US00968.7001 B2
(12) United States Patent (10) Patent No.: US 9,687,001 B2 Vujanovic et al. (45) Date of Patent: Jun. 27, 2017
(54) ENDOPHYTIC MICROBIAL SYMBIONTS IN 6,689,880 B2 2/2004 Chen et al. PLANT PRENATAL CARE 6,823,623 B2 11/2004 Minato et al. 7,037,879 B2 5, 2006 Imada et al. 7,084.331 B2 8/2006 Isawa et al. (71) Applicant: assity 9 Saskatchewan. 7,335,816 B2 2/2008 Kraus et al. askatoon (CA) 7,341,868 B2 3/2008 Chopade et al. 7,485,451 B2 2/2009 VanderGheynst et al. (72) Inventors: Vladimir Vujanovic, Saskatoon (CA); 7.555,990 B2 7/2009 Beaujot James J. Germida, Saskatoon (CA) 7,632,985 B2 12/2009 Malven et al. 7,763.420 B2 7/2010 Stritzker et al. (73) Assignee: University of Saskatchewan, 7,906,313 B2 3/2011 Henson et al. Saskatoon (CA) 7,977.550 B2 7, 2011 West et al. 8,143,045 B2 3/2012 Miansnikov et al. (*) Notice: Subject to any disclaimer, the term of this 8,455,198 B2 62013 Gao et al. patent is extended or adjusted under 35 8,455,395 B2 6/2013 Miller et al. U.S.C. 154(b) by 0 days 8,465,963 B2 6/2013 Rolston et al. M YW- 8,728.459 B2 5/2014 Isawa et al. 9,113,636 B2 8, 2015 von MaltZahn et al. (21) Appl. No.: 15/063,350 9,277,751 B2 3/2016 Sword 9,288,995 B2 3/2016 von Maltzahn et al. (22) Filed: Mar. 7, 2016 9,295,263 B2 3/2016 von MaltZahn et al. O O 2005/OO72047 A1 4/2005 Conkling et al. (65) Prior Publication Data 2006/0046246 A1 3/2006 Zeng et al. US 2016/O17457O A1 Jun. 23, 2016 2007/0028318 A1 2/2007 Livore et al. 2007/005545.6 A1 3/2007 Raftery et al. O O 2007. O142226 A1 6/2007 Franco Related U.S. Application Data 2007,0292.953 A1 12/2007 Mankin et al. 2008/0229441 A1 9/2008 Young et al. (63) Continuation of application No. 14/614,193, filed on 2008/0289060 A1 11/2008 De Beuckeleer et al. Feb. 4, 2015, which is a continuation-in-part of application No. PCT/CA2013/000091, filed on Feb. (Continued) 5, 2013. s FOREIGN PATENT DOCUMENTS (51) Int. Cl. CA 1041788 11, 1978 AOIN 63/04 (2006.01) CA 1229497 11, 1987 AOLH (700 (2006.01) (Continued) AOIN 63/02 (2006.01) B09C I/O (2006.01) AOIN 63/00 (2006.01) OTHER PUBLICATIONS CI2R L/80 (2006.01) Ravel et al 1997, Agronomie 17: 173-181.* CI2R L/46.5 (2006.01) Naik et al 2009, Microbiological Research 164: 290-296.* CI2R L/645 (2006.01) Orole et al 2011, Journal of Ecology and the Natural Environment (52) U.S. Cl. 3(9): 298-303.* CPC ...... A0IN 63/04 (2013.01); A0IH 1700 Giraldo et al 2015 Persoonia 34:10-24. (2013.01); A0IN 63/00 (2013.01); A0IN You et al 2012 Korean J. Microbiol. Biotechnol. 40(4): 287-295.* 63/02 (2013.01); B09C I/105 (2013.01): CI2R Hubbard et al 2012 Botany 90: 137-149.* I/465 (2013.01): CI2R 1/645 (2013.01): CI2R (Continued) I/80 (2013.01) (58) Field of Classification Search None Primary Examiner — David H Kruse See application file for complete search history. (74) Attorney, Agent, or Firm — Fenwick & West LLP (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT 4,940,834 A 7/1990 Hurley et al. The present disclosure provides compositions comprising 5,113,619 A 5/1992 Leps et al. novel endophytes capable of promoting germination endo 5,229,291 A 7, 1993 Nielsen et al. phytes that have a symbiotic relationship with plants. The 5,292,507 A 3/1994 Charley 5,415,672 A 5/1995 Fahey et al. present disclosure further provides methods of improving 5,730,973 A 3, 1998 Morales et al. seed vitality, biotic and abiotic stress resistance, plant health 5,919,447 A 7, 1999 Marrone et al. and yield under both stressed and unstressed environmental 5,994,117 A 11/1999 Bacon et al. conditions, comprising inoculating a seed with the novel 6,072,107 A 6, 2000 Latch et al. 6,077,505 A 6, 2000 Parke et al. endophyte strains and cultivating a plant therefrom. 6,337,431 B1 1/2002 Tricoli et al. 6,495,133 B1 12/2002 Xue 6,681, 186 B1 1/2004 Denisov et al. 4 Claims, 68 Drawing Sheets US 9,687,001 B2 Page 2
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US 9,687,001 B2 1. 2 ENDOPHYTIC MICROBAL SYMIBIONTS IN endophytic communities can herald changes not only in PLANT PRENATAL CARE pathways of nutrient (N. P. K), energy transfer in food-webs and biogeochemical cycles but also in UV-B, heat, drought CROSS REFERENCE TO RELATED or salt tolerance influencing the overall plant ecosystem APPLICATIONS establishment and stability. Despite their abundance and likely importance in all terrestrial ecosystems, nearly noth This application is a continuation of U.S. patent applica ing about the composition of endophytes in seeds or sper tion Ser. No. 14/614,193, filed on Feb. 4, 2015, which is a mosphere, their interactions, or their common response to continuation-in-part of co-pending International Application environmental changes is known. No. PCT/CA2013/000091, filed Feb. 5, 2013, which is 10 While the spermosphere represents a rapidly changing herein incorporated in its entirety by reference and microbiologically dynamic Zone of soil Surrounding a germinating seed Nelson, 2004, the rhizosphere is a micro SEQUENCE LISTING biologically active Zone of the bulk soil surrounding the plant's roots Smith and Read 2008. The rhizosphere sup The instant application contains a Sequence Listing, with 15 ports mycoheterotrophy or a plant-mycorrhiza symbiotic 19 sequences, which has been submitted via EFS-Web and relationship. The spermosphere, on the other hand, promotes is hereby incorporated by reference in its entirety. Said mycoVitality or an endophytic fungi relationship with the ASCII copy, created on Mar. 7, 2015, is named plant seeds—enhancing seed vigour, energy and uniformity 29069US sequencelisting..txt, and is 9,382 bytes in size. of germination that could be fairly predicted. Fungal endo phytes are distinct from mycorrhizae in that they can colo FIELD nize not only roots, but also other plant organs including seeds Vujanovic et al. 2000: Hubbard et al. 2011). They The present disclosure relates to synthetic preparations belong to the multicellular phyla Ascomycota and Basidi comprising a seed and a composition, where the composi omycota and form colonization symbiotic structures differ tion comprises fungal and bacterial endophytes of plants that 25 ent from those produced by unicellular or cenocytic phylum enhance seed vitality and/or plant health, conferring general Glomeromycota, known as Vesicular-arbuscular mycor improvements in the plants agricultural traits, under normal rhizal symbiosis Abdellatif et al. 2009. Endophytic bacte and stressed conditions. ria have been also found in virtually every plant studied, where they colonize an ecological niche similar to that of BACKGROUND 30 fungi. Such as the internal healthy tissues. Although most bacterial endophytes appear to originate from the rhizo Fungi and bacteria are ubiquitous microorganisms. Endo sphere or phyllosphere; some may be transmitted through phyte is the term first coined by de Bary 1866 defining the seed Ryan et al. 2008. those microbes that colonize asymptomatically plant tissues Seed germination is a vital phenophase to plants Survival Stone et al., 2000. The existence of endophytes has been 35 and reproduction in either optimal or stressful environmental known for more than one century Freeman 1904 and it conditions. Microbial endophytic colonization at the seed seems that each individual host, among the 300,000 plant state is especially critical because of the role of the seed as species, inhabits several to hundreds of endophytes Tan and a generative organ in regeneration and dispersion of flow Zou, 2001. Endophytes are microbial organisms mostly ering plants Baskin and Baskin 2004 and the role of symbiotically or mutualistically associated with living tis 40 mycobionts and symbiotically associated bacteria (bactobi Sues of plant hosts. Many are capable of conferring plant onts) as potential drivers of seedling recruitment in natu tolerance to abiotic stressors or can be used by the plant for ral undisturbed, disturbed and polluted habitats Mithl defense against pathogenic fungi and bacteria Singh et al. mann and Peintner 2000; Adriaensen et al. 2006: White and 2011. Some of these microorganisms have proven useful Tones 2010. Thus, developing methods by which seedling for very Small Subsets of agriculture (e.g., forage grass 45 emergence can be enhanced and protected under the limi growth), forestry and horticulture sectors, as well as plant tations of disease pressure, heat or drought is precious. The production of medicinally important compounds. However, use of endophytic symbionts is a promising method by no commercial endophyte seed coating products are used in which seed germination can be enhanced Vujanovic et al. the world’s largest crops including corn, wheat, rice, and 2000; Vujanovic and Vujanovic 2006; Vujanovic and barley, and Such endophyte approaches have Suffered from 50 Vujanovic 2007. The methods and compositions described high variability, inconsistent colonization, low performance herein overcome these and other limitations of the prior art. across multiple crop cultivars, and the inability to confer It was hypothesized that plant stress hardiness can be benefits to elite crop varieties under field conditions. conferred via a mycobiont-seed relationship known as Endophytes largely determine plant cell and whole plant mycoVitality—a phenomenon that had been reserved for genome regulation, including the plant's vital cycles: (i) 55 Orchidaceae Vujanovic 2008 and via bactovitality which seed pre- and post-germination events (mycoVitalism) Vu refers to a form of bactosymbiosis, using different endo janovic and Vujanovic 2007, (ii) plant nutrient uptake and phytic strains with variety of activities. growth-promoting mechanisms (mycoheterotrophism) Smith and Read 2008, and (iii) plant environmental stress SUMMARY tolerance and induced systemic resistance against diseases 60 and pests (mycosymbionticism) Wallin 1927; Margulis, The synthetic preparations and compositions described 1991. They could play a major role in plant biomass herein can benefit plant hosts, for example, but not limited production, CO sequestration, and/or yield and therefore be to, wheat, barley, corn, soybeans, alfalfa, rice, cotton, pulses, significant players in regulating the ecosphere, ensuring canola, vegetables, Sugarbeet, Sugarcane, trees, shrubs or plant health and food security. In addition, they can be 65 grasses. The benefit may come from bactovitality, mycoVi important sentinels (bioindicators) of environmental tality and mycoheterotrophy, and enhance tolerance to envi changes, as alterations in the structure and biomass of ronmental stresses, as demonstrated herein. Prenatal care in US 9,687,001 B2 3 4 agriculture is more than just seed or germinant vitality, plant vigor, nitrogen stress tolerance, enhanced Rhizobium health or vigour. It also determines what to expect before activity, enhanced nodulation frequency, and early flowering and during the germination process, seedling establishment, time. In some embodiments, the composition is disposed on and, later crop productivity or yield. an exterior Surface of the agricultural seed in an amount Several parameters of symbiotic efficacy (dormancy effective to colonize at least 1% of the cortical cells of an breakdown, germination, growth and yield) were assessed agricultural plant grown from the seed. In other embodi using efficient endophytic Saskatchewan Microbial Collec ments, the composition is disposed on an exterior Surface of tion and Database (SMCD) strain(s)-crop(s) interaction(s) the agricultural seed in an amount effective to cause a under in vitro, phytotron, greenhouse and field conditions. population of seeds inoculated with said composition to The synthetic preparations and compositions described 10 have greater germination rate, faster dormancy breakdown, herein have effects on germination, which can be assessed increased energy of germination, increased seed germination by measuring percent of germination, energy of germination vigor or increased seed vitality than a population of control and hydrothermal time required for germination, for agricultural seeds. In some embodiments, the composition is example. disposed on an exterior Surface of the agricultural seed in an Also tested was the endophyte's capacity to confer seed 15 amount effective to cause a population of seeds inoculated vitality. For both fungal and bacterial endosymbionts, with said composition to reach 50% germination faster than improved seed vitality can increase tolerance for abiotic and a population of control agricultural seeds. biotic stresses in plants that have progressed beyond the In some embodiments, the endophytes are a selected from seedling stage to the plants maturity via mycoheterotrophy. the group consisting of a spore-forming endophyte, a fac The synthetic preparations and compositions described ultative endophyte, a filamentous endophyte, an endophyte herein can improve plant traits such as increased yield, faster capable of living within another endophyte, an endophyte seedling establishment, faster growth, increased drought capable of forming hyphal coils within the plant, an endo tolerance, increased heat tolerance, increased cold tolerance, phyte capable of forming microvesicles within the plant, an increased salt tolerance, increased tolerance to pests and endophyte capable of forming micro-arbuscules within the diseases, for example increased tolerance to Fusarium infec 25 plant, an endophyte capable of forming hyphal knots within tion and to Puccinia infection, increased biomass, increased the plant, an endophyte capable of forming Hartig-like nets root length, increased fresh weight of seedlings, increased within the plant, and an endophyte capable of forming plant vigour, nitrogen stress tolerance, enhanced Rhizobium symbiosomes within the plant. In some embodiments, the activity, enhanced nodulation frequency or early flowering endophyte is in the form of at least one of conidia, chlamy time. 30 dospore, and mycelia. The synthetic preparations and compositions described In other embodiments, the composition is disposed on an herein can also modulate the expression of genes involved in exterior surface of the agricultural seed in an amount effec plant growth, genes associated with systemic acquired resis tive to colonize the cortical cells of an agricultural plant tance, or genes involved in protection from oxidative stress. grown from the seed and to produce the altered trait, wherein These genes may be involved in phytohormone production, 35 the altered trait is altered gene expression, wherein the gene for example gibberellin (GA) biosynthesis or breakdown, is selected from the group consisting of a gene involved in abscisic acid (ABA) biosynthesis or breakdown, NO pro plant growth, an acquired resistance gene, and a gene duction or breakdown, Superoxide detoxification, or could involved in protection from oxidative stress. In some be positive or negative regulators of these pathways. The embodiments, the gene is involved in phytohormone pro genes associated with systemic acquired resistance may be 40 duction. In other embodiments, the gene is a redox-regulated redox-regulated transcription factors, for example those in transcription factor. In yet other embodiments, the gene the MYB family. Non-limiting examples of such genes involved in superoxide detoxification or in NO production or include ent-kaurenoic (KAO), repression of shoot growth breakdown. (RSG), NCED, ABA8'-hydroxylase, GA3-oxidase 2, 14-3-3 In some embodiments, the agricultural plant seed is or nitric oxide (NO) genes and/or stress resistance SuperoX 45 selected from the group consisting of corn, Soy, wheat, ide dismutase (SOD), manganese SOD (MnSOD), proline cotton, rice, canola, barley and pulses. In some embodi (Pro), Myb1 and Myb2. ments, a population comprising at least 10 synthetic prepa In certain embodiments, the present disclosure provides a rations is disposed within a packaging material. synthetic preparation comprising an agricultural plant seed Further provided herein is a seed comprising an endo and a composition comprising an endophyte capable of 50 phyte or culture disclosed herein. In one embodiment, the promoting germination and an agriculturally-acceptable car seed is coated with the endophyte. In another embodiment, rier, wherein an agricultural plant grown from the seed has the seed is cultured or planted near the endophyte such that an altered trait as compared to a control agricultural plant. In the endophyte is able to colonize the seed. In one embodi certain embodiments, the endophyte capable of promoting ment, the seed planted near the endophyte is up to 4 cm away germination is a coleorhiza-activating endophyte and the 55 from the endophyte. agricultural plant seed is a monocot seed. In some embodi The endophytes used to inoculate the seeds may be ments, the composition is disposed on an exterior Surface of selected from the group consisting of a spore-forming endo the agricultural seed in an amount effective to colonize the phyte, a facultative endophyte, a filamentous endophyte, and cortical cells of an agricultural plant grown from the seed an endophyte capable of living within another endophyte. In and to produce the altered trait, wherein the altered trait is 60 Some embodiments, the endophyte is capable of forming an improved functional trait selected from the group con certain structures in the plant, where the structures are sisting of increased yield, faster seedling establishment, selected from the group consisting of hyphal coils, Hartig faster growth, increased drought tolerance, increased heat like nets, microvesicles, micro-arbuscules, hyphal knots, tolerance, increased cold tolerance, increased salt tolerance, and symbiosomes. In some embodiments, the endophyte is increased tolerance to Fusarium infection, increased toler 65 in the form of at least one of conidia, chlamydospore, and ance to Puccinia infection, increased biomass, increased mycelia. In other embodiments, the fungus or bacteria is root length, increased fresh weight of seedlings, increased capable of being part of a plant-fungus symbiotic system or US 9,687,001 B2 5 6 plant-bacteria symbiotic system that produces altered levels cetes, Sordariomycetes, or Eurotiomycetes. In some of phytohormones or anti-oxidants, as compared to a plant embodiments, the endophyte is of order Helotiales, Capno that is not in Symbiosis. In other embodiments, the plant dides, Pleosporales, Hypocreales, or Eurotiales. In some fungus symbiotic system or plant-bacterium symbiotic sys embodiments, the composition comprises an agriculturally tem has anti-aging and/or anti-senescence effects, as com- 5 acceptable carrier and a spore-forming, filamentous bacterial pared to a plant or plant organ that is not in Symbiosis. In endophyte of phylum Actinobacteria. In some embodiments, other embodiments, the plant-fungus symbiotic system or the endophyte is a bacteria of order actinomycetales. plant-bacteria Symbiotic system has increased protection In some embodiments, the present disclosure provides a against pathogens, as compared to a plant that is not in composition comprising a carrier and an endophyte of symbiosis. In some embodiments, 10 Paraconyothirium sp. strain deposited as IDAC 081111-03 The present disclosure also provides methods for improv or comprising a DNA sequence with at least 97% identity to ing seed vitality and enhancing plant health and yield under SEQ ID NO:5; an endophyte of Pseudeurotium sp. strain normal and stressed conditions. Accordingly, there is pro deposited as IDAC 081111-02 or comprising a DNA vided a method of improving seed vitality, plant health sequence with at least 97% identity to SEQ ID NO:4; an and/or plant yield comprising inoculating a seed with an 15 endophyte of Penicillium sp. strain deposited as IDAC endophyte or culture disclosed herein or a combination or 081111-01 or comprising a DNA sequence with at least 97% mixture thereof or with a composition disclosed herein. In identity to SEQID NO:3: an endophyte of Cladosporium sp. Some embodiments, the seed is cultivated into a first gen strain deposited as IDAC 2003 12-06 or comprising a DNA eration plant. sequence with at least 97% identity to SEQ ID NO:1; an In certain embodiments, provided herein are methods of 20 endophyte of Sarocladium sp. strain deposited as IDAC altering a trait in an agricultural plant seed or an agricultural 2003 12-05 or comprising a DNA sequence with at least 97% plant grown from said seed, where the methods comprise identity to SEQ ID NO:2; and/or an endophyte of Strepto inoculating the seed with a composition comprising endo myces sp. strain deposited as IDAC 081111-06 or compris phytes capable of promoting germination and an agricultur ing a DNA sequence with at least 97% sequence identity to ally-acceptable carrier, where the endophyte replicates 25 SEQ ID NO:6. In certain embodiments, the endophyte of within at least one plant tissue and colonizes the cortical Paraconyothirium sp. Strain comprises a DNA sequence cells of said plant. In some embodiments, the endophyte with at least 98% identity to SEQ ID NO:5; the endophyte colonizes at least 1% of the cortical cells of said agricultural of Pseudeurotium sp. strain comprises a DNA sequence with plant. at least 98% identity to SEQ ID NO:4; the endophyte of In Some embodiments, the trait altered by using the 30 Penicillium sp. strain comprises a DNA sequence with at method is an improved functional trait selected from the least 98% identity to SEQ ID NO:3: the endophyte of group consisting of increased yield, faster seedling estab Cladosporium sp. strain comprises a DNA sequence with at lishment, faster growth, increased drought tolerance, least 98% identity to SEQ ID NO:1; the endophyte of increased heat tolerance, increased cold tolerance, increased Sarocladium sp. strain comprises a DNA sequence with at salt tolerance, increased tolerance to Fusarium infection, 35 least 98% identity to SEQ ID NO:2; and the endophyte of increased tolerance to Puccinia infection, increased bio Streptomyces sp. Strain comprises a DNA sequence with at mass, increased root length, increased fresh weight of seed least 98% sequence identity to SEQ ID NO:6. In certain lings, increased plant vigor, nitrogen stress tolerance, embodiments, the endophyte of Paraconyothirium sp. Strain enhanced Rhizobium activity, enhanced nodulation fre comprises a DNA sequence with at least 99% identity to quency, and early flowering time. In other embodiments, the 40 SEQ ID NO:5; the endophyte of Pseudeurotium sp. strain altered trait is a seed trait selected from the group consisting comprises a DNA sequence with at least 99% identity to a greater germination rate, faster dormancy breakdown, SEQ ID NO:4; the endophyte of Penicillium sp. strain increased energy of germination, increased seed germination comprises a DNA sequence with at least 99% identity to vigor or increased seed vitality than a population of control SEQ ID NO:3: the endophyte of Cladosporium sp. strain agricultural seeds. In other embodiments, the altered trait is 45 comprises a DNA sequence with at least 99% identity to reaching 50% germination faster than a population of con SEQ ID NO:1; the endophyte of Sarocladium sp. strain trol agricultural seeds. In other embodiments, the altered comprises a DNA sequence with at least 99% identity to trait is altered gene expression, where the gene is