(12) Patent Application Publication (10) Pub. No.: US 2016/0174570 A1 Vujanovic Et Al

US 2016O174570A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0174570 A1 Vujanovic et al. (43) Pub. Date: Jun. 23, 2016

(54) ENDOPHYTIC MICROBAL SYMIBIONTS IN Publication Classification PLANT PRENATAL CARE (51) Int. Cl. (71) Applicant: University Of Saskatchewan, AOIN 63/04 (2006.01) Saskatoon (CA) (52) U.S. Cl. CPC ...... A0IN 63/04 (2013.01) (72) Inventors: Vladimir Vujanovic, Saskatoon (CA); James J. Germida, Saskatoon (CA) (57) ABSTRACT (21) Appl. No.: 15/063,350 The present disclosure provides compositions comprising (22) Filed: Mar. 7, 2016 novel endophytes capable of promoting germination endo phytes that have a symbiotic relationship with plants. The Related U.S. Application Data present disclosure further provides methods of improving (63) Continuation of application No. 14/614,193, filed on seed vitality, biotic and abiotic stress resistance, plant health Feb. 4, 2015, which is a continuation-in-part of appli and yield under both stressed and unstressed environmental cation No. PCT/CA2013/000091, filed on Feb. 5, conditions, comprising inoculating a seed with the novel 2013. endophyte strains and cultivating a plant therefrom. Patent Application Publication Jun. 23, 2016 Sheet 1 of 68 US 2016/017457.0 A1

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ENDOPHYTIC MICROBAL SYMIBIONTS IN changes not only in pathways of nutrient (N. P. K), energy PLANT PRENATAL CARE transfer in food-webs and biogeochemical cycles but also in UV-B, heat, drought or salt tolerance influencing the overall CROSS REFERENCE TO RELATED plant ecosystem establishment and stability. Despite their APPLICATIONS abundance and likely importance in all terrestrial ecosystems, 0001. This application is a continuation of U.S. patent nearly nothing about the composition of endophytes in seeds application Ser. No. 14/614,193, filed on Feb. 4, 2015, which or spermosphere, their interactions, or their common is a continuation-in-part of co-pending International Appli response to environmental changes is known. cation No. PCT/CA2013/000091, filed Feb. 5, 2013, which is 0006 While the spermosphere represents a rapidly chang herein incorporated in its entirety by reference ing and microbiologically dynamic Zone of soil Surrounding a germinating seed Nelson, 2004, the rhizosphere is a SEQUENCE LISTING microbiologically active Zone of the bulk soil Surrounding the plant's roots Smith and Read 2008. The rhizosphere sup 0002 The instant application contains a Sequence Listing, ports mycoheterotrophy or a plant-mycorrhiza symbiotic with 19 sequences, which has been submitted via EFS-Web relationship. The spermosphere, on the other hand, promotes and 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 29069US plant seeds—enhancing seed vigour, energy and uniformity 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 colonize FIELD not only roots, but also other plant organs including seeds 0003. The present disclosure relates to synthetic prepara Vujanovic et al. 2000: Hubbard et al. 2011. They belong to tions comprising a seed and a composition, where the com the multicellular phyla Ascomycota and Basidiomycota and position comprises fungal and bacterial endophytes of plants form colonization symbiotic structures different from those that enhance seed vitality and/or plant health, conferring gen produced by unicellular or cenocytic phylum Glomeromy eral improvements in the plants agricultural traits, under cota, known as vesicular-arbuscular mycorrhizal symbiosis normal and stressed conditions. Abdellatif et al. 2009. Endophytic bacteria have been also found in virtually every plant studied, where they colonize an BACKGROUND ecological niche similar to that of fungi. Such as the internal 0004 Fungi and bacteria are ubiquitous microorganisms. healthy tissues. Although most bacterial endophytes appear Endophyte is the term first coined by deBary 1866 defining to originate from the rhizosphere or phyllosphere; some may those microbes that colonize asymptomatically plant tissues be transmitted through the seed Ryan et al. 2008. Stone et al., 2000. The existence of endophytes has been 0007. Seed germination is a vital phenophase to plants known for more than one century Freeman 1904 and it survival and reproduction in either optimal or stressful envi seems that each individual host, among the 300,000 plant ronmental conditions. Microbial endophytic colonization at species, inhabits several to hundreds of endophytes Tan and the seed state is especially critical because of the role of the Zou, 2001. Endophytes are microbial organisms mostly seed as a generative organ in regeneration and dispersion of symbiotically or mutualistically associated with living tissues flowering plants Baskin and Baskin 2004 and the role of of plant hosts. Many are capable of conferring plant tolerance mycobionts and symbiotically associated bacteria (bacto to abiotic stressors or can be used by the plant for defense bionts) as potential drivers of seedling recruitment in natu against pathogenic fungi and bacteria Singh et al. 2011. ral undisturbed, disturbed and polluted habitats Mithl Some of these microorganisms have proven useful for very mann and Peintner 2000; Adriaensen et al. 2006: White and Small Subsets of agriculture (e.g., forage grass growth), for Tones 2010. Thus, developing methods by which seedling estry and horticulture sectors, as well as plant production of emergence can be enhanced and protected under the limita medicinally important compounds. However, no commercial tions of disease pressure, heat or drought is precious. The use endophyte seed coating products are used in the world’s of endophytic symbionts is a promising method by which largest crops including corn, wheat, rice, and barley, and Such seed germination can be enhanced Vujanovic et al. 2000; endophyte approaches have suffered from high variability, Vujanovic and Vujanovic 2006; Vujanovic and Vujanovic inconsistent colonization, low performance across multiple 2007. The methods and compositions described herein over crop cultivars, and the inability to confer benefits to elite crop come these and other limitations of the prior art. It was varieties under field conditions. hypothesized that plant stress hardiness can be conferred via 0005 Endophytes largely determine plant cell and whole a mycobiont-seed relationship known as mycoVitality—a plant genome regulation, including the plant's vital cycles: (i) phenomenon that had been reserved for Orchidaceae Vujan seed pre- and post-germination events (mycoVitalism) Vu ovic 2008 and via bactovitality which refers to a form of janovic and Vujanovic 2007, (ii) plant nutrient uptake and bactosymbiosis, using different endophytic strains with vari growth-promoting mechanisms (mycoheterotrophism) ety of activities. Smith and Read 2008, and (iii) plant environmental stress tolerance and induced systemic resistance against diseases SUMMARY and pests (mycosymbionticism) Wallin 1927; Margulis, 0008. The synthetic preparations and compositions 1991. They could play a major role in plant biomass produc described herein can benefit plant hosts, for example, but not tion, CO sequestration, and/or yield and therefore be signifi limited to, wheat, barley, corn, Soybeans, alfalfa, rice, cotton, cant players in regulating the ecosphere, ensuring plant health pulses, canola, Vegetables, Sugarbeet, Sugarcane, trees, and food security. In addition, they can be important sentinels shrubs or grasses. The benefit may come from bactovitality, (bioindicators) of environmental changes, as alterations in the mycoVitality and mycoheterotrophy, and enhance tolerance structure and biomass of endophytic communities can herald to environmental stresses, as demonstrated herein. Prenatal US 2016/017457.0 A1 Jun. 23, 2016 care in agriculture is more than just seed or germinant vitality, length, increased fresh weight of seedlings, increased plant health or vigour. It also determines what to expect before and vigor, nitrogen stress tolerance, enhanced Rhizobium activity, during the germination process, seedling establishment, and, enhanced nodulation frequency, and early flowering time. In later crop productivity or yield. Some embodiments, the composition is disposed on an exte 0009. Several parameters of symbiotic efficacy (dormancy rior Surface of the agricultural seed in an amount effective to breakdown, germination, growth and yield) were assessed colonize at least 1% of the cortical cells of an agricultural using efficient endophytic Saskatchewan Microbial Collec plant grown from the seed. In other embodiments, the com tion and Database (SMCD) strain(s)-crop(s) interaction(s) position is disposed on an exterior Surface of the agricultural under in vitro, phytotron, greenhouse and field conditions. seed in an amount effective to cause a population of seeds The synthetic preparations and compositions described inoculated with said composition to have greater germination herein have effects on germination, which can be assessed by rate, faster dormancy breakdown, increased energy of germi measuring percent of germination, energy of germination and nation, increased seed germination vigor or increased seed hydrothermal time required for germination, for example. Vitality than a population of control agricultural seeds. In 0010 Also tested was the endophyte's capacity to confer Some embodiments, the composition is disposed on an exte seed vitality. For both fungal and bacterial endosymbionts, rior Surface of the agricultural seed in an amount effective to improved seed vitality can increase tolerance for abiotic and cause a population of seeds inoculated with said composition biotic stresses in plants that have progressed beyond the seed to reach 50% germination faster than a population of control ling stage to the plants maturity via mycoheterotrophy. The agricultural seeds. synthetic preparations and compositions described hereincan 0013. In some embodiments, the endophytes area selected improve plant traits such as increased yield, faster seedling from the group consisting of a spore-forming endophyte, a establishment, faster growth, increased drought tolerance, facultative endophyte, a filamentous endophyte, an endo increased heat tolerance, increased cold tolerance, increased phyte capable of living within another endophyte, an endo salt tolerance, increased tolerance to pests and diseases, for phyte capable of forming hyphal coils within the plant, an example increased tolerance to Fusarium infection and to endophyte capable of forming microvesicles within the plant, Puccinia infection, increased biomass, increased root length, an endophyte capable of forming micro-arbuscules within the increased fresh weight of seedlings, increased plant vigour, plant, an endophyte capable of forming hyphal knots within nitrogen stress tolerance, enhanced Rhizobium activity, the plant, an endophyte capable of forming Hartig-like nets enhanced nodulation frequency or early flowering time. within the plant, and an endophyte capable of forming sym 0011. The synthetic preparations and compositions biosomes within the plant. In some embodiments, the endo described herein can also modulate the expression of genes phyte is in the form of at least one of conidia, chlamydospore, involved in plant growth, genes associated with systemic and mycelia. acquired resistance, or genes involved in protection from 0014. In other embodiments, the composition is disposed oxidative stress. These genes may be involved in phytohor on an exterior Surface of the agricultural seed in an amount mone production, for example gibberellin (GA) biosynthesis effective to colonize the cortical cells of an agricultural plant or breakdown, abscisic acid (ABA) biosynthesis or break grown from the seed and to produce the altered trait, wherein down, NO production or breakdown, superoxide detoxifica the altered trait is altered gene expression, wherein the gene is tion, or could be positive or negative regulators of these selected from the group consisting of a gene involved in plant pathways. The genes associated with systemic acquired resis growth, an acquired resistance gene, and a gene involved in tance may be redox-regulated transcription factors, for protection from oxidative stress. In some embodiments, the example those in the MYB family. Non-limiting examples of gene is involved in phytohormone production. In other Such genes include ent-kaurenoic (KAO), repression of shoot embodiments, the gene is a redox-regulated transcription fac growth (RSG), NCED, ABA 8-hydroxylase, GA3-oxidase 2, tor. In yet other embodiments, the gene involved in SuperoX 14-3-3 or nitric oxide (NO) genes and/or stress resistance ide detoxification or in NO production or breakdown. superoxide dismutase (SOD), manganese SOD (MnSOD), 0015. In some embodiments, the agricultural plant seed is proline (Pro), Myb1 and Myb2. selected from the group consisting of corn, soy, wheat, cotton, 0012. In certain embodiments, the present disclosure pro rice, canola, barley and pulses. In some embodiments, a popu vides a synthetic preparation comprising an agricultural plant lation comprising at least 10 synthetic preparations is dis seed and a composition comprising an endophyte capable of posed within a packaging material. promoting germination and an agriculturally-acceptable car 0016 Further provided herein is a seed comprising an rier, wherein an agricultural plant grown from the seed has an endophyte or culture disclosed herein. In one embodiment, altered trait as compared to a control agricultural plant. In the seed is coated with the endophyte. In another embodi certain embodiments, the endophyte capable of promoting ment, the seed is cultured or planted near the endophyte such germination is a coleorhiza-activating endophyte and the that the endophyte is able to colonize the seed. In one embodi agricultural plant seed is a monocot seed. In some embodi ment, the seed planted near the endophyte is up to 4 cm away ments, the composition is disposed on an exterior Surface of from the endophyte. the agricultural seed in an amount effective to colonize the 0017. The endophytes used to inoculate the seeds may be cortical cells of an agricultural plant grown from the seed and selected from the group consisting of a spore-forming endo to produce the altered trait, wherein the altered trait is an phyte, a facultative endophyte, a filamentous endophyte, and improved functional trait selected from the group consisting an endophyte capable of living within another endophyte. In of increased yield, faster seedling establishment, faster Some embodiments, the endophyte is capable of forming growth, increased drought tolerance, increased heat toler certain structures in the plant, where the structures are ance, increased cold tolerance, increased salt tolerance, selected from the group consisting of hyphal coils, Hartig increased tolerance to Fusarium infection, increased toler like nets, microvesicles, micro-arbuscules, hyphal knots, and ance to Puccinia infection, increased biomass, increased root symbiosomes. In some embodiments, the endophyte is in the US 2016/017457.0 A1 Jun. 23, 2016

form of at least one of conidia, chlamydospore, and mycelia. In some embodiments, the endophyte is in the form of at least In other embodiments, the fungus or bacteria is capable of one of conidia, chlamydospore, and mycelia. being part of a plant-fungus symbiotic system or plant-bac teria symbiotic system that produces altered levels of phyto 0022. In some embodiments, the endophyte is a fungus of hormones or anti-oxidants, as compared to a plant that is not Subphylum Pezizomycotina. In some embodiments, the in Symbiosis. In other embodiments, the plant-fungus symbi endophyte is a fungus of class Leotiomycetes, Dothideo otic system or plant-bacterium symbiotic system has anti mycetes, Sordariomycetes, or Eurotiomycetes. In some aging and/or anti-senescence effects, as compared to a plant embodiments, the endophyte is of order Helotiales, Capno or plant organ that is not in Symbiosis. In other embodiments, dides, Pleosporales, Hypocreales, or Eurotiales. In some the plant-fungus symbiotic system or plant-bacteria symbi embodiments, the composition comprises an agriculturally otic system has increased protection against pathogens, as acceptable carrier and a spore-forming, filamentous bacterial compared to a plant that is not in Symbiosis. In some embodi endophyte of phylum Actinobacteria. In some embodiments, ments, the endophyte is a bacteria of order actinomycetales. 0018. The present disclosure also provides methods for 0023. In some embodiments, the present disclosure pro improving seed vitality and enhancing plant health and yield vides a composition comprising a carrier and an endophyte of under normal and stressed conditions. Accordingly, there is Paraconyothirium sp. strain deposited as IDAC 081111-03 or provided a method of improving seed vitality, plant health comprising a DNA sequence with at least 97% identity to and/or plant yield comprising inoculating a seed with an SEQ ID NO:5; an endophyte of Pseudeurotium sp. strain endophyte or culture disclosed herein or a combination or deposited as IDAC 081111-02 or comprising a DNA mixture thereof or with a composition disclosed herein. In sequence with at least 97% identity to SEQ ID NO:4; an Some embodiments, the seed is cultivated into a first genera endophyte of Penicillium sp. strain deposited as IDAC tion plant. 081111-01 or comprising a DNA sequence with at least 97% 0019. In certain embodiments, provided herein are meth identity to SEQID NO:3: an endophyte of Cladosporium sp. ods of altering a trait in an agricultural plant seed or an strain deposited as IDAC 2003 12-06 or comprising a DNA agricultural plant grown from said seed, where the methods sequence with at least 97% identity to SEQ ID NO:1; an comprise inoculating the seed with a composition comprising endophyte of Sarocladium sp. strain deposited as IDAC endophytes capable of promoting germination and an agri 2003 12-05 or comprising a DNA sequence with at least 97% culturally-acceptable carrier, where the endophyte replicates identity to SEQID NO:2; and/or an endophyte of Streptomy within at least one plant tissue and colonizes the cortical cells ces sp. strain deposited as IDAC 081111-06 or comprising a of said plant. In some embodiments, the endophyte colonizes DNA sequence with at least 97% sequence identity to SEQID at least 1% of the cortical cells of said agricultural plant. NO:6. In certain embodiments, the endophyte of Paraco nyothirium sp. strain comprises a DNA sequence with at least 0020. In some embodiments, the trait altered by using the 98% identity to SEQID NO:5; the endophyte of Pseudeuro method is an improved functional trait selected from the tium sp. strain comprises a DNA sequence with at least 98% group consisting of increased yield, faster seedling establish identity to SEQ ID NO:4; the endophyte of Penicillium sp. ment, faster growth, increased drought tolerance, increased strain comprises a DNA sequence with at least 98% identity heat tolerance, increased cold tolerance, increased salt toler to SEQID NO:3: the endophyte of Cladosporium sp. strain ance, increased tolerance to Fusarium infection, increased comprises a DNA sequence with at least 98% identity to SEQ tolerance to Puccinia infection, increased biomass, increased ID NO:1; the endophyte of Sarocladium sp. strain comprises root length, increased fresh weight of seedlings, increased a DNA sequence with at least 98% identity to SEQID NO:2: plant vigor, nitrogen stress tolerance, enhanced Rhizobium and the endophyte of Streptomyces sp. Strain comprises a activity, enhanced nodulation frequency, and early flowering DNA sequence with at least 98% sequence identity to SEQID time. In other embodiments, the altered trait is a seed trait NO:6. In certain embodiments, the endophyte of Paraco selected from the group consisting a greater germination rate, nyothirium sp. strain comprises a DNA sequence with at least faster dormancy breakdown, increased energy of germina 99% identity to SEQID NO:5; the endophyte of Pseudeuro tion, increased seed germination vigor or increased seed vital tium sp. strain comprises a DNA sequence with at least 99% ity than a population of control agricultural seeds. In other identity to SEQ ID NO:4; the endophyte of Penicillium sp. embodiments, the altered trait is reaching 50% germination strain comprises a DNA sequence with at least 99% identity faster than a population of control agricultural seeds. In other to SEQID NO:3: the endophyte of Cladosporium sp. strain embodiments, the altered trait is altered gene expression, comprises a DNA sequence with at least 99% identity to SEQ where the gene is selected from the group consisting of a gene ID NO:1; the endophyte of Sarocladium sp. strain comprises involved in plant growth, an acquired resistance gene, and a a DNA sequence with at least 99% identity to SEQID NO:2: gene involved in protection from oxidative stress. and the endophyte of Streptomyces sp. Strain comprises a 0021. In some embodiments, the endophytes used in the DNA sequence with at least 99% sequence identity to SEQID method are a selected from the group consisting of a spore NO:6. In certain embodiments, the endophyte of Paraco forming endophyte, a facultative endophyte, a filamentous nyothirium sp. strain comprises a DNA sequence of SEQID endophyte, an endophyte capable of living within another NO:5; the endophyte of Pseudeurotium sp. strain comprises a endophyte, an endophyte capable of forming hyphal coils DNA sequence of SEQ ID NO:4; the endophyte of Penicil within the plant, an endophyte capable of forming lium sp. strain comprises a DNA sequence of SEQID NO:3: microvesicles within the plant, an endophyte capable of form the endophyte of Cladosporium sp. strain comprises a DNA ing micro-arbuscules within the plant, an endophyte capable sequence of SEQID NO:1; the endophyte of Sarocladium sp. of forming hyphal knots within the plant, an endophyte strain comprises a DNA sequence of SEQID NO:2; and the capable of forming Hartig-like nets within the plant, and an endophyte of Streptomyces sp. Strain comprises a DNA endophyte capable of forming symbiosomes within the plant. sequence of SEQID NO:6. US 2016/017457.0 A1 Jun. 23, 2016

