individually grown strains. A typical result illustrating the number of cells per mL per strain (see below) is summarized in Table 4. Microbial content was determined by Droplet Digital PCR (ddPCR) using Supermix For Probes (Bio-Rad Laboratories, Hercules, Calif.), as described in WO 2018/045004.
Production of co-cultivated microbial consortia by fermentation: Both aerobic and/or anaerobic bacteria were cultured in medium containing 2% molasses supplemented with essential elements such as phosphates, sodium, potassium and chlorides (in the form of commercially available 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 non-GMO soybean extract produced enzymatically (0.25% w/v; Ferti-Nitro Plus Plant N; Ferti-Organic, Brownsville, Tex. USA). Sodium chloride concentrations ranged from 0-4% w/v. Strains from Table 4 described above (referred to herein as AMC1) were inoculated into 2 L DASGIP bioreactors (Eppendorf North America Hauppauge, N.Y.) with a 1.5 liter working volume at a final inoculation of OD600 for each strain ranging between 6.67E-05 to 6.67E-04. Ammonium hydroxide and phosphoric acid were used as base and acid solutions respectively to maintain pH between pH 5.5 and 6.9. Temperature was controlled between 28° C. and 35° C. Anaerobic fermentations were continuously sparged with N2 gas to maintain an anaerobic environment while sparged air was used in aerobic fermentations as a source of oxygen for the microbes during the length of fermentation (typically up to 3 days). For some experiments, after fermentation batches containing different strains were pooled to generate one complete bacterial mixture of 22 strains. A typical result illustrating the number of cells per mL per strain (see below) is summarized in Table 5. Microbial content in fermentates was determined by Droplet Digital PCR (ddPCR) using Supermix For Probes (Bio-Rad Laboratories, Hercules, Calif.), as described describes in WO 2018/045004.
Production of freeze-dried microbial consortium: In some experiments, the consortium of microbes produced was freeze-dried prior to experimentation in co-formulation with agro-carriers. Consortia were produced either by pooling an equal volume of individually cultured bacteria or using co-cultured fermentates (described above). Freeze-drying was performed essentially as described in WO 2018/045004. Briefly, freeze-dried microbial formulations were produced by mixing the liquid microbial consortia with mannitol/lyoprotectant solution (OPS Diagnostics Lebanon, N.J., USA) as per manufacturer's recommendation and the microbial suspension was aliquoted into lyophilization vials (OPS Diagnostics, Lebanon, N.J., USA). After 60 minutes at −80° C., the mixtures were placed in the FreeZone 6 freeze dry system (Labconco, Kansas City, Mo.), vacuum was applied, and the water in the samples was allowed to sublimate. Samples were stored at 4° C. until needed. In some experiments, the lyoprotectant solution was prepared by adding the following chemicals to microbial cultures for a final concentration 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, Nestlé S.A, CH).
Co-formulation of microbes with agro-carriers: Liquid microbial consortia (produced from co-culture or individually grown and then pooled) or individual bacteria strains produced as described above, were impregnated onto agro-carriers such as perlite, Azomite® (Azomite, UT, USA), pumice, Monobasic Ammonium phosphate fertilizer (MAP; Mosaic, MN, USA), Muriate of potash fertilizer (MOP; Mosaic, MN, USA), and Biochar (Cool Terra®; Cool Planet Energy system, CO, USA). Depending on the carrier's water retention characteristics, different volumes of microbial consortia were used so as to saturate the carrier from as low as 35 μL for per gram up to 6 mL per gram. The microbe/agro-carrier mixture was then dried overnight at 30° C.-35° C. before storing in air tight containers for further shelf life microbial survivability studies and plant assays.
In the case of co-formulation with liquid agro-chemicals such as urea and ammonium nitrate in water (UAN32; TGI, CA, USA) or fertilizer dust control agents (DUSTROL; ArrMaz FL, USA), liquid microbial consortia, produced as detailed above, were mixed at various ratios (described in examples below) prior to storage and microbial survivability analysis. All work was performed under sanitary conditions to minimize contamination.
In some instances, freeze-dried bacterial consortia were used, in order to minimize the effects of the culture broth on the carrier's chemistry. Details are described in examples as appropriate.