selected SEQID NO:2; and the endophyte of Streptomyces sp. strain from the group consisting of a gene involved in plant comprises a DNA sequence with at least 99% sequence growth, an acquired resistance gene, and a gene involved in 50 identity to SEQ ID NO:6. In certain embodiments, the protection from oxidative stress. endophyte of Paraconyothirium sp. strain comprises a DNA In some embodiments, the endophytes used in the method sequence of SEQID NO:5; the endophyte of Pseudeurotium are a selected from the group consisting of a spore-forming sp. strain comprises a DNA sequence of SEQ ID NO:4; the endophyte, a facultative endophyte, a filamentous endo endophyte of Penicillium sp. strain comprises a DNA phyte, an endophyte capable of living within another endo- 55 sequence of SEQID NO:3: the endophyte of Cladosporium phyte, an endophyte capable of forming hyphal coils within sp. strain comprises a DNA sequence of SEQ ID NO:1; the the plant, an endophyte capable of forming microvesicles endophyte of Sarocladium sp. strain comprises a DNA within the plant, an endophyte capable of forming micro sequence of SEQ ID NO:2; and the endophyte of Strepto arbuscules within the plant, an endophyte capable of form myces sp. strain comprises a DNA sequence of SEQ ID ing hyphal knots within the plant, an endophyte capable of 60 NO:6. forming Hartig-like nets within the plant, and an endophyte In another aspect, there is provided a method of improv capable of forming symbiosomes within the plant. In some ing plant health and/or plant yield comprising treating plant embodiments, the endophyte is in the form of at least one of propagation material or a plant with an endophyte or culture conidia, chlamydospore, and mycelia. disclosed herein or a combination or mixture thereof or a In some embodiments, the endophyte is a fungus of 65 composition disclosed herein. In some embodiments, the Subphylum Pezizomycotina. In some embodiments, the plant propagation material is cultivated into a first genera endophyte is a fungus of class Leotiomycetes, Dothideomy tion plant or the plant is allowed to grow. US 9,687,001 B2 7 8 In an embodiment, the plant propagation material is any for 1000 replicates; only values that were >60% are given. plant generative/sexual (seed, generative bud or flower) and Bar indicates 0.05 nucleotide substitutions per site (nucleo vegetative? asexual (stem, cutting, root, bulb, rhizome, tuber, tide position). vegetative bud, or leaf) part that has the ability to be FIG. 7 shows left compartments of split plates (plant with cultivated into a new plant. microbial partner): healthy phenotypic appearance of wheat In an embodiment, the methods enhance landscape devel when the root is grown in contact with the microbial mats: opment and remediation. Accordingly, in one embodiment, and right-compartments of split plates (plant without micro there is provided a method of reducing soil contamination bial partner): massive formation of root hairs of wheat due comprising treating plant propagation material or a plant to the plant-fungus association made in the left compart with an endophyte or culture disclosed herein or a combi 10 ments of the split plates. nation or mixture thereof or a composition disclosed herein; FIGS. 8 (A) and (C) shows SMCD2206 discontinuous and cultivating the plant propagation material into a first colonization of wheat root (epidermis and cortex) tissue generation plant or allowing the plant to grow. In one compared to (B) and (D) which shows pathogenic Fusarium embodiment, the soil contaminant is hydrocarbons, petro graminearum’s uniform/continual cell colonization of wheat leum or other chemicals, salts, or metals, such as lead, 15 root including vascular cylinder. cadmium or radioisotopes. FIG. 9 shows Ireg index level of deviation (irregularity) Other features and advantages of the present disclosure in endophyte (SMCDs) cell form. will become apparent from the following detailed descrip FIG. 10 shows Idir index level of direction changes tion. It should be understood, however, that the detailed when colonizing living plant-host cell. description and the specific examples while indicating FIG. 11 shows endophytic hyphae in root of wheat embodiments of the disclosure are given by way of illus germinant (A-SMCD 2204: B-SMCD 2206; C-SMCD 2210; tration only, since various changes and modifications within and D-SMCD-2215) visualized with lactofuchsin staining the spirit and scope of the disclosure will become apparent and fluorescence microscopy. Symbiotic structures/organs: to those skilled in the art from this detailed description and (D) SMCD 2215 bacterial endophyte mostly formed curly respective drawings and drawing legends. 25 intercellular filaments, whereas endophytic fungi (Figures to the right) produced: SMCD 2204 intracellular coils and BRIEF DESCRIPTION OF THE DRAWINGS arbuscules, SMCD 2206 intracellular vesicules, and SMCD 2110 intracellular knots. The disclosure will now be described in relation to the FIG. 12 shows the appearance of symbiotic germinating drawings in which: 30 wheat seedlings after 10 days on moist filter paper at 21°C. FIG. 1 shows the phenotypic appearance of the endo FIG. 13 shows leaf length of germinating wheat seedlings phytic fungal strains SMCD 2204, 2004F, 2206, 2208, and after 10 days at moisture filter paper at 21° C. 2210 and bacterial strain SMCD 2215; after 10 days of FIG. 14 shows an in vitro inoculation method (A). A 5 growth on PDA at 21°C. mm agar plug, cut from the margin of the parent colony, FIG. 2A shows the inferred neighbour-joining phyloge 35 was placed hyphal side down in the centre of a 60 mm Petri netic tree of the Cladosporium sp. SMCD2204 based on ITS dish containing potato dextrose agar (PDA) media. Next, rDNA. Numbers at nodes indicate bootstrap support values five Surface-sterilized seeds were placed a distance equiva for 1000 replicates; only values that were >70% are given. lent to 48 h hyphal growth from the agar plug and germi Bar indicates 0.01 nucleotide substitutions per site (nucleo nated in the dark. The impact of three seed surface steril tide position). FIG. 2B shows the inferred neighbour-joining 40 ization methods on seed germination (B). Bars labeled with phylogenetic tree of the Sarocladium sp. SMCD 2204F one or two asterisks (*) are significantly, or highly signifi based on the sequence of the large Subunit of the nuclear cantly, different from the same endophyte grown under ribosomal RNA gene (LSU). Numbers at nodes indicate control conditions (ps0.05 or ps0.01, respectively; ANOVA, bootstrap support values for 1000 replicates. Only values followed by post-hoc LSD test). Error bars represent stan that were >70% are given. Bar indicates 0.01 nucleotide 45 dard error of the mean (SE). Substitutions per site (nucleotide position). FIG. 15 shows growth rates of free-living endophytes FIG.3 shows the inferred neighbour-joining phylogenetic SMCD 2204, 2206, 2208, 2210, and 2215 in vitro on potato tree of the Penicillium sp. SMCD 2206 based on ITS rDNA. dextrose agar (PDA) under heat stress (36°C.), drought (8% Numbers at nodes indicate bootstrap support values for 1000 polyethylene glycol (PEG) 8000) stress and control condi replicates; only values that were >70% are given. Bar 50 tions for five days and simultaneous heat (36° C.) and indicates 0.01 nucleotide substitutions per site (nucleotide drought (8% PEG) for six days. Bars labeled with one or two position). asterisks (*) are significantly, or highly significantly, differ FIG. 4 shows the inferred neighbour-joining phylogenetic ent from the same endophyte grown under control condi tree of the Pseudeurotium sp. SMCD 2208 based on ITS tions (ps0.05 or ps0.01, respectively; ANOVA, followed by rDNA. Numbers at nodes indicate bootstrap support values 55 post-hoc LSD test). Error bars represent standard error of the for 1000 replicates; only values that were >70% are given. mean (SE). Bar indicates 0.01 nucleotide substitutions per site (nucleo FIG. 16 shows percent germination and fresh weight of tide position). seedlings from initial experiments in which seeds were FIG. 5 shows the inferred neighbour-joining phylogenetic surface sterilized in 5% sodium hypochlorite for 3 min. tree of the Coniothyrium strain SMCD 2210 based on ITS 60 Percent germination of wheat seeds in vitro after three days rDNA. Numbers at nodes indicate bootstrap support values on potato dextrose agar (PDA) under heat stress (36° C.), for 1000 replicates; only values that were >70% are given. drought stress (8% polyethylene glycol (PEG) 8000) and Bar indicates 0.05 nucleotide substitutions per site (nucleo control conditions (A, B and C) with the y axis normalized tide position). to percent germination obtained under the same conditions FIG. 6 shows the inferred neighbour-joining phylogenetic 65 by seeds surface sterilized in 5% sodium hypochlorite for 1 tree of the Streptomyces sp strain SMCD 2215 based on 16S min. Fresh weight of seedlings in vitro at seven days on PDA rDNA. Numbers at nodes indicate bootstrap support values under heat stress, drought stress and control conditions (D. US 9,687,001 B2 9 10 E and F). Bars labeled with one (*) or two asterisks (**) are with one (*) or two asterisks (**) are significantly, or highly significantly, or highly significantly, different from the no significantly, different from the no endophyte stressed con endophyte control (ps0.05 or ps0.01, respectively; ANOVA, trol (ps0.05 or ps0.01, respectively; ANOVA, followed by followed by post-hoc LSD test). Error bars represent the post-hoc LSD test). standard error of the mean (SE). FIG. 24 shows pods dry weight of (A) chickpeas, (B) FIG. 17 shows percent germination over time of wheat lentils, and (C) peas in symbiosis with SMCD endophytes seeds co-cultured with the endophytes most effective at (E+) under heat stress phytotron conditions. Bars labeled conferring abiotic stress tolerance (SMCD 2206, 2210 and with one (*) or two asterisks (**) are significantly, or highly 2215) compared to uncolonized, unstressed seeds (positive significantly, different from the no endophyte stressed con control) and uncolonized, stressed seeds (negative control). 10 trol (ps0.05 or ps0.01, respectively; ANOVA, followed by Energy of germination (EG) is related to the time, in days (X post-hoc LSD test). axis) at which 50% germination (y axis) is reached. The FIG. 25 shows roots dry weight of (A) chickpeas, (B) symbols “”, “x”, “o”, “A”, and “O'” represent the positive lentils, and (C) peas in symbiosis with SMCD endophytes control, SMCD 2206 treated seeds, SMCD 2210 treated (E+) under heat stress phytotron conditions. Bars labeled seeds, SMCD 2215 treated seeds and the negative control, 15 with one (*) or two asterisks (**) are significantly, or highly respectively. Heat and drought treatments correspond to 36° significantly, different from the no endophyte stressed con C. and 8% polyethylene glycol (PEG) 8000, respectively. trol (ps0.05 or ps0.01, respectively; ANOVA, followed by Error bars represent the standard error of the mean (SE). post-hoc LSD test). Note: The seeds used in EG determination were from the FIG. 26 shows stem dry weight of (A) chickpeas, (B) second round of experiments, and hence sterilized in 5% peas, and (C) lentils under drought stress in a greenhouse. sodium hypochlorite for one minute, rather than three. Bars labeled with one (*) or two asterisks (**) are signifi FIG. 18 shows the relationship between hydrothermal cantly, or highly significantly, different from the no endo time (HTT) required to achieve 50% germination for heat phyte (E-) stressed control (ps0.05 or ps0.01, respectively; and drought alone and 5% germination for heat and drought ANOVA, followed by post-hoc LSD test). combined (X axis) and percent germination attained after 25 FIG. 27 shows dry weights of (A) chickpeas, (B) peas, seven days (y axis). Germination after seven days and HTT and (C) lentils pods in association with an endophyte (E+) were based on the results of the second round of experi under drought stress in the greenhouse. Bars labeled with ments. The symbols “”, “0” and “A” represent seeds one (*) or two asterisks (**) are significantly different from exposed to heat (36° C.), drought (8% polyethylene glycol the no endophyte (E-) stressed control (ps0.05 or ps0.01, (PEG) 8000) or both heat and drought stress, respectively. 30 respectively; ANOVA, followed by post-hoc LSD test). The R-squared values associated with the trendlines are FIG. 28 shows roots dry weight of (A) chickpeas, (B) 0.96, 0.80 and 0.18 for seeds exposed to heat, drought or peas, and (C) lentils under drought stress in the greenhouse. both heat and drought stress, respectively. Note: The seeds Bars labeled with one (*) or two asterisks (**) are signifi used to determine percent germination at seven days and cantly, or highly significantly, different from no endophyte HTT were from the second round of experiments, and hence 35 (E-) stressed control (ps0.05 or ps0.01, respectively; treated with 5% sodium hypochlorite for one minute, rather ANOVA, followed by post-hoc LSD test). than three. FIG. 29 shows (A) Chickpea Vanguard flowering plants FIG. 19 shows seeds treated or inoculated with SMCD bearing pods under drought stress in a greenhouse—left strains demonstrate improvement in all tested seed germi plant is non-Symbiotic (E-) and right plant is symbiotic with nation parameters including seed germination vigour (SGV) 40 strain SMCD 2215 (E+); (B) and (C), Chickpea Vanguard efficacy. plants bearing pods under drought stress in a greenhouse— FIG. 20 shows the relationship between drought tolerance (B) non-symbiotic and (C) symbiotic with SMCD 2215. efficiency (DTE) values in wheat (A) and barley (B) culti FIG. 30 shows root nodulation of pea varieties under heat vars without (E-) and with (E+) endophytes, based on the stress in a phytotron: Hendel (Above) and Golden (Below) average effect of symbiosis using all tested SMCD isolates, 45 inoculated (left) and uninoculated (right) with SMCD 2215. on yield exposed to drought stress in greenhouse. Note: in all samples natural infection with Rhizobium sp. FIG. 21A shows endophytic (E+) inoculants (SMCD from pea seeds has been observed. 2206, SMCD 2210, and SMCD 2215) improve kernel yield FIG.31 shows SMCD2206 and SMCD 2215 considerably in wheat genotypes compared to control (E-) treatment increase energy of seed germination (>50%) in Glamis (yield g/3 pots). FIG. 21B shows endophytic inoculants 50 (lentil) as a function of time under heat and drought in vitro. (SMCD 2206, SMCD 2210, and SMCD 2215) improve FIG. 32 shows SMCD2206 and SMCD 2215 considerably kernel yield in two row barley (B) and six row barley (B.) increase energy of seed germination (>50%) in Handel (pea) genotypes (kernel yield: 3plants/pot). as a function of time under heat and drought in vitro. FIG.22 shows (A) Barley-six row AC Metcalfe, from left FIG. 33 shows endophytic inoculants (SMCD 2206 and to the right: Drought (E-), Drought and SMCD 2206 (E+). 55 SMCD 2210) improve flax yield under drought conditions in Control (E-), Control and SMCD 2206 (E+); (B) Wheat a greenhouse. Different letters above the bars indicate sta Unity cultivar, from left to the right: Drought (E-), Drought tistically significant differences between samples (p<0.05, and SMCD 2215 (E+), Control (E-), Control and SMCD Kruskal-Wallis test). 2215 (E+); (C) Wheat-Verona cultivar, from left to the right: FIG. 34 shows endophytic inoculants (SMCD 2206, Drought (E-), Drought and SMCD 2215 (E+). Control (E-), 60 SMCD 2210, and SMCD 2215) improve canola yield under Control and SMCD 2215 (E+); and (D) Durum wheat drought conditions in a greenhouse. Different letters above TEAL, from left to the right: Drought (E-), Drought and the bars indicate statistically significant differences between SMCD 2210 (E+), Control (E-), Control and SMCD 2210 samples (p<0.05, Kruskal-Wallis test). (E+). FIG. 35 shows the survival of wheat seeds pre-inoculated FIG. 23 shows stem dry weight of (A) chickpeas, (B) 65 in-vitro (plates in above row) and wheat seedlings pre lentils, and (C) peas in symbiosis with SMCD endophytes inoculated in greenhouse (pots in below row) with endo (E+) under heat stress phytotron conditions. Bars labeled phytic SMCD 2206-showing healthy plant growth, and with US 9,687,001 B2 11 12 pathogenic Fusarium avenaceum and Fusarium FIG. 49 shows relative expression patterns of hormonal graminearum—showing disease symtoms and death of RSG and KAO regulator genes and MYB 1 and MYB 2 plants. resistance genes in coleorhiza of germinating wheat seeds FIG. 36 shows Fusarium inoculants produced on wheat for three days under cold and biological stratification. Gene kernels. expression was calculated as 2A". FIG. 37 shows that post-emergence damping-off has been FIG. 50 shows emerging radicle from wheat geminating prevented by SMCD 2206 endophyte in wheat in the green seed (A) Inverted fluorescence (B) and fluorescence imaging house. of DAF-2DA fluorescence upon reaction with NO in radicle FIG. 38 shows wheat biomass (aerial a-d and root e-f) cells (C) of AC Avonlea germinant at 5 min after treatment improved in the presence of SMCD 2206 endophyte com 10 Nakatsubo et al. 1998 with the fungal SMCD 2206 exu pared to untreated plants. (a) control plant (E-), (b) inocu date. No fluorescence reaction observed in control radicle lated plant (E+), (c) control flowering plant, (d) inoculated cells. Bar-25 um; Bar-50 um. flowering plant, (e) control plant (E-, left) compared to FIG. 51 shows DAF-2T fluorescence intensity values at 5 SMCD 2206 inoculated plant (E+, right), and (f) fluorescent min after treatment of wheat radicle from AC Avonlea microscopy of SMCD 2206 wheat root-colonization (E+). 15 germinants with the SMCD 2206 fungal exudate, fungal FIG. 39 shows aerial plant biomass/plant (left) and under exudate together with the NO scavenger cFTIO, and sterile ground (root) biomass/plant (right) in control (E-) and water. Radicle segments were incubated for 30 min in 2 ml SMCD inoculated wheat plants (E+) against F. of detection buffer (10 mM Tris-Hcl. pH 7.4, 10 mM KCl) graminearum and F. avenaceum. Vertical error bars on data containing 15 uM DAF-2DA (Sigma-Aldrich) with or with points represent the standard error of the mean. out 1 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazo FIG. 40 shows root length in control plant (CDC Teal) line-1-oxyl-3-oxide (cPTIO) as an NO scavenger. Average without SMCD endophyte compared to inoculated wheat fluorescence values are reported as a ratio of the fluores plant with SMCD strains. Bars on data points represent the cence intensity at 5 minto the fluorescence intensity at time standard error of the mean. 0. Different letters indicate statistically significant differ FIG. 41 shows dry weight of kernels/plant (TEAL culti 25 ences between samples (p<0.05, Kruskal-Wallis test). var) in wheat using the double pre-inoculation approach: a) FIG. 52 shows the change (in days) in the initial flowering SMCD endophyte--Fusarium avenaceum (Fav), and b) time of canola crops treated with the microbial compositions SMCD endophyte--Fusarium graminearum (Figr). Vertical described. Data shown is from n=4 independent replicate plotstone standard deviation. 1=Abiotic formulation con error bars on data points represent the standard error of the 30 Ca. trol, 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD 2206, FIG. 42 shows comparison of TEAL spike sizes in wheat 5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215. in the presence of pathogen (negative control) and without FIG. 53 shows the damage score due to pests of canola presence of pathogen (positive control). Left Figure—from crops treated with the microbial compositions described. left to right: i) plant+Figr, ii) plant-i-Fav, and (iii) plant; Right Data shown is from n=4 independent replicate plotstone Figure—from left to right: i) plant; ii) plant--endophyte; iii) 35 standard deviation. 1=Abiotic formulation control, plant--endophyte--F. av; and iv) plant--endophyte--F.gr. 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD 2206, FIG. 43 shows the effect of SMCD 2215 on Handel (pea) 5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215. on 10% PEG after 7 days at 21 degrees C. in darkness. (A) FIG. 54 shows the Fusarium Head Blight (FHB) inci shows the control seeds, and (B) shows the SMCD 2215 dence percentage for three spring wheat (Lillian, Unity, and treated seeds. 40 Utmost) and one durum wheat (Strongfield) varieties. Sub FIG. 44 shows (A) SOD and (B) MnSOD relative gene plots (a), (b), (e), and (f) refer to the Vanguard, Saskatch expressions in pea (Handel) exposed to PEG with and ewan, Canada test site, while Subplots (c), (d), (g), and (h) without endophytes. refer to the Stewart Valley, Saskatchewan, Canada test site. FIG. 45 shows Proline relative gene expression in pea Data shown is from n=4 independent replicate plotstone (Handel) exposed to PEG with and without endophytes. 45 standard deviation. 1=Abiotic formulation control, FIG. 46 shows germination of wheat seeds in vitro after 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD 2206, three days on potato dextrose agar (PDA). Cold stratification 5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215. was imposed by keeping seeds at 4° C. cold-room for 48 FIG.55 shows the leaf spot disease rating for three spring hours. For endophyte-indirect and endophyte-direct treat wheat (Lillian, Unity, and Utmost) and one durum wheat ments, using SMCD 2206, seeds were germinated at 50 (Strongfield) varieties. A rating of 1 is most healthy and a approximately 4 cm distance and in direct contact respec rating of 10 is most diseased. Subplots (a), (b), (e), and (f) tively. A) Percentage of germination in comparison with refers to the leaf spot disease rating on the lower leaf, while energy of germination (50% germination). B) Efficacy of Subplots (c), (d), (g), and (h) refer to the leaf spot disease germination of wheat seeds Subjected to cold and biological rating on the flag leaf. Data shown is from n=4 independent stratification. Efficacy was calculated by subtracting the 55 replicate plotstone standard deviation. 