0024. In another aspect, there is provided a method of 0033 FIG. 5 shows the inferred neighbour-joining phylo improving plant health and/or plant yield comprising treating genetic tree of the Coniothyrium strain SMCD 2210 based on plant propagation material or a plant with an endophyte or ITS rDNA. Numbers at nodes indicate bootstrap support val culture disclosed herein or a combination or mixture thereof ues for 1000 replicates; only values that were>70% are given. or a composition disclosed herein. In some embodiments, the Bar indicates 0.05 nucleotide substitutions per site (nucle plant propagation material is cultivated into a first generation otide position). plant or the plant is allowed to grow. 0034 FIG. 6 shows the inferred neighbour-joining phylo 0025. In an embodiment, the plant propagation material is genetic tree of the Streptomyces sp strain SMCD 2215 based any plant generative/sexual (seed, generative bud or flower) on 16S rDNA. Numbers at nodes indicate bootstrap support and vegetative? asexual (stem, cutting, root, bulb. rhizome, values for 1000 replicates; only values that were >60% are tuber, vegetative bud, or leaf) part that has the ability to be given. Bar indicates 0.05 nucleotide substitutions per site cultivated into a new plant. (nucleotide position). 0026. In an embodiment, the methods enhance landscape 0035 FIG.7 shows left compartments of split plates (plant development and remediation. Accordingly, in one embodi with microbial partner): healthy phenotypic appearance of ment, there is provided a method of reducing soil contamina wheat when the root is grown in contact with the microbial tion comprising treating plant propagation material or a plant mats; and right-compartments of split plates (plant without with an endophyte or culture disclosed herein or a combina microbial partner): massive formation of root hairs of wheat tion or mixture thereofora composition disclosed herein; and due to the plant-fungus association made in the left compart cultivating the plant propagation material into a first genera ments of the split plates. tion plant or allowing the plant to grow. In one embodiment, the soil contaminant is hydrocarbons, petroleum or other 0036 FIGS. 8 (A) and (C) shows SMCD2206 discontinu chemicals, salts, or metals, such as lead, cadmium or radio ous colonization of wheat root (epidermis and cortex) tissue isotopes. compared to (B) and (D) which shows pathogenic Fusarium 0027. Other features and advantages of the present disclo graminearum’s uniform/continual cell colonization of wheat sure will become apparent from the following detailed root including vascular cylinder. description. It should be understood, however, that the 0037 FIG.9 shows Ireg index level of deviation (irregu detailed description and the specific examples while indicat larity) in endophyte (SMCDs) cell form. ing embodiments of the disclosure are given by way of illus 0038 FIG. 10 shows Idir index level of direction tration only, since various changes and modifications within changes when colonizing living plant-host cell. the spirit and scope of the disclosure will become apparent to 0039 FIG. 11 shows endophytic hyphae in root of wheat those skilled in the art from this detailed description and germinant (A-SMCD 2204: B-SMCD 2206; C-SMCD 2210; respective drawings and drawing legends. and D-SMCD-2215) visualized with lactofuchsin staining and fluorescence microscopy. Symbiotic structures/organs: BRIEF DESCRIPTION OF THE DRAWINGS (D) SMCD 2215 bacterial endophyte mostly formed curly 0028. The disclosure will now be described in relation to intercellular filaments, whereas endophytic fungi (Figures to the drawings in which: the right) produced: SMCD 2204 intracellular coils and 0029 FIG. 1 shows the phenotypic appearance of the arbuscules, SMCD 2206 intracellular vesicules, and SMCD endophytic fungal strains SMCD 2204, 2004F, 2206, 2208, 2110 intracellular knots. and 2210 and bacterial strain SMCD 2215; after 10 days of 0040 FIG. 12 shows the appearance of symbiotic germi growth on PDA at 21°C. nating wheat seedlings after 10 days on moist filter paper at 0030 FIG. 2A shows the inferred neighbour-joining phy 21o C. logenetic tree of the Cladosporium sp. SMCD 2204 based on 0041 FIG. 13 shows leaf length of germinating wheat ITS rDNA. Numbers at nodes indicate bootstrap support val seedlings after 10 days at moisture filter paper at 21°C. ues for 1000 replicates; only values that were>70% are given. 0042 FIG. 14 shows an in vitro inoculation method (A). A Bar indicates 0.01 nucleotide substitutions per site (nucle 5 mm agar plug, cut from the margin of the parent colony, otideposition). FIG. 2B shows the inferred neighbour-joining was placed hyphal side down in the centre of a 60 mm Petri phylogenetic tree of the Sarocladium sp. SMCD2204F based dish containing potato dextrose agar (PDA) media. Next, five on the sequence of the large Subunit of the nuclear ribosomal Surface-sterilized seeds were placed a distance equivalent to RNA gene (LSU). Numbers at nodes indicate bootstrap Sup 48 h hyphal growth from the agar plug and germinated in the port values for 1000 replicates. Only values that were >70% dark. The impact of three seed surface sterilization methods are given. Bar indicates 0.01 nucleotide substitutions per site on seed germination (B). Bars labeled with one or two aster (nucleotide position). isks (*) are significantly, or highly significantly, different 0031 FIG.3 shows the inferred neighbour-joining phylo from the same endophyte grown under control conditions genetic tree of the Penicillium sp. SMCD 2206 based on ITS (ps0.05 or ps0.01, respectively; ANOVA, followed by post rDNA. Numbers at nodes indicate bootstrap support values hoc LSD test). Error bars represent standard error of the mean for 1000 replicates; only values that were >70% are given. (SE). Bar indicates 0.01 nucleotide substitutions per site (nucle 0043 FIG. 15 shows growth rates of free-living endo otide position). phytes SMCD 2204, 2206, 2208, 2210, and 2215 in vitro on 0032 FIG. 4 shows the inferred neighbour-joining phylo potato dextrose agar (PDA) under heat stress (36° C.), genetic tree of the Pseudeurotium sp. SMCD 2208 based on drought (8% polyethylene glycol (PEG) 8000) stress and ITS rDNA. Numbers at nodes indicate bootstrap support val control conditions for five days and simultaneous heat (36° ues for 1000 replicates; only values that were>70% are given. C.) and drought (8% PEG) for six days. Bars labeled with one Bar indicates 0.01 nucleotide substitutions per site (nucle or two asterisks (*) are significantly, or highly significantly, otide position). different from the same endophyte grown under control con US 2016/017457.0 A1 Jun. 23, 2016 ditions (ps0.05 or ps0.01, respectively; ANOVA, followed nel yield in two row barley (B) and six row barley (B.) by post-hoc LSD test). Error bars represent standard error of genotypes (kernel yield: 3plants/pot). the mean (SE). 0050 FIG. 22 shows (A) Barley-six row AC Metcalfe, 0044 FIG.16 shows percent germination and freshweight from left to the right: Drought (E-), Drought and SMCD2206 of seedlings from initial experiments in which seeds were (E+). Control (E-), Control and SMCD 2206 (E+); (B) surface sterilized in 5% sodium hypochlorite for 3 min. Per Wheat-Unity cultivar, from left to the right: Drought (E-), cent germination of wheat seeds in vitro after three days on Drought and SMCD 2215 (E+). Control (E-), Control and potato dextrose agar (PDA) under heat stress (36° C.), SMCD 2215 (E+): (C) Wheat-Verona cultivar, from left to the drought stress (8% polyethylene glycol (PEG) 8000) and right: Drought (E-), Drought and SMCD 2215 (E+). Control control conditions (A, B and C) with they axis normalized to (E-), Control and SMCD 2215 (E+); and (D) Durum wheat percent germination obtained under the same conditions by TEAL, from left to the right: Drought (E-), Drought and seeds surface sterilized in 5% sodium hypochlorite for 1 min. SMCD 2210 (E+), Control (E-), Control and SMCD 2210 Fresh weight of seedlings invitro at seven days on PDA under (E+). heat stress, drought stress and control conditions (D, E and F). 0051 FIG. 23 shows stem dry weight of (A) chickpeas, Bars labeled with one (*) or two asterisks (**) are signifi (B) lentils, and (C) peas in symbiosis with SMCD endophytes cantly, or highly significantly, different from the no endo (E+) under heat stress phytotron conditions. Bars labeled with phyte control (ps0.05 or ps0.01, respectively; ANOVA, fol one (*) or two asterisks (**) are significantly, or highly sig lowed by post-hoc LSD test). Error bars represent the nificantly, different from the no endophyte stressed control standard error of the mean (SE). (ps0.05 or ps0.01, respectively; ANOVA, followed by post 0045 FIG. 17 shows percent germination over time of hoc LSD test). wheat seeds co-cultured with the endophytes most effective at 0.052 FIG. 24 shows pods dry weight of of (A) chickpeas, conferring abiotic stress tolerance (SMCD 2206, 2210 and (B) lentils, and (C) peas in symbiosis with SMCD endophytes 2215) compared to uncolonized, unstressed seeds (positive (E+) under heat stress phytotron conditions. Bars labeled with control) and uncolonized, stressed seeds (negative control). one (*) or two asterisks (**) are significantly, or highly sig Energy of germination (EG) is related to the time, in days (X nificantly, different from the no endophyte stressed control axis) at which 50% germination (y axis) is reached. The (ps0.05 or ps0.01, respectively; ANOVA, followed by post symbols “”, “x”, “o”, “A”, and “O'” represent the positive hoc LSD test). control, SMCD 2206 treated seeds, SMCD 2210 treated 0053 FIG. 25 shows roots dry weight of (A) chickpeas, seeds, SMCD 2215 treated seeds and the negative control, (B) lentils, and (C) peas in symbiosis with SMCD endophytes respectively. Heat and drought treatments correspond to 36° (E+) under heat stress phytotron conditions. Bars labeled with C. and 8% polyethylene glycol (PEG) 8000, respectively. one (*) or two asterisks (**) are significantly, or highly sig Error bars represent the standard error of the mean (SE). nificantly, different from the no endophyte stressed control Note: The seeds used in EG determination were from the (ps0.05 or ps0.01, respectively; ANOVA, followed by post second round of experiments, and hence sterilized in 5% hoc LSD test). sodium hypochlorite for one minute, rather than three. 0054 FIG. 26 shows stem dry weight of (A) chickpeas, 0046 FIG. 18 shows the relationship between hydrother (B) peas, and (C) lentils under drought stress in agreenhouse. mal time (HTT) required to achieve 50% germination for heat Bars labeled with one (*) or two asterisks (**) are signifi and drought alone and 5% germination for heat and drought cantly, or highly significantly, different from the no endo combined (X axis) and percent germination attained after phyte (E-) stressed control (ps0.05 or ps0.01, respectively; seven days (y axis). Germination after seven days and HTT ANOVA, followed by post-hoc LSD test). were based on the results of the second round of experiments. 0055 FIG. 27 shows dry weights of (A) chickpeas, (B) The symbols “”, “0” and “A” represent seeds exposed to peas, and (C) lentils pods in association with an endophyte heat (36° C.), drought (8% polyethylene glycol (PEG) 8000) (E+) under drought stress in the greenhouse. Bars labeled or both heat and drought stress, respectively. The R-squared with one (*) or two asterisks (**) are significantly different values associated with the trendlines are 0.96, 0.80 and 0.18 from the no endophyte (E-) stressed control (ps 0.05 or ps0. for seeds exposed to heat, drought or both heat and drought 01, respectively; ANOVA, followed by post-hoc LSD test). stress, respectively. Note: The seeds used to determine per 0056 FIG. 28 shows roots dry weight of (A) chickpeas, cent germination at seven days and HTT were from the sec (B) peas, and (C) lentils under drought stress in the green ond round of experiments, and hence treated with 5% sodium house. Bars labeled with one (*) or two asterisks (**) are hypochlorite for one minute, rather than three. significantly, or highly significantly, different from no endo 0047 FIG. 19 shows seeds treated or inoculated with phyte (E-) stressed control (ps0.05 or ps0.01, respectively; SMCD strains demonstrate improvement in all tested seed ANOVA, followed by post-hoc LSD test). germination parameters including seed germination vigour 0057 FIG. 29 shows (A) Chickpea Vanguard flowering (SGV) efficacy. plants bearing pods under drought stress in a greenhouse— 0048 FIG. 20 shows the relationship between drought left plant is non-Symbiotic (E-) and right plant is symbiotic tolerance efficiency (DTE) values in wheat (A) and barley (B) with strain SMCD 2215 (E+); (B) and (C), Chickpea Van cultivars without (E-) and with (E+) endophytes, based on the guard plants bearing pods under drought stress in a green average effect of symbiosis using all tested SMCD isolates, house—(B) non-symbiotic and (C) symbiotic with SMCD on yield exposed to drought stress in greenhouse. 2215. 0049 FIG. 21A shows endophytic (E+) inoculants 0.058 FIG.30 shows root nodulation of pea varieties under (SMCD 2206, SMCD 2210, and SMCD 2215) improve ker heat stress in a phytotron: Hendel (Above) and Golden (Be nel yield in wheat genotypes compared to control (E-) treat low) inoculated (left) and uninoculated (right) with SMCD ment (yield g/3 pots). FIG.21B shows endophytic inoculants 2215. Note: in all samples natural infection with Rhizobium (SMCD 2206, SMCD 2210, and SMCD 2215) improve ker sp. from pea seeds has been observed. US 2016/017457.0 A1 Jun. 23, 2016

0059 FIG. 31 shows SMCD2206 and SMCD 2215 con (0072 FIG. 44 shows (A) SOD and (B) MnSOD relative siderably increase energy of seed germination (>50%) in gene expressions in pea (Handel) exposed to PEG with and Glamis (lentil) as a function of time under heat and drought in without endophytes. vitro. (0073 FIG. 45 shows Proline relative gene expression in 0060 FIG. 32 shows SMCD2206 and SMCD 2215 con pea (Handel) exposed to PEG with and without endophytes. siderably increase energy of seed germination (>50%) in 0074 FIG. 46 shows germination of wheat seeds in vitro Handel (pea) as a function of time under heat and drought in after three days on potato dextrose agar (PDA). Cold stratifi vitro. cation was imposed by keeping seeds at 4°C. cold-room for 0061 FIG.33 shows endophytic inoculants (SMCD2206 48 hours. For endophyte-indirect and endophyte-direct treat and SMCD 2210) improve flax yield under drought condi ments, using SMCD2206, seeds were germinated at approxi tions in agreenhouse. Different letters above the bars indicate mately 4 cm distance and in direct contact respectively. A) statistically significant differences between samples (p<0.05, Percentage of germination in comparison with energy of ger Kruskal-Wallis test). mination (50% germination). B) Efficacy of germination of 0062 FIG.34 shows endophytic inoculants (SMCD2206, wheat seeds Subjected to cold and biological stratification. SMCD 2210, and SMCD 2215) improve canola yield under Efficacy was calculated by Subtracting the germination per drought conditions in a greenhouse. Different letters above centage of control from treated seeds. the bars indicate statistically significant differences between (0075 FIG. 47 shows differential expression patterns of samples (p<0.05, Kruskal-Wallis test). gibberellin (TaGA3ox2 and 14-3-3) and ABA (TaNCED2 0063 FIG. 35 shows the survival of wheat seeds pre-in and TaABA8'OH1) genes in coleorhiza of germinating wheat oculated in-vitro (plates in above row) and wheat seedlings seeds for three days under cold and biological stratification. pre-inoculated in greenhouse (pots in below row) with endo Gene expression was calculated as 2CT. phytic SMCD 2206-showing healthy plant growth, and with (0076 FIG. 48 shows the ratio of expression levels (2 pathogenic Fusarium avenaceum and Fusarium graminearum—showing disease symtoms and death of 2-CT) of gibberellin (TaGA3ox2 and 14-3-3) and ABA plants. (TaNCED2 and TaABA8'OH1) genes in coleorhiza of germi nating wheat seeds for three days under cold and biological 0064 FIG. 36 shows Fusarium inoculants produced on stratification. wheat kernels. (0077 FIG. 49 shows relative expression patterns of hor 0065 FIG. 37 shows that post-emergence damping-off monal RSG and KAO regulator genes and MYB 1 and MYB has been prevented by SMCD2206 endophyte in wheat in the 2 resistance genes in coleorhiza of germinating wheat seeds greenhouse. for three days under cold and biological stratification. Gene 0066 FIG. 38 shows wheat biomass (aerial a-d and root expression was calculated as 2A". e-f) improved in the presence of SMCD 2206 endophyte 0078 FIG. 50 shows emerging radicle from wheat gemi compared to untreated plants. (a) control plant (E-), (b) nating seed (A) Inverted fluorescence (B) and fluorescence inoculated plant (E+), (c) control flowering plant, (d) inocu imaging of DAF-2DA fluorescence upon reaction with NO in lated flowering plant, (e) control plant (E-, left) compared to radicle cells (C) of AC Avonlea germinant at 5 min after SMCD 2206 inoculated plant (E+, right), and (f) fluorescent treatment Nakatsubo et al. 1998 with the fungal SMCD microscopy of SMCD 2206 wheat root-colonization (E+). 2206 exudate. No fluorescence reaction observed in control 0067 FIG. 39 shows aerial plant biomass/plant (left) and radicle cells. Bar-25 um; Bar-50 um. underground (root) biomass/plant (right) in control (E-) and (0079 FIG. 51 shows DAF-2T fluorescence intensity val SMCD inoculated wheat plants (E+) against F graminearum ues at 5 min after treatment of wheat radicle from AC Avonlea and F. avenaceum. Vertical error bars on data points represent germinants with the SMCD2206 fungal exudate, fungal exu the standard error of the mean. date together with the NO scavenger cRTIO, and sterile water. 0068 FIG. 40 shows root length in control plant (CDC Radicle segments were incubated for 30 min in 2 ml of detec Teal) without SMCD endophyte compared to inoculated tion buffer (10 mM Tris-Hcl, pH 7.4, 10 mMKCl) containing wheat plant with SMCD strains. Bars on data points represent 15 uM DAF-2DA (Sigma-Aldrich) with or without 1 mM the standard error of the mean. 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1- 0069 FIG. 41 shows dry weight of kernels/plant (TEAL oxyl-3-oxide (cPTIO) as an NO scavenger. Average fluores cultivar) in wheat using the double pre-inoculation approach: cence values are reported as a ratio of the fluorescence inten a) SMCD endophyte--Fusarium avenaceum (Flav), and b) sity at 5 minto the fluorescence intensity at time O. Different SMCD endophyte--Fusarium graminearum (Figr). Vertical letters indicate statistically significant differences between error bars on data points represent the standard error of the samples (p<0.05, Kruskal-Wallis test). Ca 0080 FIG. 52 shows the change (in days) in the initial 0070 FIG. 42 shows comparison of TEAL spike sizes in flowering time of canola crops treated with the microbial wheat in the presence of pathogen (negative control) and compositions described. Data shown is from n=4 independent without presence of pathogen (positive control). Left Fig replicate plotstone standard deviation. 1=Abiotic formula ure—from left to right: i) plant+Figr, ii) plant-i-Fav, and (iii) tion control, 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD plant; Right Figure—from left to right: i) plant; ii) plant-- 2206,5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215. endophyte; iii) plant--endophyte--F. av; and iv) plant--endo I0081 FIG. 53 shows the damage score due to pests of phyte--F.gr. canola crops treated with the microbial compositions (0071 FIG. 43 shows the effect of SMCD 2215 on Handel described. Data shown is from n=4 independent replicate (pea) on 10% PEG after 7 days at 21 degrees C. in darkness. plotstone standard deviation. 1=Abiotic formulation control, (A) shows the control seeds, and (B) shows the SMCD 2215 2=SMCD2204,3=SMCD 2204F, 4=SMCD 2206,5=SMCD treated seeds. 2208, 6=SMCD 2210, 7=SMCD 2215. US 2016/017457.0 A1 Jun. 23, 2016