Analysis of Bacteria Genomes for DPA Synthase Production
Microbial genomic DNA extraction: Bacterial cells of different species were grown and harvested from optimized liquid broth and culture conditions. PowerSoil DNA isolation kit (MO BIO Laboratories, Inc, Carlsbad, Calif. USA) was used for small scale genomic DNA extractions. For large scale genomic DNA extractions, the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, Mo. USA) or Qiagen Genomic DNA Buffer Set and Genomic-tip 500/G (Qiagen, Hilden, Germany) were used following the methods recommended by the manufacturer. The resulting genomic DNA was subsequently precipitated with equal volume of isopropanol, washed with 70% ethanol, air-dried, and resuspended in TE buffer.
Whole genome sequencing (WGS): Whole Genome Sequencing of biologically pure isolates was performed using PacBio RSII system (Pacific Biosciences, Menlo Park, Calif. USA) following the manufacturer's recommended method for sequence library preparation and sequencing. An average of 73,000 reads of 24 kb in length on average were generated from the microbial isolates, followed by de novo genome assembly with Hierarchical Genome Assembly Process (HGAP, Pacific Biosciences, Menlo Park, Calif. USA).
Identification of DPA synthase coding sequence in selected bacteria strains: Initial bioinformatic analyses included select members of the class Bacilli; Bacillus (Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus sp., and Bacillus flexus), as well as Lactobacillus (Lactobacillus delbrueckii). First, using previously acquired whole-genome sequences, a bacterial pan-genome analysis was performed using BPGA-Version-1.3 (iicb.res.in/bpga/index.html) to estimate the genomic diversity and to determine the core (conserved), accessory (dispensable), and unique (strain-specific) gene pool. A search was then performed for any dipicolinate synthase subunit A (DpaA) in the accessory gene set. This was followed by a complete search of the strains in Table 1 for DpaA and DpaB using a custom Bash script (Free Software Foundation, 2007).
Briefly, protein coding genes were annotated in two stages. 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 does not describe the putative gene product. These candidate genes were then compared to large databases in a hierarchical manner, starting with a smaller trustworthy database, moving to medium-sized but domain-specific databases, and finally to curated models of protein families. By default, an e-value threshold of 10−6 was used with the following series of databases:
1. All bacterial proteins in UniProt that have real protein or transcript evidence and are not a fragment. BLAST+ is used for the search.
2. All proteins from finished bacterial genomes in RefSeq for a specified genus. BLAST+ is used for the search.
3. A series of hidden Markov model profile databases, including Pfam (Punta et al., 2012) and TIGRFAMs (Haft et al., 2013). This is performed using hmmscan from the HMMER 3.1 package (Eddy, 2011).
4. If no matches can be found, label as ‘hypothetical protein’.
These data were then tabulated along with the capacity for sporulation and dry-formulation survivability (see Table 6) to establish correlations between DPA synthase and viability. Alignments of DpaA and DpaB genes were then performed using Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/) and amino acid composition and frequency statistics calculated using Seaview 4 (Gouy et al. Mol. Biol. Evol. 27:221-224, 2010). DpaA and DpaB amino acid alignments were then used to calculate consensus sequences and probability matrices in R using packages Biostrings and seqinr (Pagès et al., 2016; Charif and Lobry, Structural Approaches to Sequence Evolution, pp. 207-232, 2007; R Core Team, 2016).
Bacteria in Consortia Survivability Assay
Liquid co-formulation sample preparation: For each shelf-life timepoint, 1 mL of the co-formulation (agrochemical/bacterial consortium) was serially diluted from 10−1 to 10−5 in sterile peptone water.
Dry formulation sample preparation: For each shelf-life timepoint, 0.03-1 g of dry formulation (agro-carrier/bacterial consortium) was suspended in up to 3 mL of culture broth such as peptone water or other appropriate medium and incubated up to 1 hr at room temperature. In some instances, gentle sonication (35 khz) was used to release bacteria from dry matrices using a water bath sonicator (VWR ultrasonic cleaner). The suspension was subsequently serially diluted from 10−1 to 10−5 in sterile peptone water or other type of culture broth. In other cases, dry material was added directly to liquid media for growth.
Survivability assay: The bacterial strains which underwent treatment(s) were given the opportunity to multiply. In order 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 instances, each serial dilution produced above was spread plated onto one or more agar media in duplicate. In other cases, dry material produced above was suspended into one or more liquid media in sextuplet. For plate cultures, plates were incubated in static incubators, one set aerobically at 30° C. and the other set anaerobically at 35° C. for 3 days. For liquid cultures, tubes were incubated either shaking aerobically at 30° C. or static anaerobically at 35° C. for 7 days. Bacteria which had survived the treatment(s) grew either by forming colonies or multiplied in liquid cultures, and this growth was then be sampled and identified using droplet digital PCR (ddPCR).