1=Abiotic formula germination percentage of control from treated seeds. tion control, 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD FIG. 47 shows differential expression patterns of gibber 2206, 5–SMCD 2208, 6=SMCD 2210, 7=SMCD 2215. ellin (TaGA3ox2 and 14-3-3) and ABA (TaNCED2 and FIG. 56 shows aggregated yield data for durum wheat, TaABA8'OH1) genes in coleorhiza of germinating wheat spring wheat, barley, canola, and pulses (chickpea, pea, and 60 lentil). Each dot refers to a single plot. The "+" refers to the seeds for three days under cold and biological stratification. group mean. The data are presented as percentage gain over Gene expression was calculated as 2-C. the abiotic formulation control for each combination of crop, FIG. 48 shows the ratio of expression levels (22-CT) location and condition (irrigated or dryland). (a) refers to of gibberellin (TaGA3ox2 and 14-3-3) and ABA (TaNCED2 SMCD 2215, (b) refers to SMCD 2210, (c) refers to SMCD and TaABA8'OH1) genes in coleorhiza of germinating 65 2204, (d) refers to SMCD 2206, (e) refers to SMCD 2208, wheat seeds for three days under cold and biological strati and (f) refers to SMCD 2204F. Data shown are from n=4 fication. independent replicate plots for all Canadian sites and n=6 US 9,687,001 B2 13 14 plots for Brookings, S. Dak. sites. While no fields were molecule permeable membrane. See Table 13 for abbrevia experimentally inoculated with a pathogen, the notation tion of the names of the molecules. Data shown is average “Fus-” indicates that there was no visible occurrence of concentration in ng per gram dry tissue weight from n=3 Fusarium Head Blight (Fusarium graminearum) in that independent replicates. specific field and “Fus+ indicates that there was clear FIG. 64 shows cytokinin production data from wheat evidence of Fusarium Head Blight (Fusarium (CDC Avonlea) in seedling studies. “Control” refers to the graminearum) occurrence. N- indicates that there was no formulation control wherein SMCD 2206 was not added, nitrogen applied to the field and N-- indicates that nitrogen “A-Direct” refers to direct application of SMCD2206 to the was applied at agriculturally relevant rates. seedling, and “A-Indirect” refers to the indirect application FIG. 57 shows aggregated yield data for durum wheat, 10 of SMCD 2206 to the seedling through a small-molecule spring wheat, barley, canola, and pulses (chickpea, pea, and permeable membrane. See Table 14 for abbreviation of lentil). 95% confidence intervals for the respective formu molecules. Data shown is average concentration in ng per lation and crop are shown. The data are presented as gram dry tissue weight from n=3 independent replicates. percentage gain over the abiotic formulation control for each FIG. 65 shows auxin production data from wheat (CDC combination of crop, field trial and condition (irrigated or 15 Avonlea) in seedling studies. “Control” refers to the formu dryland). (a) refers to SMCD 2215, (b) refers to SMCD lation control wherein SMCD 2206 was not added, “A-Di 2210, (c) refers to SMCD 2204, (d) refers to SMCD 2206, rect” refers to direct application of SMCD 2206 to the (e) refers to SMCD 2208, and (0 refers to SMCD 2204F. seedling, and “A-Indirect” refers to the indirect application Data shown is from n=4 independent replicate plots for all of SMCD 2206 to the seedling through a small-molecule Canadian sites and n-6 plots for Brookings, S. Dak. As permeable membrane. Auxins were represented by the bio above, the notation "Fus-” indicates that there was no logically active indole acetic acid IAA and its conjugate with visible occurrence of Fusarium Head Blight (Fusarium aspartic acid IAA-Asp. Data shown is average concentration graminearum) in that specific field and “Fus+ indicates that in ng per gram dry tissue weight from n=3 independent there was clear evidence of Fusarium Head Blight replicates. (Fusarium graminearum) occurrence. N- indicates that 25 FIG. 66 shows symbiosomes in root of wheat germinant there was no nitrogen fertilizer applied to the field and N-- visualized with lactofuchsin staining and fluorescence indicates that nitrogen fertilizer was applied at agriculturally microscopy. A type I Symbiosome, which is composed of an relevant rates. intercellular microvesiculoid compartment formed between FIG. 58 shows the aggregated ear weight data from the two plant cell membranes (arrows), a perivesiculoid mem corn field trial in Brookings, S. Dak. 95% confidence 30 brane (large triangle) and a partially fragmented outer intervals for the respective formulation is shown. The data is vesiculoid membrane (Small triangle), is shown in A. A type presented as percentage gain over the abiotic formulation II symbiosome, which is composed of an intracellular control. “2206 refers SMCD 2206, “2204 refers to SMCD vesiculoid compartment (arrows), a perivesiculoid mem 2204, and “2215” refers to SMCD 2215. brane (large triangle) and a partially fragmented outer FIG. 59 shows data from greenhouse trials of tomato 35 vesiculoid membrane (Small triangle), is shown in B. Also inoculated with the described microbial compositions: (a) shown in B is a vesiculophore (filled arrow). The symbio shoot length, (b) shoot weight, (c) total plant biomass, (d) somes shown in A and B are both of vesicle form. Shown in root length, (e) root weight, and (f) tomato fruit weight under Care symbiosomes of knot form (type I—lower arrow; type drought conditions. Data shown is from n=3 independent II—upper arrow). replicate plantstone standard deviation. 40 FIG. 60 shows data from greenhouse trials of alfalfa DEFINITIONS treated with the described microbial compositions: (a) shoot length, (b) shoot weight, (c) total plant biomass, (d) root The term “mycoVitality” as used herein refers to the length, and (e) root weight under drought conditions. Data plant-fungus symbiosis that exists between the seeds and the shown is from n 3 independent replicate plantstone stan 45 fungi, which helps maintain the seeds capacity to live and dard deviation. develop, and leads to germination. In some embodiments, FIG. 61 shows data from greenhouse trials comparing mycoVitality may be characterized by a change in levels of normal (dark gray) and drought (light gray) water conditions certain phytohormones within the plant-fungus symbiotic for: (a) corn, (b) Sweet corn, (c) organic corn, (d) Swiss system. In some embodiments, this change may be associ chard, (e) radish, and (f) cabbage. Data shown is total 50 ated with a change in the levels of abscisic acid (ABA), biomass from n=3 independent replicate plantstone stan gibberellins (GA), auxins (IAA), and/or cytokinins. In other dard deviation. embodiments, mycoVitality may be characterized by a FIG. 62 shows gibberellin production data from wheat change in expression of the genes involved in gibberellin (CDC Avonlea) in seedling studies. “Control” refers to the (GA) biosynthesis or breakdown or in abscisic acid (ABA) formulation control wherein SMCD 2206 was not added, 55 biosynthesis or breakdown, or in positive or negative regu “A-Direct” refers to direct application of SMCD2206 to the lation of these pathways within the plant-fungus symbiotic seedling, and “A-Indirect” refers to the indirect application system. In certain embodiments, the levels of expression of of SMCD 2206 to the seedling through a small-molecule the gibberellin (GA) biosynthetic genes, such as GA3 permeable membrane. See Table 12 for abbreviation of oxidase 2, RSG, KAO, and 14-3-3 genes may be increased. molecules. Data shown is average concentration in ng per 60 In other embodiments, the levels of expression of the genes gram dry tissue weight from n=3 independent replicates. that are regulated by GAS, Such as ent-kaurenoic (KAO) and FIG. 63 shows ABA metabolite production data from repression of shoot growth (RSG), are increased. In other wheat (CDC Avonlea) in seedling studies. “Control” refers embodiments, the levels of expression of the GA degrada to the formulation control wherein SMCD 2206 was not tion genes or negative regulators of the GA biosynthesis added, “A-Direct” refers to direct application of SMCD 65 pathway, for example 14-3-3 genes, are decreased. In still 2206 to the seedling, and “A-Indirect” refers to the indirect other embodiments, mycovitality may be characterized by application of SMCD 2206 to the seedling through a small decreased levels of expression of the genes involved in the US 9,687,001 B2 15 16 ABA biosynthesis pathway, for example the NCED gene, embodiments, bactovitality is characterized by an increase within the plant-fungus symbiotic system. In other embodi in the levels of activity of the genes associated with systemic ments, the expression of the genes involved in the ABA acquired disease resistance, such as redox-regulated tran catabolic pathway, for example the 8-hydroxylase gene, are scription factors, for example those in the MYB family. In increased. In some embodiments, mycoVitality may be char some embodiments, the genes in the MYB family are Myb 1 acterized by altered levels of nitric oxide (NO) within the and Myb2. plant-fungus symbiotic system, for example as a result of a “Cold stratification' as used herein refers to the process of change in the expression of certain genes involved in NO pretreating seeds to simulate the natural winter conditions production or breakdown. In yet other embodiments, myco during which, amongst many physiological changes, the vitality may be characterized by protection of the plant 10 seed coat is softened up by frost and weathering action, fungus symbiotic system from oxidative stress. In some leading to dormancy breakdown. “Biological stratification' embodiments, mycoVitality is characterized by increased as used herein refers to the process of treating seeds with levels of expression of the genes involved in Superoxide biological components to release seed dormancy and thereby detoxification within the plant-fungus symbiotic system. In promoting germination. In some embodiments, the biologi Some embodiments, the genes associated with Superoxide 15 cal components may be endophytes. Therefore, as compared detoxification encode superoxide dismutase (SOD) or man to cold stratification, in which an abiotic stimulation is used, ganese SOD (MnSOD), and in other cases the levels of the biological stratification uses a biotic stimulation. As for cold amino acid proline (Pro) are elevated. In some embodi stratification, biological stratification may increase the rate ments, mycoVitality is characterized by an increase in the of germination in seeds. In both cases, the progress of levels of activity of the genes associated with systemic stratification and dormancy breakdown may be associated acquired disease resistance, such as redox-regulated tran with an increase in levels of GA and a decrease in levels of scription factors, for example those in the MYB family. In ABA. In certain embodiments, the levels of expression of some embodiments, the genes in the MYB family are Myb 1 gibberellin (GA) biosynthetic genes, such as GA3-oxidase 2 and Myb2. and 14-3-3 genes, are increased. In other embodiments, the The term “bactovitality” as used herein refers to the 25 levels of expression of the genes that are regulated by GAS, plant-bacterium symbiosis that exists between the seeds and Such as ent-kaurenoic (KAO) and repression of shoot growth the bacteria, which helps maintain the seeds capacity to live (RSG), are increased. In other embodiments, the levels of and develop, and leads to germination. In some embodi expression of GA degradation genes or negative regulators ments, bactovitality may be characterized by a change in of the GA biosynthesis pathway, for example 14-3-3 genes, levels of certain phytohormones within the plant-bacterium 30 are decreased. In still other embodiments, the levels of symbiotic system. In some embodiments, this change may expression of the genes involved in the ABA biosynthesis be associated with a change in the levels of abscisic acid pathway, for example the NCED gene, are decreased. In (ABA), gibberellins (GA), auxins (IAA), and/or cytokinins. other embodiments, the expression of genes involved in the In other embodiments, bactovitality may be characterized by ABA catabolic pathway, for example the 8-hydroxylase a change in expression of the genes involved in gibberellin 35 gene, is increased. (GA) biosynthesis or breakdown or in abscisic acid (ABA) "Anti-aging or “anti-senescence' as used herein refers to biosynthesis or breakdown, or in positive or negative regu a process within a seed or plant that protects the seed or plant lation of these pathways within the plant-bacteria symbiotic from aging and senescence or that results in delayed aging system. In certain embodiments, the levels of expression of or senescence of the seed or plant. In some embodiments, the the gibberellin (GA) biosynthetic genes, such as GA3 40 anti-aging or anti-senescence effects of endophytes are char oxidase 2, RSG, KAO, and 14-3-3 genes may be increased. acterized by increased levels of nitric oxide (NO) within the In other embodiments, the levels of expression of the genes plant-fungus or plant-bacterium symbiotic system, for that are regulated by GAs. Such as ent-kaurenoic (KAO) and example as a result of a change in the expression of certain repression of shoot growth (RSG), are increased. In other genes involved in NO production or breakdown. In certain embodiments, the levels of expression of the GA degrada 45 embodiments, the anti-aging or anti-senescence effects of tion genes or negative regulators of the GA biosynthesis endophytes may be characterized by a change in levels of pathway, for example 14-3-3 genes, are decreased. In still certain phytohormones within the plant-fungus or plant other embodiments, bactovitality may be characterized by bacterium symbiotic system. In some embodiments, this decreased levels of expression of the genes involved in the change may be associated with decreased by levels of ABA biosynthesis pathway, for example the NCED gene, 50 abscisic acid (ABA), increased levels of gibberellins (GA) within the plant-bacterium symbiotic system. In other or increased levels of auxins. In some embodiments, myco embodiments, the expression of the genes involved in the Vitality may be characterized by a change in expression of ABA catabolic pathway, for example the 8-hydroxylase the genes involved in gibberellin (GA) biosynthesis or gene, are increased. In some embodiments, bactovitality breakdown or in abscisic acid (ABA) biosynthesis or break may be characterized by altered levels of nitric oxide (NO) 55 down, or in positive or negative regulation of these pathways within the plant-bacterium symbiotic system, for example as within the plant-fungus or plant-bacterium symbiotic sys a result of a change in the expression of certain genes tem. In certain embodiments, the levels of expression of the involved in NO production or breakdown. In yet other gibberellin (GA) biosynthetic genes, such as GA3-oxidase 2, embodiments, bactovitality may be characterized by protec RSG, KAO, and 14-3-3 genes may be increased. In other tion of the plant-bacterium symbiotic system from oxidative 60 embodiments, the levels of expression of the genes that are stress. In some embodiments, bactovitality is characterized regulated by GAS, such as ent-kaurenoic (KAO) and repres by increased levels of expression of the genes involved in sion of shoot growth (RSG), are increased. In other embodi Superoxide detoxification within the plant-bacterium sym ments, the levels of expression of the GA degradation genes biotic system. In some embodiments, the genes associated or negative regulators of the GA biosynthesis pathway, for with Superoxide detoxification encode Superoxide dismutase 65 example 14-3-3 genes, are decreased. In still other embodi (SOD) or manganese SOD (MnSOD), and in other cases the ments, the anti-aging or anti-senescence effects of endo levels of the amino acid proline (Pro) are elevated. In some phytes may be characterized by decreased levels of expres US 9,687,001 B2 17 18 sion of the genes involved in the ABA biosynthesis pathway, fungi that are “capable of forming spores' or “spore-forming for example the NCED gene, within the plant-fungus or endophytes' are those bacteria and fungi containing the plant-bacterium symbiotic system. In other embodiments, genes and other necessary abilities to produce spores under the expression of the genes involved in the ABA catabolic Suitable environmental conditions. pathway, for example the 8-hydroxylase gene, are The term “filamentous fungi as used herein are fungi that increased. form hyphae, and includes taxa that have both filamentous As used herein, “symbiosome' or “symbiotic organs’ and yeast-like stages in their life cycle. refers to the new compartment that is formed within the The term “facultative endophytes' as used herein are plant cell when bacteria or fungi colonize the plant. In type endophytes capable of Surviving in the soil, on the plant I symbiosomes, the new structure is an intercellular 10 Surface, inside a plant and/or on artificial nutrients. Facul microvesiculoid compartment formed between two plant tative endophytes may also have the capacity to Survive cell membranes. A “microvesiculoid” compartment is a inside a variety of different plant species. structure that has the form of a microvesicle. In type II The term “endophyte capable of living within another symbiosomes, the new compartment is localized intracellu endophyte' as used herein refers to an endophytic bacterium larly and can be described as an intracellular structure in the 15 or fungus that can live within another endophyte. Such form of a vesicle, or “intracellular vesiculoid compartment.” endophytic bacteria may also be able to live autonomously Both types of symbiosomes are further characterized by the in the Soil, on the plant Surface, inside a plant and/or on presence of a “perivesiculoid membrane,” which is the artificial nutrients. plasma membrane that Surrounds the vesicles, and a partially The term "endophyte capable of promoting germination' fragmented “outer vesiculoid membrane,” which is an outer as used herein refers to endophytes that have the capacity to membrane in the form of a vesicle. In this context, a colonize a seed or part of a seed and alter the seeds symbiosome is not limited to the structure that is formed physiology Such that the seed or a population of seeds shows during nitrogen fixation. a faster dormancy breakdown, greater germination rate, “Mycoheterotrophy’ as used herein refers to a symbiotic earlier germination, increased energy of germination, greater relationship between a plant and a fungus that allows the 25 rate of germination, greater uniformity of germination, plant to obtain water, minerals, and carbohydrates more including greater uniformity of rate of germination and efficiently. In this context, the plant may be any plant, even greater uniformity of timing of germination, and/or a fully photosynthetic plant, that may derive a benefit via its increased vigor and energy of germination. In some embodi association with the fungus. ments, the endophyte capable of promoting germination is As used herein, the term “microarbuscule” refers to 30 an endophyte that is capable of activating the coleorhiza of intracellular, multiarbuscular, microsized (~10 um), bush a monocot seed, and can be called a “coleorhiza-activating like haustorial structures. endophyte'. The term “vitality,” as used herein means the capacity to The term 'agricultural plant’ means a plant that is typi live and develop. cally used in agriculture. The agricultural plant may be a The term “hydrothermal time' refers to parameters of 35 monocot or dicot plant, and may be planted for the produc water, temperature and time by which seed germination can tion of an agricultural product, for example grain, food, be described under various environmental conditions. The fiber, etc. The plant may be a cereal plant. The term “plant” parameters enable germination strategies to be compared in as used herein refers to a member of the Plantae Kingdom different environments and to assess the effects of endo and includes all stages of the plant life cycle, including phytes on germination relative to other variables. 40 without limitation, seeds, and includes all plant parts. The In some embodiments, the endophyte is chosen from the plant can be selected from, but not limited to, the following group consisting of a spore-forming endophyte, a facultative list: endophyte, a filamentous endophyte, and an endophyte Food crops: Cereals including Maize? corn (Zea mays), capable of living within another endophyte. In some Sorghum (Sorghum spp.), Millet (Panicum miliaceum, P. embodiments, the endophyte is capable of forming certain 45 Sumatrense), Rice (Oryza sativa indica, Oryza sativa structures in the plant, where the structures are selected from japonica), Wheat (Triticum sativa), Barley (Hordeum vul the group consisting of hyphal coils, Hartig-like nets, gare), Rye (Secale cereale), Triticale (Triticunx...Secale), microvesicles, micro-arbuscules, hyphal knots, and Symbio Oats (Avena fatua); Somes. In some embodiments, the endophyte is in the form leafy vegetables (brassicaceous plants such as cabbages, of at least one of conidia, chlamydospore, and mycelia. In 50 broccoli, bok choy, rocket, Salad greens such as spinach, other embodiments, the fungus or bacteria is capable of cress, lettuce); being part of a plant-fungus symbiotic system or plant fruiting and flowering vegetables (e.g. avocado, Sweet bacteria symbiotic system that produces altered levels of corn, artichokes, curcubits e.g. squash, cucumbers, melons, phytohormones or anti-oxidants, as compared to a plant that courgettes, pumpkins; Solononaceous vegetables/fruits e.g. is not in Symbiosis. In other embodiments, the plant-fungus 55 tomatoes, eggplant, capsicums); symbiotic system or plant-bacterium symbiotic system has podded vegetables (groundnuts, peas, beans, lentils, anti-aging and/or anti-senescence effects, as compared to a chickpea, okra); plant or plant organ that is not in Symbiosis. In other bulbed and stem vegetables (asparagus, celery, Allium embodiments, the plant-fungus symbiotic system or plant crops e.g. garlic, onions, leeks); bacteria symbiotic system has increased protection against 60 roots and tuberous vegetables (carrots, beet, bamboo pathogens, as compared to a plant that is not in Symbiosis. shoots, cassava, yams, ginger, Jerusalem artichoke, parsnips, A “spore' or a population of “spores' refers to bacterial radishes, potatoes, Sweet potatoes, taro, turnip, wasabi); or fungal structures that are more resilient to environmental Sugar crops including Sugar beet (Beta vulgaris), Sugar influences Such as heat and bacteriocidal agents and fungi cane (Saccharum officinarum); cides than vegetative forms of the same bacteria or fungi. 