Methods and compositions for improving plant traits

Abstract

Methods and systems are provided for generating and utilizing a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.

What is claimed is: 
     
         1 . A method of increasing nitrogen fixation in a non-leguminous plant, comprising:
 applying to the plant a plurality of non-intergeneric bacteria, said plurality comprising non-intergeneric bacteria that:   i. 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; and   ii. produce fixed N of at least about 1×10 −17  mmol N per bacterial cell per hour, and   wherein the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant, and   wherein each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.   
     
     
         2 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise bacteria that: 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. 
     
     
         3 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise bacteria that: produce fixed N of at least about 1×10 −17  mmol N per bacterial cell per hour. 
     
     
         4 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise bacteria that: 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 and produce fixed N of at least about 1×10 −17  mmol N per bacterial cell per hour. 
     
     
         5 . The method according to  claim 1 , wherein the at least one genetic variation comprises an introduced control sequence operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network. 
     
     
         6 . The method according to  claim 1 , wherein the at least one genetic variation comprises a promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network. 
     
     
         7 . The method according to  claim 1 , wherein the at least one genetic variation comprises an inducible promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network. 
     
     
         8 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene of the nitrogen fixation or assimilation genetic regulatory network. 
     
     
         9 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene in the nif gene cluster. 
     
     
         10 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation. 
     
     
         11 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria applied to the plant do not stimulate an increase in the uptake of exogenous non-atmospheric nitrogen. 
     
     
         12 . The method according to  claim 1 , wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight. 
     
     
         13 . The method according to  claim 1 , wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight, and wherein the nitrogen-containing fertilizer comprises ammonium or an ammonium containing molecule. 
     
     
         14 . The method according to  claim 1 , wherein the exogenous nitrogen is selected from fertilizer comprising one or more of: glutamine, ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing molecules, nitrate-containing molecules, and nitrite-containing molecules. 
     
     
         15 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant. 
     
     
         16 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant. 
     
     
         17 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, fix atmospheric nitrogen in non-nitrogen-limiting conditions. 
     
     
         18 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation. 
     
     
         19 . The method according to  claim 1 , wherein the fixed nitrogen produced by the plurality of non-intergeneric bacteria is measured through dilution of enriched fertilizer by atmospheric N 2  gas in plant tissue. 
     
     
         20 . The method according to  claim 1 , wherein the at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme. 
     
     
         21 . The method according to  claim 1 , wherein the at least one genetic variation is a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD. 
     
     
         22 . The method according to  claim 1 , wherein the at least one genetic variation is selected from: (A) a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion. 
     
     
         23 . The method according to  claim 1 , wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene. 
     
     
         24 . The method according to  claim 1 , wherein the at least one genetic variation is a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain. 
     
     
         25 . The method according to  claim 1 , wherein the at least one genetic variation is a mutated amtB gene that results in the lack of expression of said amtB gene. 
     
     
         26 . The method according to  claim 1 , wherein the at least one genetic variation is selected from: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof. 
     
     
         27 . The method according to  claim 1 , wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain. 
     
     
         28 . The method according to  claim 1 , wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated amtB gene that results in the lack of expression of said amtB gene. 
     
     
         29 . The method according to  claim 1 , wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome. 
     
     
         30 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are formulated into a composition. 
     
     
         31 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are formulated into a composition comprising an agriculturally acceptable carrier. 
     
     
         32 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are applied into furrows in which seeds of said plant are planted. 
     
     
         33 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are formulated into a liquid in-furrow composition comprising bacteria at a concentration of about 1×10 7  to about 1×10 10  cfu per milliliter. 
     
     
         34 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are applied onto a seed of said plant. 
     
     
         35 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are formulated into a seed coating and are applied onto a seed of said plant. 
     
     
         36 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are formulated into a seed coating and are applied onto a seed of said plant, at a concentration of about 1×10 5  to about 1×10 7  cfu per seed. 
     
     
         37 . The method according to  claim 1 , wherein the plant is a cereal crop. 
     
     
         38 . The method according to  claim 1 , wherein the plant is selected from the group consisting of: corn, rice, wheat, barley,  sorghum , millet, oat, rye, and triticale. 
     
     
         39 . The method according to  claim 1 , wherein the plant is corn. 
     
     
         40 . The method according to  claim 1 , wherein the plant is an agricultural crop plant. 
     
     
         41 . The method according to  claim 1 , wherein the plant is a genetically modified organism. 
     
     
         42 . The method according to  claim 1 , wherein the plant is not a genetically modified organism. 
     
     
         43 . The method according to  claim 1 , wherein the plant has been genetically engineered or bred for efficient nitrogen use. 
     
     
         44 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise at least two different species of bacteria. 
     
     
         45 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise at least two different strains of the same species of bacteria. 
     
     
         46 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise bacteria selected from:  Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter  sp.,  Azospirillum lipoferum, Kosakonia sacchari , and combinations thereof. 
     
     
         47 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria are endophytic, epiphytic, or rhizospheric. 
     
     
         48 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: a bacteria deposited as PTA-122293, a bacteria deposited as PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria deposited as NCMA 201708004, a bacteria deposited as NCMA 201708003, a bacteria deposited as NCMA 201708002, a bacteria deposited as NCMA 201712001, a bacteria deposited as NCMA 201712002, and combinations thereof. 
     
     
         49 . The method according to  claim 1 , wherein the bacterium produces fixed N of at least about 1×10 −17  mmol N per bacterial cell per hour when in the presence of exogenous nitrogen. 
     
     
         50 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant. 
     
     
         51 . The method accordingly to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant. 
     
     
         52 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant. 
     
     
         53 . The method according to  claim 1 , wherein the plurality of non-intergeneric bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant. 
     
     
         54 . The method according to  claim 1 , wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10 −7  mmol N per gram of fresh weight of plant root tissue per hour. 
     
     
         55 . The method according to  claim 1 , wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10 −6  mmol N per gram of fresh weight of plant root tissue per hour.