Genomic DNA was extracted from harvested cells using the DNeasy PowerLyzer PowerSoil kit (Qiagen, Inc., Germantown, Md. USA) per the manufacturer's recommendations. DNA was then quantified using the Quantas Fluorometer and the QuantiFluor dsDNA (Promega Corporation, Madison, Wis. USA) and processed for identification and quantification using strain specific probes in conjunction with ddPCR (Dreo et al., Anal. Bioanal. Chem. 406:6513-6528, 2014; Yin, et al., Journal of Microbiological Methods 65:21-31, 2006). Briefly, ddPCR reactions were prepared by combining DNA sample, primers, and probes (designed using unique sequences from the 16S genes and/or unique coding gene sequences identified from WGS genome assemblies as previously described in WO 2018/045004, incorporated herein by reference) with Bio-Rad's ddPCR Supermix for Probes per the manufacturer's recommendations. Droplets were then generated using either the QX200™ droplet generator or the AutoDG™ Instrument per the manufacturer's recommendations. Polymerase chain reaction (PCR) was carried out using the Eppendorf Mastercycler® nexus gradient using the recommended thermal cycling conditions from Bio-Rad's ddPCR Supermix for Probes. Following the PCR protocol, reactions were read using the QX200 droplet reader. Finally, concentrations were analyzed with QuantaSoft™ software.
In the case of single strain survivability identification after co-formulation with liquid or dry agro-carrier, simple plating was performed using agar medium best suited of the given strains, as described in Table 2.
Example 2 Identification of DPA Synthase Coding Sequence in Selected Bacterial StrainsDipicolinate synthase subunit A (DpaA): An amino acid alignment of DpaA from the bacterial strains revealed that it is divergent, with only 28.4% of amino acids conserved. Results are summarized in Tables 7-9 and
Dipicolinate synthase subunit B (DpaB): An amino acid alignment of DpaB from the bacterial strains revealed that it is more conserved that DpaA, with 45.5% of the amino acids conserved. Results are summarized in Tables 10-12 and
Correlation between production of DPA (in assays described above) and presence of DPA genes: As noted in Table 6, Clostridium beijerinckii was detected as a DPA-producing strain via the Terbium-DPA fluorescence assay, but neither DpaA or DpaB were identifiable in its genome. We found that C. beijerinckii possesses an iron-sulfur flavoprotein (Isf) that is structurally related to EtfA, which has been previously implicated in DPA production (Orsburn et al., Mol. Microbiol. 75:178-186, 2010). Six copies of the iron-sulfur flavoprotein were detected in the genome of C. beijerinckii. These sequences were then aligned with isf sequences from four diverse bacteria: Archaeoglobus fulgidus, Methanocaldococcus jannaschii, Methanosarcina thermophila, and Peptoclostridium difficile (
An evaluation of bacterial survivability in Azomite® impregnated with individual strains was performed. Four strains were selected based on their ability to produce DPA in lab assays and/or on the results of DPA gene identification. The impregnated material was stored dry for 5 days before evaluating bacteria survivability. Results are as indicated in Table 13. 100% of strains with detectable production of DPA (2/2) remained viable for 5 days. For strains with undetectable DPA production, 50% (1/2 strains) remained viable for 5 days.
In a second experiment, the number of strains was expanded. An evaluation of bacteria survivability in Azomite® following impregnation was performed over a 1-month period. Briefly, Azomite® was impregnated with individual isogenic strains, dried, and samples were taken on days 3, 7, 14, 21, and 28 for bacteria viability analysis. Results are as indicated in Table 14 and show that 100% (4/4) bacteria with identified DPA genes and producing DPA remained viable for 1 month. For strains with no identified DPA genes and undetectable DPA production, 40% (2/5 strains) remained viable for 1 month.
A second agro-carrier (perlite) was then tested. A microbial survivability evaluation was performed over a 1-month period, as previously was described with Azomite®. The results are as indicated in Table 15 and show that 100% (4/4) of bacteria with identified DPA genes and producing DPA remained viable for 1 month. For strains with no detected DPA genes and undetectable DPA production, 60% (3/5 strains) remained viable for 1 month.