65 crops grown for the production of non-alcoholic bever Spores are typically capable of germination and out-growth ages and stimulants (coffee, black, herbal and green teas, giving rise to vegetative forms of the species. Bacteria and cocoa, tobacco); US 9,687,001 B2 19 20 fruit crops such as true berry fruits (e.g. kiwifruit, grape, plants used as direct or indirect feedstocks for the pro currants, gooseberry, guava, feijoa, pomegranate), citrus duction of biofuels i.e. after chemical, physical (e.g. thermal fruits (e.g. oranges, lemons, limes, grapefruit), epigynous or catalytic) or biochemical (e.g. enzymatic pre-treatment) fruits (e.g. bananas, cranberries, blueberries), aggregate fruit or biological (e.g. microbial fermentation) transformation (blackberry, raspberry, boysenberry), multiple fruits (e.g. during the production of biofuels, industrial solvents or pineapple, fig), Stone fruit crops (e.g. apricot, peach, cherry, chemical products e.g. ethanol or butanol, propane diols, or plum), pip-fruit (e.g. apples, pears) and others such as other fuel or industrial material including Sugar crops (e.g. strawberries, sunflower seeds; beet, Sugar cane), starch-producing crops (e.g. C3 and C4 culinary and medicinal herbs e.g. rosemary, basil, bay cereal crops and tuberous crops), cellulosic crops such as laurel, coriander, mint, dill, Hypericum, foxglove, alovera, 10 forest trees (e.g. Pines, Eucalypts) and Graminaceous and rosehips); Poaceous plants such as bamboo, Switch grass, miscanthus, crop plants producing spices e.g. black pepper, cumin crops used in energy, biofuel or industrial chemical pro cinnamon, nutmeg, ginger, cloves, Saffron, cardamom, duction by gasification and/or microbial or catalytic conver mace, paprika, masalas, star anise; sion of the gas to biofuels or other industrial raw materials crops grown for the production of nuts and oils e.g. 15 Such as solvents or plastics, with or without the production almonds and walnuts, Brazil nut, cashew nuts, coconuts, of biochar (e.g. biomass crops such as coniferous, eucalypt, chestnut, macadamia nut, pistachio nuts; peanuts, pecan tropical or broadleaf forest trees, graminaceous and poa nuts, soybean, cotton, olives, Sunflower, Sesame, lupin spe ceous crops such as bamboo, Switch grass, miscanthus, cies and brassicaeous crops (e.g. canolafoilseed rape); and, Sugar cane, or hemp or softwoods such as poplars, willows: crops grown for production of beers, wines and other and, alcoholic beverages e.g. grapes, hops; biomass crops used in the production of biochar, edible fungi e.g. white mushrooms, Shiitake and oyster Crops Producing Natural Products Useful for the Phar mushrooms; maceutical, Agricultural Nutraceutical and Cosmeceutical Plants Used in Pastoral Agriculture: legumes: Trifolium Industries: crops producing pharmaceutical precursors or species, Medicago species, and Lotus species; White clover 25 compounds or nutraceutical and cosmeceutical compounds (T. repens); Red clover (T. pratense); Caucasian clover (T. and materials for example, star anise (shikimic acid), Japa ambigum); subterranean clover (T. subterraneum); Alfalfa/ nese knotweed (resveratrol), kiwifruit (soluble fiber, pro Lucerne (Medicago sativum); annual medics; barrel medic; teolytic enzymes): black medic; Sainfoin (Onobrychis viciifolia); Birdsfoot Floricultural, Ornamental and Amenity Plants Grown for trefoil (Lotus corniculatus); Greater Birdsfoot trefoil (Lotus 30 their Aesthetic or Environmental Properties: Flowers such as pedunculatus); roses, tulips, chrysanthemums; Forage and Amenity grasses: Temperate grasses such as Ornamental shrubs such as Buxus, Hebe, Rosa, Rhodo Lolium species; Festuca species; Agrostis spp., Perennial dendron, Hedera, ryegrass (Lolium perenne); hybrid ryegrass (Lolium hybri Amenity plants such as Platanus, Choisya, Escallonia, dum); annual ryegrass (Lolium multiflorum), tall fescue 35 Euphorbia, Carex, (Festuca arundinacea); meadow fescue (Festuca pratensis); Mosses such as Sphagnum moss; and red fescue (Festuca rubra); Festuca ovina; Festuloliums Plants Grown for Bioremediation: Helianthus, Brassica, (LoliumxFestuca crosses); CockSfoot (Dactylis glomerata); Salix, Populus, and Eucalyptus. Kentucky bluegrass Poa pratensis, Poa palustris, Poa nem A "host plant' includes any plant, particularly an agricul oralis, Poa trivialis, Poa compresa, Bromus species; 40 tural plant, which an endophytic microbe Such as an endo Phalaris (Phleum species); Arrhenatherum elatius, Agropy phyte capable of promoting germinations can colonize. As ron species; Avena Strigosa, Setaria italic, used herein, a microbe is said to “colonize' a plant or seed Tropical grasses such as: Phalaris species; Brachiaria when it can be stably detected within the plant or seed over species; Eragrostis species; Panicum species; Bahai grass a period time, such as one or more days, weeks, months or (Paspalum notatum); Brachypodium species; and, 45 years. In other words, a colonizing microbe is not transiently Grasses used for biofuel production Such as Switchgrass associated with the plant or seed. (Panicum virgatum) and Miscanthus species; As used herein, an "agricultural seed' is a seed used to Fiber Crops: hemp, jute, coconut, sisal, flax (Linum spp.), grow a plant typically used in agriculture (an 'agricultural New Zealand flax (Phormium spp.); plantation and natural plant'). The seed may be of a monocot or dicot plant, and forest species harvested for paper and engineered wood fiber 50 may be planted for the production of an agricultural product, products such as coniferous and broadleafed forest species; for example grain, food, fiber, etc. The seed may be of a Tree and Shrub Species Used in Plantation Forestry and cereal plant. As used herein, an agricultural seed is a seed Bio Fuel Crops: Pine (Pinus species); Fir (Pseudotsuga that is prepared for planting, for example, in farms for species); Spruce (Picea species); Cypress (Cupressus spe growing. cies); Wattle (Acacia species); Alder (Alnus species); Oak 55 As used herein, a “control agricultural plant’ or “control species (Quercus species); Redwood (Sequoiadendron spe seed' is an agricultural plant or seed of the same species, cies); willow (Salix species); birch (Betula species); Cedar strain, or cultivar to which a treatment, formulation, com (Cedurus species); Ash (Fraxinus species); Larch (Larix position or endophyte preparation as described herein is not species); Eucalyptus species; Bamboo (Bambu.seae species) administered/contacted. A control agricultural plant or con and Poplars (Populus species). 60 trol seed, therefore, is identical to the treated plant or seed Plants Grown for Conversion to Energy, Biofuels or with the exception of the presence of the endophyte and can Industrial Products by Extractive, Biological, Physical or serve as a control for detecting the effects of the endophyte Biochemical Treatment: Oil-producing plants such as oil that is conferred to the plant. palm, jatropha, linseed; A population' of plants or seeds, as used herein, can refer Latex-producing plants such as the Para Rubber tree, 65 to a plurality of plants or seeds that were subjected to the Hevea brasiliensis and the Panama Rubber Tree Castilla same inoculation methods described herein, or a plurality of elastica, plants or seeds that are progeny of a plant or group of seeds US 9,687,001 B2 21 22 that were subjected to the inoculation methods. In addition, inoculation comprises foliar application or soil application a population of plants can be a group of plants that are grown of the endophyte or combination thereof with any solid or from coated seeds. The plants or seeds within a population liquid carrier at any growing stage of the plant. will typically be of the same species, and will also typically The term "enhancing seed vitality” as used herein refers share a common genetic derivation. to plant prenatal care improving the ability of the seed to The term “endophyte' as used herein refers to a fungal or germinate and produce a plant under normal and/or stressed bacterial organism that can live symbiotically in a plant and conditions and includes, without limitation, any one or more is also referred to herein as “endosymbiont'. A fungal of the following: breaking dormancy, providing seed strati endophyte may be in the form of a spore, hypha, or mycelia. fication, increasing seed germination, modulating gene A bacterial endophyte may be a cell or group of cells. The 10 expression, decreasing time to reach energy of germination, term “endophyte' as used herein includes progeny of the protecting against biotic stresses, protecting against abiotic strains recited herein. stresses, reducing hydrothermal time required for germina In some cases, the present invention contemplates the use tion, increasing seed germination vigour, increasing seed of microbes that are “compatible” with agricultural chemi germination efficacy, increasing uniformity of seed germi cals, for example, a fungicide, an anti-bacterial compound, 15 nation, ameliorating drought/heat tolerance efficacy, increas or any other agent widely used in agricultural which has the ing the weight of seedlings, and increasing the yield of effect of killing or otherwise interfering with optimal growth seedlings. Drought/Heat Tolerance Efficiency (DTE/THE) is of microbes. As used herein, a microbe is “compatible' with the term opposed (antonym) to susceptibility. an agricultural chemical when the microbe is modified. Such Energy of germination is defined as 50% of germination, as by genetic modification, e.g., contains a transgene that relative to the number of seeds tested. The seed germination confers resistance to an herbicide, or is adapted to grow in, vigour shows the difference between total percentage of or otherwise Survive, the concentration of the agricultural germinating treated seeds and germinating untreated seeds. chemical used in agriculture. For example, a microbe dis The hydrothermal time postulates that an individual seed posed on the surface of a seed is compatible with the begins to germinate when the Sum of both temperatures and fungicide metalaxyl if it is able to survive the concentrations 25 water potential are sufficiently accumulated over a period of that are applied on the seed surface. time allowing germination. Germination efficacy is defined As used herein, a “colony-forming unit” (“CFU’) is used as the percentage of treated seeds germinating after a set as a measure of viable microorganisms in a sample. A CFU time period after planting, relative to the number of seeds is an individual viable cell capable of forming on a solid tested in an untreated control. Biological Stratification is medium a visible colony whose individual cells are derived 30 defined as releasing seed dormancy by a symbiont in pro by cell division from one parental cell. moting germination. Uniformity of seed germination repre In some embodiments, the invention uses microbes that sents the maximum percentage of seed germination within a are heterologous to a seed or plant in making synthetic minimal time of incubation. combinations or agricultural formulations. A microbe is The terms “decreased”, “fewer”, “slower' and considered heterologous to the seed or plant if the seed or 35 “increased “faster "enhanced’ “greater as used herein seedling that is unmodified (e.g., a seed or seedling that is refers to a decrease or increase in a characteristic of the not treated with a population of endophytes capable of endophyte treated seed or resulting plant compared to an promoting germination described herein) does not contain untreated seed or resulting plant. For example, a decrease in detectable levels of the microbe. For example, the invention a characteristic may be at least 1%, at least 2%, at least 3%, contemplates the synthetic combinations of seeds or seed 40 at least 4%, at least 5%, at least 10%, at least 15%, at least lings of agricultural plants and an endophytic microbe 20%, at least 25%, at least 30%, at least 35%, at least 40%, population (e.g., an endophyte capable of promoting germi at least 45%, at least 50%, at least 75%, at least 100%, or at nation), in which the microbe population is "heterologously least 200% or more lower than the untreated control and an disposed on the exterior surface of or within a tissue of the increase may be at least 1%, at least 2%, at least 3%, at least agricultural seed or seedling in an amount effective to 45 4%, at least 5%, at least 10%, at least 15%, at least 20%, at colonize the plant. A microbe is considered "heterologously least 25%, at least 30%, at least 35%, at least 40%, at least disposed on the surface or within a plant (or tissue) when 45%, at least 50%, at least 75%, at least 100%, or at least the microbe is applied or disposed on the plant in a number 200% or more higher than the untreated control. that is not found on that plant before application of the The term “increased yield’ refers to increased seed microbe. For example, population of endophytes capable of 50 weight, seed size, seed number per plant, seed number per promoting germination that is disposed on an exterior Sur unit area (i.e. seeds, or weight of seeds, per acre), bushels per face or within the seed can be an endophyte that may be acre, tons per acre, kilo per hectare, increased grain yield, associated with the mature plant, but is not found on the increased dry weight of grain, increased seed weight, surface of or within the seed. As such, a microbe is deemed increased dry weight of wheat Spikes and increased biomass. heterologously disposed when applied on the plant that 55 “Biomass” means the total mass or weight (fresh or dry), at either does not naturally have the microbe on its surface or a given time, of a plant tissue, plant tissues, an entire plant, within the particular tissue to which the microbe is disposed, or population of plants. Biomass is usually given as weight or does not naturally have the microbe on its surface or per unit area. Increased biomass includes without limitation within the particular tissue in the number that is being increased pod biomass, stem biomass, and root biomass. applied. The term “exogenous can be used interchangeably 60 In certain embodiments, the plant is cultivated under with "heterologous.” abiotic or biotic stressed conditions. The term "abiotic The phrase “inoculating a seed as used herein refers to stress' as used herein refers to a non-living stress that applying, infecting, co-planting, spraying, immersing, dust typically affects seed vitality and plant health and includes, ing, dipping or coating the seed with the endophyte. Tech without limitation, heat, drought, nitrogen, cold, salt and niques for inoculating the seed are known in the art, for 65 osmotic stress. In one embodiment, the abiotic stress is heat example, as disclosed by Hynes and Boyetchko (2006, Soil stress. In another embodiment, the abiotic stress is drought Biology & Biochemistry 38: 845-84). In an embodiment, stress, osmotic stress or salt stress. The term “biotic stress' US 9,687,001 B2 23 24 as used herein refers to a living stress that typically affects physiological properties of plant species. Our classification seed vitality and plant health, and includes without limita based on studies involving a large range of agricultural tion, insect infestation, nematode infestation, bacterial infec plants distinguishes the chosen endophytes from those endo tion, fungal infection, oomycete infection, protozoal infec phytes that interact with only some classes of plant species. tion, viral infection, and herbivore grazing, or any 5 Endophytes classified in this way can include fungi and combination thereof. In one embodiment, the biotic stress is bacteria and the classification highlights that the fungi and a Fusarium infection. bacteria have informational systems in common. The infor As used herein an "agriculturally compatible carrier' mational systems programming the plant-endophyte inter refers to any material, other than water, which can be added actions are complex and comprise signaling systems, mul to a seed or a seedling without causing or having an adverse 10 tiple networks and pathways that underpin growth of many effect on the seed (e.g., reducing seed germination) or the types of plant cells and organs as well as endophyte cells. plant that grows from the seed, or the like. They are thus best described by the outcomes of the plant The term "plant propagation material” as used herein endophyte interactions. refers to any plant generative/sexual and vegetative? asexual The endophyte class described herein provides the unique part that has the ability to be cultivated into a new plant. In 15 ability to confer mycovitalism to a large number of diverse an embodiment, the plant propagation material is generative plant hosts, as well as to confer stress tolerance and seed, generative bud or flower, and vegetative stem, cutting, increased yield. Specifically, this endophyte class is able to, root, bulb, rhizome, tuber, vegetative bud, or leaf parts. when coated onto the outside of a seed or placed in its In some cases, the present invention contemplates the use proximity, increase expression of key genes related to seed of microbes (e.g., endophytes) that are “compatible' with 20 germination, vigor, and stress tolerance. The endophytes are agricultural chemicals, for example, a fungicide, an anti then able to penetrate the cortical layer of the seed and plant bacterial compound, or any other agent widely used in in order to enter the plants internal tissues and replicate agriculture that has the effect of killing or otherwise inter within at least one tissue in the host and establish symbiotic fering with optimal growth of microbes. As used herein, a organs comprising microstructures that allow intimate com microbe such as a seed bacterial endophyte is “compatible’ 25 munication between the endophyte and the plants intra with an agricultural chemical when the microbe is modified, and/or intercellular spaces. These endophytes further act in Such as by genetic modification, e.g., contains a transgene symbiosis with the host to improve stress tolerance of the that confers resistance to an herbicide, or is adapted to grow seedling and adult plant and to increase yield. in, or otherwise Survive, the concentration of the agricultural Germination of mature, dry seeds is a process that is chemical used in agriculture. For example, a microbe dis- 30 conserved across angiosperms, being regulated by water, posed on the surface of a seed is compatible with the temperature, the hormones gibberellic acid, abscisic acid fungicide metalaxyl if it is able to survive the concentrations and ethylene, amongst other vital molecules, and involves that are applied on the seed surface. changes to cell walls, breakdown of food reserves and their The term “phytoremediation” as used herein refers to the conversion to new molecules and structures that define root use of plants for removal, reduction or neutralization of 35 and shoot growth. The group of endophytes revealed here is Substances, wastes or hazardous material from a site so as to readily characterized by its ability to stimulate seed germi prevent or minimize any adverse effects on the environment. nation or make germination more uniform when any of its The term “phytoreclamation” as used herein refers to the use members are present as a synthetic preparation that physi of plants for reconverting disturbed land to its former or cally interacts with a seed from monocot or dicot plants. In other productive uses. 40 other embodiments, the group of endophytes is recognized as capable of altering plant flowering time and/or increasing DETAILED DESCRIPTION tolerance to biotic and abiotic stresses and many other traits. All of these features support the conclusion that members of The present invention identifies a small, unique family of this group of endophytes can be physically complexed with endophytes that can be placed into synthetic combination 45 monocot and dicot seeds to achieve multiple agricultural with a variety of plant hosts and work Synergistically with benefits due to their particular informational systems that the plant hosts to exhibit a Surprising number of altered and interact with those conserved in plants. improved biological processes. This family of endophytes represents a Surprising discov Plants across the Angiosperms have many features in ery in their ability to engage in synthetic associations with common that emanate from having evolved from a common 50 plants, leading to a number of altered physiological pro ancestor. This is true for the many systems that control cesses across the lifespan of the plant-endophyte composite growth and development and also tolerance to abiotic and association. Notably, the synthetic associations between this biotic stresses. Plants have co-evolved with endosymbionts Small family of endophytes and both monocot and dicot and in consequence these latter organisms, fungi and bac plants are characterized by the activation of multiple plant teria, can possess features that enable them to interact with 55 genes and hormones during seed germination, seedling plants. It is well accepted that microrganisms can be clas development, and responses to environmental and biotic sified on the basis of their taxonomy or cladistics groupings, StreSSes. as well as based on key morphological, functional, and Novel Compositions and Seeds ecological roles. Here, by screening hundreds of synthetic Accordingly, the present disclosure provides a composi associations between endophytes and plants, we discovered 60 tion comprising at least one endophyte capable of promoting a family of endophytes based on their ability to interact with germination or comprising a combination or mixture a variety of plant species to create agricultural value. These thereof, and an agriculturally-acceptable carrier. In some endophytes possess systems that enable them to physically embodiments, the at least one endophyte capable of pro and chemically interact with a broad range of agricultural moting germination are coleorhiza-activating endophytes. In plants bred by man, including monocots and dicots, endors 65 Some embodiments, a synthetic preparation is made using ing the conclusion that when living together with the plant the composition and an agricultural plant seed. In some they interact intimately with the conserved genetic and cases, plants are inoculated with at least one endophyte that US 9,687,001 B2 25 26 is heterologous to the inoculated agricultural plant seed or structures in the plant, where the structures are selected from the agricultural plant grown from the agricultural seed. In the group consisting of hyphal coils, Hartig-like nets, Some embodiments, the at least one endophyte capable of microvesicles, micro-arbuscules, hyphal knots, and Symbio promoting germination are disposed on the Surface or within Somes. In some embodiments, the at least one endophyte is a tissue of the agricultural seed or seedling. In some embodi in the form of at least one of conidia, chlamydospore, and ments, a plant grown from a seed inoculated with this mycelia. In other embodiments, the fungus or bacteria is composition has an improved functional trait as compared to capable of being part of a plant-fungus symbiotic system or a control plant. In some embodiments, the improved func plant-bacteria symbiotic system that produces altered levels tional trait is resistance to biotic or abiotic stress. In some of phytohormones or anti-oxidants, as compared to a plant embodiments, the improved functional trait is selected from 10 that is not in Symbiosis. In other embodiments, the plant the group consisting of increased yield, faster seedling fungus symbiotic system or plant-bacterium symbiotic sys establishment, faster growth, increased photosynthetic rate, tem has anti-aging and/or anti-senescence effects, as com increased carbon dioxide assimilation rate, increased pared to a plant or plant organ that is not in Symbiosis. In drought tolerance, increased heat tolerance, increased cold other embodiments, the plant-fungus symbiotic system or tolerance, increased salt tolerance, increased tolerance to 15 plant-bacteria Symbiotic system has increased protection pests and diseases, increased biomass, increased root and/or against pathogens, as compared to a plant that is not in shoot length or weight, increased fresh weight of seedlings, symbiosis. increased seed or fruit number, increased plant vigour, In some embodiments, the at least one endophyte is a nitrogen stress tolerance, enhanced Rhizobium activity, fungus of Subphylum Pezizomycotina. In some embodi enhanced nodulation frequency, early flowering time, or any ments, the at least one endophyte is a fungus of class combination thereof. In some embodiments, the increased Leotiomycetes, Dothideomycetes, Sordariomycetes, or