I0082 FIG. 54 shows the Fusarium Head Blight (FHB) shoot length, (b) shoot weight, (c) total plant biomass, (d) root incidence percentage for three spring wheat (Lillian, Unity, length, (e) root weight, and (f) tomato fruit weight under and Utmost) and one durum wheat (Strongfield) varieties. drought conditions. Data shown is from n=3 independent Subplots (a), (b), (e), and (f) refer to the Vanguard, replicate plantstone standard deviation. Saskatchewan, Canada test site, while Subplots (c), (d), (g), I0088 FIG. 60 shows data from greenhouse trials of alfalfa and (h) refer to the Stewart Valley, Saskatchewan, Canada test treated with the described microbial compositions: (a) shoot site. Data shown is from n 4 independent replicate plotstone length, (b) shoot weight, (c) total plant biomass, (d) root standard deviation. 1=Abiotic formulation control, 2=SMCD length, and (e) root weight under drought conditions. Data 2204, 3=SMCD 2204F, 4=SMCD 2206, 5–SMCD 2208, shown is from n 3 independent replicate plantstone standard 6=SMCD 2210, 7=SMCD 2215. deviation. I0083 FIG.55 shows the leaf spot disease rating for three I0089 FIG. 61 shows data from greenhouse trials compar spring wheat (Lillian, Unity, and Utmost) and one durum ing normal (dark gray) and drought (light gray) water condi wheat (Strongfield) varieties. A rating of 1 is most healthy and tions for: (a) corn, (b) sweet corn, (c) organic corn, (d) Swiss a rating of 10 is most diseased. Subplots (a), (b), (e), and (f) chard, (e) radish, and (f) cabbage. Data shown is total biomass refers to the leaf spot disease rating on the lower leaf, while from n 3 independent replicate plantstone standard devia Subplots (c), (d), (g), and (h) refer to the leaf spot disease tion. rating on the flag leaf. Data shown is from n=4 independent (0090 FIG. 62 shows gibberellin production data from replicate plotstone standard deviation. 1=Abiotic formula wheat (CDC Avonlea) in seedling studies. “Control” refers to tion control, 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD the formulation control wherein SMCD2206 was not added, 2206,5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215. “A-Direct” refers to direct application of SMCD 2206 to the 0084 FIG. 56 shows aggregated yield data for durum seedling, and A-Indirect” refers to the indirect application of wheat, spring wheat, barley, canola, and pulses (chickpea, SMCD 2206 to the seedling through a small-molecule per pea, and lentil). Each dot refers to a single plot. The "+" refers meable membrane. See Table 12 for abbreviation of mol to the group mean. The data are presented as percentage gain ecules. Data shown is average concentration in ng per gram over the abiotic formulation control for each combination of dry tissue weight from n=3 independent replicates. crop, location and condition (irrigated or dryland). (a) refers (0091 FIG. 63 shows ABA metabolite production data to SMCD 2215, (b) refers to SMCD 2210, (c) refers to SMCD from wheat (CDC Avonlea) in seedling studies. “Control 2204, (d) refers to SMCD2206, (e) refers to SMCD2208, and refers to the formulation control wherein SMCD 2206 was (f) refers to SMCD 2204F. Data shown are from n=4 inde not added, 'A-Direct” refers to direct application of SMCD pendent replicate plots for all Canadian sites and n=6 plots for 2206 to the seedling, and A-Indirect” refers to the indirect Brookings, S. Dak. sites. While no fields were experimentally application of SMCD 2206 to the seedling through a small inoculated with a pathogen, the notation “Fus-” indicates that molecule permeable membrane. See Table 13 for abbrevia there was no visible occurrence of Fusarium Head Blight tion of the names of the molecules. Data shown is average (Fusarium graminearum) in that specific field and “Fus+” concentration in ng per gram dry tissue weight from n=3 indicates that there was clear evidence of Fusarium Head independent replicates. Blight (Fusarium graminearum) occurrence. N- indicates 0092 FIG. 64 shows cytokinin production data from that there was no nitrogen applied to the field and N-- indi wheat (CDC Avonlea) in seedling studies. “Control” refers to cates that nitrogen was applied at agriculturally relevant rates. the formulation control wherein SMCD2206 was not added, 0085 FIG. 57 shows aggregated yield data for durum “A-Direct” refers to direct application of SMCD 2206 to the wheat, spring wheat, barley, canola, and pulses (chickpea, seedling, and A-Indirect” refers to the indirect application of pea, and lentil). 95% confidence intervals for the respective SMCD 2206 to the seedling through a small-molecule per formulation and crop are shown. The data are presented as meable membrane. See Table 14 for abbreviation of mol percentage gain over the abiotic formulation control for each ecules. Data shown is average concentration in ng per gram combination of crop, field trial and condition (irrigated or dry tissue weight from n=3 independent replicates. dryland). (a) refers to SMCD 2215, (b) refers to SMCD 2210, 0093 FIG. 65 shows auxin production data from wheat (c) refers to SMCD2204, (d) refers to SMCD2206, (e) refers (CDC Avonlea) in seedling studies. “Control refers to the to SMCD 2208, and (0 refers to SMCD2204F. Data shown is formulation control wherein SMCD 2206 was not added, from n=4 independent replicate plots for all Canadian sites “A-Direct” refers to direct application of SMCD 2206 to the and n=6 plots for Brookings, S. Dak. As above, the notation seedling, and A-Indirect” refers to the indirect application of “Fus-” indicates that there was no visible occurrence of SMCD 2206 to the seedling through a small-molecule per Fusarium Head Blight (Fusarium graminearum) in that spe meable membrane. Auxins were represented by the biologi cific field and “Fus+ indicates that there was clear evidence cally active indole acetic acid IAA and its conjugate with of Fusarium Head Blight (Fusarium graminearum) occur aspartic acid IAA-Asp. Data shown is average concentration rence. N- indicates that there was no nitrogen fertilizer in ng per gram dry tissue weight from n 3 independent rep applied to the field and N-- indicates that nitrogen fertilizer licates. was applied at agriculturally relevant rates. 0094 FIG. 66 shows symbiosomes in root of wheat ger I0086 FIG. 58 shows the aggregated ear weight data from minant visualized with lactofuchsin staining and fluores the corn field trial in Brookings, S. Dak. 95% confidence cence microscopy. A type I Symbiosome, which is composed intervals for the respective formulation is shown. The data is of an intercellular microvesiculoid compartment formed presented as percentage gain over the abiotic formulation between two plant cell membranes (arrows), a perivesiculoid control. “2206 refers SMCD 2206, “2204 refers to SMCD membrane (large triangle) and a partially fragmented outer 2204, and “2215” refers to SMCD 2215. vesiculoid membrane (Small triangle), is shown in A. A type 0087 FIG.59 shows data from greenhouse trials of tomato II symbiosome, which is composed of an intracellular vesicu inoculated with the described microbial compositions: (a) loid compartment (arrows), a perivesiculoid membrane (large US 2016/017457.0 A1 Jun. 23, 2016 triangle) and a partially fragmented outer vesiculoid mem ments, bactovitality may be characterized by a change in brane (Small triangle), is shown in B. Also shown in B is a expression of the genes involved in gibberellin (GA) biosyn vesiculophore (filled arrow). The symbiosomes shown in A thesis or breakdown or in abscisic acid (ABA) biosynthesis or and B are both of vesicle form. Shown in C are symbiosomes breakdown, or in positive or negative regulation of these of knot form (type I—lower arrow; type II—upper arrow). pathways within the plant-bacteria symbiotic system. In cer tain embodiments, the levels of expression of the gibberellin DEFINITIONS (GA) biosynthetic genes, such as GA3-oxidase 2, RSG, 0095. The term “mycovitality” as used herein refers to the KAO, and 14-3-3 genes may be increased. In other embodi plant-fungus symbiosis that exists between the seeds and the ments, the levels of expression of the genes that are regulated fungi, which helps maintain the seeds capacity to live and by GAS, Such as ent-kaurenoic (KAO) and repression of shoot develop, and leads to germination. In some embodiments, growth (RSG), are increased. In other embodiments, the lev mycoVitality may be characterized by a change in levels of els of expression of the GA degradation genes or negative certain phytohormones within the plant-fungus symbiotic regulators of the GA biosynthesis pathway, for example system. In some embodiments, this change may be associated 14-3-3 genes, are decreased. In still other embodiments, bac with a change in the levels of abscisic acid (ABA), gibberel tovitality may be characterized by decreased levels of expres lins (GA), auxins (IAA), and/or cytokinins. In other embodi sion of the genes involved in the ABA biosynthesis pathway, ments, mycoVitality may be characterized by a change in for example the NCED gene, within the plant-bacterium sym expression of the genes involved in gibberellin (GA) biosyn biotic system. In other embodiments, the expression of the thesis or breakdown or in abscisic acid (ABA) biosynthesis or genes involved in the ABA catabolic pathway, for example breakdown, or in positive or negative regulation of these the 8-hydroxylase gene, are increased. In some embodi pathways within the plant-fungus symbiotic system. In cer ments, bactovitality may be characterized by altered levels of tain embodiments, the levels of expression of the gibberellin nitric oxide (NO) within the plant-bacterium symbiotic sys (GA) biosynthetic genes, such as GA3-oxidase 2, RSG, tem, for example as a result of a change in the expression of KAO, and 14-3-3 genes may be increased. In other embodi certain genes involved in NO production or breakdown. In yet ments, the levels of expression of the genes that are regulated other embodiments, bactovitality may be characterized by by GAS, Such as ent-kaurenoic (KAO) and repression of shoot protection of the plant-bacterium symbiotic system from oxi growth (RSG), are increased. In other embodiments, the lev dative stress. In some embodiments, bactovitality is charac els of expression of the GA degradation genes or negative terized by increased levels of expression of the genes involved regulators of the GA biosynthesis pathway, for example in Superoxide detoxification within the plant-bacterium sym 14-3-3 genes, are decreased. In still other embodiments, biotic system. In some embodiments, the genes associated mycovitality may be characterized by decreased levels of with Superoxide detoxification encode Superoxide dismutase expression of the genes involved in the ABA biosynthesis (SOD) or manganese SOD (MnSOD), and in other cases the pathway, for example the NCED gene, within the plant-fun levels of the amino acid proline (Pro) are elevated. In some gus symbiotic system. In other embodiments, the expression embodiments, bactovitality is characterized by an increase in of the genes involved in the ABA catabolic pathway, for the levels of activity of the genes associated with systemic example the 8'-hydroxylase gene, are increased. In some acquired disease resistance, Such as redox-regulated tran embodiments, mycovitality may be characterized by altered scription factors, for example those in the MYB family. In levels of nitric oxide (NO) within the plant-fungus symbiotic some embodiments, the genes in the MYB family are Myb 1 system, for example as a result of a change in the expression and Myb2. of certain genes involved in NO production or breakdown. In 0097. “Cold stratification as used herein refers to the yet other embodiments, mycoVitality may be characterized by process of pretreating seeds to simulate the natural winter protection of the plant-fungus Symbiotic system from oxida conditions during which, amongst many physiological tive stress. In some embodiments, mycoVitality is character changes, the seed coat is softened up by frost and weathering ized by increased levels of expression of the genes involved in action, leading to dormancy breakdown. "Biological Stratifi Superoxide detoxification within the plant-fungus symbiotic cation' as used herein refers to the process of treating seeds system. In some embodiments, the genes associated with with biological components to release seed dormancy and Superoxide detoxification encode Superoxide dismutase thereby promoting germination. In some embodiments, the (SOD) or manganese SOD (MnSOD), and in other cases the biological components may be endophytes. Therefore, as levels of the amino acid proline (Pro) are elevated. In some compared to cold stratification, in which an abiotic stimula embodiments, mycoVitality is characterized by an increase in tion is used, biological stratification uses a biotic stimulation. the levels of activity of the genes associated with systemic As for cold stratification, biological stratification may acquired disease resistance, Such as redox-regulated tran increase the rate of germination in seeds. In both cases, the scription factors, for example those in the MYB family. In progress of Stratification and dormancy breakdown may be some embodiments, the genes in the MYB family are Myb 1 associated with an increase in levels of GA and a decrease in and Myb2. levels of ABA. In certain embodiments, the levels of expres 0096. The term “bactovitality” as used herein refers to the sion of gibberellin (GA) biosynthetic genes, such as GA3 plant-bacterium symbiosis that exists between the seeds and oxidase 2 and 14-3-3 genes, are increased. In other embodi the bacteria, which helps maintain the seeds capacity to live ments, the levels of expression of the genes that are regulated and develop, and leads to germination. In some embodiments, by GAS, Such as ent-kaurenoic (KAO) and repression of shoot bactovitality may be characterized by a change in levels of growth (RSG), are increased. In other embodiments, the lev certain phytohormones within the plant-bacterium symbiotic els of expression of GA degradation genes or negative regu system. In some embodiments, this change may be associated lators of the GA biosynthesis pathway, for example 14-3-3 with a change in the levels of abscisic acid (ABA), gibberel genes, are decreased. In still other embodiments, the levels of lins (GA), auxins (IAA), and/or cytokinins. In other embodi expression of the genes involved in the ABA biosynthesis US 2016/017457.0 A1 Jun. 23, 2016