In the following experiment, 22 bacteria were grown individually and subsequently mixed to produce a consortium, as described above. Dry Azomite® in a pelletized form was impregnated with 0.23-0.4 mL of consortium (to preserve the integrity of the pellet), dried and stored for two weeks before analyzing which bacteria survived using the assay for assaying microbial viability from a consortium (Example 1). Two independent trials were conducted, and the results are summarized in Table 16. We observed that 83% and 92% of bacteria strains with identified DPA genes and/or producing DPA remained viable 2 weeks after impregnation on Azomite®. The strain which did not appear to show consistent viability in the two trials was O. oncorhynchi. As mentioned above, O. oncorhynchi DPA synthase subunit A appears more divergent than most other examined strains, with a 58% percent identity with the consensus sequence using BLOSUM62. For strains with no identified DPA genes and no detectable DPA production, the results varied between 60% and 70% of bacteria remaining viable within the same period. Reproducibility between the two trials was low and may be attributed to different metabolic states of this set of microbes at the time of impregnation.
Similar to the experiment with Azomite® described above, the 22 bacteria were grown individually and subsequently mixed to produce a consortium. Perlite was impregnated with 2-6 mL of consortium, dried and stored for 2 weeks before analyzing which bacteria survived using the assay for assaying microbial viability from a consortium, (Example 1). Two independent trials were conducted, and the results are summarized in Table 17. We observed that 58% and 100% of bacteria strains with identified DPA genes and/or producing DPA remain viable two 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 bacteria remaining viable within the same period. Compared with the Azomite® experiment, the bacteria with identified DPA genes and/or producing DPA showed good reproducibility even using a different impregnation substrate. They also outperformed those with no identified DPA genes or with no detectable DPA production in our assay.
In the experiments below, bacterial strains of interest were grown on semi-solid agar plates using media as described above (see Table 2). Dust control chemical/bacteria co-formulations were generated by suspending a 1 μL loopful scraped from an agar plate in 200 μL of sterile peptone water, and then mixing with dust control chemicals at a rate of 5% v/v. Survivability of each strain in co-formulation was then determined by inoculating fresh media (Table 3) with the bacteria/agrochemical mixture and scoring for signs of growth.
Survivability of bacteria co-formulated with MDC-200: An evaluation of bacterial viability following co-formulation with the dust control agent MDC-200 (ArrMaz, FL, USA) was performed over a 1-month period. Results are as indicated in Table 18. We observed that 92% (11/12) of bacteria with identified DPA genes and/or producing DPA remained viable for 1 month. For strains with no identified DPA genes and undetectable DPA production, 0% (0/10 strains) remained viable for 1 month.
Survivability of bacteria co-formulated with DUSTROL® 3275: An evaluation of bacterial viability following co-formulation with DUSTROL® 3275 (ArrMaz, FL, USA) was performed over a 1-month period. Results are as indicated in Table 18. We observed that 100% (12/12) of bacteria with identified DPA genes and/or producing DPA remained viable for 1 month. For strains with no identified DPA genes and undetectable DPA production, 0% (0/10 strains) remained viable for 1 month.
Survivability of bacteria co-formulated with DUSTROL® 3133: An evaluation of bacterial viability following co-formulation with DUSTROL® 3133 (ArrMaz, FL, USA) was performed over a 1-month period. Results are as indicated in Table 18. We observed that 100% (12/12) of bacteria with identified DPA genes and/or producing DPA remained viable for 1 month. For strains with no identified DPA genes and undetectable DPA production, 0% (0/10 strains) remained viable for 1 month.
Survivability of bacteria co-formulated with DUSTROL® 3139: An evaluation of bacterial viability following co-formulation with DUSTROL® 3139 (ArrMaz, FL, USA) was performed over a 1-month period. Results are as indicated in Table 18. We observed that 92% (11/12) of bacteria with identified DPA genes and/or producing DPA remained viable for 1 month. For strains with no identified DPA genes and undetectable DPA production, 0% (0/10 strains) remained viable for 1 month.
Survivability of bacteria co-formulated with DUSTROL® 3001: An evaluation of bacterial viability following co-formulation with DUSTROL® 3001 (ArrMaz, FL, USA) was performed over a 1-month period. Results are as indicated in Table 18. We observed that 33% (4/12) of bacteria with identified DPA genes and/or producing DPA remained viable for 1 month. Strains with no identified DPA genes and undetectable DPA production, 0% (0/10 strains) remained viable for 1 month. The reduced survival in this experiment may be due to incompatibility of microbes (whether or not they produce DPA) with one or more components of DUSTROL® 3001.