tolerance to disease is increased tolerance to Fusarium Eurotiomycetes. In some embodiments, the at least one infection, increased tolerance to Septoria infection, and/or endophyte is of order Helotiales, Capnodides, Pleosporales, increased tolerance to Puccinia infection. In some embodi Hypocreales, or Eurotiales. In some embodiments, the at ments, yield is measured on a population of plants grown in 25 least one endophyte is selected from one of the following the field and is calculated via combine harvesting or mea families: Acarosporaceae, Adelococcaceae, Agyriaceae, Suring ear weight. For all altered traits, the change can be at Aigialaceae, Ajellomycetaceae, Amniculicolaceae. Amor least 1%, for example at least 2%, at least 3%, at least 4%, phothecaceae, Amphisphaeriaceae, Amplistromataceae, at least 5%, at least 10%, at least 20%, at least 30%, at least Anamylopsoraceae, Annulatascaceae, Anteagloniaceae, 40%, at least 50%, at least 60%, at least 75%, at least 100%, 30 Antennulariellaceae, Aphanopsidaceae, Apiosporaceae, or more, when compared with a control agricultural seed or Apiosporaceae, Arachnomycetaceae, Arctomiaceae, Arma plant. In some embodiments, the improved trait is heritable tellaceae, Arthoniaceae, Arthopyreniaceae, Arthrodermata by progeny of the agricultural plant grown from the seed. ceae, Arthrorhaphidaceae, Ascobolaceae, Ascocorticiaceae, In some embodiments, the agricultural seed is a seed of a Ascodesmidaceae, Ascodichaenaceae, Ascosphaeraceae, monocot plant. In some embodiments, the agricultural seed 35 Asterinaceae, Aulographaceae, Australiascaceae, Baeomyc is a seed of a cereal plant. In some embodiments, the etaceae, Bambusicolaceae, Batistiaceae, Bertiaceae, Biator agricultural seed is a seed of a corn, wheat, barley, rice, ellaceae, Biatriosporaceae, Bionectriaceae, Boliniaceae, Sorghum, millet, oats, rye or triticale. In some embodiments, Brigantiaeaceae, Bulgariaceae, BySSolomataceae, Calici the agricultural seed is a seed of a dicot plant. In some aceae, Caloscyphaceae, Calosphaeriaceae, Calycidiaceae, embodiments, the agricultural seed is a seed of cotton, 40 Candelariaceae, Capnodiaceae, Carbomycetaceae, Carboni canola, Soybean or a pulse. colaceae, Catabotrydaceae, Catillariaceae, Celotheliaceae, In some embodiments, a synthetic preparation is made Cephalothecaceae, Ceratocystidaceae, Ceratomycetaceae, comprising a canola seed and a composition comprising at Ceratostomataceae, Chadefaudiellaceae, Chaetomiaceae, least one endophyte capable of promoting germination and Chaetosphaerellaceae, Chaetosphaeriaceae, Chaetosphaeri an agriculturally-acceptable carrier, and a canola plant 45 aceae, Chaetothyriaceae, Chorioactidaceae, grown from the seed flowers earlier as compared to a control Chrysotrichaceae, Cladoniaceae, Cladosporiaceae, Clavi canola plant. In some embodiments, a synthetic preparation cipitaceae, Clypeosphaeriaceae, Coccocarpiaceae, Cocco is made comprising a tomato, alfalfa, corn, Swiss chard, diniaceae, Coccoideaceae, Coccotremataceae, Coenogoni radish, or cabbage seed and a composition comprising at aceae, Collemataceae, Coniocessiaceae, Coniochaetaceae, least one endophyte capable of promoting germination and 50 Coniocybaceae, Coniothyriaceae, Cordycipitaceae, Corono an agriculturally-acceptable carrier, and a tomato, alfalfa, phoraceae, Coryneliaceae, Corynesporascaceae, Crocyni corn, Swiss chard, radish, or cabbage plant grown under aceae, Cryphonectriaceae, Cryptomycetaceae, Cucurbitari drought conditions from the seed has higher biomass as aceae, Cudoniaceae, Cyphellophoraceae, Cyttariaceae, compared to a control plant grown under drought conditions. Dactylosporaceae, Davidiellaceae, Delitschiaceae, Der In some embodiments, the composition is disposed on an 55 mateaceae, Diademaceae, Diaporthaceae, Diatrypaceae, exterior Surface of the agricultural seed in an amount effec Didymellaceae, Didymosphaeriaceae, Discinaceae, Disso tive to colonize at least 0.1%, at least 1%, at least 2%, at least coniaceae, Dothideaceae, Dothidotthiaceae, Dothioraceae, 3%, at least 4%, at least 5%, at least 10%, at least 20%, at Ectolechiaceae, Elaphomycetaceae, Elixiaceae, Elsino least 30%, at least 40%, at least 50%, at least 60%, at least aceae, Eremascaceae, Eremithallaceae, Erysiphaceae, Euc 70% or at least 80% of cortical cells of a plant grown from 60 eratomycetaceae, Extremaceae, Fissurinaceae, Fuscide the seed. aceae, Geoglossaceae, Glaziellaceae, Gloeoheppiaceae, In some embodiments, the composition comprises a car Glomerellaceae, Glomerellaceae, Gnomoniaceae, Gomphil rier and at least one endophyte chosen from the group laceae, Gondwanamycetaceae, Graphidaceae, Graphostro consisting of a spore-forming endophyte, a facultative endo mataceae, Gyalectaceae, Gymnoascaceae, Gypsoplacaceae, phyte, a filamentous endophyte, and an endophyte capable 65 Haematommataceae, Halojulellaceae, Halosphaeriaceae, of living within another endophyte. In some embodiments, Halotthiaceae, Harknessiaceae, Helminthosphaeriaceae, the at least one endophyte is capable of forming certain Helotiaceae, Helvellaceae, Hemiphacidiaceae, Heppiaceae, US 9,687,001 B2 27 28 Herpomycetaceae, Herpotrichiellaceae, Hyaloscyphaceae, SEQ ID NO:5; an endophyte of Pseudeurotium sp. strain Hymeneliaceae, Hypocreaceae, Hyponectriaceae, Hyp deposited as IDAC 081111-02 or comprising a DNA Sostromataceae, Icmadophilaceae, Jobellisiaceae, Juncigen sequence with at least 97% identity to SEQ ID NO:4; an aceae, Karstenellaceae, Kathistaceae, Koerberiaceae, Kora endophyte of Penicillium sp. strain deposited as IDAC lionastetaceae, Laboulbeniaceae, Lachnaceae, 081111-01 or comprising a DNA sequence with at least 97% Lasiosphaeriaceae, Lecanoraceae, Lecideaceae, Lentith identity to SEQID NO:3: an endophyte of Cladosporium sp. eciaceae, Leotiaceae, Leprocaulaceae, Leptosphaeriaceae, strain deposited as IDAC 2003 12-06 or comprising a DNA Letrouiltiaceae, Lichinaceae, Lindgomycetaceae, Lobari sequence with at least 97% identity to SEQ ID NO:1; an aceae, Lophiostomataceae, Lophiotremataceae, Loramyc endophyte of Sarocladium sp. strain deposited as IDAC etaceae, Lulworthiaceae, Lyrommataceae, Magna 10 2003 12-05 or comprising a DNA sequence with at least 97% porthaceae, Malmideaceae, Massariaceae, Massarinaceae, identity to SEQ ID NO:2; and/or an endophyte of Strepto Megalariaceae, Megalosporaceae, Megasporaceae, Melan myces sp. strain deposited as IDAC 081111-06 or compris conidaceae, Melanommataceae, Melaspileaceae, Meli ing a DNA sequence with at least 97% sequence identity to olaceae, Metacapnodiaceae, Microascaceae, Miltideaceae, SEQ ID NO:6. In certain embodiments, the endophyte of Monascaceae, Monoblastiaceae, Montagnulaceae, Morchel 15 Paraconyothirium sp. Strain comprises a DNA sequence laceae, Morosphaeriaceae, Mycoblastaceae, Mycocalici with at least 98% identity to SEQ ID NO:5; the endophyte aceae, Mycosphaerellaceae, Myeloconidaceae, Myrian of Pseudeurotium sp. strain comprises a DNA sequence with giaceae, Myxotrichaceae, Nannizziopsidaceae, Nectriaceae, at least 98% identity to SEQ ID NO:4; the endophyte of Nephromataceae, Niessliaceae, Nitschkiaceae, Obryzaceae, Penicillium sp. strain comprises a DNA sequence with at Ochrolechiaceae, Odontotremataceae, Onygenaceae, Oph least 98% identity to SEQ ID NO:3: the endophyte of iocordycipitaceae, Ophioparmaceae, Ophiostomataceae, Cladosporium sp. Strain comprises a DNA sequence with at Orbiliaceae, Pachyascaceae, Pannariaceae, Pannariaceae, least 98% identity to SEQ ID NO:1; the endophyte of Papulosaceae, Parmeliaceae, Parmulariaceae, Peltigeraceae, Sarocladium sp. strain comprises a DNA sequence with at Peltulaceae, Pertusariaceae, Pezizaceae, Phacidiaceae, least 98% identity to SEQ ID NO:2; and the endophyte of Phaeochoraceae, Phaeococcomycetaceae, Phaeosphaeri 25 Streptomyces sp. Strain comprises a DNA sequence with at aceae, Phaeotrichaceae, Phaneromycetaceae, Phlyctidaceae, least 98% sequence identity to SEQ ID NO:6. In certain Phyllachoraceae, Physciaceae, Piedraiaceae, Pilocarpaceae, embodiments, the endophyte of Paraconyothirium sp. Strain Placynthiaceae, Platystomaceae, Plectosphaerellaceae, Pleo comprises a DNA sequence with at least 99% identity to massariaceae, Pleosporaceae, Pleurostomataceae, Pori SEQ ID NO:5; the endophyte of Pseudeurotium sp. strain naceae, Porpidiaceae, Protothelenellaceae, Pseudoplagiosto 30 comprises a DNA sequence with at least 99% identity to mataceae, Pseudovalsaceae, Psoraceae, Pycnoraceae, SEQ ID NO:4; the endophyte of Penicillium sp. strain Pyrenulaceae, Pyronemataceae, Pyxidiophoraceae, Ramali comprises a DNA sequence with at least 99% identity to naceae, Requienellaceae, Reticulascaceae, Rhizinaceae, SEQ ID NO:3: the endophyte of Cladosporium sp. strain Rhizocarpaceae, Rhynchostomataceae, Rhytismataceae, comprises a DNA sequence with at least 99% identity to Roccellaceae, Roccellographaceae, Ropalosporaceae, Rous 35 SEQ ID NO:1; the endophyte of Sarocladium sp. strain Soellaceae, Rutstroemiaceae, Sagiolechiaceae, Salsug comprises a DNA sequence with at least 99% identity to ineaceae, Sarcoscyphaceae, Sarcosomataceae, Sarramean SEQID NO:2; and the endophyte of Streptomyces sp. strain aceae, Schaereriaceae, Schizoparmaceae, comprises a DNA sequence with at least 99% sequence Schizoparmeaceae, Sclerotiniaceae, Scoliciosporaceae, identity to SEQ ID NO:6. In certain embodiments, the Scortechiniaceae, Shiraiaceae, Sordariaceae, Spathulospo 40 endophyte of Paraconyothirium sp. strain comprises a DNA raceae, Sphaerophoraceae, Sphinctrinaceae, Sporastati sequence of SEQID NO:5; the endophyte of Pseudeurotium aceae, Sporormiaceae, Stereocaulaceae, Stictidaceae, sp. strain comprises a DNA sequence of SEQ ID NO:4; the Strigulaceae, Sydowiellaceae, Sympoventuriaceae, Teicho endophyte of Penicillium sp. strain comprises a DNA sporaceae, Teloschistaceae, Teratosphaeriaceae, Testudi sequence of SEQID NO:3: the endophyte of Cladosporium naceae, Tetraplosphaeriaceae. Thelebolaceae. Thelenel 45 sp. strain comprises a DNA sequence of SEQ ID NO:1; the laceae. Thelocarpaceae. Thermoascaceae, Thyridariaceae, endophyte of Sarocladium sp. strain comprises a DNA Thyridiaceae, Thyridiaceae. Togniniaceae, Trapeliaceae, sequence of SEQ ID NO:2; and the endophyte of Strepto Trematosphaeriaceae, Trichocomaceae, Trichomeriaceae, myces sp. strain comprises a DNA sequence of SEQ ID Trichosphaeriaceae, Tuberaceae, Tubeufiaceae, Umbilicari NO:6. aceae, Vahliellaceae, Valsaceae, Venturiaceae, Verrucari 50 In some embodiments, the present disclosure provides a aceae, Vezdaeaceae, Vialaeaceae, Vibrisseaceae, Xanthopy synthetic preparation comprising an agricultural plant seed reniaceae, Xylariaceae, Xylonomycetaceae, and Zopfiaceae. and a composition comprising endophytes capable of pro In some embodiments, the composition comprises an moting germination and an agriculturally-acceptable carrier, agriculturally-acceptable carrier and at least one spore wherein the synthetic preparation has altered gene expres forming, filamentous bacterial endophyte of phylum Acti 55 sion in a plant grown from a seed inoculated with said nobacteria. In some embodiments, the at least one endophyte composition, as compared to a control plant. In some is a bacteria of order actinomycetales. In some embodi embodiments, the composition is disposed on an exterior ments, the at least one endophyte is selected from one of the Surface of an agricultural seed in an amount effective to following families: Actinomycetaceae, Actinopoly colonize the cortical cells of an agricultural plant grown sporineae, Catenulisporineae, Corynebacterineae, Franki 60 from the seed and to alter the expression of genes involved neae, Glycomycineae, Kineosporineae, Micrococcineae, in plant growth, genes associated with systemic acquired Micromonosporineae, Propionibacterineae, Pseudonocar resistance, or genes involved in protection from oxidative dineae, Streptomycineae, and Streptosporangineae. stress. In some embodiments, these genes may be involved In some embodiments, the present disclosure provides a in phytohormone production, for example in gibberellin composition comprising a carrier and an endophyte of 65 (GA) biosynthesis or breakdown, abscisic acid (ABA) bio Paraconyothirium sp. strain deposited as IDAC 081111-03 synthesis or breakdown, NO production or breakdown, or comprising a DNA sequence with at least 97% identity to Superoxide detoxification, or are positive or negative regu US 9,687,001 B2 29 30 lators of these pathways. In other embodiments, the genes least 5%, at least 10%, at least 20%, at least 30%, at least associated with systemic acquired resistance are redox 40%, at least 50% at least 60%, at least 70%, or at least 80% regulated transcription factors. In still other embodiments, of cortex cells of a plant grown from a seed inoculated with the redox-regulated transcription factors belong to the MYB said composition and wherein said plant has an improved family of genes. In some embodiments, the gene with altered 5 trait as compared to a control plant. In certain embodiments, expression is selected from the group consisting of P5CS, the plant grown from seed inoculated with the composition SOD, MnSOD, GA3-oxidase 2, 14-3-3, NCED2, has an improved trait selected from the group consisting of ABA8'OH1, RSG, KAO, Myb1 and Myb2. In some embodi increased yield, faster seedling establishment, faster growth, ments, the change in gene expression can be at least 1%, for increased drought tolerance, increased heat tolerance, example at least 2%, at least 3%, at least 4%, at least 5%, at 10 increased cold tolerance, increased salt tolerance, increased least 10%, at least 20%, at least 30%, at least 40%, at least tolerance to Fusarium infection, increased biomass, 50%, at least 60%, at least 75%, at least 100%, or more, increased root length, increased fresh weight of Seedlings, when compared with a control agricultural seed or plant. In increased plant vigour, nitrogen stress tolerance, enhanced Some embodiments, said composition is disposed on an Rhizobium activity, enhanced nodulation frequency and exterior Surface of an agricultural seed in an amount effec 15 early flowering time compared to a control plant. tive to colonize at least 0.1%, at least 1%, at least 2%, at least In another embodiment, the synthetic preparations and 3%, at least 4%, at least 5%, at least 10%, at least 20%, at compositions described herein comprise two or more (e.g., least 30%, at least 40%, at least 50%, at least 60%, at least 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or 70%, or at least 80% of the cortical cells of an agricultural more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or plant grown from the seed and to alter the expression of more, or greater than 25) different endophytes capable of genes involved in plant growth, genes associated with sys promoting germination, e.g., obtained from different fami temic acquired resistance, or genes involved in protection lies or different genera of fungi or bacteria, or from the same from oxidative stress. genera but different species of fungi or bacteria. In embodi In some embodiments, the present disclosure provides a ments in which two or more endophytes capable of promot composition comprising at least one endophyte capable of 25 ing germination are used, each of the endophytes capable of promoting germination and an agriculturally-acceptable car promoting germination can have different properties or rier, wherein said composition is disposed on an exterior activities, confer different beneficial traits, or colonize dif Surface of an agricultural seed in an amount effective to ferent parts of a plant (e.g., leaves, stems, flowers, fruits, cause a population of seeds inoculated with said composi seeds, or roots). For example, one endophyte capable of tion to have a faster dormancy breakdown, greater germi 30 promoting germination can colonize a first tissue and a nation rate, earlier germination, increased energy of germi second endophyte capable of promoting germination can nation, greater rate of germination, greater uniformity of colonize a tissue that differs from the first tissue. Alterna germination, including greater uniformity of rate of germi tively, each of the endophytes capable of promoting germi nation and greater uniformity of timing of germination, nation can have similar properties or activities, confer simi and/or increased energy of germination as compared to a 35 lar beneficial traits, or colonize different parts of a plant. population of control seeds. In some embodiments, the The synthetic combination or preparation of the present composition is disposed on the Surface or within a tissue of invention contemplates the presence of an endophyte on the an agricultural seed or seedling in an amount effective to surface of the seed of the first plant. In one embodiment, the cause a population of seeds inoculated with said composi seed of the first plant is coated with at least 10 CFU of the tion to reach 50% germination faster than a population of 40 endophyte per seed, for example, at least 20 CFU, at least 50 control seeds or to cause increased NO accumulation in a CFU, at least 100 CFU, at least 200 CFU, at least 300 CFU, plant grown from a seed inoculated with said composition, at least 500 CFU, at least 1,000 CFU, at least 3,000 CFU, at as compared to a control plant. In other embodiments, the least 10,000 CFU, or at least 30,000 CFU or more per seed. composition is disposed on an exterior Surface of an agri In another embodiment, the seed is coated with at least 10, cultural seed an in an amount effective to cause altered levels 45 for example, at least 20, at least 50, at least 100, at least 200, of phytohormones to be produced in an agricultural plant at least 300, at least 500, at least 1,000, at least 3,000, at least grown from the seed, as compared to a control agricultural 10,000, at least 30,000, at least 100,000, at least 300,000, at plant. In some embodiments, the phytohormones that are least 1,000,000 or more of the endophyte as determined by altered are gibberellins, abscisic acid, or cytokinins. In the number of copies of a particular endophyte gene further embodiments, the gibberellins may be gibberellin 1, 50 detected, for example, by quantitative PCR. 19, 44 or 53. In still further embodiments, the cytokinin may Further provided herein is a seed inoculated with any of be zeatin. For all these altered traits (a faster dormancy the compositions described herein. In one embodiment, the breakdown, greater germination rate, earlier germination, seed is inoculated by soil-based inoculation. In another increased energy of germination, greater rate of germination, embodiment, the seed is coated with an endophyte or culture greater uniformity of germination, including greater unifor 55 thereof. In yet another embodiment, the seed is sprayed, mity of rate of germination and greater uniformity of timing injected, inoculated, grafted, coated or treated with the of germination, increased energy of germination, 50% ger endophyte or culture thereof. In an embodiment, the seed is mination, increased NO accumulation, and altered levels of planted near an endophyte. In one embodiment, the seed phytohormones), the change can be at least 1%, for example planted near the endophyte is about 4 cm away from the at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, 60 endophyte. at least 20%, at least 30%, at least 40%, at least 50%, at least In another aspect, the invention provides a population of 60%, at least 75%, at least 100%, or more, when compared at least 10 synthetic preparations, each synthetic preparation with a control agricultural seed or plant. comprising an agricultural plant seed and a composition In some embodiments, the present disclosure provides a comprising at least one endophyte capable of promoting composition comprising at least one endophyte and a carrier, 65 germination and an agriculturally-acceptable carrier, where wherein said composition is capable of colonizing at least the population is comprised within a packaging material. 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at The packaging material can be selected from a bag, box, bin, US 9,687,001 B2 31 32 envelope, carton, or container. In an embodiment, the Syn In one embodiment, spontaneous isolates of microbes that thetic preparation can be disposed within a package and is are compatible with agrichemicals can be used to inoculate shelf stable. In another embodiment, the invention features the plants according to the methods described herein. For an agricultural product that includes a predetermined num example, fungal microbes which are compatible with agri ber of seeds or a predetermined weight of seeds. In an 5 culturally employed fungicides can be isolated by plating a embodiment, the bag or container contains at least 1000 culture of the microbes on a petri dish containing an effec seeds, wherein the packaging material optionally comprises tive concentration of the fungicide, and isolating colonies of a dessicant, and wherein the synthetic preparation optionally the microbe that are compatible with the fungicide. In comprises an anti-fungal agent. another embodiment, a microbe that is compatible with a In yet another aspect, the invention features an article of 10 fungicide is used for the methods described herein. For manufacture that includes packaging material; one or more example, the endophyte can be compatible with at least one plant seeds within the packaging material, and at least one of the fungicides selected from the group consisting of species of endophytes capable of promoting germination 2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, associated with the seeds. The article can include two or 8-hydroxyquinoline Sulfate, ametoctradin, amisulbrom, anti 15 mycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, more species of endophytes capable of promoting germina Bacillus subtilis, benalaxyl, benomyl, benthiavalicarb-iso tion. propyl, benzylaminobenzene-sulfonate (BABS) salt, bicar In another aspect, the invention features an agricultural bonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasti product that includes a predetermined number of seeds or a cidin-S, borax, Bordeaux mixture, boScalid, bromuconazole, predetermined weight of seeds. In an embodiment, the bag bupirimate, calcium polysulfide, captafol, captan, carben or container contains at least 1000 seeds of a synthetic dazim, carboxin, carpropamid, carvone, chloroneb, chloro preparation produced by the step of inoculating a plurality of thalonil, chloZolinate, Coniothyrium minitans, copper plant seeds with a formulation comprising a fungal or hydroxide, copper octanoate, copper oxychloride, copper bacterial population at a concentration of at least 1 CFU per Sulfate, copper Sulfate (tribasic), cuprous oxide, cyaZofamid, agricultural plant seed, wherein at least 10% of the CFUs 25 cyflufenamid, cymoxanil, cyproconazole, cyprodinil, present in the formulation are one or more endophytes dazomet, debacarb, diammonium ethylenebis-(dithiocar capable of promoting germination, under conditions such bamate), dichlofluanid, dichlorophen, diclocymet, diclom that the formulation is associated with the surface of the eZine, dichloran, diethofencarb, difenoconazole, difenzo seeds in a manner effective for the endophytes capable of quat ion, diflumetorim, dimethomorph, dimoxystrobin, promoting germination to confer a benefit to the seeds or to 30 diniconazole, diniconazole-M, dinobuton, dinocap, diphe a crop comprising a plurality of agricultural plants produced nylamine, dithianon, dodemorph, dodemorph acetate, from the seeds. The endophytes capable of promoting ger dodine, dodine free base, edifenphos, enestrobin, epoxicon mination can be present in a concentration of from about 10 azole, ethaboxam, ethoxyquin, etridiazole, famoxadone, to about 10 CFU/ml or from about 10 to about 10 fenamidone, fenarimol, fenbuconazole, fenfuram, fenhex CFU/seed. The formulation can be a liquid and the fungal or 35 amid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, bacterial concentration can be from about 10 to about 10' fentin, fentin acetate, fentin hydroxide, ferbam, ferimZone, CFU/ml. The formulation can be a gel or powder and the fluaZinam, fludioxonil, flumorph, fluopicolide, fluopyram, fungal or bacterial concentration can be from about 10 to fluoroimide, fluoxastrobin, fluguinconazole, flusilazole, flu about 10' CFU/gm. sulfamide, flutianil, flutolanil, flutriafol, fluxapyroxad, fol In some cases, the endophytic microbe can be modified. 