pathway, for example the NCED gene, are decreased. In other 0101. As used herein, the term “microarbuscule” refers to embodiments, the expression of genes involved in the ABA intracellular, multiarbuscular, microsized (~10 um), bush catabolic pathway, for example the 8'-hydroxylase gene, is like haustorial structures. increased. 0102 The term “vitality, as used herein means the capac 0098 “Anti-aging or “anti-senescence” as used herein ity to live and develop. refers to a process within a seed or plant that protects the seed 0103) The term “hydrothermal time' refers to parameters or plant from aging and senescence or that results in delayed of water, temperature and time by which seed germination aging or senescence of the seed or plant. In some embodi can be described under various environmental conditions. ments, the anti-aging or anti-senescence effects of endo The parameters enable germination strategies to be compared phytes are characterized by increased levels of nitric oxide in different environments and to assess the effects of endo (NO) within the plant-fungus or plant-bacterium symbiotic phytes on germination relative to other variables. system, for example as a result of a change in the expression 0104. In some embodiments, the endophyte is chosen of certain genes involved in NO production or breakdown. In from the group consisting of a spore-forming endophyte, a certain embodiments, the anti-aging or anti-senescence facultative endophyte, a filamentous endophyte, and an endo effects of endophytes may be characterized by a change in phyte capable of living within another endophyte. In some levels of certain phytohormones within the plant-fungus or embodiments, the endophyte is capable of forming certain plant-bacterium symbiotic system. In some embodiments, structures in the plant, where the structures are selected from this change may be associated with decreased by levels of the group consisting of hyphal coils, Hartig-like nets, abscisic acid (ABA), increased levels of gibberellins (GA) or microvesicles, micro-arbuscules, hyphal knots, and Symbio increased levels of auxins. In some embodiments, mycoVital Somes. In some embodiments, the endophyte is in the form of ity may be characterized by a change in expression of the at least one of conidia, chlamydospore, and mycelia. In other genes involved in gibberellin (GA) biosynthesis or break embodiments, the fungus or bacteria is capable of being part down or in abscisic acid (ABA) biosynthesis or breakdown, of a plant-fungus symbiotic system or plant-bacteria symbi or in positive or negative regulation of these pathways within otic system that produces altered levels of phytohormones or the plant-fungus or plant-bacterium symbiotic system. In cer anti-oxidants, as compared to a plant that is not in Symbiosis. tain embodiments, the levels of expression of the gibberellin In other embodiments, the plant-fungus symbiotic system or (GA) biosynthetic genes, such as GA3-oxidase 2, RSG, plant-bacterium symbiotic system has anti-aging and/or anti KAO, and 14-3-3 genes may be increased. In other embodi senescence effects, as compared to a plant or plant organ that ments, the levels of expression of the genes that are regulated is not in symbiosis. In other embodiments, the plant-fungus by GAS, Such as ent-kaurenoic (KAO) and repression of shoot symbiotic system or plant-bacteria symbiotic system has growth (RSG), are increased. In other embodiments, the lev increased protection against pathogens, as compared to a els of expression of the GA degradation genes or negative plant that is not in Symbiosis. regulators of the GA biosynthesis pathway, for example 0105. A “spore' or a population of “spores' refers to bac terial or fungal structures that are more resilient to environ 14-3-3 genes, are decreased. In still other embodiments, the mental influences such as heat and bacteriocidal agents and anti-aging or anti-senescence effects of endophytes may be fungicides than vegetative forms of the same bacteria or characterized by decreased levels of expression of the genes fungi. Spores are typically capable of germination and out involved in the ABA biosynthesis pathway, for example the growth giving rise to vegetative forms of the species. Bacteria NCED gene, within the plant-fungus or plant-bacterium sym and fungi that are “capable of forming spores' or “spore biotic system. In other embodiments, the expression of the forming endophytes' are those bacteria and fungi containing genes involved in the ABA catabolic pathway, for example the genes and other necessary abilities to produce spores the 8'-hydroxylase gene, are increased. under Suitable environmental conditions. 0099. As used herein, “symbiosome” or “symbiotic 0106 The term “filamentous fungi as used herein are organs’ refers to the new compartment that is formed within fungi that form hyphae, and includes taxa that have both the plant cell whenbacteria or fungicolonize the plant. In type filamentous and yeast-like stages in their life cycle. I Symbiosomes, the new structure is an intercellular microve 0107 The term “facultative endophytes' as used herein Siculoid compartment formed between two plant cell mem are endophytes capable of Surviving in the soil, on the plant branes. A “microvesiculoid” compartment is a structure that Surface, inside a plant and/or on artificial nutrients. Faculta has the form of a microvesicle. In type II symbiosomes, the tive endophytes may also have the capacity to Survive inside new compartment is localized intracellularly and can be a variety of different plant species. described as an intracellular structure in the form of a vesicle, 0108. The term “endophyte capable of living within or “intracellular vesiculoid compartment.” Both types of another endophyte' as used herein refers to an endophytic symbiosomes are further characterized by the presence of a bacterium or fungus that can live within another endophyte. “perivesiculoid membrane,” which is the plasma membrane Such endophytic bacteria may also be able to live autono that Surrounds the vesicles, and a partially fragmented "outer mously in the Soil, on the plant Surface, inside a plant and/or vesiculoid membrane,” which is an outer membrane in the on artificial nutrients. form of a vesicle. In this context, a symbiosome is not limited 0109 The term “endophyte capable of promoting germi to the structure that is formed during nitrogen fixation. nation” as used herein refers to endophytes that have the 0100 “Mycoheterotrophy’ as used herein refers to a sym capacity to colonize a seed or part of a seed and alter the biotic relationship between a plant and a fungus that allows seeds physiology Such that the seed or a population of seeds the plant to obtain water, minerals, and carbohydrates more shows a faster dormancy breakdown, greater germination efficiently. In this context, the plant may be any plant, even a rate, earlier germination, increased energy of germination, fully photosynthetic plant, that may derive a benefit via its greater rate of germination, greater uniformity of germina association with the fungus. tion, including greater uniformity of rate of germination and US 2016/017457.0 A1 Jun. 23, 2016 greater uniformity of timing of germination, and/or increased clover (T repens); Red clover (T. pratense); Caucasian clover vigor and energy of germination. In some embodiments, the (T. ambigum); Subterranean clover (T. subterraneum); endophyte capable of promoting germination is an endophyte Alfalfa/Lucerne (Medicago sativum); annual medics; barrel that is capable of activating the coleorhiza of a monocot seed, medic; black medic; Sainfoin (Onobrychis viciifolia); Birds and can be called a “coleorhiza-activating endophyte'. foot trefoil (Lotus corniculatus); Greater Birdsfoot trefoil 0110. The term "agricultural plant’ means a plant that is (Lotus pedunculatus); typically used in agriculture. The agricultural plant may be a 0.126 Forage and Amenity grasses: Temperate grasses monocot or dicot plant, and may be planted for the production Such as Lolium species; Festuca species; Agrostis spp., Peren of an agricultural product, for example grain, food, fiber, etc. nial ryegrass (Lolium perenne); hybrid ryegrass (Lolium The plant may be a cereal plant. The term “plant’ as used hybridum); annual ryegrass (Lolium multiflorum), tall fescue herein refers to a member of the Plantae Kingdom and (Festuca arundinacea); meadow fescue (Festuca pratensis); includes all stages of the plant life cycle, including without red fescue (Festuca rubra); Festuca ovina; Festuloliums (LO limitation, seeds, and includes all plant parts. The plant can be lium X Festuca crosses); CockSfoot (Dactylis glomerata); selected from, but not limited to, the following list: Kentucky bluegrass Poa pratensis, Poa palustris, Poa nem 0111 Food crops: Cereals including Maize/corn (Zea oralis, Poa trivialis, Poa compresa, Bromus species; mays), Sorghum (Sorghum spp.), Millet (Panicum miliaceum, Phalaris (Phleum species); Arrhenatherum elatius, Agropy P Sumatrense), Rice (Oryza sativa indica, Oryza sativa ron species; Avena Strigosa, Setaria italic, japonica), Wheat (Triticum sativa), Barley (Hordeum vul I0127 Tropical grasses such as: Phalaris species; Bra gare), Rye (Secale cereale), Triticale (Triticum X Secale), chiaria species; Eragrostis species; Panicum species; Bahai Oats (Avena fatua); grass (Paspalum notatum); Brachypodium species; and, 0112 leafy vegetables (brassicaceous plants such as cab I0128 Grasses used for biofuel production such as Switch bages, broccoli, bok choy, rocket; salad greens such as spin grass (Panicum virgatum) and Miscanthus species; ach, cress, lettuce); 0113 fruiting and flowering vegetables (e.g. avocado, I0129. Fiber Crops: hemp, jute, coconut, sisal, flax (Linum Sweet corn, artichokes, curcubits e.g. squash, cucumbers, spp.), New Zealand flax (Phormium spp.); plantation and melons, courgettes, pumpkins; Solononaceous vegetables/ natural forest species harvested for paper and engineered fruits e.g. tomatoes, eggplant, capsicums); wood fiber products such as coniferous and broadleafed for 0114 podded vegetables (groundnuts, peas, beans, lentils, est species; chickpea, okra): I0130 Tree and Shrub Species Used in Plantation Forestry 0115 bulbed and stem vegetables (asparagus, celery, and Bio Fuel Crops: Pine (Pinus species); Fir (Pseudotsuga Allium crops e.g. garlic, onions, leeks); species); Spruce (Picea species); Cypress (Cupressus spe 0116 roots and tuberous vegetables (carrots, beet, bam cies); Wattle (Acacia species); Alder (Alnus species); Oak boo shoots, cassava, yams, ginger, Jerusalem artichoke, pars species (Quercus species); Redwood (Sequoiadendron spe nips, radishes, potatoes, Sweet potatoes, taro, turnip, wasabi); cies); willow (Salix species); birch (Betula species); Cedar 0117 Sugar crops including Sugar beet (Beta vulgaris), (Cedurus species); Ash (Fraxinus species); Larch (Larix spe Sugar cane (Saccharum officinarum); cies); Eucalyptus species; Bamboo (Bambu.seae species) and 0118 crops grown for the production of non-alcoholic Poplars (Populus species). beverages and stimulants (coffee, black, herbal and green I0131 Plants Grown for Conversion to Energy, Biofuels or teas, cocoa, tobacco); Industrial Products by Extractive, Biological, Physical or 0119 fruit crops such as true berry fruits (e.g. kiwifruit, Biochemical Treatment: Oil-producing plants such as oil grape, currants, gooseberry, guava, feijoa, pomegranate), cit palm, jatropha, linseed; rus fruits (e.g. oranges, lemons, limes, grapefruit), epigynous 0.132. Latex-producing plants such as the Para Rubber fruits (e.g. bananas, cranberries, blueberries), aggregate fruit tree, Hevea brasiliensis and the Panama Rubber Tree Castilla (blackberry, raspberry, boysenberry), multiple fruits (e.g. elastica, pineapple, fig), Stone fruit crops (e.g. apricot, peach, cherry, 0.133 plants used as direct or indirect feedstocks for the plum), pip-fruit (e.g. apples, pears) and others such as Straw production of biofuels i.e. after chemical, physical (e.g. ther berries, sunflower seeds; mal or catalytic) or biochemical (e.g. enzymatic pre-treat 0120 culinary and medicinal herbs e.g. rosemary, basil, ment) or biological (e.g. microbial fermentation) transforma bay laurel, coriander, mint, dill, Hypericum, foxglove, tion during the production of biofuels, industrial solvents or alovera, rosehips); chemical products e.g. ethanol or butanol, propane diols, or 0121 crop plants producing spices e.g. black pepper, other fuel or industrial material including Sugar crops (e.g. cumin cinnamon, nutmeg, ginger, cloves, Saffron, cardamom, beet, Sugar cane), starch-producing crops (e.g. C3 and C4 mace, paprika, masalas, star anise; cereal crops and tuberous crops), cellulosic crops such as 0122 crops grown for the production of nuts and oils e.g. forest trees (e.g. Pines, Eucalypts) and Graminaceous and almonds and walnuts, Brazil nut, cashew nuts, coconuts, Poaceous plants such as bamboo, Switch grass, miscanthus; chestnut, macadamianut, pistachio nuts; peanuts, pecan nuts, 0.134 crops used in energy, biofuel or industrial chemical Soybean, cotton, olives, Sunflower, sesame, lupin species and production by gasification and/or microbial or catalytic con brassicaeous crops (e.g. canola/oilseed rape); and, version of the gas to biofuels or other industrial raw materials 0123 crops grown for production of beers, wines and Such as solvents or plastics, with or without the production of other alcoholic beverages e.g. grapes, hops; biochar (e.g. biomass crops such as coniferous, eucalypt, 0.124 edible fungi e.g. white mushrooms, Shiitake and tropical or broadleaf forest trees, graminaceous and poaceous oyster mushrooms; crops such as bamboo, Switch grass, miscanthus, Sugarcane, 0.125 Plants Used in Pastoral Agriculture: legumes: Trifo or hemp or softwoods such as poplars, willows; and, lium species, Medicago species, and Lotus species; White 0.135 biomass crops used in the production of biochar; US 2016/017457.0 A1 Jun. 23, 2016

0.136 Crops Producing Natural Products Useful for the fied, such as by genetic modification, e.g., contains a trans Pharmaceutical, Agricultural Nutraceutical and Cosmeceuti gene that confers resistance to an herbicide, or is adapted to cal Industries: crops producing pharmaceutical precursors or grow in, or otherwise Survive, the concentration of the agri compounds or nutraceutical and cosmeceutical compounds cultural chemical used in agriculture. For example, a microbe and materials for example, star anise (shikimic acid), Japa disposed on the surface of a seed is compatible with the nese knotweed (resveratrol), kiwifruit (soluble fiber, pro fungicide metalaxyl if it is able to survive the concentrations teolytic enzymes): that are applied on the seed Surface. 0.137 Floricultural, Ornamental and Amenity Plants 0.148. As used herein, a “colony-forming unit” (“CFU) is Grown for their Aesthetic or Environmental Properties: Flow used as a measure of viable microorganisms in a sample. A ers such as roses, tulips, chrysanthemums; CFU is an individual viable cell capable of forming on a solid 0138 Ornamental shrubs such as Buxus, Hebe, Rosa, medium a visible colony whose individual cells are derived Rhododendron, Hedera, by cell division from one parental cell. 0139 Amenity plants such as Platanus, Choisya, Escal 0149. In some embodiments, the invention uses microbes lonia, Euphorbia, Carex, that are heterologous to a seed or plant in making synthetic 0140 Mosses such as Sphagnum moss; and combinations or agricultural formulations. A microbe is con 0141 Plants Grown for Bioremediation: Helianthus, sidered heterologous to the seed or plant if the seed or seed Brassica, Salix, Populus, and Eucalyptus. ling that is unmodified (e.g., a seed or seedling that is not 0142. A “host plant' includes any plant, particularly an treated with a population of endophytes capable of promoting agricultural plant, which an endophytic microbe Such as an germination described herein) does not contain detectable endophyte capable of promoting germinations can colonize. levels of the microbe. For example, the invention contem As used herein, a microbe is said to “colonize' a plant or seed plates the synthetic combinations of seeds or seedlings of when it can be stably detected within the plant or seed over a agricultural plants and an endophytic microbe population period time, such as one or more days, weeks, months or (e.g., an endophyte capable of promoting germination), in years. In other words, a colonizing microbe is not transiently which the microbe population is "heterologously disposed associated with the plant or seed. on the exterior surface of or within a tissue of the agricultural 0143. As used herein, an "agricultural seed' is a seed used seed or seedling in an amount effective to colonize the plant. to grow a plant typically used in agriculture (an "agricultural A microbe is considered "heterologously disposed on the plant”). The seed may be of a monocot ordicot plant, and may surface or within a plant (or tissue) when the microbe is be planted for the production of an agricultural product, for applied or disposed on the plant in a number that is not found example grain, food, fiber, etc. The seed may be of a cereal on that plant before application of the microbe. For example, plant. As used herein, an agricultural seed is a seed that is population of endophytes capable of promoting germination prepared for planting, for example, in farms for growing. that is disposed on an exterior Surface or within the seed can 0144. As used herein, a “control agricultural plant’ or be an endophyte that may be associated with the mature plant, “control seed' is an agricultural plant or seed of the same but is not found on the surface of or within the seed. As such, species, strain, or cultivar to which a treatment, formulation, a microbe is deemed heterologously disposed when applied composition or endophyte preparation as described herein is on the plant that either does not naturally have the microbe on not administered/contacted. A control agricultural plant or its surface or within the particular tissue to which the microbe control seed, therefore, is identical to the treated plant or seed is disposed, or does not naturally have the microbe on its with the exception of the presence of the endophyte and can surface or within the particular tissue in the number that is serve as a control for detecting the effects of the endophyte being applied. The term “exogenous can be used inter that is conferred to the plant. changeably with "heterologous.” 0145 A "population of plants or seeds, as used herein, can refer to a plurality of plants or seeds that were subjected 0150. The phrase “inoculating a seed as used herein to the same inoculation methods described herein, or a plu refers to applying, infecting, co-planting, spraying, immers rality of plants or seeds that are progeny of a plant or group of ing, dusting, dipping or coating the seed with the endophyte. seeds that were subjected to the inoculation methods. In addi Techniques for inoculating the seed are known in the art, for tion, a population of plants can be a group of plants that are example, as disclosed by Hynes and Boyetchko (2006, Soil grown from coated seeds. The plants or seeds within a popu Biology & Biochemistry 38: 845-84). In an embodiment, lation will typically be of the same species, and will also inoculation comprises foliar application or soil application of typically share a common genetic derivation. the endophyte or combination thereof with any solid or liquid 0146 The term “endophyte' as used herein refers to a carrier at any growing stage of the plant. fungal or bacterial organism that can live symbiotically in a 0151. The term "enhancing seed vitality” as used herein plant and is also referred to herein as “endosymbiont'. A refers to plant prenatal care improving the ability of the seed fungal endophyte may be in the form of a spore, hypha, or to germinate and produce a plant under normal and/or mycelia. Abacterial endophyte may be a cell or group of cells. stressed conditions and includes, without limitation, any one The term “endophyte' as used herein includes progeny of the or more of the following: breaking dormancy, providing seed strains recited herein. stratification, increasing seed germination, modulating gene 0147 In some cases, the present invention contemplates expression, decreasing time to reach energy of germination, the use of microbes that are “compatible' with agricultural protecting against biotic stresses, protecting against abiotic chemicals, for example, a fungicide, an anti-bacterial com stresses, reducing hydrothermal time required for germina pound, or any other agent widely used in agricultural which tion, increasing seed germination vigour, increasing seed ger has the effect of killing or otherwise interfering with optimal mination efficacy, increasing uniformity of seed germination, growth of microbes. As used herein, a microbe is "compat ameliorating drought/heat tolerance efficacy, increasing the ible' with an agricultural chemical when the microbe is modi weight of seedlings, and increasing the yield of seedlings. US 2016/017457.0 A1 Jun. 23, 2016

Drought/Heat Tolerance Efficiency (DTE/THE) is the term asexual part that has the ability to be cultivated into a new opposed (antonym) to susceptibility. plant. In an embodiment, the plant propagation material is 0152 Energy of germination is defined as 50% of germi generative seed, generative bud or flower, and vegetative nation, relative to the number of seeds tested. The seed ger stem, cutting, root, bulb. rhizome, tuber, vegetative bud, or mination vigour shows the difference between total percent leaf parts. age of germinating treated seeds and germinating untreated 0158. In some cases, the present invention contemplates seeds. The hydrothermal time postulates that an individual the use of microbes (e.g., endophytes) that are “compatible” seed begins to germinate when the Sum of both temperatures with agricultural chemicals, for example, a fungicide, an anti and water potential are sufficiently accumulated over a period bacterial compound, or any other agent widely used in agri of time allowing germination. Germination efficacy is defined culture that has the effect of killing or otherwise interfering as the percentage of treated seeds germinating after a set time with optimal growth of microbes. As used herein, a microbe period after planting, relative to the number of seeds tested in such as a seed bacterial endophyte is “compatible' with an an untreated control. Biological stratification is defined as agricultural chemical when the microbe is modified, such as releasing seed dormancy by a symbiont in promoting germi by genetic modification, e.g., contains a transgene that con nation. Uniformity of seed germination represents the maxi fers resistance to an herbicide, or is adapted to grow in, or mum percentage of seed germination within a minimal time otherwise Survive, the concentration of the agricultural of incubation. chemical used in agriculture. For example, a microbe dis 0153. The terms “decreased”, “fewer”, “slower and posed on the Surface of a seed is compatible with the fungi “increased “faster "enhanced’ “greater as used herein cide metalaxyl if it is able to survive the concentrations that refers to a decrease or increase in a characteristic of the are applied on the seed surface. endophyte treated seed or resulting plant compared to an 0159. The term “phytoremediation” as used herein refers untreated seed or resulting plant. For example, a decrease in a to the use of plants for removal, reduction or neutralization of characteristic may be at least 1%, at least 2%, at least 3%, at Substances, wastes or hazardous material from a site so as to least 4%, at least 5%, at least 10%, at least 15%, at least 20%, prevent or minimize any adverse effects on the environment. at least 25%, at least 30%, at least 35%, at least 40%, at least The term “phytoreclamation” as used herein refers to the use 45%, at least 50%, at least 75%, at least 100%, or at least of plants for reconverting disturbed land to its former or other 200% or more lower than the untreated control and an productive uses. increase may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at DETAILED DESCRIPTION least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100%, or at least 0160 The present invention identifies a small, unique 200% or more higher than the untreated control. family of endophytes that can be placed into synthetic com 0154 The term “increased yield’ refers to increased seed bination with a variety of plant hosts and work Synergistically weight, seed size, seed number per plant, seed number per with the plant hosts to exhibit a suprising number of altered unit area (i.e. seeds, or weight of seeds, per acre), bushels per and improved biological proceses. acre, tons per acre, kilo per hectare, increased grain yield, 0.161 Plants across the Angiosperms have many features increased dry weight of grain, increased seed weight, in common that emanate from having evolved from a com increased dry weight of wheat spikes and increased biomass. mon ancestor. This is true for the many systems that control “Biomass” means the total mass or weight (fresh or dry), at a growth and development and also tolerance to abiotic and given time, of a plant tissue, plant tissues, an entire plant, or biotic stresses. Plants have co-evolved with endosymbionts population of plants. Biomass is usually given as weight per and in consequence these latter organisms, fungi and bacteria, unit area. Increased biomass includes without limitation can possess features that enable them to interact with plants. increased pod biomass, stem biomass, and root biomass. It is well accepted that microrganisms can be classified on the 0155. In certain embodiments, the plant is cultivated under basis of their taxonomy or cladistics groupings, as well as abiotic or biotic stressed conditions. The term "abiotic stress' based onkey morphological, functional, and ecological roles. as used herein refers to a non-living stress that typically Here, by Screening hundreds of synthetic associations affects seed vitality and plant health and includes, without between endophytes and plants, we discovered a family of limitation, heat, drought, nitrogen, cold, salt and osmotic endophytes based on their ability to interact with a variety of stress. In one embodiment, the abiotic stress is heat stress. In plant species to create agricultural value. These endophytes another embodiment, the abiotic stress is drought stress, possess systems that enable them to physically and chemi osmotic stress or salt stress. The term “biotic stress' as used cally interact with a broad range of agricultural plants bred by herein refers to a living stress that typically affects seed vital man, including monocots and dicots, endorsing the conclu ity and plant health, and includes without limitation, insect sion that when living together with the plant they interact infestation, nematode infestation, bacterial infection, fungal intimately with the conserved genetic and physiological infection, oomycete infection, protozoal infection, viral properties of plant species. Our classification based on studies infection, and herbivore grazing, or any combination thereof. involving a large range of agricultural plants distinguishes the In one embodiment, the biotic stress is a Fusarium infection. chosen endophytes from those endophytes that interact with 0156. As used herein an "agriculturally compatible car only some classes of plant species. Endophytes classified in rier refers to any material, other than water, which can be this way can include fungi and bacteria and the classification added to a seed or a seedling without causing or having an highlights that the fungi and bacteria have informational sys adverse effect on the seed (e.g., reducing seed germination) or tems in common. The informational systems programing the the plant that grows from the seed, or the like. plant-endophyte interactions are complex and comprise sig 0157. The term “plant propagation material as used naling systems, multiple networks and pathways that under herein refers to any plant generative/sexual and vegetative/ pin growth of many types of plant cells and organs as well as US 2016/017457.0 A1 Jun. 23, 2016