Survivability of bacteria co-formulated with DUSTROL® 3010: An evaluation of bacterial viability following co-formulation with DUSTROL® 3010 (ArrMaz, FL, USA) was performed over a 1-month period. Results are as indicated in Table 18. We observed that 75% (9/12) of bacteria with identified DPA genes and/or producing DPA remained viable for 1 month. For strains with no identified DPA genes and undetectable DPA production, 20% (2/10 strains) remained viable for 1 month.
An evaluation of bacterial viability following co-formulation with liquid UAN fertilizer was performed at various timepoints over a 1-month period. Briefly, each isogenic strain was grown in liquid medium (as described above) either aerobically or anaerobically. Cultures were then mixed in equal ratios, and the mixture was then combined with UAN32 at a ratio of 1:180 (microbes:UAN). Microbial survivability was assessed on days 0, 7, 14, and 28 and is summarized in Table 19. For the Day 0 timepoint (˜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 for the Day 0 timepoint. By the Day 7 timepoint, 67% (8/12) of bacteria strains with identified DPA genes and/or producing DPA remained viable remained viable. Following Day 7, there was no change in viability for this set of strains. For bacteria with no detectable DPA production, only 10% (1/10 strains) remained viable for the Day 7 timepoint.
In this experiment, the 22 bacteria shown in Table 4 were grown individually and subsequently mixed to produce a consortium, as described above. One gram of Biochar (Cool Terra®; Cool Planet Energy system, CO, USA) was impregnated with ˜0.5 mL of consortium, dried, and stored for 7 days before analyzing which bacteria survived using the survivability assay previously described. The results are summarized in Table 20. 66.7% (7/12 strains) of the bacteria with identified DPA genes and/or producing DPA remained viable 7 days after 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.
In this experiment, the 22 bacteria shown in Table 4 were grown individually and subsequently mixed to produce a consortium and impregnated on MOP (0-0-60) dry fertilizer at a rate of 35 μL/g, as described above. The fertilizer was then stored at room temperature and periodically sampled to assess microbial survivability as previously described. The results are summarized in Table 21. 66.7% (8/12 strains) of the bacteria with identified DPA genes and/or producing DPA remained viable 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.
The 22 bacteria shown in Table 4 were also grown individually and subsequently mixed to produce a consortium and impregnated on MAP (11-52-0) at a rate of 35 μL/g, as described above. The fertilizer was then stored at room temperature and periodically sampled to assess microbial survivability as previously described. The results are summarized in Table 22. In this case, 58.3% (7/12 strains) of the bacteria with identified DPA genes and/or producing DPA remained viable 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.
In this experiment, the 22 bacteria shown in Table 4 were grown individually and subsequently mixed to produce a consortium, as described above. The consortium was then stored at room temperature and periodically sampled to assess microbial survivability, as previously described. The results are summarized in Table 23.
On week 2, 83% (10/12 strains) of the bacteria with identified DPA genes and/or producing DPA remained viable. For strains with no identified DPA genes and no detectable DPA production, 60% (6/10 strains) remained viable within the same time frame. After 7.5 weeks, that 58% (7/12 strains) of the bacteria with identified DPA genes and/or producing DPA remained viable. For strains with no identified DPA genes and no detectable DPA production, 50% (5/10 strains) remained viable within the period.
