Methods and compositions for improving plant traits
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.
1 . A method for increasing the available nitrogen for a non-leguminous plant in a field, said method comprising applying to said non-leguminous plant in said field a plurality of engineered diazotrophic bacteria, wherein:
said engineered diazotrophic bacteria have a product of (i) colonization ability under field conditions and (ii) fixed N produced per bacterial cell per hour of at least about 2.5×10 −8 mmol N per gram of fresh weight of plant root tissue per hour; and
said engineered diazotrophic bacteria comprises one or more genetic variations, wherein the one or more genetic variations comprise a complete or partial nifL deletion and insertion of a constitutive promoter operably linked to a nifA gene.
2 . The method of claim 1 , wherein said plurality of engineered diazotrophic bacteria, in planta, produce 5% or more of the available nitrogen in said non-leguminous plant.
3 . The method of claim 1 , wherein said engineered diazotrophic bacteria comprise a bacterium from a species selected from the group consisting of Kosakonia sacchari, Rahnella aquatilis, Klebsiella variicola , and Kosakonia pseudosacchari.
4 . The method of claim 3 , wherein said Kosakonia sacchari bacterium is a bacterium deposited under Patent Depository Designation No. 201701002, 201708004, 201708003, or 201708002.
5 . The method of claim 3 , wherein said Rahnella aquatilis bacterium is a bacterium deposited under Accession No. PTA-122293.
6 . The method of claim 3 , wherein said Klebsiella variicola bacterium is a bacterium deposited under Patent Depository Designation No. 201712002.
7 . A non-leguminous plant colonized with a plurality of engineered diazotrophic bacteria, wherein:
said engineered diazotrophic bacteria have a product of (i) colonization ability under field conditions and (ii) N produced per bacterial cell per hour of at least about 2.5×10 −8 mmol N per gram of fresh weight of plant root tissue per hour; and
said engineered diazotrophic bacteria comprise one or more genetic variations, wherein the one or more genetic variations comprise a complete or partial nifL deletion and insertion of a constitutive promoter operably linked to a nifA gene.
8 . The non-leguminous plant of claim 7 , wherein said non-leguminous plant is selected from the group consisting of corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
9 . A composition comprising:
a) a plurality of engineered diazotrophic bacteria, wherein:
said engineered diazotrophic bacteria have a product of (i) colonization ability under field conditions and (ii) N produced per bacterial cell per hour of at least about 2.5×10 −8 mmol N per gram of fresh weight of non-leguminous plant root tissue per hour; and
said engineered diazotrophic bacteria comprise one or more genetic variations, wherein the one or more genetic variations comprise a complete or partial nifL deletion and insertion of a constitutive promoter operably linked to a nifA gene; and
b) a non-leguminous plant seed, wherein said plurality of engineered diazotrophic bacteria can provide at least one pound of nitrogen per acre per season to a non-leguminous plant produced from said plant seed in a field.
10 . The composition of claim 9 , wherein said plurality of engineered diazotrophic bacteria comprises two or more bacterial strains.
11 . The composition of claim 9 , wherein said plurality of engineered diazotrophic bacteria comprises a bacterium from a species selected from the group consisting of Kosakonia sacchari, Rahnella aquatilis, Klebsiella variicola , and Kosakonia pseudosacchari.
12 . The composition of claim 11 , wherein said Kosakonia sacchari bacterium is a bacterium deposited under Patent Depository Designation No. 201701002, 201708004, 201708003, or 201708002.
13 . The composition of claim 11 , wherein said Rahnella aquatilis bacterium is a bacterium deposited under Accession No. PTA-122293.
14 . The composition of claim 11 , wherein said Klebsiella variicola bacterium is a bacterium deposited under Patent Depository Designation No. 201712002.
15 . The method of claim 1 , wherein said plurality of engineered diazotrophic bacteria comprises a bacterium from a genus selected from the group consisting of Kosakonia, Rahnella , and Klebsiella.
16 . The method of claim 1 , wherein said plurality of engineered diazotrophic bacteria comprises two or more bacterial strains.
17 . The method of claim 16 , wherein said two or more bacterial strains are from a genus independently selected from the group consisting of Kosakonia, Rahnella , and Klebsiella.
18 . The method of claim 16 , wherein at least two bacterial strains of said two or more bacterial strains are from a genus independently selected from the group consisting of Kosakonia and Rahnella.
19 . The method of claim 1 , wherein said one or more genetic variations comprise a partial deletion of nifL and further comprise a partial deletion of glnE.
20 . The method of claim 19 , wherein said partial deletion of glnE results in deletion of the nucleotides encoding the adenylyl-removing domain of the GlnE protein.
21 . The non-leguminous plant of claim 7 , wherein said engineered diazotrophic bacteria comprise a bacterium from a species selected from the group consisting of Kosakonia sacchari, Rahnella aquatilis, Klebsiella variicola , and Kosakonia pseudosacchari.
22 . The non-leguminous plant of claim 21 , wherein said Kosakonia sacchari bacterium is a bacterium deposited under Patent Depository Designation No. 201701002, 201708004, 201708003, or 201708002.
23 . The non-leguminous plant of claim 21 , wherein said Rahnella aquatilis bacterium is a bacterium deposited under Accession No. PTA-122293.
24 . The non-leguminous plant of claim 21 , wherein said Klebsiella variicola bacterium is a bacterium deposited under Patent Depository Designation No. 201712002.
25 . The non-leguminous plant of claim 7 , wherein said plurality of engineered diazotrophic bacteria comprises two or more bacterial strains.
26 . The non-leguminous plant of claim 7 , wherein said plurality of engineered diazotrophic bacteria comprises a bacterium from a genus selected from the group consisting of Kosakonia, Rahnella , and Klebsiella.
27 . The non-leguminous plant of claim 26 , wherein said two or more bacterial strains are from a genus independently selected from the group consisting of Kosakonia, Rahnella , and Klebsiella.
28 . The non-leguminous plant of claim 26 , wherein at least two bacterial strains of said two or more bacterial strains are from a genus independently selected from the group consisting of Kosakonia and Rahnella.
29 . The non-leguminous plant of claim 7 , wherein said one or more genetic variations comprise a partial deletion of nifL and further comprise a partial deletion of glnE.
30 . The non-leguminous plant of claim 29 , wherein said partial deletion of glnE results in deletion of the nucleotides encoding the adenylyl-removing domain of the GlnE protein.
31 . The composition of claim 9 , wherein said plurality of engineered diazotrophic bacteria comprises a bacterium from a genus selected from the group consisting of Kosakonia, Rahnella , and Klebsiella.
32 . The composition of claim 10 , wherein said two or more bacterial strains are from a genus independently selected from the group consisting of Kosakonia, Rahnella , and Klebsiella.
33 . The composition of claim 10 , wherein at least two bacterial strains of said two or more bacterial strains are from a genus independently selected from the group consisting of Kosakonia and Rahnella.
34 . The composition of claim 9 , wherein said one or more genetic variations comprise a partial deletion of nifL and further comprise a partial deletion of glnE.
35 . The composition of claim 34 , wherein said partial deletion of glnE results in deletion of the nucleotides encoding the adenylyl-removing domain of the GlnE protein.
36 . The method of claim 1 , wherein said one or more genetic variations further comprise a complete or partial amtB deletion.
37 . The non-leguminous plant of claim 7 , wherein said one or more genetic variations further comprise a complete or partial amtB deletion.
38 . The composition of claim 9 , wherein said one or more genetic variations further comprise a complete or partial amtB deletion.