endophyte cells. They are thus best described by the outcomes tance to biotic or abiotic stress. In some embodiments, the of the plant-endophyte interactions. improved functional trait is selected from the group consist 0162 The endophyte class described herein provides the ing of increased yield, faster seedling establishment, faster unique ability to confer mycoVitalism to a large number of growth, increased photosynthetic rate, increased carbon diox diverse plant hosts, as well as to confer stress tolerance and ide assimilation rate, increased drought tolerance, increased increased yield. Specifically, this endophyte class is able to, heat tolerance, increased cold tolerance, increased salt toler when coated onto the outside of a seed or placed in its proX ance, increased tolerance to pests and diseases, increased imity, increase expression of key genes related to seed ger biomass, increased root and/or shoot length or weight, mination, vigor, and stress tolerance. The endophytes are then increased fresh weight of seedlings, increased seed or fruit able to penetrate the cortical layer of the seed and plant in number, increased plant vigour, nitrogen stress tolerance, order to enter the plants internal tissues and replicate within enhanced Rhizobium activity, enhanced nodulation fre at least one tissue in the host and establish symbiotic organs quency, early flowering time, or any combination thereof. In comprising microstructures that allow intimate communica Some embodiments, the increased tolerance to disease is tion between the endophyte and the plants intra- and/or inter increased tolerance to Fusarium infection, increased toler cellular spaces. These endophytes further act in Symbiosis ance to Septoria infection, and/or increased tolerance to Puc with the host to improve stress tolerance of the seedling and cinia infection. In some embodiments, yield is measured on a adult plant and to increase yield. population of plants grown in the field and is calculated via 0163 Germination of mature, dry seeds is a process that is combine harvesting or measuring ear weight. For all altered conserved across angiosperms, being regulated by water, traits, the change can be at least 1%, for example at least 2%, temperature, the hormones gibberellic acid, abscisic acid and at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, ethylene, amongst other vital molecules, and involves at least 30%, at least 40%, at least 50%, at least 60%, at least changes to cell walls, breakdown of food reserves and their 75%, at least 100%, or more, when compared with a control conversion to new molecules and structures that define root agricultural seed or plant. In some embodiments, the and shoot growth. The group of endophytes revealed here is improved trait is heritable by progeny of the agricultural plant readily characterized by its ability to stimulate seed germina grown from the seed. tion or make germination more uniform when any of its 0166 In some embodiments, the agricultural seed is a seed members are present as a synthetic preparation that physi of a monocot plant. In some embodiments, the agricultural cally interacts with a seed from monocot or dicot plants. In seed is a seed of a cereal plant. In some embodiments, the other embodiments, the group of endophytes is recognized as agricultural seed is a seed of a corn, wheat, barley, rice, capable of altering plant flowering time and/or increasing Sorghum, millet, oats, rye or triticale. In some embodiments, tolerance to biotic and abiotic stresses and many other traits. the agricultural seed is a seed of a dicot plant. In some All of these features support the conclusion that members of embodiments, the agricultural seed is a seed of cotton, canola, this group of endophytes can be physically complexed with Soybean or a pulse. monocot and dicot seeds to achieve multiple agricultural ben 0167. In some embodiments, a synthetic preparation is efits due to their particular informational systems that interact made comprising a canola seed and a composition compris with those conserved in plants. ing at least one endophyte capable of promoting germination 0164. This family of endophytes represents a suprising and an agriculturally-acceptable carrier, and a canola plant discovery in their ability to engage in synthetic associations grown from the seed flowers earlier as compared to a control with plants, leading to a number of altered physiological canola plant. In some embodiments, a synthetic preparation is processes across the lifespan of the plant-endophyte compos made comprising a tomato, alfalfa, corn, Swiss chard, radish, ite association. Notably, the synthetic associations between or cabbage seed and a composition comprising at least one this small family of endophytes and both monocot and dicot endophyte capable of promoting germination and an agricul plants are characterized by the activation of multiple plant turally-acceptable carrier, and a tomato, alfalfa, corn, Swiss genes and hormones during seed germination, seedling devel chard, radish, or cabbage plant grown under drought condi opment, and responses to environmental and biotic stresses. tions from the seed has higher biomass as compared to a control plant grown under drought conditions. Novel Compositions and Seeds 0.168. In some embodiments, the composition is disposed 0.165 Accordingly, the present disclosure provides a com on an exterior Surface of the agricultural seed in an amount position comprising at least one endophyte capable of pro effective to colonize at least 0.1%, at least 1%, at least 2%, at moting germination or comprising a combination or mixture least 3%, at least 4%, at least 5%, at least 10%, at least 20%, thereof, and an agriculturally-acceptable carrier. In some at least 30%, at least 40%, at least 50%, at least 60%, at least embodiments, the at least one endophyte capable of promot 70% or at least 80% of cortical cells of a plant grown from the ing germination are coleorhiza-activing endophytes. In some seed. embodiments, a synthetic preparation is made using the com 0169. In some embodiments, the composition comprises a position and an agricultural plant seed. In some cases, plants carrier and at least one endophyte chosen from the group are inoculated with at least one endophyte that is heterolo consisting of a spore-forming endophyte, a facultative endo gous to the inoculated agricultural plant seed or the agricul phyte, a filamentous endophyte, and an endophyte capable of tural plant grown from the agricultural seed. In some embodi living within another endophyte. In some embodiments, theat ments, the at least one endophyte capable of promoting least one endophyte is capable of forming certainstructures in germination are disposed on the Surface or within a tissue of the plant, where the structures are selected from the group the agricultural seed or seedling. In some embodiments, a consisting of hyphal coils, Hartig-like nets, microvesicles, plant grown from a seed inoculated with this composition has micro-arbuscules, hyphal knots, and symbiosomes. In some an improved functional trait as compared to a control plant. In embodiments, the at least one endophyte is in the form of at Some embodiments, the improved functional trait is resis least one of conidia, chlamydospore, and mycelia. In other US 2016/017457.0 A1 Jun. 23, 2016

embodiments, the fungus or bacteria is capable of being part Kathistaceae, Koerberiaceae, Koralionastetaceae, Laboulbe of a plant-fungus symbiotic system or plant-bacteria symbi niaceae, Lachnaceae, Lasiosphaeriaceae, Lecanoraceae, otic system that produces altered levels of phytohormones or Lecideaceae, Lentitheciaceae, Leotiaceae, Leprocaulaceae, anti-oxidants, as compared to a plant that is not in Symbiosis. Leptosphaeriaceae, Letrouiltiaceae, Lichinaceae, Lindgo In other embodiments, the plant-fungus symbiotic system or mycetaceae, Lobariaceae, Lophiostomataceae, Lophiotrem plant-bacterium symbiotic system has anti-aging and/or anti ataceae, Loramycetaceae, Lulworthiaceae, Lyrommataceae, senescence effects, as compared to a plant or plant organ that Magnaporthaceae, Malmideaceae, Massariaceae, Massari is not in Symbiosis. In other embodiments, the plant-fungus naceae, Megalariaceae, Megalosporaceae, Megasporaceae, symbiotic system or plant-bacteria symbiotic system has Melanconidaceae, Melanommataceae, Melaspileaceae, increased protection against pathogens, as compared to a Meliolaceae, Metacapnodiaceae, Microascaceae, Miltide plant that is not in Symbiosis. aceae, Monascaceae, Monoblastiaceae, Montagnulaceae, Morchellaceae. Morosphaeriaceae, Mycoblastaceae, Myco 0170 In some embodiments, the at least one endophyte is caliciaceae, Mycosphaerellaceae, Myeloconidaceae, Myri a fungus of Subphylum Pezizomycotina. In some embodi angiaceae, Myxotrichaceae, Nannizziopsidaceae, Nectri ments, the at least one endophyte is a fungus of class Leotio aceae, Nephromataceae, Niessliaceae, Nitschkiaceae, mycetes, Dothideomycetes, Sordariomycetes, or Eurotio Obryzaceae, Ochrolechiaceae, Odontotremataceae, Onygen mycetes. In some embodiments, the at least one endophyte is aceae, Ophiocordycipitaceae, Ophioparmaceae, Ophiosto of order Helotiales, Capnodides, Pleosporales, Hypocreales, mataceae, Orbiliaceae, Pachyascaceae, Pannariaceae, Pan or Eurotiales. In some embodiments, the at least one endo nariaceae, Papulosaceae, Parmeliaceae, Parmulariaceae, phyte is selected from one of the following families: Peltigeraceae, Peltulaceae, Pertusariaceae, Pezizaceae, Pha Acarosporaceae, Adelococcaceae, Agyriaceae, Aigialaceae, cidiaceae, Phaeochoraceae, Phaeococcomycetaceae, Ajellomycetaceae, Amniculicolaceae. Amorphothecaceae, Phaeosphaeriaceae, Phaeotrichaceae, Phaneromycetaceae, Amphisphaeriaceae, Amplistromataceae, Anamylopsora Phlyctidaceae, Phyllachoraceae, Physciaceae, Piedraiaceae, ceae, Annulatascaceae, Anteagloniaceae, Antennulariel Pilocarpaceae, Placynthiaceae, Platystomaceae, laceae, Aphanopsidaceae, Apiosporaceae, Apiosporaceae, Plectosphaerellaceae, Pleomassariaceae, Pleosporaceae, Arachnomycetaceae, Arctomiaceae, Armatellaceae, Arthoni Pleurostomataceae, Porinaceae, Porpidiaceae, Protothelenel aceae, Arthopyreniaceae, Arthrodermataceae, Arthrorhaphi laceae, Pseudoplagiostomataceae, Pseudovalsaceae, Psora daceae, Ascobolaceae, Ascocorticiaceae, Ascodesmidaceae, ceae, Pycnoraceae, Pyrenulaceae, Pyronemataceae, Pyxidio Ascodichaenaceae, Ascosphaeraceae, Asterinaceae, phoraceae, Ramalinaceae, Requienellaceae, Aulographaceae, Australiascaceae, Baeomycetaceae, Bam Reticulascaceae, Rhizinaceae, Rhizocarpaceae, Rhynchosto busicolaceae, Batistiaceae, Bertiaceae, Biatorellaceae, Bia mataceae, Rhytismataceae, Roccellaceae, Roccel triosporaceae, Bionectriaceae, Boliniaceae, Brigantiaeaceae, lographaceae, Ropalosporaceae, Roussoellaceae, Rutstro Bulgariaceae, BySSolomataceae, Caliciaceae, Calos emiaceae, Sagiolechiaceae, Salsugineaceae, cyphaceae, Calosphaeriaceae, Calycidiaceae, Candelari Sarcoscyphaceae, Sarcosomataceae, Sarrameanaceae, aceae, Capnodiaceae, Carbomycetaceae, Carbonicolaceae, Schaereriaceae, Schizoparmaceae, Schizoparmeaceae, Scle Catabotrydaceae, Catillariaceae, Celotheliaceae, Cephaloth rotiniaceae, Scoliciosporaceae, Scortechiniaceae, Shirai ecaceae, Ceratocystidaceae, Ceratomycetaceae, Ceratosto aceae, Sordariaceae, Spathulosporaceae, Sphaerophoraceae, mataceae, Chadefaudiellaceae, Chaetomiaceae, Chaeto Sphinctrinaceae, Sporastatiaceae, Sporormiaceae, Stereo sphaerellaceae, Chaetosphaeriaceae, Chaetosphaeriaceae, caulaceae, Stictidaceae, Strigulaceae, Sydowiellaceae, Sym Chaetothyriaceae, Chorioactidaceae, Chrysotrichaceae, Cla poventuriaceae, Teichosporaceae, Teloschistaceae, Ter doniaceae, Cladosporiaceae, Clavicipitaceae, Cly atosphaeriaceae, Testudinaceae, Tetraplosphaeriaceae, peosphaeriaceae, Coccocarpiaceae, Coccodiniaceae, Coc Thelebolaceae. Thelenellaceae. Thelocarpaceae. Thermoas coideaceae, Coccotremataceae, Coenogoniaceae, caceae, Thyridariaceae, Thyridiaceae, Thyridiaceae. Tog Collemataceae, Coniocessiaceae, Coniochaetaceae, Conio niniaceae, Trapeliaceae, Trematosphaeriaceae, Trichoco cybaceae, Coniothyriaceae, Cordycipitaceae, Coronophora maceae, Trichomeriaceae, Trichosphaeriaceae, Tuberaceae, ceae, Coryneliaceae, Corynesporascaceae, Crocyniaceae, Tubeufiaceae, Umbilicariaceae, Vahliellaceae, Valsaceae, Cryphonectriaceae, Cryptomycetaceae, Cucurbitariaceae, Venturiaceae, Verrucariaceae, Vezdaeaceae, Vialaeaceae, Cudoniaceae, Cyphellophoraceae, Cyttariaceae, Dactylospo Vibrisseaceae, Xanthopyreniaceae, Xylariaceae, Xylono raceae, Davidiellaceae, Delitschiaceae, Dermateaceae, Dia demaceae, Diaporthaceae, Diatrypaceae, Didymellaceae, mycetaceae, and Zopfiaceae. Didymosphaeriaceae, Discinaceae, Dissoconiaceae, Dot 0171 In some embodiments, the composition comprises hideaceae, Dothidotthiaceae, Dothioraceae, Ectolechiaceae, an agriculturally-acceptable carrier and at least one spore Elaphomycetaceae, Elixiaceae, Elsinoaceae, Eremascaceae, forming, filamentous bacterial endophyte of phylum Actino Eremithallaceae, Erysiphaceae, Euceratomycetaceae, bacteria. In some embodiments, the at least one endophyte is Extremaceae, Fissurinaceae, Fuscideaceae, Geoglossaceae, a bacteria of order actinomycetales. In some embodiments, Glaziellaceae, Gloeoheppiaceae, Glomerellaceae, Glomerel the at least one endophyte is selected from one of the follow laceae, Gnomoniaceae, Gomphillaceae, Gondwanamyceta ing families: Actinomycetaceae, Actinopolysporineae, ceae, Graphidaceae, Graphostromataceae, Gyalectaceae, Catenulisporineae, Corynebacterineae, Frankineae, Glyco Gymnoascaceae, Gypsoplacaceae, Haematommataceae, mycineae, Kineosporiineae, Micrococcineae, Micromono Halojulellaceae, Halosphaeriaceae, Halotthiaceae, Harknes sporineae, Propionibacterineae, Pseudonocardineae, Strepto siaceae, Helminthosphaeriaceae, Helotiaceae, Helvellaceae, mycineae, and Streptosporangineae. Hemiphacidiaceae, Heppiaceae, Herpomycetaceae, Herpot 0172. In some embodiments, the present disclosure pro richiellaceae, Hyaloscyphaceae, Hymeneliaceae, Hypocre vides a composition comprising a carrier and an endophyte of aceae, Hyponectriaceae, Hypsostromataceae, Icmado Paraconyothirium sp. strain deposited as IDAC 081111-03 or philaceae, Jobellisiaceae, Juncigenaceae, Karstenellaceae, comprising a DNA sequence with at least 97% identity to US 2016/017457.0 A1 Jun. 23, 2016