40 pet, formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, For example, the endophytic microbe can be genetically furalaxyl, furametpyr, guaZatine, guaZatine acetates, GY-81, modified by introduction of a transgene that stably integrates hexachlorobenzene, hexaconazole, hymexaZol, imazalil, into its genome. In another embodiment, the endophytic imazalil Sulfate, imibenconazole, iminoctadine, iminocta microbe can be modified to harbor a plasmid or episome dine triacetate, iminoctadine tris(albesilate), ipconazole, containing a transgene. In still another embodiment, the 45 iprobenfos, iprodione, iprovalicarb, isoprothiolane, isopyra microbe can be modified by repeated passaging under selec Zam, isotianil, kasugamycin, kasugamycin hydrochloride tive conditions. hydrate, kresoxim-methyl, mancopper, mancoZeb, mandip The microbe can be modified to exhibit altered charac ropamid, maneb, mepanipyrim, mepronil, mercuric chlo teristics. In one embodiment, the endophytic microbe is ride, mercuric oxide, mercurous chloride, metalaxyl, modified to exhibit increased compatibility with chemicals 50 mefenoxam, metalaxyl-M, metam, metam-ammonium, commonly used in agriculture. Agricultural plants are often metam-potassium, metam-Sodium, metconazole, methasul treated with a vast array of agrichemicals, including fungi focarb, methyl iodide, methyl isothiocyanate, metiram, cides, biocides (anti-bacterial and anti-fungal agents), her metominostrobin, metrafenone, mildiomycin, myclobutanil, bicides, insecticides, nematicides, rodenticides, fertilizers, nabam, nitrothal-isopropyl. nuarimol, octhillinone, ofurace, and other agents. Many Such agents can affect the ability of 55 oleic acid (fatty acids), orysastrobin, oxadixyl, oxine-cop an endophytic microbe to grow, divide, and/or otherwise per, Oxpoconazole fumarate, oxycarboxin, pefurazoate, pen confer beneficial traits to the plant. conazole, pencycuron, penflufen, pentachlorophenol, pen In some cases, it can be important for the microbe to be tachlorophenyl laurate, penthiopyrad, phenylmercury compatible with agrichemicals, particularly those with fun acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin gicidal or antibacterial properties, in order to persist in the 60 B, polyoxins, polyoxorim, potassium bicarbonate, potas plant although, as mentioned earlier, there are many Such sium hydroxyquinoline Sulfate, probenazole, prochloraZ. fungicidal or antibacterial agents that do not penetrate the procymidone, propamocarb, propamocarb hydrochloride, plant, at least at a concentration Sufficient to interfere with propiconazole, propineb, produinazid, prothioconazole, the microbe. Therefore, where a systemic fungicide or pyraclostrobin, pyrametostrobin, pyraoxystrobin, pyrazo antibacterial agent is used in the plant, compatibility of the 65 phos, pyribencarb, pyributicarb, pyrifenox, pyrimethanil, microbe to be inoculated with such agents will be an pyroquilon, quinoclamine, quinoxyfen, quintoZene, Reynou important criterion. tria Sachalinensis extract, sedaxane, silthiofam, Simecon US 9,687,001 B2 33 34 azole, Sodium 2-phenylphenoxide, Sodium bicarbonate, eZolid, PosiZolid, Radezolid, Torezolid, Amoxicillin, sodium pentachlorophenoxide, spiroxamine, sulfur, SYP Ampicillin, AZlocillin, Carbenicillin, Cloxacillin, Dicloxa Z071, SYP-Z048, tar oils, tebuconazole, tebufloquin, tecna cillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Zene, tetraconazole, thiabendazole, thifluZamide, thiophan Oxacillin, Penicillin G, Penicillin V. Piperacillin, Penicillin ate-methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, G, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampi triadimefon, triadimenol, triaZOxide, tricyclazole, tride cillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavu morph, trifloxystrobin, triflumizole, triforine, triticonazole, lanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, validamycin, Valifenalate, Valiphenal, VincloZolin, Zineb, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxi Ziram, Zoxamide, Candida oleophila, Fusarium oxysporum, floxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trova Gliocladium spp., Phlebiopsis gigantea, Streptomyces gris 10 floxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, eoviridis, Trichoderma spp., (RS) N-(3,5-dichlorophenyl)- Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, 2-(methoxymethyl)-Succinimide, 1,2-dichloropropane, 1.3- Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sul dichloro-1, 1,3,3-tetrafluoroacetone hydrate, 1-chloro-2,4- fanilimide (archaic), Sulfasalazine, Sulfisoxazole, dinitronaphthalene, 1-chloro-2-nitropropane, 2-(2- Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP heptadecyl-2-imidazolin-1-yl)ethanol, 2,3-dihydro-5- 15 SMX), Sulfonamidochrysoidine (archaic), Demeclocycline, phenyl-1,4-dithi-line 1.1.4,4-tetraoxide, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, 2-methoxyethylmercury acetate, 2-methoxyethylmercury Clofazimine, Dapsone, Capreomycin, Cycloserine, Etham chloride, 2-methoxyethylmercury silicate, 3-(4-chlorophe butol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin nyl)-5-methylrhodanine, 4-(2-nitroprop-1-enyl)phenyl thio (Rifampin in US), Rifabutin, Rifapentine, Streptomycin, cyanateme, ampropylfos, anilazine, azithiram, barium poly Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, sulfide, Bayer 32394, benodanil, benquinox, bentaluron, Metronidazole, Mupirocin, Platensimycin, Quinupristin/ benzamacril; benzamacril-isobutyl, benzamorf, binapacryl, Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, and bis(methylmercury) sulfate, bis(tributyltin) oxide, buthio Trimethoprim. bate, cadmium calcium copper Zinc chromate Sulfate, car Fungicide compatible microbes can also be isolated by bamorph, CECA, chlobenthiazone, chloraniformethan, 25 selection on liquid medium. The culture of microbes can be chlorfenazole, chlorquinox, climbazole, cyclafuramid, plated on petri dishes without any forms of mutagenesis; cypendazole, cyprofuram, decafentin, dichlone, dichloZo alternatively, the microbes can be mutagenized using any line, diclobutraZol, dimethirimol, dinocton, dinosulfon, means known in the art. For example, microbial cultures can dinoterbon, dipyrithione, ditalimfos, dodicin, draZOXolon, be exposed to UV light, gamma-irradiation, or a chemical EBP, ESBP. etaconazole, etem, ethirim, fenaminosulf, fena 30 mutagen such as ethylmethanesulfonate (EMS) prior to panil, fenitropan, 5-fluorocytosine and profungicides selection on fungicide containing media. Finally, where the thereof, fluotrimazole, furcarbanil, furconazole, furcon mechanism of action of a particular fungicide is known, the azole-cis, furmecyclox, furophanate, glyodine, griseofulvin, target gene can be specifically mutated (either by gene halacrinate, Hercules 3944, hexylthiofos, ICIA0858, iso deletion, gene replacement, site-directed mutagenesis, etc.) pamphos, isovaledione, mebenil, mecarbinZid, metaZOXo 35 to generate a microbe that is resilient against that particular lon, methfuroxam, methylmercury dicyandiamide, metSul fungicide. It is noted that the above-described methods can foVax, milineb, mucochloric anhydride, myclozolin, N-3,5- be used to isolate fungi that are compatible with both dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, fungistatic and fungicidal compounds. natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel It will also be appreciated by one skilled in the art that a bis(dimethyldithiocarbamate), OCH, phenylmercury dim 40 plant may be exposed to multiple types of fungicides or ethyldithiocarbamate, phenylmercury nitrate, phosdiphen, antibacterial compounds, either simultaneously or in Suc picolinamide UK-2A and derivatives thereof, prothiocarb: cession, for example at different stages of plant growth. prothiocarb hydrochloride, pyracarbolid, pyridinitril, Where the target plant is likely to be exposed to multiple pyroxychlor, pyroxyfur, quinacetol; quinacetol Sulfate, qui fungicidal and/or antibacterial agents, a microbe that is naZamid, quinconazole, rabenzazole, salicylanilide, SSF 45 compatible with many or all of these agrichemicals can be 109, Sultropen, tecoram, thiadifluor, thicyofen, thiochlorfen used to inoculate the plant. A microbe that is compatible phim, thiophanate, thioquinox, tioxymid, triamiphos, with several fungicidal agents can be isolated, for example, triarimol, triazbutil, trichlamide, urbacid, XRD-563, and by serial selection. A microbe that is compatible with the Zarilamide, IK-1140 first fungicidal agent is isolated as described above (with or In still another embodiment, an endophyte that is com 50 without prior mutagenesis). A culture of the resulting patible with an antibacterial compound is used for the microbe can then be selected for the ability to grow on liquid methods described herein. For example, the endophyte can or Solid media containing the second antifungal compound be compatible with at least one of the antibiotics selected (again, with or without prior mutagenesis). Colonies isolated from the group consisting of Amikacin, Gentamicin, from the second selection are then tested to confirm its Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromo 55 compatibility to both antifungal compounds. mycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaxi Likewise, bacterial microbes that are compatible to bio min, Streptomycin, Loracarbef, Ertapenem, Doripenem, Imi cides (including herbicides such as glyphosate or antibac penem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, terial compounds, whether bacteriostatic or bactericidal) that Cefalotin or Cefalothin, Cefalexin, Cefaclor, Cefamandole, are agriculturally employed can be isolated using methods Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, 60 similar to those described for isolating fungicide compatible Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Cef microbes. In one embodiment, mutagenesis of the microbial tazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, population can be performed prior to selection with an Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomy antibacterial agent. In another embodiment, selection is cin, Telavancin, Clindamycin, Lincomycin, Daptomycin, performed on the microbial population without prior muta Azithromycin, Clarithromycin, Dirithromycin, Erythromy 65 genesis. In still another embodiment, serial selection is cin, Roxithromycin, Troleandomycin, Telithromycin, Spi performed on a microbe: the microbe is first selected for ramycin, Aztreonam, Furazolidone, Nitrofurantoin, Lin compatibility to a first antibacterial agent. The isolated US 9,687,001 B2 35 36 compatible microbe is then cultured and selected for com increased drought tolerance, increased heat tolerance, patibility to the second antibacterial agent. Any colony thus increased cold tolerance, increased salt tolerance, increased isolated is tested for compatibility to each, or both antibac tolerance to pests and diseases, increased biomass, increased terial agents to confirm compatibility with these two agents. root and/or shoot length or weight, increased fresh weight of The selection process described above can be repeated to seedlings, increased seed or fruit number, increased plant identify isolates of the microbe that are compatible with a vigour, nitrogen stress tolerance, enhanced Rhizobium activ multitude of antifungal or antibacterial agents. Candidate ity, enhanced nodulation frequency, early flowering time, or isolates can be tested to ensure that the selection for any combination thereof. In some embodiments, the agrichemical compatibility did not result in loss of a desired increased tolerance to disease is increased tolerance to microbial bioactivity. Isolates of the microbe that are com 10 Fusarium infection, increased tolerance to Septoria infec patible with commonly employed fungicides can be selected tion, increased tolerance to Puccinia infection. In some as described above. The resulting compatible microbe can be embodiments, yield is measured on a population of plants compared with the parental microbe on plants in its ability grown in the field and is calculated via combine harvesting to promote germination. or measuring ear weight. In another aspect, the altered trait Methods 15 is a seed trait selected from the group consisting a greater Further provided herein are methods of enhancing seed germination rate, faster dormancy breakdown, increased Vitality, plant health and/or yield comprising inoculating a energy of germination, increased seed germination vigor or seed with an endophyte or culture disclosed herein or a increased seed vitality. In yet another embodiment, the combination or mixture thereof or with a composition dis altered trait is altered gene expression, wherein the gene is closed herein. In some embodiments, a first generation plant selected from the group consisting of a gene involved in is cultivated from the seed. gibberellin production, a gene involved in abscisic acid In one aspect, the invention provides a method of altering production, a gene involved in plant growth, an acquired a trait in an agricultural plant seed or an agricultural plant resistance gene, and a gene involved in protection from grown from said seed, said method comprising inoculating oxidative stress. In some embodiments, the genes may be said seed with a composition comprising endophytes 25 involved in phytohormone production. In some embodi capable of promoting germination and an agriculturally ments, the phytohormone is altered in the plant-fungus or acceptable carrier, wherein the endophyte replicates within plant-bacterial symbiotic system. In some embodiments, the at least one plant tissue and colonizes the cortical cells of method further comprises planting the agricultural plant said plant. In one embodiment, the endophyte capable of seed. In another embodiment, the method further comprises promoting germination is a coleorhiza-activating endophyte, 30 selecting a plant seed or plant that has the altered trait. For and the seed is a monocot seed. In another embodiment, the all altered traits, the change can be at least 1%, for example endophyte capable of promoting germination is heterolo at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, gous to the seed. at least 20%, at least 30%, at least 40%, at least 50%, at least In some embodiments, the endophytes are a selected from 60%, at least 75%, at least 100%, or more, when compared the group consisting of a spore-forming endophyte, a fac 35 with a control agricultural seed or plant. ultative endophyte, a filamentous endophyte, and an endo In one embodiment, the invention provides methods of phyte capable of living within another endophyte. In some improving the 50% germination rate of a population of seeds embodiments, the endophyte is capable of forming certain comprising inoculating said population of seeds with a structures in the plant, where the structures are selected from composition as described herein. In one embodiment, the the group consisting of hyphal coils, Hartig-like nets, 40 method is a method of improving the 50% germination rate microvesicles, micro-arbuscules, hyphal knots, and Symbio of a population of seeds and of improving a trait in plants Somes. In some embodiments, the endophyte is in the form grown from the seeds, comprising inoculating said popula of at least one of conidia, chlamydospore, and mycelia. In tion of seeds with a composition as described herein. In one other embodiments, the fungus or bacteria is capable of embodiment, the improved trait is selected from the group being part of a plant-fungus symbiotic system or plant 45 consisting of increased yield, faster seedling establishment, bacteria symbiotic system that produces altered levels of faster growth, increased drought tolerance, increased heat phytohormones or anti-oxidants, as compared to a plant that tolerance, increased cold tolerance, increased salt tolerance, is not in Symbiosis. In other embodiments, the plant-fungus increased tolerance to pests and diseases, increased biomass, symbiotic system or plant-bacterium symbiotic system has increased root and/or shoot length or weight, increased fresh anti-aging and/or anti-senescence effects, as compared to a 50 weight of seedlings, increased seed or fruit number, plant or plant organ that is not in Symbiosis. In other increased plant vigour, nitrogen stress tolerance, enhanced embodiments, the plant-fungus symbiotic system or plant Rhizobium activity, enhanced nodulation frequency, early bacteria symbiotic system has increased protection against flowering time, or any combination thereof. In some pathogens, as compared to a plant that is not in Symbiosis. embodiments, the increased tolerance to disease is increased In other aspects, the endophyte colonizes at least 0.1%, at 55 tolerance to Fusarium infection, increased tolerance to Sep least 1%, at least 2%, at least 3%, at least 4%, at least 5%, toria infection, increased tolerance to Puccinia infection. In at least 10%, at least 20%, at least 30%, at least 40%, at least Some embodiments, yield is measured on a population of 50%, at least 60%, at least 70%, or at least 80% of the plants grown in the field and is calculated via combine cortical cells of said agricultural plant. harvesting or measuring ear weight. For all altered traits, the In yet another aspect, the invention provides a method of 60 change can be at least 1%, for example at least 2%, at least altering a trait in an agricultural plant seed or an agricultural 3%, at least 4%, at least 5%, at least 10%, at least 20%, at plant grown from said seed, said method comprising inocu least 30%, at least 40%, at least 50%, at least 60%, at least lating said seed with a composition comprising endophytes 75%, at least 100%, or more, when compared with a control capable of promoting germination and an agriculturally agricultural seed or plant. In some embodiments, the method acceptable carrier, wherein the altered trait is an improved 65 further comprises planting the agricultural plant seed. In functional trait selected from the group consisting of another embodiment, the method further comprises selecting increased yield, faster seedling establishment, faster growth, a plant seed or plant that has the altered trait. US 9,687,001 B2 37 38 In one embodiment, the method is a method of improving In one embodiment, the method is a method of increasing the 50% germination rate of a population of seeds and the germination rate, speeding up dormancy breakdown, altering the gene expression in a plant grown from the seeds, increasing the energy of germination, increasing the germi comprising inoculating said population of seeds with a nation vigour, speeding up germination, increasing the composition as described herein. In some embodiments, the energy of germination, producing greater uniformity of gene is altered in the plant-fungus or plant-bacterial symbi germination, including greater uniformity of rate of germi otic system. In some embodiments, the gene with altered nation and greater uniformity of timing of germination, or expression is a gene involved in plant growth, an acquired increasing the vitality of a seed, comprising inoculating resistance gene, and a gene involved in protection from seeds or a population of seeds with a composition as 10 described herein. In some embodiments, the method further oxidative stress. In some embodiments, these genes may be comprises planting the agricultural plant seed. involved in phytohormone production, such as those In one embodiment, the invention provides a method of involved in GA biosynthesis or breakdown, abscisic acid releasing a seed from dormancy, said method comprising (ABA) biosynthesis or breakdown, NO production or break inoculating said seed with a composition comprising endo down, Superoxide detoxification, or are positive or negative 15 phytes capable of promoting germination and an agricultur regulators of these pathways. In other embodiments, the ally-acceptable carrier. In some embodiments, the endo genes associated with systemic acquired resistance are phytes capable of promoting germination are coleorhiza redox-regulated transcription factors. In still other embodi activating endophytes. ments, the redox-regulated transcription factors belong to In one embodiment, the invention provides a method of the MYB family of genes. In some embodiments, the gene improving the 50% germination rate of a population of seeds with altered expression is selected from the group consisting and increasing NO accumulation in a plant grown from the of P5CS, SOD, MnSOD, GA3-oxidase 2, 14-3-3, NCED2, seeds, comprising inoculating seeds with a composition as ABA8'OH1, RSG, KAO, Myb1 and Myb2. In some embodi described herein. In some embodiments, the method further ments, the change in gene expression can be at least 1%, for comprises planting the agricultural plant seed. example at least 2%, at least 3%, at least 4%, at least 5%, at 25 In another embodiment, a method of altering a trait in an least 10%, at least 20%, at least 30%, at least 40%, at least agricultural plant seed or an agricultural plant grown from 50%, at least 60%, at least 75%, at least 100%, or more, said seed is disclosed, comprising obtaining a synthetic when compared with a control agricultural seed or plant. In preparation comprising an agricultural plant seed and a Some embodiments, the method further comprises planting composition comprising endophytes capable of promoting the agricultural plant seed. In another embodiment, the 30 germination and an agriculturally-acceptable carrier and method further comprises selecting a plant seed or plant that planting the synthetic preparation. In some embodiments, has the altered trait. the method further comprises planting the agricultural plant In one embodiment, a method of improving the 50% seed. In another embodiment, the method further comprises germination rate of a population of seeds and providing at selecting a plant seed or plant that has the altered trait. least 