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(12) Patent Application Publication (10) Pub. No.: US 2017/0164619 A1 Von Maltzahn Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2017/0164619 A1 Von Maltzahn Et Al US 201701 64619A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0164619 A1 VOn MaltZahn et al. (43) Pub. Date: Jun. 15, 2017 (54) METHODS OF USE OF SEED-ORIGIN continuation of application No. 14/316,469, filed on ENDOPHYTE POPULATIONS Jun. 26, 2014, now Pat. No. 9,113,636. (60) Provisional application No. 61/935,761, filed on Feb. (71) Applicant: Indigo Ag., Inc., Boston, MA (US) 4, 2014, provisional application No. 61/959,854, filed (72) Inventors: Geoffrey von Maltzahn, Boston, MA on Sep. 4, 2013, provisional application No. 61/959, (US); Richard Bailey Flavell, 870, filed on Sep. 4, 2013, provisional application No. Thousand Oaks, CA (US); Gerardo V. 61/959,859, filed on Sep. 4, 2013, provisional appli Toledo, Belmont, MA (US); Slavica cation No. 61/959,861, filed on Sep. 4, 2013, provi Djonovic, Malden, MA (US); Luis sional application No. 61/959,847, filed on Sep. 4, Miguel Marquez, Belmont, MA (US); 2013, provisional application No. 61/959,858, filed David Morris Johnston, Cambridge, on Sep. 4, 2013, provisional application No. 61/957, MA (US); Yves Alain Millet, 255, filed on Jun. 26, 2013. Newtonville, MA (US); Jeffrey Lyford, Hollis, NH (US); Alexander Naydich, Publication Classification Cambridge, MA (US); Craig Sadowski, (51) Int. Cl. Somerville, MA (US) AOIN 63/00 (2006.01) (52) U.S. Cl. (21) Appl. No.: 15/436,592 CPC .................................... A0IN 63/00 (2013.01) (57) ABSTRACT (22) Filed: Feb. 17, 2017 This application relates to methods and materials for pro viding a benefit to a seed or seedling of an agricultural plant Related U.S. Application Data (e.g., an agricultural grass plant), or the agricultural plant (63) Continuation of application No. 15/145,687, filed on derived from the seed or seedling. For example, this appli May 3, 2016, now Pat. No. 9,622,485, which is a cation provides purified bacterial populations that include continuation of application No. 15/017.531, filed on novel seed-origin bacterial endophytes, and synthetic com Feb. 5, 2016, now Pat. No. 9,532,573, which is a binations of seeds and/or seedlings (e.g., cereal seeds and/or continuation of application No. 14/704,891, filed on seedlings) with heterologous seed-derived bacterial endo May 5, 2015, now Pat. No. 9,288,995, which is a phytes. Patent Application Publication Jun. 15, 2017 Sheet 1 of 5 US 2017/O164619 A1 FG. A FG. B 8 taggex. $88: 88 ags: 8888 8s 88sis 8x8 text 3:3: 8 ixs 888 8x8ts: 888: sex:8: 8888: is: 8s 888 Patent Application Publication Jun. 15, 2017 Sheet 2 of 5 US 2017/O164619 A1 FG 2 Sait-Stressed Conditions FIG. 3 8:38 (giiitists -88 Stress Sait Stress s 388-38::::::s Patent Application Publication Jun. 15, 2017 Sheet 3 of 5 US 2017/O164619 A1 FIG. 4 Sit Stress eat Stress FG, 5 Sait-Stressed Conditions Patent Application Publication Jun. 15, 2017 Sheet 4 of 5 US 2017/O164619 A1 F.G. 6 carts: Sy:8: sittic: SYS383 ca: 38:8 388-z 38 S388:33 S385F8 Patent Application Publication Jun. 15, 2017 Sheet 5 of 5 US 2017/O164619 A1 F.G. 8 US 2017/01 64619 A1 Jun. 15, 2017 METHODS OF USE OF SEED-ORIGIN in more arid climates is driving a critical need for innova ENDOPHYTE POPULATIONS tions in crop water use efficiency and temperature tolerance. 0005 Today, crop performance is optimized primarily via CROSS-REFERENCE TO RELATED technologies directed towards the interplay between crop APPLICATIONS genotype (e.g., plant breeding, genetically-modified (GM) 0001. This application is a continuation of U.S. applica crops) and its Surrounding environment (e.g., fertilizer, tion Ser. No. 15/145,687, filed May 3, 2016, which is a synthetic herbicides, pesticides). While these paradigms continuation of U.S. application Ser. No. 15/017.531, filed have assisted in doubling global food production in the past Feb. 5, 2016, now U.S. Pat. No. 9,532,573, issued Jan. 3, fifty years, yield growth rates have stalled in many major 2017, which is a continuation of U.S. application Ser. No. crops, and shifts in the climate have been linked to produc 14/704,891, filed May 5, 2015, now U.S. Pat. No. 9,288,995, tion instability and declines in important crops such as issued Mar. 22, 2016, which is a continuation of U.S. wheat, driving an urgent need for novel Solutions to crop application Ser. No. 14/316,469, filed Jun. 26, 2014, now yield improvement. In addition to their long development U.S. Pat. No. 9,113,636, issued Aug. 25, 2015, which claims and regulatory timelines, public fears of GM-crops and priority to Provisional Application No. 61/957.255, filed synthetic chemicals has challenged their use in many key Jun. 26, 2013: Provisional Application No. 61/959,859, filed crops and countries, resulting in a complete lack of accep Sep. 4, 2013: Provisional Application No. 61/959,847, filed tance for GM traits in wheat and the exclusion of