# US 10,499,652 B2 · Methods of use of seed-origin endophyte populations > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,499,652 B2 - Document ID: US10499652B2 - Status: Granted - Kind: B2 - Application number: 16/024,029 - Filed: 2018-06-29 - Published: 2018-12-27 - Granted: 2019-12-10 - Earliest claimed: 2013-06-26 - Assignees: Indigo N.V. - Applicants: Indigo Ag, Inc. - Inventors: Geoffrey von Maltzahn; Richard Bailey Flavell; Gerardo V. Toledo; Slavica Djonovic; Luis Miguel Marquez; David Morris Johnston; Yves Alain Millet; Jeffrey Lyford; Alexander Naydich; Craig Sadowski - Web page: https://patentgrep.com/patent/US10499652B2 ## CPC - A01N 63/20: Bacteria; Substances produced thereby or obtained therefrom - A01C 1/06: Coating or dressing seed - G01N 33/5097: {involving plant cells (immunoassays of plant cells G01N33/56961)} - A01C 1/00: Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - A01C: PLANTING; SOWING; FERTILISING (parts, details or accessories of agricultural machines or implements, in general A01B51/00 - A01B75/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract This application relates to methods and materials for providing a benefit to a seed or seedling of an agricultural plant (e.g., an agricultural grass plant), or the agricultural plant derived from the seed or seedling. For example, this application provides purified bacterial populations that include novel seed-origin bacterial endophytes, and synthetic combinations of seeds and/or seedlings (e.g., cereal seeds and/or seedlings) with heterologous seed-derived bacterial endophytes. ## Claims ### Claim 1 A method of treating seeds of a cereal agricultural plant, comprising mechanically or manually inoculating a plurality of cereal agricultural plant seeds with an agricultural formulation comprising an agriculturally acceptable carrier and a purified bacterial population that comprises a seed bacterial endophyte of the genus Enterobacter that is heterologous to the seed, and has a 16S nucleic acid sequence that is at least 99% identical to a 16S nucleic acid sequence selected from the group consisting of SEQ ID NO: 541, 595, 540, and 550, and is capable of producing an auxin or acetoin, wherein the endophyte is heterologously disposed to the seed in an amount effective to colonize a plant germinated from the inoculated seed, wherein the plant germinated from the inoculated seed has increased growth as compared to a plant germinated from a reference seed sowed under the same conditions, and wherein the cereal agricultural plant is selected from the group consisting of: sorghum, millet, oat, triticale, rye, and barley. ### Claim 2 (depends on 1) The method of claim 1, wherein the bacterial endophyte is obtainable from an interior seed compartment. ### Claim 3 (depends on 1) The method of claim 1, wherein the bacterial endophyte is obtainable from an exterior surface of a seed. ### Claim 4 (depends on 1) The method of claim 1, wherein the bacterial endophyte is obtainable from a different cultivar, variety or crop as compared to the seed. ### Claim 5 (depends on 1) The method of claim 1, wherein the bacterial endophyte colonizes the roots of a seedling germinated from the inoculated seed. ### Claim 6 (depends on 1) The method of claim 1, wherein the bacterial population includes two or more bacterial endophytes. ### Claim 7 (depends on 1) The method of claim 1, wherein the bacterial endophyte is obtained from rice seed, maize seed, wheat seed, or barley seed. ### Claim 8 (depends on 1) The method of claim 1, wherein the agricultural formulation further comprises a purified fungal population. ### Claim 9 (depends on 1) The method of claim 1, wherein the increased growth occurs under conditions of biotic stress. ### Claim 10 (depends on 9) The method of claim 9, wherein the biotic stress is selected from the group consisting of nematode stress, insect herbivory stress, fungal pathogen stress, bacterial pathogen stress, and viral pathogen stress. ### Claim 11 (depends on 1) The method of claim 1, wherein the plant germinated from the inoculated seed has a larger amount of lateral roots and root-hairs as compared to the plant germinated from the reference seed. ### Claim 12 (depends on 1) The method of claim 1, wherein the plant germinated from the inoculated seed has increased shoot length as compared to the plant germinated from the reference seed. ### Claim 13 (depends on 1) The method of claim 1, wherein the plant germinated from the inoculated seed has increased seedling weight as compared to the plant germinated from the reference seed. ### Claim 14 (depends on 1) The method of claim 1, wherein the plant germinated from the inoculated seed has increased shoot biomass as compared to the plant germinated from the reference seed. ### Claim 15 (depends on 1) The method of claim 1, wherein the plant germinated from the inoculated seed has increased nitrogen use efficiency as compared to the plant germinated from the reference seed. ### Claim 16 (depends on 1) The method of claim 1, wherein the plant germinated from the inoculated seed has increased root biomass as compared to the plant germinated from the reference seed. ### Claim 17 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence selected from the group consisting of SEQ ID NO: 541, 595, 540, and 550. ### Claim 18 (depends on 1) The method of claim 1, wherein the effective amount is at least 103 CFU/seed on the surface of the seed. ### Claim 19 (depends on 1) The method of claim 1, wherein the cereal agricultural plant is oat. ### Claim 20 (depends on 1) The method of claim 1, wherein the cereal agricultural plant is barley. ### Claim 21 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is at least 99% identical to the 16S nucleic acid sequence of SEQ ID NO: 541. ### Claim 22 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is at least 99% identical to the 16S nucleic acid of SEQ ID NO: 595. ### Claim 23 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is at least 99% identical to the 16S nucleic acid sequence of SEQ ID NO: 540. ### Claim 24 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is at least 99% identical to the 16S nucleic acid sequence of SEQ ID NO: 550. ### Claim 25 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 541. ### Claim 26 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid of SEQ ID NO: 595. ### Claim 27 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 540. ### Claim 28 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 550. ### Claim 29 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 541, and wherein the cereal agricultural plant is oat. ### Claim 30 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid of SEQ ID NO: 595, and wherein the cereal agricultural plant is oat. ### Claim 31 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 540, and wherein the cereal agricultural plant is oat. ### Claim 32 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 550, and wherein the cereal agricultural plant is oat. ### Claim 33 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 541, and wherein the cereal agricultural plant is barley. ### Claim 34 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid of SEQ ID NO: 595, and wherein the cereal agricultural plant is barley. ### Claim 35 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 540, and wherein the cereal agricultural plant is barley. ### Claim 36 (depends on 1) The method of claim 1, wherein the seed bacterial endophyte of the genus Enterobacter has a 16S nucleic acid sequence that is 100% identical to the 16S nucleic acid sequence of SEQ ID NO: 550, and wherein the cereal agricultural plant is barley.