SEQ ID NO:5; an endophyte of Pseudeurotium sp. strain are positive or negative regulators of these pathways. In other deposited as IDAC 081111-02 or comprising a DNA embodiments, the genes associated with systemic acquired sequence with at least 97% identity to SEQ ID NO:4; an resistance are redox-regulated transcription factors. In still endophyte of Penicillium sp. strain deposited as IDAC other embodiments, the redox-regulated transcription factors 081111-01 or comprising a DNA sequence with at least 97% belong to the MYB family of genes. In some embodiments, identity to SEQID NO:3: an endophyte of Cladosporium sp. the gene with altered expression is selected from the group strain deposited as IDAC 2003 12-06 or comprising a DNA consisting of P5CS, SOD, MnSOD, GA3-oxidase 2, 14-3-3, sequence with at least 97% identity to SEQ ID NO:1; an NCED2, ABA8'OH1, RSG, KAO, Myb1 and Myb2. In some endophyte of Sarocladium sp. strain deposited as IDAC embodiments, the change in gene expression can be at least 2003 12-05 or comprising a DNA sequence with at least 97% 1%, for example at least 2%, at least 3%, at least 4%, at least identity to SEQID NO:2; and/or an endophyte of Streptomy 5%, at least 10%, at least 20%, at least 30%, at least 40%, at ces sp. strain deposited as IDAC 081111-06 or comprising a least 50%, at least 60%, at least 75%, at least 100%, or more, DNA sequence with at least 97% sequence identity to SEQID when compared with a control agricultural seed or plant. In NO:6. In certain embodiments, the endophyte of Paraco Some embodiments, said composition is disposed on an exte nyothirium sp. strain comprises a DNA sequence with at least rior Surface of an agricultural seed in an amount effective to 98% identity to SEQID NO:5; the endophyte of Pseudeuro colonize at least 0.1%, at least 1%, at least 2%, at least 3%, at tium sp. strain comprises a DNA sequence with at least 98% least 4%, at least 5%, at least 10%, at least 20%, at least 30%, identity to SEQ ID NO:4; the endophyte of Penicillium sp. at least 40%, at least 50%, at least 60%, at least 70%, or at strain comprises a DNA sequence with at least 98% identity least 80% of the cortical cells of an agricultural plant grown to SEQ ID NO:3: the endophyte of Cladosporium sp. strain from the seed and to alter the expression of genes involved in comprises a DNA sequence with at least 98% identity to SEQ plant growth, genes associated with systemic acquired resis ID NO:1; the endophyte of Sarocladium sp. strain comprises tance, or genes involved in protection from oxidative stress. a DNA sequence with at least 98% identity to SEQID NO:2: 0.174. In some embodiments, the present disclosure pro and the endophyte of Streptomyces sp. Strain comprises a vides a composition comprising at least one endophyte DNA sequence with at least 98% sequence identity to SEQID capable of promoting germination and an agriculturally-ac NO:6. In certain embodiments, the endophyte of Paraco ceptable carrier, wherein said composition is disposed on an nyothirium sp. strain comprises a DNA sequence with at least exterior Surface of an agricultural seed in an amount effective 99% identity to SEQID NO:5; the endophyte of Pseudeuro to cause a population of seeds inoculated with said composi tium sp. strain comprises a DNA sequence with at least 99% tion to have a faster dormancy breakdown, greater germina identity to SEQ ID NO:4; the endophyte of Penicillium sp. tion rate, earlier germination, increased energy of germina strain comprises a DNA sequence with at least 99% identity tion, greater rate of germination, greater uniformity of to SEQ ID NO:3: the endophyte of Cladosporium sp. strain germination, including greater uniformity of rate of germina comprises a DNA sequence with at least 99% identity to SEQ tion and greater uniformity of timing of germination, and/or ID NO:1; the endophyte of Sarocladium sp. strain comprises increased energy of germination as compared to a population a DNA sequence with at least 99% identity to SEQID NO:2: of control seeds. In some embodiments, the composition is and the endophyte of Streptomyces sp. Strain comprises a disposed on the Surface or within a tissue of an agricultural DNA sequence with at least 99% sequence identity to SEQID seed or seedling in an amount effective to cause a population NO:6. In certain embodiments, the endophyte of Paraco of seeds inoculated with said composition to reach 50% ger nyothirium sp. strain comprises a DNA sequence of SEQID mination faster than a population of control seeds or to cause NO:5; the endophyte of Pseudeurotium sp. strain comprises a increased NO accumulation in a plant grown from a seed DNA sequence of SEQ ID NO:4; the endophyte of Penicil inoculated with said composition, as compared to a control lium sp. strain comprises a DNA sequence of SEQID NO:3: plant. In other embodiments, the composition is disposed on the endophyte of Cladosporium sp. strain comprises a DNA an exterior Surface of an agricultural seed an in an amount sequence of SEQID NO:1; the endophyte of Sarocladium sp. effective to cause altered levels of phytohormones to be pro strain comprises a DNA sequence of SEQID NO:2; and the duced in an agricultural plant grown from the seed, as com endophyte of Streptomyces sp. Strain comprises a DNA pared to a control agricultural plant. In some embodiments, sequence of SEQID NO:6. the phytohormones that are altered are gibberellins, abscisic 0173. In some embodiments, the present disclosure pro acid, or cytokinins. In further embodiments, the gibberellins vides a synthetic preparation comprising an agricultural plant may be gibberellin 1, 19, 44 or 53. In still further embodi seed and a composition comprising endophytes capable of ments, the cytokinin may be zeatin. For all these altered traits promoting germination and an agriculturally-acceptable car (a faster dormancy breakdown, greater germination rate, ear rier, wherein the synthetic preparation has altered gene lier germination, increased energy of germination, greater expression in a plant grown from a seed inoculated with said rate of germination, greater uniformity of germination, composition, as compared to a control plant. In some embodi including greater uniformity of rate of germination and ments, the composition is disposed on an exterior Surface of greater uniformity of timing of germination, increased energy an agricultural seed in an amount effective to colonize the of germination, 50% germination, increased NO accumula cortical cells of an agricultural plant grown from the seed and tion, and altered levels of phytohormones), the change can be to alter the expression of genes involved in plant growth, at least 1%, for example at least 2%, at least 3%, at least 4%, genes associated with Systemic acquired resistance, or genes at least 5%, at least 10%, at least 20%, at least 30%, at least involved in protection from oxidative stress. In some embodi 40%, at least 50%, at least 60%, at least 75%, at least 100%, ments, these genes may be involved in phytohormone pro or more, when compared with a control agricultural seed or duction, for example in gibberellin (GA) biosynthesis or plant. breakdown, abscisic acid (ABA) biosynthesis or breakdown, 0.175. In some embodiments, the present disclosure pro NO production or breakdown, superoxide detoxification, or vides a composition comprising at least one endophyte and a US 2016/017457.0 A1 Jun. 23, 2016 carrier, wherein said composition is capable of colonizing at preparation comprising an agricultural plant seed and a com least 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at position comprising at least one endophyte capable of pro least 5%, at least 10%, at least 20%, at least 30%, at least 40%, moting germination and an agriculturally-acceptable carrier, at least 50% at least 60%, at least 70%, or at least 80% of where the population is comprised within a packaging mate cortex cells of a plant grown from a seed inoculated with said rial. The packaging material can be selected from a bag, box, composition and wherein said plant has an improved trait as bin, envelope, carton, or container. In an embodiment, the compared to a control plant. In certain embodiments, the synthetic preparation can be disposed within a package and is plant grown from seed inoculated with the composition has an shelfstable. In another embodiment, the invention features an improved trait selected from the group consisting of increased agricultural product that includes a predetermined number of yield, faster seedling establishment, faster growth, increased seeds or a predetermined weight of seeds. In an embodiment, drought tolerance, increased heat tolerance, increased cold the bag or container contains at least 1000 seeds, wherein the tolerance, increased salt tolerance, increased tolerance to packaging material optionally comprises a dessicant, and Fusarium infection, increased biomass, increased root wherein the synthetic preparation optionally comprises an length, increased fresh weight of seedlings, increased plant anti-fungal agent. vigour, nitrogen stress tolerance, enhanced Rhizobium activ 0180. In yet another aspect, the invention features an ity, enhanced nodulation frequency and early flowering time article of manufacture that includes packaging material; one compared to a control plant. or more plant seeds within the packaging material, and at least 0176). In another embodiment, the synthetic preparations one species of endophytes capable of promoting germination and compositions described herein comprise two or more associated with the seeds. The article can include two or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 species of endophytes capable of promoting germination. or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or 0181. In another aspect, the invention features an agricul more, or greater than 25) different endophytes capable of tural product that includes a predetermined number of seeds promoting germination, e.g., obtained from different families or a predetermined weight of seeds. In an embodiment, the or different genera of fungi or bacteria, or from the same bag or container contains at least 1000 seeds of a synthetic genera but different species of fungi or bacteria. In embodi preparation produced by the step of inoculating a plurality of ments in which two or more endophytes capable of promoting plant seeds with a formulation comprising a fungal or bacte germination are used, each of the endophytes capable of rial population at a concentration of at least 1 CFU per agri promoting germination can have different properties or cultural plant seed, wherein at least 10% of the CFUs present activities, confer different beneficial traits, or colonize differ in the formulation are one or more endophytes capable of ent parts of a plant (e.g., leaves, stems, flowers, fruits, seeds, promoting germination, under conditions such that the for or roots). For example, one endophyte capable of promoting mulation is associated with the Surface of the seeds in a germination can colonize a first tissue and a second endo manner effective for the endophytes capable of promoting phyte capable of promoting germination can colonize a tissue germination to confer a benefit to the seeds or to a crop that differs from the first tissue. Alternatively, each of the comprising a plurality of agricultural plants produced from endophytes capable of promoting germination can have simi the seeds. The endophytes capable of promoting germination lar properties or activities, confer similar beneficial traits, or can be present in a concentration of from about 10° to about colonize different parts of a plant. 10 CFU/ml or from about 10 to about 10 CFU/seed. The 0177. The synthetic combination or preparation of the formulation can be a liquid and the fungal or bacterial con present invention contemplates the presence of an endophyte centration can be from about 10 to about 10' CFU/ml. The on the surface of the seed of the first plant. In one embodi formulation can be a gel or powder and the fungal or bacterial ment, the seed of the first plant is coated with at least 10 CFU concentration can be from about 10 to about 10' CFU/gm. of the endophyte per seed, for example, at least 20 CFU, at 0182. In some cases, the endophytic microbe can be modi least 50 CFU, at least 100 CFU, at least 200 CFU, at least 300 fied. For example, the endophytic microbe can be genetically CFU, at least 500 CFU, at least 1,000 CFU, at least 3,000 modified by introduction of a transgene that stably integrates CFU, at least 10,000 CFU, or at least 30,000 CFU or more per into its genome. In another embodiment, the endophytic seed. In another embodiment, the seed is coated with at least microbe can be modified to harbor a plasmid or episome 10, for example, at least 20, at least 50, at least 100, at least containing a transgene. In still another embodiment, the 200, at least 300, at least 500, at least 1,000, at least 3,000, at microbe can be modified by repeated passaging under selec least 10,000, at least 30,000, at least 100,000, at least 300,000, tive conditions. at least 1,000,000 or more of the endophyte as determined by 0183. The microbe can be modified to exhibit altered char the number of copies of a particular endophyte gene detected, acteristics. In one embodiment, the endophytic microbe is for example, by quantitative PCR. modified to exhibit increased compatibility with chemicals 0178. Further provided herein is a seed inoculated with commonly used in agriculture. Agricultural plants are often any of the compositions described herein. In one embodi treated with a vast array of agrichemicals, including fungi ment, the seed is inoculated by soil-based inoculation. In cides, biocides (anti-bacterial and anti-fungal agents), herbi another embodiment, the seed is coated with an endophyte or cides, insecticides, nematicides, rodenticides, fertilizers, and culture thereof. In yet another embodiment, the seed is other agents. Many Such agents can affect the ability of an sprayed, injected, inoculated, grafted, coated or treated with endophytic microbe to grow, divide, and/or otherwise confer the endophyte or culture thereof. In an embodiment, the seed beneficial traits to the plant. is planted near an endophyte. In one embodiment, the seed 0184. In some cases, it can be important for the microbe to planted near the endophyte is about 4 cm away from the be compatible with agrichemicals, particularly those with endophyte. fungicidal orantibacterial properties, in order to persist in the 0179. In another aspect, the invention provides a popula plant although, as mentioned earlier, there are many Such tion of at least 10 synthetic preparations, each synthetic fungicidal or antibacterial agents that do not penetrate the US 2016/017457.0 A1 Jun. 23, 2016

plant, at least at a concentration sufficient to interfere with the orim, potassium bicarbonate, potassium hydroxyquinoline microbe. Therefore, where a systemic fungicide or antibac Sulfate, probenazole, prochloraz, procymidone, propam terial agent is used in the plant, compatibility of the microbe ocarb, propamocarb hydrochloride, propiconazole, propineb, to be inoculated with Such agents will be an important crite produinazid, prothioconazole, pyraclostrobin, pyrameto 1O. strobin, pyraoxystrobin, pyrazophos, pyribencarb, pyributi carb, pyrifenox, pyrimethanil, pyroquilon, quinoclamine, 0185. In one embodiment, spontaneous isolates of quinoxyfen, quintoZene, Reynoutria Sachalinensis extract, microbes that are compatible with agrichemicals can be used sedaxane, silthiofam, Simeconazole, Sodium 2-phenylphe to inoculate the plants according to the methods described noxide, sodium bicarbonate, Sodium pentachlorophenoxide, herein. For example, fungal microbes which are compatible spiroxamine, sulfur, SYP-Z071, SYP-Z048, tar oils, tebu with agriculturally employed fungicides can be isolated by conazole, tebufloquin, tecnaZene, tetraconazole, thiabenda plating a culture of the microbes on a petri dish containing an Zole, thifluZamide, thiophanate-methyl, thiram, tiadinil, tol effective concentration of the fungicide, and isolating colo clofos-methyl, tolylfluanid, triadimefon, triadimenol, nies of the microbe that are compatible with the fungicide. In triazoxide, tricyclazole, tridemorph, trifloxystrobin, triflumi another embodiment, a microbe that is compatible with a Zole, triforine, triticonazole, validamycin, Valifenalate, fungicide is used for the methods described herein. For Valiphenal, VincloZolin, Zineb, Ziram, Zoxamide, Candida example, the endophyte can be compatible with at least one of Oleophila, Fusarium oxysporum, Gliocladium spp., Phlebi the fungicides selected from the group consisting of: 2-(thio opsis gigantea, Streptomyces griseoviridis, Trichoderma cyanatomethylthio)-benzothiazole, 2-phenylphenol, 8-hy spp., (RS) N-(3,5-dichlorophenyl)-2-(methoxymethyl)- droxyquinoline Sulfate, ametoctradin, amisulbrom, antimy Succinimide, 1,2-dichloropropane, 1,3-dichloro-1, 1.3.3-tet cin, Ampelomyces quisqualis, azaconazole, azoxystrobin, rafluoroacetone hydrate, 1-chloro-2,4-dinitronaphthalene, Bacillus subtilis, benalaxyl, benomyl, benthiavalicarb-iso 1-chloro-2-nitropropane, 2-(2-heptadecyl-2-imidazolin-1- propyl, benzylaminobenzene-sulfonate (BABS) salt, bicar yl)ethanol, 2,3-dihydro-5-phenyl-1,4-dithi-line 1,1,4,4-tet bonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasti raoxide, 2-methoxyethylmercury acetate, 2-methoxyethylm cidin-S, borax, Bordeaux mixture, boScalid, bromuconazole, ercury chloride, 2-methoxyethylmercury silicate, 3-(4- bupirimate, calcium polysulfide, captafol, captan, carbenda chlorophenyl)-5-methylrhodanine, 4-(2-nitroprop-1-enyl) Zim, carboxin, carpropamid, carvone, chloroneb, chlorotha phenyl thiocyanateme, ampropylfos, anilazine, azithiram, lonil, chloZolinate, Coniothyrium minitans, copper hydrox barium polysulfide, Bayer 32394, benodanil, benquinox, ide, copper octanoate, copper oxychloride, copper Sulfate, bentaluron, benzamacril; benzamacril-isobutyl, benzamorf, copper sulfate (tribasic), cuprous oxide, cyazofamid, cyflufe binapacryl, bis(methylmercury) sulfate, bis(tributyltin) namid, cymoxanil, cyproconazole, cyprodinil, dazomet, oxide, buthiobate, cadmium calcium copper Zinc chromate debacarb, diammonium ethylenebis-(dithiocarbamate), sulfate, carbamorph, CECA, chlobenthiazone, chlorani dichlofluanid, dichlorophen, diclocymet, diclomeZine, formethan, chlorfenazole, chlorquinox, climbazole, cycla dichloran, diethofencarb, difenoconazole, difenZoquation, furamid, cypendazole, cyprofuram, decafentin, dichlone, diflumetorim, dimethomorph, dimoxystrobin, diniconazole, dichlozoline, diclobutrazol, dimethirimol, dinocton, dinosul diniconazole-M, dinobuton, dinocap, diphenylamine, fon, dinoterbon, dipyrithione, ditalimfos, dodicin, draZOXo dithianon, dodemorph, dodemorph acetate, dodine, dodine lon, EBP, ESBP. etaconazole, etem, ethirim, fenaminosulf, free base, edifenphos, enestrobin, epoxiconazole, fenapanil, fenitropan, 5-fluorocytosine and profungicides ethaboxam, ethoxyquin, etridiazole, famoxadone, fenami thereof, fluotrimazole, furcarbanil, furconazole, furconazole done, fenarimol, fenbuconazole, fenfuram, fenhexamid, cis, furmecyclox, furophanate, glyodine, griseofulvin, hala fenoxanil, fenpiclonil, fenpropidin, fempropimorph, fentin, crinate, Hercules 3944, hexylthiofos, ICIA0858, isopam fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazi phos, isovaledione, mebenil, mecarbinzid, metaZOXolon, nam, fludioxonil, flumorph, fluopicolide, fluopyram, fluor methfuroxam, methylmercury dicyandiamide, metSulfovax, oimide, fluoxastrobin, fluguinconazole, flusilaZole, flusulfa milineb, mucochloric anhydride, myclozolin, N-3,5-dichlo mide, flutianil, flutolanil, flutriafol, fluxapyroxad, folpet, rophenyl-Succinimide, N-3-nitrophenylitaconimide, natamy formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, cin, N-ethylmercurio-4-toluenesulfonanilide, nickelbis(dim furalaxyl, furametpyr, guaZatine, guaZatine acetates, GY-81, ethyldithiocarbamate), OCH, phenylmercury hexachlorobenzene, hexaconazole, hymexaZol, imazalil, dimethyldithiocarbamate, phenylmercury nitrate, phos imazalil Sulfate, imibenconazole, iminoctadine, iminoctadine diphen, picolinamide UK-2A and derivatives thereof, pro triacetate, iminoctadine tris(albesilate), ipconazole, iproben thiocarb; prothiocarb hydrochloride, pyracarbolid, pyridini fos, iprodione, iprovalicarb, isoprothiolane, isopyrazam, iso tril, pyroxychlor, pyroxyfur, quinacetol; quinacetol Sulfate, tianil, kasugamycin, kasugamycin hydrochloride hydrate, quinaZamid, quinconazole, rabenzazole, salicylanilide, SSF kresoxim-methyl, mancopper, mancoZeb, mandipropamid, 109, Sultropen, tecoram, thiadifluor, thicyofen, thiochlorfen maneb, mepanipyrim, mepronil, mercuric chloride, mercuric phim, thiophanate, thioquinox, tioxymid, triamiphos, triari oxide, mercurous chloride, metalaxyl, mefenoxam, metal axyl-M, metam, metam-ammonium, metam-potassium, mol, triazbutil, trichlamide, urbacid, XRD-563, and metam-Sodium, metconazole, methasulfocarb, methyl Zarilamide, IK-1 140 iodide, methyl isothiocyanate, metiram, metominostrobin, 0186. In still another embodiment, an endophyte that is metrafenone, mildiomycin, myclobutanil, nabam, nitrothal compatible with an antibacterial compound is used for the isopropyl, nuarimol, octhillinone, ofurace, oleic acid (fatty methods described herein. For example, the endophyte can be acids), orysastrobin, oxadixyl, oxine-copper, Oxpoconazole compatible with at least one of the antibiotics selected from fumarate, oxycarboxin, pefurazoate, penconazole, pencycu the group consisting of Amikacin, Gentamicin, Kanamycin, ron, penflufen, pentachlorophenol, pentachlorophenyl lau Neomycin, Netilmicin, Tobramycin, Paromomycin, Specti rate, penthiopyrad, phenylmercury acetate, phosphonic acid, nomycin, Geldanamycin, Herbimycin, Rifaximin, Strepto phthalide, picoxystrobin, polyoxin B, polyoxins, polyox mycin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cil US 2016/017457.0 A1 Jun. 23, 2016

