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(12) Patent Application Publication (10) Pub. No.: US 2015/0373993 A1 Von Maltzahn Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2015/0373993 A1 Von Maltzahn Et Al US 20150373993A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0373993 A1 VOn MaltZahn et al. (43) Pub. Date: Dec. 31, 2015 (54) ENDOPHYTES, ASSOCIATED (60) Provisional application No. 62/017.796, filed on Jun. COMPOSITIONS, AND METHODS OF USE 26, 2014, provisional application No. 62/017,809, THEREOF filed on Jun. 26, 2014, provisional application No. 62/017.816, filed on Jun. 26, 2014, provisional appli (71) Applicant: Symbiota, LLC, Cambridge, MA (US) cation No. 62/017,813, filed on Jun. 26, 2014, provi (72) Inventors: Geoffrey von Maltzahn, Boston, MA sional application No. 62/017,815, filed on Jun. 26, (US); Richard Bailey Flavell, Thosand 2014, provisional application No. 62/017.818, filed on Oaks, CA (US); Gerardo V. Toledo, Jun. 26, 2014. Belmont, MA (US); Jonathan W. Leff, Cambridge, MA (US); Philip Samayoa, Cambridge, MA (US); Luis Miguel Publication Classification Marquez, Belmont, MA (US); David Morris Johnston, Cambridge, MA (US); (51) Int. Cl. Slavica Djonovic, Malden, MA (US); AOIN 63/04 (2006.01) Yves Alain Millet, Newtonville, MA AOIN 63/00 (2006.01) (US); Craig Sadowski. Somerville, MA C05F II/08 (2006.01) (US); Jeffrey Lyford, Hollis, NH (US); (52) U.S. Cl. Karen V. Ambrose, Cambridge, MA CPC ................ A0IN 63/04 (2013.01); C05F II/08 (US); Xuecheng Zhang, Brookline, MA (2013.01); A0IN 63/00 (2013.01) (US) (21) Appl. No.: 14/752,829 (57) ABSTRACT (22) Filed: Jun. 26, 2015 Materials and methods for improving plant traits and for Related U.S. Application Data providing plant benefits are provided. In some embodiments, (63) Continuation-in-part of application No. PCT/US2014/ the materials, and methods employing the same, can com 044427, filed on Jun. 26, 2014. prise endophytes. Patent Application Publication Dec. 31, 2015 Sheet 1 of 4 US 2015/0373993 A1 F.G. 2 -O.25 OOO 0.25 O.50 MDS1 B) O O 0.2 CN CO 2 0.0 -O2 O Patent Application Publication Dec. 31, 2015 Sheet 2 of 4 US 2015/0373993 A1 F.G. 2 A) OOOO50 o 0.00025-O 2 0.00000 -0.00025: 2 6 Patent Application Publication Dec. 31, 2015 Sheet 3 of 4 US 2015/0373993 A1 FG. 3 Sesii 8::fae risie icies FIG. 4 : t 4 - is 3 - & ; 2 - -- f f aiiace tier Patent Application Publication Dec. 31, 2015 Sheet 4 of 4 US 2015/0373993 A1 F.G. 5 isosis acitage i::::e: 83e33; F.G. 6 8. s fic 38iace 838er US 2015/0373993 A1 Dec. 31, 2015 ENDOPHYTES, ASSOCIATED and synthetic chemicals has challenged their use in many key COMPOSITIONS, AND METHODS OF USE crops and countries, resulting in a complete lack of accep THEREOF tance for GM traits in wheat and the exclusion of GM crops and many synthetic chemistries from European markets. CROSS REFERENCE TO RELATED Thus, there is a significant need for innovative, effective, and APPLICATIONS publically-acceptable approaches to improving the intrinsic 0001. This application claims the benefit of U.S. Provi yield and resilience of crops to severe stresses. sional Application No. 62/017.796, filed Jun. 26, 2014, and 0006. Like humans, which benefit from a complement of U.S. Provisional Application No. 62/017,809, filed Jun. 26, beneficial microbial symbionts, plants have been purported to 2014, and U.S. Provisional Application No. 62/017.816, filed benefit somewhat from the vast array of bacteria and fungi Jun. 26, 2014, and U.S. Provisional Application No. 62/017. that live both within and around their tissues to support their 813, filed Jun. 26, 2014, and U.S. Provisional Application No. health and growth. Endophytes are fungal or bacterial organ 62/017,815, filed Jun. 26, 2014, and U.S. Provisional Appli isms that live within plants. Bacterial and fungal endophytes cation No. 62/017,818, filed Jun. 26, 2014, each of which is appear to inhabit various host plant tissues and have been hereby incorporated in its entirety by reference. isolated from plant leaves, stems, or roots. 0007. A small number of these symbiotic endophyte-host SEQUENCE LISTING relationships have been purported in limited studies to pro vide agronomic benefits to model host plants within con 0002 The instant application contains a Sequence Listing trolled laboratory settings, such as enhancement of biomass which has been submitted via EFS-Web and is hereby incor production (i.e., yield) and nutrition, increased tolerance to porated by reference in its entirety. Said ASCII copy, created stress such as drought, and/or pests. Yet, such endophytes on Jun. 26, 2015, is named 29868US CRF sequencelisting. have been demonstrated to be ineffective in conferring ben txt, and is 2,970,000 bytes in size. efits to a variety of agriculturally-important plants; as such, they do not adequately address the need to provide improved FIELD OF THE INVENTION yield and tolerance to environmental stresses present in many 0003. This invention relates to compositions and methods agricultural