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, 35 In another embodiment, the invention provides a method at least 5%, at least 10%, at least 20%, at least 30%, at least for treating seeds comprising contacting the Surface of an 40%, at least 50%, at least 60%, at least 70%, or at least 80% agricultural plant seed with a formulation comprising a colonization in cortex cells of plants grown from the seeds microbial population that comprises an endophyte capable is provided. of promoting germination that is heterologous to the seed, In one embodiment, methods of obtaining at least 0.1%, 40 wherein the endophyte capable of promoting germination is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, present in the formulation in an amount effective to alter the at least 10%, at least 20%, at least 30%, at least 40%, at least level of at least one gene within the seed, seedlings derived 50%, at least 60%, at least 70%, or at least 80% endophyte from the seed or agricultural plants derived from the seed. In colonization in the cortex cells of a plant and of improving Some embodiments, the gene with altered expression is a a trait in the plant are disclosed, comprising inoculating the 45 gene involved in phytohormone production, an acquired seed of said plant with a composition as described herein. In resistance gene, and a gene involved in protection from one embodiment, the improved trait is selected from the oxidative stress. In some embodiments, these genes are group consisting of increased yield, faster seedling estab those involved in GA biosynthesis or breakdown, abscisic lishment, faster growth, increased drought tolerance, acid (ABA) biosynthesis or breakdown, NO production or increased heat tolerance, increased cold tolerance, increased 50 breakdown, Superoxide detoxification, or are positive or salt tolerance, increased tolerance to pests and diseases, negative regulators of these pathways. In other embodi increased biomass, increased root and/or shoot length or ments, the genes associated with systemic acquired resis weight, increased fresh weight of Seedlings, increased seed tance are redox-regulated transcription factors. In still other or fruit number, increased plant vigour, nitrogen stress embodiments, the redox-regulated transcription factors tolerance, enhanced Rhizobium activity, enhanced nodula 55 belong to the MYB family of genes. In some embodiments, tion frequency, early flowering time, or any combination the gene with altered expression is selected from the group thereof. In some embodiments, the increased tolerance to consisting of P5CS, SOD, MnSOD, GA3-oxidase 2, 14-3-3, disease is increased tolerance to Fusarium infection, NCED2, ABA8'OH1, RSG, KAO, Myb1 and Myb2. increased tolerance to Septoria infection, increased toler In another embodiment, the invention provides a method ance to Puccinia infection. In some embodiments, yield is 60 for treating seeds comprising contacting the Surface of an measured on a population of plants grown in the field and is agricultural plant seed with a formulation comprising a calculated via combine harvesting or measuring ear weight. microbial population that comprises an endophyte capable For all altered traits, the change can be at least 1%, for of promoting germination that is heterologous to the seed, example at least 2%, at least 3%, at least 4%, at least 5%, at wherein the endophyte capable of promoting germination is least 10%, at least 20%, at least 30%, at least 40%, at least 65 present in the formulation in an amount effective to alter the 50%, at least 60%, at least 75%, at least 100%, or more, level of at least one phytohormone within the seed, seedlings when compared with a control agricultural seed or plant. derived from the seed or agricultural plants derived from the US 9,687,001 B2 39 40 seed. In some embodiments, the phytohormones that are include at least one member selected from the group con altered are gibberellins, abscisic acid, or cytokinins. In sisting of a tackifier, a microbial stabilizer, a fungicide, an further embodiments, the gibberellins may be gibberellin 1, antibacterial agent, an herbicide, a nematicide, an insecti 19, 44 or 53. In still further embodiments, the cytokinin may cide, a plant growth regulator, a rodenticide, a dessicant, and be zeatin. For these altered phytohormone levels, the change a nutrient. can be at least 1%, for example at least 2%, at least 3%, at In some cases, the purified bacterial or fungal population least 4%, at least 5%, at least 10%, at least 20%, at least is mixed with an agriculturally compatible carrier. The 30%, at least 40%, at least 50%, at least 60%, at least 75%, carrier can be a solid carrier or liquid carrier, and in various at least 100%, or more, when compared with a control forms including microspheres, powders, emulsions and the agricultural seed or plant. 10 like. The carrier may be any one or more of a number of In another embodiment, the invention provides a method carriers that confer a variety of properties, such as increased for treating seeds comprising contacting the Surface of an stability, wettability, or dispersability. Wetting agents such agricultural plant seed with a formulation comprising a as natural or synthetic Surfactants, which can be nonionic or microbial population that comprises an endophyte capable ionic Surfactants, or a combination thereof can be included of promoting germination that is heterologous to the seed, 15 in a composition of the invention. Water-in-oil emulsions wherein the endophyte capable of promoting germination is can also be used to formulate a composition that includes the present in the formulation in an amount effective to improve purified bacterial or fungal population (see, for example, a trait in the seed or a plant grown from the seed. In some U.S. Pat. No. 7,485,451, which is incorporated herein by embodiments, the improved trait is selected from the group reference in its entirety). Suitable formulations that may be consisting of increased yield, faster seedling establishment, prepared include wettable powders, granules, gels, agar faster growth, increased drought tolerance, increased heat strips or pellets, thickeners, and the like, microencapsulated tolerance, increased cold tolerance, increased salt tolerance, particles, and the like, liquids such as aqueous flowables, increased tolerance to pests and diseases, increased biomass, aqueous Suspensions, water-in-oil emulsions, etc. The for increased root and/or shoot length or weight, increased fresh mulation may include grain or legume products, for weight of seedlings, increased seed or fruit number, 25 example, ground grain or beans, broth or flour derived from increased plant vigour, nitrogen stress tolerance, enhanced grain or beans, starch, Sugar, or oil. Rhizobium activity, enhanced nodulation frequency, early In some embodiments, the agricultural carrier may be soil flowering time, or any combination thereof. In some or a plant growth medium. Other agricultural carriers that embodiments, the increased tolerance to disease is increased may be used include water, fertilizers, plant-based oils, tolerance to Fusarium infection, increased tolerance to Sep 30 humectants, or combinations thereof. Alternatively, the agri toria infection, increased tolerance to Puccinia infection. In cultural carrier may be a solid, Such as diatomaceous earth, some embodiments, yield is measured on a population of loam, silica, alginate, clay, bentonite, vermiculite, seed plants grown in the field and is calculated via combine cases, other plant and animal products, or combinations, harvesting or measuring ear weight. For all altered traits, the including granules, pellets, or Suspensions. Mixtures of any change can be at least 1%, for example at least 2%, at least 35 of the aforementioned ingredients are also contemplated as 3%, at least 4%, at least 5%, at least 10%, at least 20%, at carriers, such as but not limited to, pesta (flour and kaolin least 30%, at least 40%, at least 50%, at least 60%, at least clay), agar or flour-based pellets in loam, sand, or clay, etc. 75%, at least 100%, or more, when compared with a control Formulations may include food sources for the cultured agricultural seed or plant. In some embodiments, the method organisms, such as barley, rice, or other biological materials further comprises planting the agricultural plant seed. In 40 Such as seed, plant parts, Sugar cane bagasse, hulls or stalks another embodiment, the method further comprises selecting from grain processing, ground plant material or wood from a plant seed or plant that has the altered trait. building site refuse, sawdust or Small fibers from recycling In another aspect, there is provided a method of improv of paper, fabric, or wood. Other suitable formulations will be ing plant health and/or plant yield comprising treating plant known to those skilled in the art. propagation material or a plant with a composition disclosed 45 In one embodiment, the formulation can include a tacki herein; and cultivating the plant propagation material into a fier or adherent. Such agents are useful for combining the first generation plant or allowing the plant to grow. bacterial or fungal population of the invention with carriers In another embodiment, the methods reduce the effects of that can contain other compounds (e.g., control agents that stress, such as heat, drought and/or biotic stress. are not biologic), to yield a coating composition. Such In an embodiment, the methods enhance landscape devel 50 compositions help create coatings around the plant or seed opment and remediation. to maintain contact between the microbe and other agents Accordingly, in one embodiment, there is provided a with the plant or plant part. In one embodiment, adherents method of phytoremediation or phytoreclamation of a con are selected from the group consisting of alginate, gums, taminated site comprising treating plant propagation mate starches, lecithins, formononetin, polyvinyl alcohol, alkali rial or a plant with a composition disclosed herein, and 55 formononetinate, hesperetin, polyvinyl acetate, cephalins, cultivating the plant propagation material into a first gen Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene eration plant or allowing the plant to grow; thereby reme Glycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galac diating or reclaiming the site. tan, Methyl Cellulose, PEG 400, Chitosan, Polyacrylamide, In one embodiment, the site is soil. Such as at a landfill. Polyacrylate, Polyacrylonitrile, Glycerol, Triethylene gly In an embodiment, the Substances, wastes or hazardous 60 col, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl, materials comprise hydrocarbons, petroleum or other chemi Carboxymethyl cellulose, Gum Ghatti, and polyoxyethyl cals, salts, or metals, such as lead, cadmium or radioiso ene-polyoxybutylene block copolymers. Other examples of topes. adherent compositions that can be used in the synthetic Formulations/Seed Coating Compositions preparation include those described in EP 0818135, CA The purified endophytes described herein can be formu 65 1229497, WO 2013090628, EP 0192342, WO 2008103422 lated using an agriculturally compatible carrier. The formu and CA 1041788, each of which is incorporated herein by lation useful for these embodiments generally typically reference in its entirety. US 9,687,001 B2 41 42 The formulation can also contain a Surfactant. Non glycerol, ethylene glycol, polyethylene glycol, propylene limiting examples of Surfactants include nitrogen-surfactant glycol, polypropylene glycol, etc. blends such as Prefer 28 (Cenex), Surf-N(US), Inhance In one particular embodiment, the formulation is ideally (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified seed Suited for coating of the endophytic microbial population oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), onto seeds. The bacterial or fungal endophytic populations Hasten (Wilfarm) and Mes-100 (Drexel); and organo-sili described in the present invention are capable of conferring cone surfactants include Silwet L77 (UAP), Silikin (Terra), many fitness benefits to the host plants. The ability to confer Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wil Such benefits by coating the bacterial or fungal populations bur-Ellis) and Century (Precision). In one embodiment, the on the Surface of seeds has many potential advantages, surfactant is present at a concentration of between 0.01% V/v 10 particularly when used in a commercial (agricultural) scale. to 10% V/v. In another embodiment, the surfactant is present The bacterial or fungal endophytic populations herein can at a concentration of between 0.1% V/v to 1% V/v. be combined with one or more of the agents described above In certain cases, the formulation includes a microbial to yield a formulation suitable for combining with an stabilizer. Such an agent can include a desiccant. As used agricultural seed or seedling. The bacterial or fungal popu herein, a “desiccant can include any compound or mixture 15 lation can be obtained from growth in culture, for example, of compounds that can be classified as a desiccant regardless using a synthetic growth medium. In addition, the microbe of whether the compound or compounds are used in Such can be cultured on Solid media, for example on petri dishes, concentrations that they in fact have a desiccating effect on scraped off and Suspended into the preparation. Microbes at the liquid inoculant. Such desiccants are ideally compatible different growth phases can be used. For example, microbes with the bacterial or fungal population used, and should at lag phase, early-log phase, mid-log phase, late-log phase, promote the ability of the microbial population to survive stationary phase, early death phase, or death phase can be application on the seeds and to Survive desiccation. used. Examples of Suitable desiccants include one or more of The formulations comprising the bacterial or fungal endo trehalose. Sucrose, glycerol, and Methylene glycol. Other phytic population of the present invention typically contains Suitable desiccants include, but are not limited to, non 25 between about 0.1 to 95% by weight, for example, between reducing Sugars and Sugar alcohols (e.g., mannitol or Sor about 1% and 90%, between about 3% and 75%, between bitol). The amount of desiccant introduced into the formu about 5% and 60%, or between about 10% and 50% in wet lation can range from about 5% to about 50% by weight/ weight of the bacterial or fungal population of the present volume, for example, between about 10% to about 40%, invention. It is preferred that the formulation contains at between about 15% and about 35%, or between about 20% 30 least about 10 CFU per ml of formulation, for example, at and about 30%. least about 10, at least about 10, at least about 10, at least In some cases, it is advantageous for the formulation to 107 CFU, at least 10 CFU per ml of formulation. contain agents such as a fungicide, an antibacterial agent, an Populations of Seeds herbicide, a nematicide, an insecticide, a plant growth regu In another aspect, the invention provides for a Substan lator, a rodenticide, or a nutrient. Such agents are ideally 35 tially uniform population of seeds comprising a plurality of compatible with the agricultural seed or seedling onto which seeds comprising the population of endophytes capable of the formulation is applied (e.g., it should not be deleterious promoting germination, as described herein above. Substan to the growth or health of the plant). Furthermore, the agent tial uniformity can be determined in many ways. In some is ideally one that does not cause safety concerns for human, cases, at least 10%, for example, at least 20%, at least 30%, animal or industrial use (e.g., no safety issues, or the 40 at least 40%, at least 50%, at least 60%, at least 70%, at least compound is sufficiently labile that the commodity plant 75%, at least 80%, at least 90%, at least 95% or more of the product derived from the plant contains negligible amounts seeds in the population, contain the endophytic population in of the compound). an amount effective to colonize the plant disposed on the In the liquid form, for example, Solutions or Suspensions, surface of the seeds. In other cases, at least 10%, for the bacterial or fungal endophytic populations of the present 45 example, at least 20%, at least 30%, at least 40%, at least invention can be mixed or Suspended in water or in aqueous 50%, at least 60%, at least 70%, at least 75%, at least 80%, solutions. Suitable liquid diluents or carriers include water, at least 90%, at least 95% or more of the seeds in the aqueous Solutions, petroleum distillates, or other liquid population, contain at least 1, at least 10, or at least 100 CFU carriers. on the seed surface or per gram of seed, for example, at least Solid compositions can be prepared by dispersing the 50 200 CFU, at least 300 CFU, at least 1,000 CFU, at least bacterial or fungal endophytic populations of the invention 3,000 CFU, at least 10,000 CFU, at least 30,000 CFU, at in and on an appropriately divided solid carrier, Such as peat, least 100,000 CFU, at least 300,000 CFU, or at least wheat, bran, Vermiculite, clay, talc, bentonite, diatomaceous 1,000,000 CFU per seed or more. earth, fuller's earth, pasteurized soil, and the like. When In a particular embodiment, the population of seeds is Such formulations are used as wettable powders, biologi 55 packaged in a bag or container Suitable for commercial sale. cally compatible dispersing agents such as non-ionic, Such a bag contains a unit weight or count of the seeds anionic, amphoteric, or cationic dispersing and emulsifying comprising the bacterial or fungal endophytic population as agents can be used. described herein, and further comprises a label. In one The solid carriers used upon formulation include, for embodiment, the bag or container contains a predetermined example, mineral carriers such as kaolin clay, pyrophyllite, 60 number of seeds. In an embodiment, the bag or container bentonite, montmorillonite, diatomaceous earth, acid white contains at least 1,000 seeds, for example, at least 5,000 soil, Vermiculite, and pearlite, and inorganic salts such as seeds, at least 10,000 seeds, at least 20,000 seeds, at least ammonium Sulfate, ammonium phosphate, ammonium 30,000 seeds, at least 50,000 seeds, at least 70,000 seeds, at nitrate, urea, ammonium chloride, and calcium carbonate. least 80,000 seeds, at least 90,000 seeds or more. In another Also, organic fine powders such as wheat flour, wheat bran, 65 embodiment, the bag or container can comprise a discrete and rice bran may be used. The liquid carriers include weight of seeds, for example, at least 1 lb, at least 2 lbs, at vegetable oils such as soybean oil and cottonseed oil, least 5 lbs, at least 10 lbs, at least 30 lbs, at least 50 lbs, at US 9,687,001 B2 43 44 least 70 lbs or more. The bag or container may comprise a In one embodiment, seeds may be treated with composi label describing the seeds and/or said bacterial or fungal tion(s) described herein in several ways but preferably via endophytic population. The label can contain additional spraying or dripping. Spray and drip treatment may be information, for example, the information selected from the conducted by formulating compositions described herein group consisting of net weight, lot number, geographic and spraying or dripping the composition(s) onto a seed(s) origin of the seeds, test date, germination rate, inert matter via a continuous treating system (which is calibrated to content, and/or the amount of noxious weeds, if any. Suit apply treatment at a predefined rate in proportion to the able containers or packages include those traditionally used continuous flow of seed), such as a drum-type of treater. in plant seed commercialization. The invention also con Batch systems, in which a predetermined batch size of seed templates other containers with more Sophisticated Storage 10 and composition(s) as described herein are delivered into a capabilities (e.g., with microbiologically tight wrappings or mixer, may also be employed. Systems and apparati for with gas- or water-proof containments). performing these processes are commercially available from In some cases, a Sub-population of seeds comprising the numerous Suppliers, e.g., Bayer CropScience (Gustafson). bacterial or fungal endophytic population is further selected In another embodiment, the treatment entails coating on the basis of increased uniformity, for example, on the 15 seeds. One such process involves coating the inside wall of basis of uniformity of microbial population. For example, a round container with the composition(s) described herein, individual seeds of pools collected from individual cobs, adding seeds, then rotating the container to cause the seeds individual plants, individual plots (representing plants to contact the wall and the composition(s), a process known inoculated on the same day) or individual fields can be tested in the art as "container coating. Seeds can be coated by for uniformity of microbial density, and only those pools combinations of coating methods. Soaking typically entails meeting specifications (e.g., at least 80% of tested seeds using liquid forms of the compositions described. For have minimum density, as determined by quantitative meth example, seeds can be soaked for about 1 minute to about 24 ods described elsewhere) are combined to provide the agri hours (e.g., for at least 1 min, at least 5 min, at least 10 min, cultural seed Sub-population. at least 20 min, at least 40 min, at least 80 min, at least 3 hr, The methods described herein can also comprise a vali 25 at least 6 hr, at least 12 hr., or at least 24 hr). dating step. The validating step can entail, for example, Increased Uniformity in Populations of Plants/Agricultural growing some seeds collected from the inoculated plants Fields into mature agricultural plants, and testing those individual A major focus of crop improvement efforts has been to plants for uniformity. Such validating step can be performed select varieties with traits that give, in addition to the highest on individual seeds collected from cobs, individual plants, 30 return, the greatest homogeneity and uniformity. While individual plots (representing plants inoculated on the same inbreeding can yield plants with Substantial genetic identity, day) or individual fields, and tested as described above to heterogeneity with respect to plant height, flowering time, identify pools meeting the required specifications. and time to seed, remain impediments to obtaining a homo In some embodiments, methods described herein include geneous field of plants. The inevitable plant-to-plant vari planting a synthetic combination described herein. Suitable 35 ability is caused by a multitude of factors, including uneven planters include an air seeder and/or fertilizer apparatus used environmental conditions and management practices. in agricultural operations to apply particulate materials Another possible source of variability can, in some cases, be including one or more of the following, seed, fertilizer due to the heterogeneity of the microbial population inhab and/or inoculants, into soil during the planting operation. iting the plants. By providing bacterial or fungal endophytic Seeder/fertilizer devices can include a tool bar having 40 populations onto seeds and seedlings, the resulting plants ground-engaging openers thereon, behind which is towed a generated by germinating the seeds and seedlings have a wheeled cart that includes one or more containment tanks or more consistent microbial composition, and thus are bins and associated metering means to respectively contain expected to yield a more uniform population of plants. and meter therefrom particulate materials. See, e.g., U.S. Therefore, in another aspect, the invention provides a Pat. No. 7,555,990. 