In all instances tested, spore forming bacteria with identified DPA genes and/or producing DPA outperformed strains with no identified DPA genes and no detectable DPA production in terms of survivability in co-formulation with agro-carriers over time, as either liquid or dry formulations (Table 24 and
Cucumber seeds purchased from The Seed Kingdom (Lubbock, Tex.) were pre-germinated for 4 days at 22-24° C. in rolled germination paper (Anchor Paper, Saint Paul, Minn.) impregnated with a dilute mixture of liquid fertilizer (25 ppm NPK in water). Potting medium (Sunshine Mix) was pre-treated with a Hoagland solution (Hoagland, Calif. Agric. Exp. Stn. Bull. 347:36-39, 1938), 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 rate of 1 L per pound of potting medium was used. To each pot, 1 mL of a 10% w/v urea solution was added before pre-germinated cucumber seedlings with similar length were transplanted. For each treatment (including control) 17-18 plants were randomized in flats in defined growth conditions, controlling for temperature (16-24° C.) and 12 hours photoperiod. The control pots contained 2 g of untreated perlite. The experimental pots were treated with perlite impregnated with 2 mL of the 22 bacteria shown in Table 4. The impregnated perlite had been stored dry for two weeks prior to use in this assay. The flats were watered 3 times a week with modified Hoagland solution. After 28 to 32 days, shoots were dried, and weights were recorded for each plant. The data were analyzed by One-way ANOVA (Analysis Of Variance) and with post-hoc Tukey test to compare samples within the experiment. Shoot dry weight was increased in plants treated with the microbe-impregnated perlite, though it did not reach statistical significance (
To identify suitable strains for use in designing an additional DPA producing consortium, an in-house microbial collection was screened. Strains derived from genera that are known to sporulate were revived from bacterial glycerol stocks on appropriate media and allowed to grow for up to three days. Single colonies were then selected and passaged at least three times to verify purity. Strains were tested for DPA production using the 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 Illumina NovaSeq 6000 sequencing system (Illumina, Inc., San Diego, Calif. USA) following the manufacturer's recommended method for sequence library preparation and sequencing. An average of 5,133,928 reads of 87 bp in length on average 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). Assembled contigs were then annotated with PROKKA (Seemann, Bioinformatics 30(14):2068-2069, 2014). 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 DpaA and DpaB genes and lacked Isf genes. All strains that produced DPA and lacked DpaA and DpaB possessed one or more copies of Isf genes. In three cases strains possessed all three (DpaA, DpaB, and Isf) genes. Therefore, detection of DPA production via the terbium-DPA fluorescence assay was perfectly correlated with the presence of DpaA, DpaB, and/or Isf genes.
For strains that tested positive for DPA production, potential microbial metabolic activities (such as but not limited to nitrogen metabolism, sulfur metabolism, salt tolerance, mineral salt solubilization, cellulose degradation, chitin degradation, phytohormone production, iron metabolism, dephosphorylation of organic matter) were assessed using laboratory assays as described in WO 2018/045004 and Example 1. Strains possessing the greatest number of metabolic activities were selected for co-fermentation in microbial consortia along with 5 strains that lack DPA production which were selected for their metabolic activities (see Example 1). Those strains that proved to be amenable to co-fermentation in consortium (see Table 26), also referred to herein as Dry Formulation Consortium (DFC), were selected for co-formulation with a carrier such as bentonite, perlite and/or urea.
All consortia included in this example were fermented as follows. Both aerobic and anaerobic bacteria strains were cultured on medium 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), non-GMO soybean extract produced enzymatically (0.1-0.5% w/v; Ferti-Nitro Plus Plant N; Ferti-Organic, Brownsville, Tex. USA), spirulina (0.1-0.5% w/v), and/or peptone (0.1-0.5% w/v). When needed, additional vitamins and micronutrients were provided by kelp extract (0.1-0.5% w/v), purified B-vitamins (Sigma), and/or Wolfe's trace metal solution. When needed, additional salts were added as phosphate buffered saline solution and/or sodium chloride addition (0-4% w/v). Strains from AMC1 (described above) were inoculated into 2 L DASGIP bioreactors (Eppendorf North America Hauppauge, N.Y.) with a 1.5 liter working volume. The pH during fermentation was maintained between 5.0 and 7.0. Aeration conditions during fermentation were controlled by varying agitation, gas composition (air and/or nitrogen gas) and gas flow rates to obtain target oxygen transfer rate (estimated by using kLa) and ranged from having a kLa (per hour) of 0 to 110. Temperature was controlled between 28° C. and 35° C.
To produce co-formulations, DFC fermentate was applied to bentonite or perlite, and then dried for approximately 24 hours. In some cases, fermentate was concentrated via centrifugation prior to application. Co-formulations were then tested for viability. In addition, certain co-formulations were tested in the plant growth room trials to verify plant beneficial characteristics.
Viability of DFC and AMC1 with carriers: In preparation for field trials that included DFC liquid, AMC1 liquid, DFC-impregnated perlite, AMC1-impregnated perlite, DFC-impregnated bentonite, and AMC1-impregnated bentonite, DFC and AMC1 were each co-fermented and perlite or bentonite carriers were impregnated with 2 mL/g or 1 mL/g respectively. The impregnated perlite and bentonite were stored dry for one to three weeks, at which point the viability assay was performed to determine the number of strains that were viable at the time of application. In addition, liquid was stored for one week, at which point the viability assay was performed to determine the number of strains that were viable at the time of application. In liquid, 16 out of 23 DFC strains were viable after one week versus 14 out of 22 AMC1 strains (Table 27).
On impregnated perlite, 17 out of 23 DFC strains versus 14 out of 22 AMC1 strains were viable after up to three weeks (Table 28).