astatin, Meropenem, Cefadroxil, Cefazolin, Cefalotin or 0189 Likewise, bacterial microbes that are compatible to Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, biocides (including herbicides such as glyphosate or antibac Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, terial compounds, whether bacteriostatic or bactericidal) that CefoperaZone, Cefotaxime, Cefpodoxime, Ceftazidime, Cef are agriculturally employed can be isolated using methods tibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline similar to those described for isolating fungicide compatible fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, microbes. In one embodiment, mutagenesis of the microbial Clindamycin, Lincomycin, Daptomycin, Azithromycin, population can be performed prior to selection with an anti Clarithromycin, Dirithromycin, Erythromycin, Roxithromy bacterial agent. In another embodiment, selection is per cin, Troleandomycin, Telithromycin, Spiramycin, AZtre formed on the microbial population without prior mutagen onam, Furazolidone, Nitrofurantoin, Linezolid, PosiZolid, esis. In still another embodiment, serial selection is Radezolid, Torezolid, Amoxicillin, Ampicillin, AZlocillin, performed on a microbe: the microbe is first selected for Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, compatibility to a first antibacterial agent. The isolated com Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, patible microbe is then cultured and selected for compatibil Penicillin V. Piperacillin, Penicillin G. Temocillin, Ticarcil ity to the second antibacterial agent. Any colony thus isolated lin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piper is tested for compatibility to each, or both antibacterial agents acillin?taZobactam, Ticarcillin/clavulanate, Bacitracin, to confirm compatibility with these two agents. Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxa 0190. The selection process described above can be cin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic repeated to identify isolates of the microbe that are compat acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, ible with a multitude of antifungal or antibacterial agents. Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sul Candidate isolates can be tested to ensure that the selection fadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfame for agrichemical compatibility did not result in loss of a thizole, Sulfamethoxazole, Sulfanilimide (archaic), Sul desired microbial bioactivity. Isolates of the microbe that are fasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole compatible with commonly employed fungicides can be (Co-trimoxazole) (TMP-SMX), Sulfonamidochrysoidine selected as described above. The resulting compatible (archaic), Demeclocycline, Doxycycline, Minocycline, microbe can be compared with the parental microbe on plants Oxytetracycline, Tetracycline, Clofazimine, Dapsone, in its ability to promote germination. Capreomycin, Cycloserine, Ethambutol, Ethionamide, Iso niazid, Pyrazinamide, Rifampicin (Rifampin in US), Rifabu Methods tin, Rifapentine, Streptomycin, Arsphenamine, Chloram phenicol, Fosfomycin, Fusidic acid, Metronidazole, (0191) Further provided herein are methods of enhancing Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thia seed vitality, plant health and/or yield comprising inoculating a seed with an endophyte or culture disclosed herein or a mphenicol, Tigecycline, Tinidazole, and Trimethoprim. combination or mixture thereof or with a composition dis 0187 Fungicide compatible microbes can also be isolated closed herein. In some embodiments, a first generation plant by selection on liquid medium. The culture of microbes can is cultivated from the seed. be plated on petri dishes without any forms of mutagenesis; 0.192 In one aspect, the invention provides a method of alternatively, the microbes can be mutagenized using any altering a trait in an agricultural plant seed or an agricultural means known in the art. For example, microbial cultures can plant grown from said seed, said method comprising inocu be exposed to UV light, gamma-irradiation, or a chemical lating said seed with a composition comprising endophytes mutagen such as ethylmethanesulfonate (EMS) prior to selec capable of promoting germination and an agriculturally-ac tion on fungicide containing media. Finally, where the ceptable carrier, wherein the endophyte replicates within at mechanism of action of a particular fungicide is known, the least one plant tissue and colonizes the cortical cells of said target gene can be specifically mutated (either by gene dele plant. In one embodiment, the endophyte capable of promot tion, gene replacement, site-directed mutagenesis, etc.) to ing germination is a coleorhiza-activating endophyte, and the generate a microbe that is resilient against that particular seed is a monocot seed. In another embodiment, the endo fungicide. It is noted that the above-described methods can be phyte capable of promoting germination is heterologous to used to isolate fungi that are compatible with both fungistatic the seed. and fungicidal compounds. 0193 In some embodiments, the endophytes area selected 0188 It will also be appreciated by one skilled in the art from the group consisting of a spore-forming endophyte, a that a plant may be exposed to multiple types of fungicides or facultative endophyte, a filamentous endophyte, and an endo antibacterial compounds, either simultaneously or in Succes phyte capable of living within another endophyte. In some sion, for example at different stages of plant growth. Where embodiments, the endophyte is capable of forming certain the target plant is likely to be exposed to multiple fungicidal structures in the plant, where the structures are selected from and/or antibacterial agents, a microbe that is compatible with the group consisting of hyphal coils, Hartig-like nets, many or all of these agrichemicals can be used to inoculate the microvesicles, micro-arbuscules, hyphal knots, and Symbio plant. A microbe that is compatible with several fungicidal Somes. In some embodiments, the endophyte is in the form of agents can be isolated, for example, by serial selection. A at least one of conidia, chlamydospore, and mycelia. In other microbe that is compatible with the first fungicidal agent is embodiments, the fungus or bacteria is capable of being part isolated as described above (with or without prior mutagen of a plant-fungus symbiotic system or plant-bacteria symbi esis). A culture of the resulting microbe can then be selected otic system that produces altered levels of phytohormones or for the ability to grow on liquid or Solid media containing the anti-oxidants, as compared to a plant that is not in Symbiosis. second antifungal compound (again, with or without prior In other embodiments, the plant-fungus symbiotic system or mutagenesis). Colonies isolated from the second selection are plant-bacterium symbiotic system has anti-aging and/or anti then tested to confirm its compatibility to both antifungal senescence effects, as compared to a plant or plant organ that compounds. is not in Symbiosis. In other embodiments, the plant-fungus US 2016/017457.0 A1 Jun. 23, 2016 symbiotic system or plant-bacteria symbiotic system has plant vigour, nitrogen stress tolerance, enhanced Rhizobium increased protection against pathogens, as compared to a activity, enhanced nodulation frequency, early flowering plant that is not in Symbiosis. In other aspects, the endophyte time, or any combination thereof. In some embodiments, the colonizes at least 0.1%, at least 1%, at least 2%, at least 3%, increased tolerance to disease is increased tolerance to at least 4%, at least 5%, at least 10%, at least 20%, at least Fusarium infection, increased tolerance to Septoria infection, 30%, at least 40%, at least 50%, at least 60%, at least 70%, or increased tolerance to Puccinia infection. In some embodi at least 80% of the cortical cells of said agricultural plant. ments, yield is measured on a population of plants grown in 0194 In yet another aspect, the invention provides a the field and is calculated via combine harvesting or measur method of altering a trait in an agricultural plant seed or an ing ear weight. For all altered traits, the change can be at least agricultural plant grown from said seed, said method com 1%, for example at least 2%, at least 3%, at least 4%, at least prising inoculating said seed with a composition comprising 5%, at least 10%, at least 20%, at least 30%, at least 40%, at endophytes capable of promoting germination and an agri least 50%, at least 60%, at least 75%, at least 100%, or more, culturally-acceptable carrier, wherein the altered trait is an when compared with a control agricultural seed or plant. In improved functional trait selected from the group consisting Some embodiments, the method further comprises planting of increased yield, faster seedling establishment, faster the agricultural plant seed. In another embodiment, the growth, increased drought tolerance, increased heat toler method further comprises selecting a plant seed or plant that ance, increased cold tolerance, increased salt tolerance, has the altered trait. increased tolerance to pests and diseases, increased biomass, 0196. In one embodiment, the method is a method of increased root and/or shoot length or weight, increased fresh improving the 50% germination rate of a population of seeds weight of seedlings, increased seed or fruit number, increased and altering the gene expression in a plant grown from the plant vigour, nitrogen stress tolerance, enhanced Rhizobium seeds, comprising inoculating said population of seeds with a activity, enhanced nodulation frequency, early flowering composition as described herein. In some embodiments, the time, or any combination thereof. In some embodiments, the gene is altered in the plant-fungus or plant-bacterial symbi increased tolerance to disease is increased tolerance to otic system. In some embodiments, the gene with altered Fusarium infection, increased tolerance to Septoria infection, expression is a gene involved in plant growth, an acquired increased tolerance to Puccinia infection. In some embodi resistance gene, and a gene involved in protection from oxi ments, yield is measured on a population of plants grown in dative stress. In some embodiments, these genes may be the field and is calculated via combine harvesting or measur involved in phytohormone production, such as those involved ing ear weight. In another aspect, the altered trait is a seed trait in GA biosynthesis or breakdown, abscisic acid (ABA) bio selected from the group consisting a greater germination rate, synthesis or breakdown, NO production or breakdown, super faster dormancy breakdown, increased energy of germina oxide detoxification, or are positive or negative regulators of tion, increased seed germination vigor or increased seed vital these pathways. In other embodiments, the genes associated ity. In yet another embodiment, the altered trait is altered gene with systemic acquired resistance are redox-regulated tran expression, wherein the gene is selected from the group con Scription factors. In still other embodiments, the redox-regu sisting of a gene involved in gibberellin production, a gene lated transcription factors belong to the MYB family of involved in abscisic acid production, a gene involved in plant genes. In some embodiments, the gene with altered expres growth, an acquired resistance gene, and a gene involved in sion is selected from the group consisting of P5CS, SOD, protection from oxidative stress. In some embodiments, the MnSOD, GA3-oxidase 2, 14-3-3, NCED2, ABA8'OH1, genes may be involved in phytohormone production. In some RSG, KAO. Myb1 and Myb2. In some embodiments, the embodiments, the phytohormone is altered in the plant-fun change in gene expression can be at least 1%, for example at gus or plant-bacterial symbiotic system. In some embodi least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at ments, the method further comprises planting the agricultural least 20%, at least 30%, at least 40%, at least 50%, at least plant seed. In another embodiment, the method further com 60%, at least 75%, at least 100%, or more, when compared prises selecting a plant seed or plant that has the altered trait. with a control agricultural seed or plant. In some embodi For all altered traits, the change can be at least 1%, for ments, the method further comprises planting the agricultural example at least 2%, at least 3%, at least 4%, at least 5%, at plant seed. In another embodiment, the method further com least 10%, at least 20%, at least 30%, at least 40%, at least prises selecting a plant seed or plant that has the altered trait. 50%, at least 60%, at least 75%, at least 100%, or more, when 0.197 In one embodiment, a method of improving the 50% compared with a control agricultural seed or plant. germination rate of a population of seeds and providing at 0.195. In one embodiment, the invention provides methods least 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at of improving the 50% germination rate of a population of least 5%, at least 10%, at least 20%, at least 30%, at least 40%, seeds comprising inoculating said population of seeds with a at least 50%, at least 60%, at least 70%, or at least 80% composition as described herein. In one embodiment, the colonization in cortex cells of plants grown from the seeds is method is a method of improving the 50% germination rate of provided. a population of seeds and of improving a trait in plants grown 0.198. In one embodiment, methods of obtaining at least from the seeds, comprising inoculating said population of 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at least seeds with a composition as described herein. In one embodi 5%, at least 10%, at least 20%, at least 30%, at least 40%, at ment, the improved trait is selected from the group consisting least 50%, at least 60%, at least 70%, or at least 80% endo of increased yield, faster seedling establishment, faster phyte colonization in the cortex cells of a plant and of improv growth, increased drought tolerance, increased heat toler ing a trait in the plant are disclosed, comprising inoculating ance, increased cold tolerance, increased salt tolerance, the seed of said plant with a composition as described herein. increased tolerance to pests and diseases, increased biomass, In one embodiment, the improved trait is selected from the increased root and/or shoot length or weight, increased fresh group consisting of increased yield, faster seedling establish weight of seedlings, increased seed or fruit number, increased ment, faster growth, increased drought tolerance, increased US 2016/017457.0 A1 Jun. 23, 2016 20 heat tolerance, increased cold tolerance, increased salt toler In some embodiments, these genes are those involved in GA ance, increased tolerance to pests and diseases, increased biosynthesis or breakdown, abscisic acid (ABA) biosynthesis biomass, increased root and/or shoot length or weight, or breakdown, NO production or breakdown, superoxide increased fresh weight of seedlings, increased seed or fruit detoxification, or are positive or negative regulators of these number, increased plant vigour, nitrogen stress tolerance, pathways. In other embodiments, the genes associated with enhanced Rhizobium activity, enhanced nodulation fre systemic acquired resistance are redox-regulated transcrip quency, early flowering time, or any combination thereof. In tion factors. In still other embodiments, the redox-regulated Some embodiments, the increased tolerance to disease is transcription factors belong to the MYB family of genes. In increased tolerance to Fusarium infection, increased toler Some embodiments, the gene with altered expression is ance to Septoria infection, increased tolerance to Puccinia infection. In some embodiments, yield is measured on a selected from the group consisting of P5CS, SOD, MnSOD, population of plants grown in the field and is calculated via GA3-oxidase 2, 14-3-3, NCED2, ABA8'OH1, RSG, KAO, combine harvesting or measuring ear weight. For all altered Myb1 and Myb2. traits, the change can be at least 1%, for example at least 2%, 0204. In another embodiment, the invention provides a at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, method for treating seeds comprising contacting the Surface at least 30%, at least 40%, at least 50%, at least 60%, at least of an agricultural plant seed with a formulation comprising a 75%, at least 100%, or more, when compared with a control microbial population that comprises an endophyte capable of agricultural seed or plant. promoting germination that is heterologous to the seed, 0199. In one embodiment, the method is a method of wherein the endophyte capable of promoting germination is increasing the germination rate, speeding up dormancy present in the formulation in an amount effective to alter the breakdown, increasing the energy of germination, increasing level of at least one phytohormone within the seed, seedlings the germination vigour, speeding up germination, increasing derived from the seed or agricultural plants derived from the the energy of germination, producing greater uniformity of seed. In some embodiments, the phytohormones that are germination, including greater uniformity of rate of germina altered are gibberellins, abscisic acid, or cytokinins. In further tion and greater uniformity of timing of germination, or embodiments, the gibberellins may be gibberellin 1, 19, 44 or increasing the vitality of a seed, comprising inoculating seeds 53. In still further embodiments, the cytokinin may be zeatin. or a population of seeds with a composition as described For these altered phytohormone levels, the change can be at herein. In some embodiments, the method further comprises least 1%, for example at least 2%, at least 3%, at least 4%, at planting the agricultural plant seed. least 5%, at least 10%, at least 20%, at least 30%, at least 40%, 0200. In one embodiment, the invention provides a at least 50%, at least 60%, at least 75%, at least 100%, or method of releasing a seed from dormancy, said method com more, when compared with a control agricultural seed or prising inoculating said seed with a composition comprising plant. endophytes capable of promoting germination and an agri 0205. In another embodiment, the invention provides a culturally-acceptable carrier. In some embodiments, the method for treating seeds comprising contacting the Surface endophytes capable of promoting germination are of an agricultural plant seed with a formulation comprising a coleorhiza-activating endophytes. microbial population that comprises an endophyte capable of 0201 In one embodiment, the invention provides a promoting germination that is heterologous to the seed, method of improving the 50% germination rate of a popula wherein the endophyte capable of promoting germination is tion of seeds and increasing NO accumulation in a plant present in the formulation in an amount effective to improve grown from the seeds, comprising inoculating seeds with a a trait in the seed or a plant grown from the seed. In some composition as described herein. In some embodiments, the embodiments, the improved trait is selected from the group method further comprises planting the agricultural plant seed. consisting of increased yield, faster seedling establishment, 0202 In another embodiment, a method of altering a trait faster growth, increased drought tolerance, increased heat in an agricultural plant seed or an agricultural plant grown tolerance, increased cold tolerance, increased salt tolerance, from said seed is disclosed, comprising obtaining a synthetic increased tolerance to pests and diseases, increased biomass, preparation comprising an agricultural plant seed and a com increased root and/or shoot length or weight, increased fresh position comprising endophytes capable of promoting germi weight of seedlings, increased seed or fruit number, increased nation and an agriculturally-acceptable carrier and planting plant vigour, nitrogen stress tolerance, enhanced Rhizobium the synthetic preparation. In some embodiments, the method activity, enhanced nodulation frequency, early flowering further comprises planting the agricultural plant seed. In time, or any combination thereof. In some embodiments, the another embodiment, the method further comprises selecting increased tolerance to disease is increased tolerance to a plant seed or plant that has the altered trait. Fusarium infection, increased tolerance to Septoria infection, 0203. In another embodiment, the invention provides a increased tolerance to Puccinia infection. In some embodi method for treating seeds comprising contacting the Surface ments, yield is measured on a population of plants grown in of an agricultural plant seed with a formulation comprising a the field and is calculated via combine harvesting or measur microbial population that comprises an endophyte capable of ing ear weight. For all altered traits, the change can be at least promoting germination that is heterologous to the seed, 1%, for example at least 2%, at least 3%, at least 4%, at least wherein the endophyte capable of promoting germination is 5%, at least 10%, at least 20%, at least 30%, at least 40%, at present in the formulation in an amount effective to alter the least 50%, at least 60%, at least 75%, at least 100%, or more, level of at least one gene within the seed, seedlings derived when compared with a control agricultural seed or plant. In from the seed or agricultural plants derived from the seed. In Some embodiments, the method further comprises planting Some embodiments, the gene with altered expression is a gene the agricultural plant seed. In another embodiment, the involved in phytohormone production, an acquired resistance method further comprises selecting a plant seed or plant that gene, and a gene involved in protection from oxidative stress. has the altered trait. US 2016/017457.0 A1 Jun. 23, 2016