GM crops Sep. 4, 2013: Provisional Application No. 61/959,858, filed and many synthetic chemistries from European markets. Sep. 4, 2013: Provisional Application No. 61/959,854, filed Thus, there is a significant need for innovative, effective, Sep. 4, 2013: Provisional Application No. 61/959,861, filed environmentally-sustainable, and publically-acceptable Sep. 4, 2013: Provisional Application No. 61/959,870, filed approaches to improving the yield and resilience of crops to Sep. 4, 2013; and Provisional Application No. 61/935,761, severe drought and heat stresses. filed Feb. 4, 2014, the disclosures of which are incorporated 0006 Improvement of crop resilience to heat and drought by reference in their entirety. stress has proven challenging for conventional genetic and chemical paradigms for crop improvement. This challenge is SEQUENCE LISTING in part due to the complex, network-level changes that arise 0002 The instant application contains a Sequence Listing during exposure to these stresses. For example, plants under which has been submitted electronically in ASCII format Such stress can Succumb to a variety of physiological and and is hereby incorporated by reference in its entirety. Said developmental damages, including dehydration, elevated ASCII copy, created on Feb. 15, 2017, is named 36337 reactive oxygen species, impairment of photosynthetic car US CRF Sequence Listing..txt, includes 1470 sequences, bon assimilation, inhibition of translocation of assimilates, and is 1,952.080 bytes in size. increased respiration, reduced organ size due to a decrease in the duration of developmental phases, disruption of seed TECHNICAL FIELD development, and a reduction in fertility. 0003. This application relates to methods and materials 0007 Like humans, who utilize a complement of benefi for providing a benefit to a seed or seedling of an agricultural cial microbial symbionts, plants have been purported to plant Such as an agricultural grass plant, particularly a derive a benefit from the vast array of bacteria and fungi that cereal, or an agricultural plant Such as an agricultural grass live both within and around their tissues in order to support plant derived from the seed or seedling. For example, this the plants health and growth. As described in detail herein, application provides purified bacterial populations that endophytes are fungal or bacterial organisms that live within include novel seed-origin bacterial endophytes, and Syn plants. Bacterial endophytes, such as Firmicutes, Actinobac thetic combinations of seeds and/or seedlings with heterolo teria, Proteobacteria, Bacteroidetes, and Verrucomicrobia, gous seed-derived bacterial endophytes. Such seed-origin appear to inhabit various host plant tissues and have been bacterial endophytes can provide beneficial properties to the isolated from plant leaves, stems, and roots. seed, seedling, or the agricultural plant derived from the 0008 To date, a small number of these symbiotic endo seed or seedling, including metabolism, transcription, pro phyte-host relationships have been analyzed in limited stud teome alterations, morphology, and the resilience to a vari ies to provide fitness benefits to model host plants within ety of environmental stresses, and combination of Such controlled laboratory settings, such as enhancement of bio properties. mass production (i.e., yield) and nutrition, increased toler ance to stress such as drought and pests. Yet, such endo BACKGROUND phytes have been demonstrated to be ineffective or of limited 0004 Economically-, environmentally-, and socially efficacy in conferring benefits to a variety of agriculturally Sustainable approaches to agriculture and food production important plants such as modern cereals; as such, they do not are required to meet the needs of a growing global popula adequately address the need to provide improved yield and tion. By 2050 the United Nations Food and Agriculture tolerance to environmental stresses present in many agricul Organization projects that total food production must tural situations for Such crops, particularly drought and heat. increase by 70% to meet the needs of the growing popula 0009. Thus, there is a need for compositions and methods tion, a challenge that is exacerbated by numerous factors, of providing cereal crops with improved yield and resistance including diminishing freshwater resources, increasing com to various environmental stresses. Provided herein are novel petition for arable land, rising energy prices, increasing compositions of symbionts, bacterial and fungal endophytes, input costs, and the likely need for crops to adapt to the as well as novel symbiont-plant compositions, created based pressures of a drier, hotter, and more extreme global climate. on the analysis of the key properties that enhance the utility The need to grow nearly twice as much food with less water and commercialization of an endophytic composition. US 2017/01 64619 A1 Jun.
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