On impregnated bentonite, 17 out of 23 DFC strains versus 14 out of 22 AMC1 strains were viable after up to three weeks (Table 29). This illustrates how DPA-producing strains maintain improved viability when stored in liquid, as well as when dried on a carrier such as perlite or bentonite.
Following 3 months of aging, DFC-impregnated perlite showed 15 strains as viable, whereas AMC1-impregnated perlite showed only 11 strains as viable and only one of which was not DPA producing (Tables 30 and 31).
Following the same 3 months of aging, DFC-impregnated bentonite showed 19 strains as viable, whereas AMC1-impregnated perlite showed on 13 strains as viable and only three of which were not DPA producing (Tables 30 and 31).
Evaluation of DFC Plant Beneficial Activity: Cucumber seeds purchased from The Seed Kingdom (Lubbock, Tex.) were pre-germinated for 4 days at 22° C. in rolled germination paper (Anchor Paper, Saint Paul, Minn.) impregnated with a dilute mixture of liquid fertilizer (25 ppm NPK in water). At the time of seed preparation, the potting medium (Sunshine Mix) was prepared with a pre-treatment of 67.60 kg Tricalcium phosphate (TCP) ha−1 and a modified Hoagland solution (Hoagland, Calif. Agric. Exp. Stn. Bull. 347:36-39, 1938). This 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, which was applied a rate of 1 L per pound of potting medium. The 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 however those carriers were impregnated with 2 mL/g of DFC or 1 mL/g of DFC, respectively. The impregnated perlite and bentonite had been stored dry for two weeks prior to use in this assay, at which point the viability assay was performed to determine the number of strains from DFC that were viable at the time of application. The large majority of strains (15-18 out of 23 strains viable) survived the co-formulation and aging process.
Following the pre-germination of the cucumber seeds, similar length seedling were selected and one was transplanted into each pot. For each treatment (including control) 18 plants were randomized in flats in defined growth conditions, controlling for temperature (16-24° C.) and 12 hours photoperiod. The flats were watered for the first time three days after transplanting with a modified Hoagland solution, PK+ which contains 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 3 times a week with NK+ Hoagland solution. After 32 days, shoots were harvested, and dried weights were recorded for each plant. The data were analyzed by One-way ANOVA (Analysis Of Variance) and with a post-hoc Tukey test to compare samples within the experiment. These trials were performed twice on two separate occasions.
The results of the initial trial showed a significant increase in shoot weight for both DFC impregnated perlite and DFC impregnated bentonite when compared to controls (
Microbe seed treatments were applied to corn and soybean seed using a batch treater SGS (SGS North America, Brookings, S. Dak. USA). One kg of seed was treated for each treatment. In separate seed treatments, microbes were either applied directly to untreated seed, applied as an overcoat to seed previously treated with an insecticide/fungicide package, or mixed with the insecticide/fungicide slurry prior to seed application. For corn, insecticide/fungicide treatment consisted of either an Acceleron mix containing metalaxyl, trifloxystrobin, ipconazole, and clothianidin, or a CruiserMaxx mix containing Cruiser (thiamethoxam), fludioxonil, mefenoxam, azoxystrobin, and thiabendazole. For soybean, insecticide/fungicide treatment consisted of either an Acceleron mix containing metalaxyl, pyraclostrobin, imidacloprid, and fluxapyroxad, or a CrusierMaxx mix containing Cruiser (thiamethoxam), mefenoxam, fludioxonil, and sedexane.
Following seed treatment, viability was tested (as in Example 1) within 48 hours and three weeks thereafter. The overwhelming majority of strains (19-20 out of 23 strains viable) survived the initial co-formulation process. After three weeks, only a slight reduction in viability was observed (17-18 out of 23 strains viable;
In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
Claims
1. A composition comprising cells of microbial species including or consisting of each of 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.
2. The composition of claim 1, comprising cells of microbial species including or consisting of microbes with 16S rDNA nucleic acid sequences having at least 99% sequence identity to each of SEQ ID NOs: 3-25.
3. The composition of claim 2, comprising cells of microbial species including or consisting of microbes with 16S rDNA nucleic acid sequences of each of SEQ ID NOs: 3-25.
4. A composition comprising American Type Culture Collection deposit number PTA-125924.
5. The composition of claim 1, further comprising one or more of chitin, chitosan, glucosamine, amino acids, and liquid fertilizer.
