Agricultural compositions comprising remodeled nitrogen fixing microbes
Inventors
REISINGER, MARK • SANDERS, ERNEST • BROGLIE, RICHARD • Temme, Karsten
Assignees
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Publication Number
US-12268212-B2
Publication Date
2025-04-08
Expiration Date
Abstract
The present disclosure provides non-intergeneric remodeled microbes that are able to fix atmospheric nitrogen and deliver such to plants in a targeted, efficient, and environmentally sustainable manner. The utilization of the taught microbial products will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived nitrogen fertilizer. The remodeled microbes taught herein are able to be combined with leading agricultural chemistry and elite germplasm. Furthermore, the disclosure provides seed treatments comprising remodeled microbes.
Core Innovation
The document describes a composition comprising a plurality of non-intergeneric remodeled bacteria and at least one herbicide, where the non-intergeneric remodeled bacteria produce fixed N and have an average colonization ability per unit of plant root tissue. The disclosed arrangement targets nitrogen fixation regulation so that nitrogen fixation activity is maintained in fertilized fields while fixed N contribution and plant nitrogen production are provided by colonizing bacteria.
The non-intergeneric remodeled bacteria are remodeled to alter nitrogen fixation and/or nitrogen assimilation regulation through changes in a nitrogen fixation or assimilation genetic regulatory network. The description identifies nitrogen regulation pathway targets including nifA, nifL, glnE, glnD, gln, ntrB, ntrC, draT, amtB, and linked activities, and also includes heterologous promoter operably linked to genes from the network, promoter/allele modifications, markerless deletions, and genetic changes in genes or non-coding polynucleotides.
The herbicide component is limited to specified chemical families and, in specific embodiments, to selected named herbicide actives, including glufosinate. The document further frames the composition in seed treatment, agricultural composition, and plant-protectant composition contexts, and describes microbial identification and performance measurement using junction-based qPCR detection, in planta transcriptomics, acetylene reduction assay, isotope tracing approaches, and nitrogen delivery quantification per acre-season.
Claims Coverage
The provided claim content centers on a herbicide-containing composition with a plurality of non-intergeneric remodeled bacteria meeting fixed-N production and plant-root colonization thresholds. The inventive features combine functional performance thresholds with herbicide family or active selection, and further refinement of nitrogen fixation or assimilation genetic regulatory network features.
Non-intergeneric remodeled bacteria with fixed N production and root colonization
A composition comprising a plurality of non-intergeneric remodeled bacteria, wherein the non-intergeneric remodeled bacteria produce fixed N of at least about 1×10^-17 mmol N per bacterial cell per hour, and wherein the non-intergeneric remodeled bacteria have an average colonization ability per unit of plant root tissue of at least about 1.0×10^4 bacterial cells per gram of fresh weight of plant root tissue.
Herbicide selection limited to specified chemical families
The at least one herbicide belongs to a chemical family selected from the group consisting of cyclohexanediones, aryloxyphenoxypropionates, phenylpyrazolins, imidazolinones, sulfonylureas, triazopyrimidine, sulfonylaminocarbonyltriazolinones, dinitroanilines, benzamide, arylpicolinate, phenoxyacetic acids, benzoic acids, pyridines, triazines, triazinone, phenyl-carbamates, uracils, benzothiadiazoles, nitriles, phenylureas, thiocarbamates, organophosphorus, isoxazolidinone, diphenylether, N-phenylphthalimide, aryl triazolinone, pyrazoles, pyrimidinedione, acetamides, benzofuranes, semicarbazone, bipyridiliums, isoxazole, pyrazolone, and triketone.
Specific herbicide active selection
The at least one herbicide is selected from the group consisting of 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bicyclopyrone, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam-methyl, cycloate, dimethyl tetrachloroterephthalate (DCPA), desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron disodium methylarsonate (DSMA), endothall, S-ethyl dipropyl carbamothioate (EPTC), ethalfluralin, ethofumesate, fenoxaprop, fluazifop, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaflutole, lactofen, linuron, 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-(4-chloro-2-methylphenoxy) butanoic acid (MCPB), mesotrione, metribuzin, indaziflam, metsulfuron, molinate, monosodium methanearsonate (MSMA), napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithioac, quinclorac, quizalofop, rimsulfuron, S-metolachlor, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, tembotrione, terbacil, thiazopyr, thifensulfuron, thiobencarb, topramezone, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, and triflusulfuron.
Heterologous promoter operably linked to nitrogen fixation or assimilation regulatory genes
At least one of the plurality of non-intergeneric remodeled bacteria contains a heterologous promoter operably linked to genes from a nitrogen fixation or assimilation genetic regulatory network.
Genetic modifications in nitrogen fixation or assimilation regulatory network genes or non-coding polynucleotides
The plurality of non-intergeneric remodeled bacteria include genetic changes in genes or non-coding polynucleotides of a nitrogen fixation or assimilation genetic regulatory network that shift expression/activity of specific nitrogen regulation and enzyme activities.
Genetic modification of nif genes and glutamine-related genes
The composition includes at least one genetic modification to the nifL, glnE, or glnD gene in at least one of the plurality of non-intergeneric remodeled bacteria.
The inventive concept is a herbicide-containing composition in which non-intergeneric remodeled bacteria meet defined fixed N production and plant-root colonization thresholds. The claim themes also include nitrogen fixation or assimilation genetic regulatory network control, including heterologous promoters and genetic modifications involving nifL, glnE, or glnD, together with herbicide restriction to specified families or named actives.
Stated Advantages
Improved colonization and plant nitrogen contribution/yield under fertilized field conditions.
Nitrogenase derepression.
Increased ammonia excretion.
Quantification of N delivered per acre-season is supported by the document’s datasets and analyses.
Documented Applications
Use in association with fertilized field conditions with reported improvements in colonization and plant nitrogen contribution/yield.
Measurement/validation of non-intergeneric remodeled bacteria via assays and datasets including colonization assays, junction-based qPCR detection, in planta transcriptomics, and nitrogen delivery quantification per acre-season.
Evaluation of fixed N delivery using nitrogenase derepression and reported nitrogen-related performance outputs, including plant nitrogen contribution and yield.
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