45 Substantially uniform population of plants. The population In certain embodiments, a composition described herein can include at least 100 plants, for example, at least 300 may be in the form of a liquid, a slurry, a Solid, or a powder plants, at least 1,000 plants, at least 3,000 plants, at least (wettable powder or dry powder). In another embodiment, a 10,000 plants, at least 30,000 plants, at least 100,000 plants composition may be in the form of a seed coating. Compo or more. The plants are grown from the seeds comprising the sitions in liquid, slurry, or powder (e.g., wettable powder) 50 bacterial and/or fungal endophytic population as described form may be suitable for coating seeds. When used to coat herein. The increased uniformity of the plants can be mea seeds, the composition may be applied to the seeds and sured in a number of different ways. allowed to dry. In embodiments wherein the composition is In one embodiment, there is an increased uniformity with a powder (e.g., a wettable powder), a liquid, such as water, respect to the microbes within the plant population. For may need to be added to the powder before application to a 55 example, in one embodiment, a Substantial portion of the seed. population of plants, for example at least 10%, at least 20%, In still another embodiment, the methods can include at least 30%, at least 40%, at least 50%, at least 60%, at least introducing into the Soil an inoculum of one or more of the 70%, at least 75%, at least 80%, at least 90%, at least 95% endophyte populations described herein. Such methods can or more of the seeds or plants in a population, contains a include introducing into the soil one or more of the com 60 threshold number of the bacterial or fungal endophytic positions described herein. The inoculum(s) or compositions population. The threshold number can be at least 10 CFU, at may be introduced into the Soil according to methods known least 100 CFU, for example at least 300 CFU, at least 1,000 to those skilled in the art. Non-limiting examples include CFU, at least 3,000 CFU, at least 10,000 CFU, at least in-furrow introduction, spraying, coating seeds, foliar intro 30,000 CFU, at least 100,000 CFU or more, in the plant or duction, etc. In a particular embodiment, the introducing 65 a part of the plant. Alternatively, in a Substantial portion of step comprises in-furrow introduction of the inoculum or the population of plants, for example, in at least 1%, at least compositions described herein. 10%, at least 20%, at least 30%, at least 40%, at least 50%, US 9,687,001 B2 45 46 at least 60%, at least 70%, at least 75%, at least 80%, at least Commodity Plant Product 90%, at least 95% or more of the plants in the population, the The present invention provides a commodity plant prod bacterial or fungal endophyte population that is provided to uct, as well as methods for producing a commodity plant the seed or seedling represents at least 0.1%, at least 1%, at product, that is derived from a plant of the present invention. least 5%, at least 10%, at least 20%, at least 30%, at least As used herein, a “commodity plant product” refers to any 40%, at least 50%, at least 60%, at least 70%, at least 80%, composition or product that is comprised of material derived at least 90%, at least 95%, at least 99%, or 100% of the total from a plant, seed, plant cell, or plant part of the present microbe population in the plant/seed. invention. Commodity plant products may be sold to con In one embodiment, there is increased genetic uniformity Sumers and can be viable or nonviable. Nonviable commod of a substantial proportion or all detectable microbes within 10 ity products include but are not limited to nonviable seeds the taxa, genus, or species of the microbe relative to an and grains; processed seeds, seed parts, and plant parts; uninoculated control. This increased uniformity can be a dehydrated plant tissue, frozen plant tissue, and processed result of the microbe being of monoclonal origin or other plant tissue; seeds and plant parts processed for animal feed wise deriving from a microbial population comprising a for terrestrial and/or aquatic animal consumption, oil, meal, 15 flour, flakes, bran, fiber, paper, tea, coffee, silage, crushed of more uniform genome sequence and plasmid repertoire than whole grain, and any other food for human or animal would be present in the microbial population a plant that consumption; and biomasses and fuel products; and raw derives its microbial community largely via assimilation of material in industry. Industrial uses of oils derived from the diverse soil symbionts. agricultural plants described herein include ingredients for In another embodiment, there is an increased uniformity paints, plastics, fibers, detergents, cosmetics, lubricants, and with respect to a physiological parameter of the plants biodiesel fuel. Soybean oil may be split, inter-esterified, within the population. In some cases, there can be an Sulfurized, epoxidized, polymerized, ethoxylated, or increased uniformity in the height of the plants when com cleaved. Designing and producing soybean oil derivatives pared with a population of reference agricultural plants with improved functionality and improved oliochemistry is grown under the same conditions. For example, there can be 25 a rapidly growing field. The typical mixture of triglycerides a reduction in the standard deviation in the height of the is usually split and separated into pure fatty acids, which are plants in the population of at least 5%, for example, at least then combined with petroleum-derived alcohols or acids, 10%, at least 15%, at least 20%, at least 30%, at least 40%, nitrogen, Sulfonates, chlorine, or with fatty alcohols derived at least 50%, at least 60% or more, when compared with a from fats and oils to produce the desired type of oil or fat. population of reference agricultural plants grown under the 30 Commodity plant products also include industrial com same conditions. In other cases, there can be a reduction in pounds, such as a wide variety of resins used in the formu the standard deviation in the flowering time of the plants in lation of adhesives, films, plastics, paints, coatings and foams. The above disclosure generally describes the present the population of at least 5%, for example, at least 10%, at application. A more complete understanding can be obtained least 15%, at least 20%, at least 30%, at least 40%, at least 35 by reference to the following specific examples. These 50%, at least 60% or more, when compared with a popula examples are described solely for the purpose of illustration tion of reference agricultural plants grown under the same and are not intended to limit the scope of the disclosure. conditions. Changes in form and Substitution of equivalents are con Decreased Uniformity in Populations of Plants/Agricultural templated as circumstances might Suggest or render expe Fields 40 dient. Although specific terms have been employed herein, In certain circumstances, decreased uniformity in a popu Such terms are intended in a descriptive sense and not for lation can be desirable. For example, plants within a popu purposes of limitation. lation that are not all at the same developmental stage may The following non-limiting examples are illustrative of not all be negatively affected by a biotic or an abiotic stress the present disclosure: event, and as a result, the population as a whole may show 45 a beneficial trait such as increased yield. As another EXAMPLES example, a lack of uniformity may allow for the selection of plants/seeds with a trait that is not present in the other Dormancy and germination depend on several processes members of the population. Therefore, in another embodi and factors. To ensure seedling establishment and Success, it ment, there is a decreased uniformity with respect to a 50 is important to control the underlying processes or condi physiological parameter of the plants within the population. tions. The role of plant genetics, hormones, and different In some cases, there can be a decreased uniformity in the seed tissues have been relatively well studied. The present height of the plants when compared with a population of examples study the endophyte-plant seed relationship, trans reference agricultural plants grown under the same condi mitting into a root symbiotic stage towards plant maturation. tions. For example, there can be an increase in the standard 55 deviation in the height of the plants in the population of at Example 1 least 5%, for example, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% Taxonomy and Physical Properties of the or more, when compared with a population of reference Endophytes agricultural plants grown under the same conditions. In 60 other cases, there can be an increase intracellular vesiculoid The endophytes used in the synthetic compositions in the standard deviation in the flowering time of the plants described herein have been deposited as follows: Interna in the population of at least 5%, for example, at least 10%, tional Depositary Authority of Canada—IDAC (original at least 15%, at least 20%, at least 30%, at least 40%, at least strains deposited IDAC, National Microbiology Labora 50%, at least 60% or more, when compared with a popula 65 tory, Public Health Agency of Canada, 1015 Arlington tion of reference agricultural plants grown under the same Street, Winnipeg, Manitoba, Canada, R3E 3R2; receipts and conditions. viability in Appendix A) and Saskatchewan Microbial Col US 9,687,001 B2 47 48 lection and Database—SMCD (copies of strains deposited) SMCD2215 is also a facultative, spore-forming endo International Depository Authority of Canada—IDAC phyte that is capable of living within another endophyte. It (original strains deposited) and Saskatchewan Microbial is capable of forming hyphal coils and intracellular or Hartig Collection and Database SMCD (copies of strains depos net-like structures within a plant. SMCD2215 is a bacterium ited), (see FIGS. 1-6 and Table 1). Strains: that is capable of being part of a plant-fungus symbiont that (a) IDAC 081111-06=SMCD 2215; produces altered levels of phytohormones, and/or altered (b) IDAC 081111-03=SMCD 2210; levels of anti-oxidants, as compared to a plant that is not in (c) IDAC 081111-02-SMCD 2208; symbiosis. This endophyte is also capable of being part of a (d) IDAC 081111-01=SMCD 2206; plant-fungus symbiont that shows decreased aging and/or (e) IDAC 200312-06=SMCD 2204; and 10 senescence, and/or increased protection against pathogens, (f) IDAC 200312-05=SMCD 2204.F. as compared to a plant or plant organ that is not in Symbiosis. SMCD 2215 strain was originally isolated as an endo phytic bacterium of Phyalocephala sensu lato plant endo Example 2 phytic SMCD fungus. Classification according to Labeda et al. 2012. This phylogenetic study examines almost all 15 Symbiotic Microbe-Plant Association and Level of described species (615 taxa) within the family Streptomy Compatibility cetaceae based on 16S rDNA gene sequences and illustrates the species diversity within this family, which is observed to The level of microbe-plant compatibility was assessed contain 130 statistically supported clades. The present 16S using a slightly modified method of Abdellatif et al. 2009. rDNA sequence data confirm that Streptomyces sp. Strain In a bicompartmental agar 10 cm plate without nutrients SMCD 2215 can be assigned to a separate unknown clade (FIG. 7), the plants health and the formation of root according to Labeda et al 2012 but separate species from hairs—the absorbants of water and minerals—were charac Streptomyces lividans. Within a plant, it is capable of terized in co-culture, with and without microbial partners. In forming intercellular hyphae-like filaments and intracellular FIG. 7, the left compartment of each split plate shows a individual spore-like cells. 25 culture with the microbial partner, and the right compart SMCD2204 is a filamentous, spore-forming facultative ment of each split plate shows a culture without the micro endophyte. Within a plant, it is capable of forming hyphal bial partner. The experiment was repeated twice in three coils, microvesicles, microarbuscules, hyphal knots and replicates. intracellular or Hartig net-like structures. SMCD2204 is a As shown in the left compartment of each split plate, fungus that is capable of being part of a plant-fungus 30 healthy plant tissue formed even when the plant roots were symbiont that produces altered levels of phytohormones, grown directly on the dense microbial mats. The biomass of and/or altered levels of anti-oxidants, as compared to a plant root hairs is enhanced to about twice as much compared to that is not in Symbiosis. This endophyte is also capable of the right compartment of each split plate where the micro being part of a plant-fungus symbiont that shows decreased bial partner is absent (see left compartments). aging and/or senescence, and/or increased protection against 35 The plant efficacy to establish symbiotic association is pathogens, as compared to a plant or plant organ that is not dependent on the type of endophyte distribution within the in Symbiosis. root endodermis. Typical endophytic root colonization is SMCD2204F is filamentous, spore-forming facultative discontinuous and partial with a lower number of occupied endophyte that is capable of living within another endo cells<50% (Table 2) compared to the colonization of fungal phyte. It is capable of forming hyphal coils and intracellular 40 pathogens which is characterized by a uniform/continual or Hartig net-like structures within a plant. This endophyte (frequency: 60-80%) colonization of cells (FIG. 8). is also capable of being part of a plant-fungus symbiont that An endophyte's performance should not only be assessed shows decreased aging and/or senescence, as compared to a by measuring biomass production, because what underlies plant or plant organ that is not in Symbiosis. the visibly increased yield is the endophyte's efficiency in SMCD2206 is a filamentous, spore-forming facultative 45 colonizing the plant. This can be assessed by characterizing endophyte. Within a plant, it is capable of forming hyphal their association with plant cells, tissues, or organs (i.e. seed coils, microvesicles, microarbuscules, and hyphal knots. and radicles) using mathematical Indices which have been SMCD2206 is a fungus that is capable of being part of a developed Abdellatif et al. 2009 and applied in this study plant-fungus symbiont that produces altered levels of phy (FIG. 9 and FIG. 10). tohormones, and/or altered levels of anti-oxidants, as com 50 These Indices are based on the following observations: pared to a plant that is not in Symbiosis. This endophyte is Endophytic symbionts show different radicle (root)-coloni also capable of being part of a plant-fungus symbiont that Zation patterns (regularity or level of deviation in endophyte shows decreased aging and/or senescence, and/or increased cell form-Ireg and direction-Idir when colonizing living cell) protection against pathogens, as compared to a plant or plant compared to dead radicle-cell (which usually remain colo organ that is not in Symbiosis. 55 nized by true saprophytes). SMCD2208 is a spore-forming facultative endophyte. High Ireg and Idir index values determine mutualistic SMCD2210 is a facultative, spore-forming endophyte that (beneficial) plant-Symbiont relationships. In conclusion, the is capable of living within another endophyte. Within a results show that the symbiotic microbe-plant association is plant, it is capable of forming hyphal coils and microve characterised by a high level of compatibility between the sicles. SMCD2210 is a fungus that is capable of being part 60 two partners, leading to an equilibrated (<50% of colonized of a plant-fungus symbiont that produces altered levels of cortex cells) and discontinuous root colonisation by the phytohormones, and/or altered levels of anti-oxidants, as microbial endophytes measured using mathematical indices compared to a plant that is not in Symbiosis. This endophyte Abdellatif et al. 2009. This mutualistic partnership is is also capable of being part of a plant-fungus symbiont that further characterised by the direct effect of endophytic shows decreased aging and/or senescence, and/or increased 65 microbes on plant healthy growth (bacto- and mycodepen protection against pathogens, as compared to a plant or plant dency) when the plant is challenged to use the microbial organ that is not in Symbiosis. partners as the only source of nutrients or energy for growth. US 9,687,001 B2 49 50 In addition, the enhancement of the root hairs biomass by the Materials and Methods endophytes was observed and measured even in roots in Hydrothermal Time Model of Germination and Energy of distal compartments of split plates where microbial partners Germination were absent, indicating a possible systemic plant growth The hydrothermal time (HTT) model Gummerson 1986 promoting function of the endophytes. postulates that an individual seed begins to germinate when two conditions are met. First, the Sum of daily temperatures, Example 3 above a minimum cardinal value (T), accumulated over a period of time, must pass a threshold value (0), measured in degree days. Second, the seed must accumulate Sufficient Symbiotic Organs of Endophytes on Wheat water potential (0) per degree-day. Thus, HTT (0) can be 10 expressed as: Each taxonomical group of endophytes establishes a unique type of mycoVitalism, consequently forming differ 0 (0(0). (Equation 1) ent symbiotic organs. Characterization of the mycoVitalism According to Köchy and Tielbörger 2007. was done using Abdellatif et al. 2009 methodology, con 91 (Tsubstrate-Tain) (Equation 2) sisting of in vitro seed and microbe co-cultures assessing an 15 early stage of the microbe-plant symbiotic association. The with t representing the time elapsed in days, and diversity of microbial symbiotic organs formed by SMCD OHP substrate-Pain (Equation 3) 2204, 2206, 2210, and 2215 on wheat germinants is shown in a constant environment assuming that T is equal to in FIG. 11. or less than the optimal temperature for seed germination. In In Summary, the results show differential types of sym Equation 3, l's, and , represent the Water potential biotic organs formed in wheat root by each endophyte likely of the substrate and the minimum water potential at which related to their different symbiotic functions. An equilibrated germination is possible, in MPa, respectively. Consistent with Bradford 2002, equations 2 and 3 can be substituted colonization abundance, patchy colonization patterns, into equation 1 to yield: increased hypha septation in living root cells, as well as 25 formation of arbuscules, knots, coils and vesicles—putative OH T('substrate-Pain)(Tsubstrate-Tain) (Equation 4). symbiotic functional organs—may indicate local specializa However, in the present study, the temperature exceeds tion within the fungal endophytes to promote plant myco the optimal temperature for the germination of wheat re vitality and mycoheterotrophy. Bactovitality is mostly char viewed by McMaster (2009), necessitating the consider acterized by Streptomyces intercellular curly filaments. 30 ation of a maximum temperature (T) above which ger FIG. 66 shows symbiosomes formed in wheat root. The mination cannot occur. Thus, equation 2 was modified to: symbiosome is the new compartment that is formed in the plant cell when bacteria or fungi enter it. Symbiosomes can 0–VI(T.substrate-Tain)(Tsubstrate-Tina)) (Equation 5) be classified into two types: I and II. Both types are where TsTasT. If equation 5 is Substituted for composed of a perivesiculoid membrane and a partially 35 2 in equation 4, the following results: fragmented outer vesiculoid membrane. Type I Symbio Out ('substrate-ni, VITsubstrate-Tain)(Tsubstrate Somes are additionally composed of an intercellular Tina.) It (Equation 6) microvesiculoid compartment formed between two plant where TinisTsubstratesTina. cell membranes, while type II symbiosomes are additionally Energy of germination (EG) can be defined in several composed of an intracellular vesiculoid compartment. Both 40 ways, including the percentage of seeds germinating after a types can be seed in the form of vesicles (A and B) and knots set time period after planting, relative to the number of seeds (C). tested Ruan et al. 2002: Dong-dong et al. 2009, or 50% of Symbiosis at the seed level resulted in increased wheat germination attained Allen 1958. In order to integrate EG germinants after 10 days of co-innoculation (FIG. 12 and with the HTT model of germination the latter definition was FIG. 13). 45 used, meaning that EG is equal to t in Equation 2. Example 4 Estimation of Parameters The estimation of T, and T for wheat was based on Endophytes Improve Wheat Seed Germination both information available in the literature and the present inventors’ own observations. McMaster 2009 summarizes Under Heat and Drought Stress 50 data originating from Friend et al. 1962, Cao and Moss Seed germination is a critical life stage for plant Survival 1989, and Jame et al. indicating the existence of a curvi and timely seedling establishment especially in stressful linear relationship between wheat development rate and environments. It was hypothesized that endophytes would temperature. Since germination and development of wheat does not take place below 0°C. or above 40°C. T., and improve wheat seed germination under heat and drought 55 T. were assigned the values of 0° C. and 40° C., respec stress. The hydrothermal time (HTT) model of germination tively. is a conceptual model useful for predicting the timing and The parameter up was estimated in vitro by germinating energy of germination (EG) under a given set of conditions. wheat seeds grown on potato dextrose agar (PDA; Difico) The HTT and EG are applied to determine if one or more media containing a range of polyethylene glycol (PEG) 8000 compatible endophytes enhance heat or drought tolerance in 60 wheat. Endophytes tested dramatically increased the percent concentrations (Amresco Inc.). The water activity (a) of PDA alone and PDA containing 8%, 12% and 16% PEG was of germination, improved EG and HTT values, and dimin measured using the AquaLab 4TE, Series 4 Quick Start, ished wheat Susceptibility to heat and drought as measured Decagon Devices. Water activity was converted to water by fresh weight of seedlings. When colonised by the most potential () using the relationship adapted from Bloom and effective endophyte, the values of the parameters tested in 65 wheat seeds exposed to heat stress resembled those of Richard 2002: unstressed seeds. (Equation 7)