6. A method comprising contacting soil, plants, plant parts, or seeds with the composition of claim 1.
7. The method of claim 6, further comprising contacting the soil, plants, plant parts, or seeds with:
- one or more of chitin, chitosan, glucosamine, and amino acids;
- a liquid fertilizer; and/or
- 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 elicitors, or combinations of two or more thereof.
8-9. (canceled)
10. The method of claim 6, further comprising activating the microbial species in the composition prior to contacting the soil, plants, plant parts, or seeds with the composition.
11. A composition comprising the composition of claim 1 and a carrier or a seed.
12. The composition of claim 11, wherein the carrier comprises urea, potash, ammonium phosphate, ammonium nitrate, clay, peat, coal, inorganic soil, charcoal, sawdust, wheat/soy/oat brain, compost, coco coir, perlite, vermiculite, bentonite, Azomite®, kaolin, silicates, pumice, talc, a liquid fertilizer or a liquid dust control chemical.
13. The composition of claim 11, wherein the seed comprises corn seed, sunflower seed, canola seed, wheat seed, cucumber seed, tomato seed, rice seed, and/or cotton seed.
14. The composition of claim 11, further comprising one or more insecticide and/or fungicide.
15. A method for co-formulating a microbe with a carrier or seed, comprising:
- identifying a microbe that comprises one or more dipicolinic acid (DPA) synthase genes, a microbe that expresses one or more DPA synthase proteins, and/or a microbe that produces DPA;
- selecting the microbe for co-formulation with a carrier or seed; and
- co-formulating the selected microbe with the carrier or the seed.
16. (canceled)
17. The method of claim 15, wherein the one or more DPA synthase genes comprises a DPA synthase subunit A (DpaA) gene and/or or a DPA synthase subunit B (DpaB) gene.
18. The method of claim 17, wherein:
- the DpaA gene encodes a DpaA protein with at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 26-41 or wherein the DpaB gene encodes a DpaB protein with at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 42-56;
- the DpaA gene encodes a DpaA protein with at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 26-41 or wherein the DpaB gene encodes a DpaB protein with at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 42-56; or
- the DpaA gene encodes a DpaA protein comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 26-41 or wherein the DpaB gene encodes a DpaB protein comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 42-56.
19-24. (canceled)
25. The method of claim 15, wherein identifying a microbe that comprises one or more dipicolinic acid (DPA) synthase genes comprises detecting nucleic acids or nucleic acid sequences that encode the one or more DPA synthase genes.
26-27. (canceled)
28. The method of claim 15, wherein identifying a microbe that expresses the one or more DPA synthase proteins comprises an immunoassay or mass spectrometry.
29. (canceled)
30. The method of claim 15, wherein identifying a microbe that produces DPA comprises detecting DPA in the microbe or in medium containing the microbe.
31-32. (canceled)
33. The method of claim 15, wherein identifying a microbe that produces DPA comprises identifying a microbe that comprises one or more EtfA or Isf gene and/or expresses one or more EtfA or Isf proteins.
34. The method of claim 33, wherein the Isf protein:
- comprises at least 20% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 57-66;
- comprises at least 60% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 57-66; or
- comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 57-66.
35-36. (canceled)
37. The method of claim 15, wherein the selected microbe has increased viability when co-formulated with a carrier or seed compared to a microbe that does not comprise one or more dipicolinic acid (DPA) synthase genes, does not express one or more DPA synthase proteins, and/or does not produce DPA.
38. The method of claim 15, wherein co-formulating the selected microbe with the carrier or seed comprises contacting one or more selected microbes with the carrier or seed.
39. The method of claim 38, wherein the one or more selected microbes are in a liquid medium or are in a solid or dry form.
40. (canceled)
41. The method of claim 38, further comprising contacting the carrier or seed with one or more microbes that do not comprise one or more DPA synthase genes, do not express one or more DPA proteins, and/or do not produce DPA.
42. The method of claim 15, wherein the carrier comprises a dry or solid carrier or a liquid carrier.
43. The method of claim 42, wherein the dry or solid carrier comprises a dry fertilizer, a soil-derived substance, an organic substance, an inert material, or a mixture of two or more thereof or wherein the liquid carrier comprises a liquid fertilizer or a liquid dust control chemical.
44-47. (canceled)
Type: Application
Filed: Jun 5, 2019
Publication Date: Oct 21, 2021
Inventors: Benjamin Gordon (Davis, CA), Frederic Kendirgi (Woodland, CA)
Application Number: 15/734,484