situations for Such crops. for improving the cultivation of plants, particularly agricul 0008 Thus, there is a need for compositions and methods tural plants. For example, this invention describes beneficial of providing agricultural crops with improved yield and resis bacteria and fungi that are capable of living in a plant, which tance to various environmental stresses. Provided herein are may be used to impart improved agronomic traits to plants. novel compositions of bacterial and fungal endophytes and The disclosed invention also describes methods of improving synthetic endophyte-plant compositions based on the analy plant characteristics by introducing Such beneficial bacteria sis of the key properties that enhance the utility and commer and/or fungi to those plants. Further, this invention also pro cialization of an endophytic composition. vides methods of treating seeds and other plant elements with beneficial bacteria and/or fungi that are capable of living SUMMARY OF THE INVENTION within a plant, to impart improved agronomic characteristics 0009. The disclosures of PCT/US2014/044427, filed Jun. to plants, particularly agricultural plants. 26, 2014, and U.S. application Ser. No. 14/316,469, filed Jun. 26, 2014, are incorporated by reference in their entirety, BACKGROUND including the sequence listing containing SEQ ID NOS: 0004 Agriculture faces numerous challenges that are 1-1448. making it increasingly difficult to provide food, materials, 0010. The present invention is based on the surprising and fuels to the world's population. Population growth and discovery that a number of bacterial and fungal taxa of endo changes in diet associated with rising incomes are increasing phytes microbes are conserved across diverse species and/or global food demand, while many key resources for agricul cultivars of agricultural plants, and can be derived therefrom ture are becoming increasingly scarce. By 2050, the FAO and heterologously associated with diverse new cultivars to projects that total food production must increase by 70% to provide benefits. The present invention is also based on the meet the needs of the growing population, a challenge that is discovery that a plant element of a plant can be effectively exacerbated by numerous factors, including diminishing augmented by coating its Surface with Such endophytes in an freshwater resources, increasing competition for arable land, amount that is not normally found on the plant element. The rising energy prices, increasing input costs, and the likely endophytes can be isolated from inside the same plant or a need for crops to adapt to the pressures of a more extreme different plant, or from inside a part or tissue of the same plant global climate. The need to grow nearly twice as much food in or different plant. The plant element thus coated with the more uncertain climates is driving a critical need for new endophyte can be used to confer improved agronomic trait or innovations. traits to the seed or the plant that is grown from the plant 0005 Today, crop performance is optimized via of tech element. nologies directed towards the interplay between crop geno 0011. The inventors have postulated that attempts to select type (e.g., plant breeding, genetically-modified (GM) crops) for cultivars with certain improved traits and alterations in the and its surrounding environment (e.g., fertilizer, synthetic environmental and chemical conditions of agriculture have herbicides, pesticides). While these paradigms have assisted led to the inadvertent loss of microbes in modern varieties that in doubling global food production in the past fifty years, can provide beneficial traits to agricultural plants. The present yield growth rates have stalled in many major crops and shifts invention is based on the Surprising discovery that many in the climate have been linked to production declines in modern cultivars of agricultural plants display striking dis important crops such as wheat. In addition to their long devel tinctions in their microbial communities when compared with opment and regulatory timelines, public fears of GM-crops ancestral varieties. The present invention is also based on the US 2015/0373993 A1 Dec. 31, 2015 observation that, in some cases, providing the microbial taxa mature seeds per plant, increased chlorophyll content, present in Such ancestral cultivars but are absent or underrep increased number of pods perplant, increased length of pods resented in modern varieties can lead to dramatic improve per plant, reduced number of wilted leaves perplant, reduced ments in a number of agronomic traits in the modern cultivars. number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant. SUMMARY 0015 The seed or plant can be a dicot, e.g., soybean, 0012 Described herein are methods for preparing an agri cotton, tomato and pepper or a monocot, e.g., corn, wheat, cultural seed composition comprising contacting the Surface barley and rice.
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