0206. In another aspect, there is provided a method of barley, rice, or other biological materials such as seed, plant improving plant health and/or plant yield comprising treating parts, Sugar cane bagasse, hulls or stalks from grain process plant propagation material or a plant with a composition ing, ground plant material or wood from building site refuse, disclosed herein; and cultivating the plant propagation mate sawdust or Small fibers from recycling of paper, fabric, or rial into a first generation plant or allowing the plant to grow. wood. Other suitable formulations will be known to those 0207. In another embodiment, the methods reduce the skilled in the art. effects of stress. Such as heat, drought and/or biotic stress. 0214. In one embodiment, the formulation can include a 0208. In an embodiment, the methods enhance landscape tackifier or adherent. Such agents are useful for combining development and remediation. the bacterial or fungal population of the invention with carri 0209. Accordingly, in one embodiment, there is provided ers that can contain other compounds (e.g., control agents that a method of phytoremediation or phytoreclamation of a con are not biologic), to yield a coating composition. Such com taminated site comprising treating plant propagation material positions help create coatings around the plant or seed to ora plant with a composition disclosed herein, and cultivating maintain contact between the microbe and other agents with the plant propagation material into a first generation plant or the plant or plant part. In one embodiment, adherents are allowing the plant to grow; thereby remediating or reclaiming selected from the group consisting of alginate, gums, the site. starches, lecithins, formononetin, polyvinyl alcohol, alkali 0210. In one embodiment, the site is soil, such as at a formononetinate, hesperetin, polyvinyl acetate, cephalins, landfill. In an embodiment, the Substances, wastes or hazard Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene Gly ous materials comprise hydrocarbons, petroleum or other col (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, chemicals, salts, or metals, such as lead, cadmium or radio Methyl Cellulose, PEG 400, Chitosan, Polyacrylamide, Poly isotopes. acrylate, Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl, Car Formulations/Seed Coating Compositions boxymethyl cellulose, Gum Ghatti, and polyoxyethylene 0211. The purified endophytes described herein can be polyoxybutylene block copolymers. Other examples of formulated using an agriculturally compatible carrier. The adherent compositions that can be used in the synthetic prepa formulation useful for these embodiments generally typically ration include those described in EP 0818135, CA 1229497, include at least one member selected from the group consist WO 2013090628, EP 0192342, WO 2008103422 and CA ing of a tackifier, a microbial stabilizer, a fungicide, an anti 1041788, each of which is incorporated herein by reference in bacterial agent, an herbicide, a nematicide, an insecticide, a its entirety. plant growth regulator, a rodenticide, a dessicant, and a nutri 0215. The formulation can also contain a surfactant. Non ent. limiting examples of Surfactants include nitrogen-surfactant 0212. In some cases, the purified bacterial or fungal popu blends such as Prefer 28 (Cenex), Surf-N(US), Inhance lation is mixed with an agriculturally compatible carrier. The (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified seed carrier can be a solid carrier or liquid carrier, and in various oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), forms including microspheres, powders, emulsions and the Hasten (Wilfarm) and Mes-100 (Drexel); and organo-silicone like. The carrier may be any one or more of a number of surfactants include Silwet L77 (UAP), Silikin (Terra), Dyne carriers that confer a variety of properties, such as increased Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) stability, wettability, or dispersability. Wetting agents such as and Century (Precision). In one embodiment, the surfactant is natural or synthetic Surfactants, which can be nonionic or present at a concentration of between 0.01% V/v to 10% V/v. ionic Surfactants, or a combination thereof can be included in In another embodiment, the Surfactant is present at a concen a composition of the invention. Water-in-oil emulsions can tration of between 0.1% w/v to 1% V/v. also be used to formulate a composition that includes the 0216. In certain cases, the formulation includes a micro purified bacterial or fungal population (see, for example, U.S. bial stabilizer. Such an agent can include a desiccant. As used Pat. No. 7,485,451, which is incorporated herein by reference herein, a “desiccant can include any compound or mixture of in its entirety). Suitable formulations that may be prepared compounds that can be classified as a desiccant regardless of include wettable powders, granules, gels, agar Strips or pel whether the compound or compounds are used in Such con lets, thickeners, and the like, microencapsulated particles, centrations that they in fact have a desiccating effect on the and the like, liquids such as aqueous flowables, aqueous Sus liquid inoculant. Such desiccants are ideally compatible with pensions, water-in-oil emulsions, etc. The formulation may the bacterial or fungal population used, and should promote include grain or legume products, for example, ground grain the ability of the microbial population to survive application or beans, broth or flour derived from grain or beans, starch, on the seeds and to Survive desiccation. Examples of suitable Sugar, or oil. desiccants include one or more of trehalose, Sucrose, glyc 0213. In some embodiments, the agricultural carrier may erol, and Methylene glycol. Other suitable desiccants be soil or a plant growth medium. Other agricultural carriers include, but are not limited to, non-reducing Sugars and Sugar that may be used include water, fertilizers, plant-based oils, alcohols (e.g., mannitol or Sorbitol). The amount of desiccant humectants, or combinations thereof. Alternatively, the agri introduced into the formulation can range from about 5% to cultural carrier may be a solid, Such as diatomaceous earth, about 50% by weight/volume, for example, between about loam, silica, alginate, clay, bentonite, Vermiculite, seed cases, 10% to about 40%, between about 15% and about 35%, or other plant and animal products, or combinations, including between about 20% and about 30%. granules, pellets, or Suspensions. Mixtures of any of the 0217. In some cases, it is advantageous for the formulation aforementioned ingredients are also contemplated as carriers, to contain agents such as a fungicide, an antibacterial agent, Such as but not limited to, pesta (flour and kaolin clay), agar or an herbicide, a nematicide, an insecticide, a plant growth flour-based pellets in loam, sand, or clay, etc. Formulations regulator, a rodenticide, or a nutrient. Such agents are ideally may include food sources for the cultured organisms, such as compatible with the agricultural seed or seedling onto which US 2016/017457.0 A1 Jun. 23, 2016 22 the formulation is applied (e.g., it should not be deleterious to Populations of Seeds the growth or health of the plant). Furthermore, the agent is ideally one that does not cause safety concerns for human, 0224. In another aspect, the invention provides for a sub animal or industrial use (e.g., no safety issues, or the com stantially uniform population of seeds comprising a plurality pound is sufficiently labile that the commodity plant product of seeds comprising the population of endophytes capable of derived from the plant contains negligible amounts of the promoting germination, as described herein above. Substan compound). tial uniformity can be determined in many ways. In some cases, at least 10%, for example, at least 20%, at least 30%, at 0218. In the liquid form, for example, solutions or suspen least 40%, at least 50%, at least 60%, at least 70%, at least sions, the bacterial or fungal endophytic populations of the 75%, at least 80%, at least 90%, at least 95% or more of the present invention can be mixed or Suspended in water or in seeds in the population, contain the endophytic population in aqueous solutions. Suitable liquid diluents or carriers include an amount effective to colonize the plant disposed on the water, aqueous Solutions, petroleum distillates, or other liquid surface of the seeds. In other cases, at least 10%, for example, carriers. at least 20%, at least 30%, at least 40%, at least 50%, at least 0219 Solid compositions can be prepared by dispersing 60%, at least 70%, at least 75%, at least 80%, at least 90%, at the bacterial or fungal endophytic populations of the inven least 95% or more of the seeds in the population, contain at tion in and on an appropriately divided Solid carrier, Such as least 1, at least 10, or at least 100 CFU on the seed surface or peat, wheat, bran, Vermiculite, clay, talc, bentonite, diatoma per gram of seed, for example, at least 200 CFU, at least 300 ceous earth, fuller's earth, pasteurized soil, and the like. CFU, at least 1,000 CFU, at least 3,000 CFU, at least 10,000 When such formulations are used as wettable powders, bio CFU, at least 30,000 CFU, at least 100,000 CFU, at least logically compatible dispersing agents such as non-ionic, 300,000 CFU, or at least 1,000,000 CFU per seed or more. anionic, amphoteric, or cationic dispersing and emulsifying 0225. In a particular embodiment, the population of seeds agents can be used. is packaged in a bag or container Suitable for commercial sale. 0220. The solid carriers used upon formulation include, Such a bag contains a unit weight or count of the seeds for example, mineral carriers such as kaolin clay, pyrophyl comprising the bacterial or fungal endophytic population as lite, bentonite, montmorillonite, diatomaceous earth, acid described herein, and further comprises a label. In one white soil, Vermiculite, and pearlite, and inorganic salts such embodiment, the bag or container contains a predetermined as ammonium sulfate, ammonium phosphate, ammonium number of seeds. In an embodiment, the bag or container nitrate, urea, ammonium chloride, and calcium carbonate. contains at least 1,000 seeds, for example, at least 5,000 Also, organic fine powders such as wheat flour, wheat bran, seeds, at least 10,000 seeds, at least 20,000 seeds, at least and rice bran may be used. The liquid carriers include Veg 30,000 seeds, at least 50,000 seeds, at least 70,000 seeds, at etable oils such as soybean oil and cottonseed oil, glycerol, least 80,000 seeds, at least 90,000 seeds or more. In another ethylene glycol, polyethylene glycol, propylene glycol, embodiment, the bag or container can comprise a discrete polypropylene glycol, etc. weight of seeds, for example, at least 11b, at least 2 lbs, at least 5 lbs, at least 10 lbs, at least 30 lbs, at least 50 lbs, at least 70 0221. In one particular embodiment, the formulation is lbs or more. The bag or container may comprise a label ideally Suited for coating of the endophytic microbial popu describing the seeds and/or said bacterial or fungal endo lation onto seeds. The bacterial or fungal endophytic popula phytic population. The label can contain additional informa tions described in the present invention are capable of con tion, for example, the information selected from the group ferring many fitness benefits to the host plants. The ability to consisting of net weight, lot number, geographic origin of the confer Such benefits by coating the bacterial or fungal popu seeds, test date, germination rate, inert matter content, and/or lations on the Surface of seeds has many potential advantages, the amount of noxious weeds, if any. Suitable containers or particularly when used in a commercial (agricultural) scale. packages include those traditionally used in plant seed com 0222. The bacterial or fungal endophytic populations mercialization. The invention also contemplates other con herein can be combined with one or more of the agents tainers with more Sophisticated storage capabilities (e.g., described above to yield a formulation suitable for combining with microbiologically tight wrappings or with gas- or water with an agricultural seed or seedling. The bacterial or fungal proof containments). population can be obtained from growth in culture, for 0226. In some cases, a Sub-population of seeds comprising example, using a synthetic growth medium. In addition, the the bacterial or fungal endophytic population is further microbe can be cultured on Solid media, for example on petri selected on the basis of increased uniformity, for example, on dishes, scraped off and Suspended into the preparation. the basis of uniformity of microbial population. For example, Microbes at different growth phases can be used. For individual seeds of pools collected from individual cobs, example, microbes at lag phase, early-log phase, mid-log individual plants, individual plots (representing plants inocu phase, late-log phase, stationary phase, early death phase, or lated on the same day) or individual fields can be tested for death phase can be used. uniformity of microbial density, and only those pools meeting 0223) The formulations comprising the bacterial or fungal specifications (e.g., at least 80% of tested seeds have mini endophytic population of the present invention typically con mum density, as determined by quantitative methods tains between about 0.1 to 95% by weight, for example, described elsewhere) are combined to provide the agricultural between about 1% and 90%, between about 3% and 75%, seed Sub-population. between about 5% and 60%, or between about 10% and 50% 0227. The methods described herein can also comprise a in wet weight of the bacterial or fungal population of the validating step. The validating step can entail, for example, present invention. It is preferred that the formulation contains growing some seeds collected from the inoculated plants into at least about 10 CFU per ml of formulation, for example, at mature agricultural plants, and testing those individual plants least about 10, at least about 10, at least about 10, at least for uniformity. Such validating step can be performed on 107 CFU, at least 10 CFU per ml of formulation. individual seeds collected from cobs, individual plants, indi US 2016/017457.0 A1 Jun. 23, 2016 vidual plots (representing plants inoculated on the same day) Increased Uniformity in Populations of Plants/Agricultural or individual fields, and tested as described above to identify Fields pools meeting the required specifications. 0233. A major focus of crop improvement efforts has been 0228. In some embodiments, methods described herein to select varieties with traits that give, in addition to the include planting a synthetic combination described herein. highest return, the greatest homogeneity and uniformity. Suitable planters include an air seeder and/or fertilizer appa While inbreeding can yield plants with substantial genetic ratus used in agricultural operations to apply particulate identity, heterogeneity with respect to plant height, flowering time, and time to seed, remain impediments to obtaining a materials including one or more of the following, seed, fer homogeneous field of plants. The inevitable plant-to-plant tilizer and/or inoculants, into Soil during the planting opera variability is caused by a multitude of factors, including tion. Seeder/fertilizer devices can include a tool bar having uneven environmental conditions and management practices. ground-engaging openers thereon, behind which is towed a Another possible source of variability can, in some cases, be wheeled cart that includes one or more containment tanks or due to the heterogeneity of the microbial population inhabit bins and associated metering means to respectively contain ing the plants. By providing bacterial or fungal endophytic and meter therefrom particulate materials. See, e.g., U.S. Pat. populations onto seeds and seedlings, the resulting plants No. 7,555,990. generated by germinating the seeds and seedlings have a more 0229. In certain embodiments, a composition described consistent microbial composition, and thus are expected to herein may be in the form of a liquid, a slurry, a solid, or a yield a more uniform population of plants. powder (wettable powder or dry powder). In another embodi 0234. Therefore, in another aspect, the invention provides ment, a composition may be in the form of a seed coating. a Substantially uniform population of plants. The population can include at least 100 plants, for example, at least 300 Compositions in liquid, slurry, or powder (e.g., wettable pow plants, at least 1,000 plants, at least 3,000 plants, at least der) form may be suitable for coating seeds. When used to 10,000 plants, at least 30,000 plants, at least 100,000 plants or coat seeds, the composition may be applied to the seeds and more. The plants are grown from the seeds comprising the allowed to dry. In embodiments wherein the composition is a bacterial and/or fungal endophytic population as described powder (e.g., a wettable powder), a liquid. Such as water, may herein. The increased uniformity of the plants can be mea need to be added to the powder before application to a seed. sured in a number of different ways. 0230. In still another embodiment, the methods can 0235. In one embodiment, there is an increased uniformity include introducing into the soil an inoculum of one or more with respect to the microbes within the plant population. For of the endophyte populations described herein. Such methods example, in one embodiment, a Substantial portion of the can include introducing into the Soil one or more of the population of plants, for example at least 10%, at least 20%, compositions described herein. The inoculum(s) or compo at least 30%, at least 40%, at least 50%, at least 60%, at least sitions may be introduced into the soil according to methods 70%, at least 75%, at least 80%, at least 90%, at least 95% or known to those skilled in the art. Non-limiting examples more of the seeds or plants in a population, contains a thresh include in-furrow introduction, spraying, coating seeds, old number of the bacterial or fungal endophytic population. foliar introduction, etc. In a particular embodiment, the intro The threshold number can be at least 10 CFU, at least 100 ducing step comprises in-furrow introduction of the inoculum CFU, for example at least 300 CFU, at least 1,000 CFU, at or compositions described herein. least 3,000 CFU, at least 10,000 CFU, at least 30,000 CFU, at least 100,000 CFU or more, in the plant or a part of the plant. 0231. In one embodiment, seeds may be treated with com Alternatively, in a substantial portion of the population of position(s) described herein in several ways but preferably plants, for example, in at least 1%, at least 10%, at least 20%, via spraying or dripping. Spray and drip treatment may be at least 30%, at least 40%, at least 50%, at least 60%, at least conducted by formulating compositions described herein and 70%, at least 75%, at least 80%, at least 90%, at least 95% or spraying or dripping the composition(s) onto a seed(s) via a more of the plants in the population, the bacterial or fungal continuous treating system (which is calibrated to apply treat endophyte population that is provided to the seed or seedling ment at a predefined rate in proportion to the continuous flow represents at least 0.1%, at least 1%, at least 5%, at least 10%, of seed). Such as a drum-type of treater. Batch systems, in at least 20%, at least 30%, at least 40%, at least 50%, at least which a predetermined batch size of seed and composition(s) 60%, at least 70%, at least 80%, at least 90%, at least 95%, at as described herein are delivered into a mixer, may also be least 99%, or 100% of the total microbe population in the employed. Systems and apparati for performing these pro plant/seed. cesses are commercially available from numerous Suppliers, 0236. In one embodiment, there is increased genetic uni e.g., Bayer CropScience (Gustafson). formity of a substantial proportion or all detectable microbes 0232. In another embodiment, the treatment entails coat within the taxa, genus, or species of the microbe relative to an ing seeds. One such process involves coating the inside wall uninoculated control. This increased uniformity can be a of a round container with the composition(s) described result of the microbe being of monoclonal origin or otherwise herein, adding seeds, then rotating the container to cause the deriving from a microbial population comprising a more uni seeds to contact the wall and the composition(s), a process form genome sequence and plasmid repertoire than would be known in the art as “container coating. Seeds can be coated present in the microbial population a plant that derives its by combinations of coating methods. Soaking typically microbial community largely via assimilation of diverse soil entails using liquid forms of the compositions described. For symbionts. example, seeds can be soaked for about 1 minute to about 24 0237. In another embodiment, there is an increased uni hours (e.g., for at least 1 min, at least 5 min, at least 10 min, at formity with respect to a physiological parameter of the plants least 20 min, at least 40 min, at least 80 min, at least 3 hr, at within the population. In some cases, there can be an least 6 hr, at least 12 hr., or at least 24 hr). increased uniformity in the height of the plants when com US 2016/017457.0 A1 Jun. 23, 2016 24 pared with a population of reference agricultural plants grown sel fuel. Soybean oil may be split, inter-esterified, sulfurized, under the same conditions. For example, there can be a reduc epoxidized, polymerized, ethoxylated, or cleaved. Designing tion in the standard deviation in the height of the plants in the and producing soybean oil derivatives with improved func population of at least 5%, for example, at least 10%, at least tionality and improved oliochemistry is a rapidly growing 15%, at least 20%, at least 30%, at least 40%, at least 50%, at field. The typical mixture of triglycerides is usually split and least 60% or more, when compared with a population of separated into pure fatty acids, which are then combined with reference agricultural plants grown under the same condi petroleum-derived alcohols or acids, nitrogen, Sulfonates, tions. In other cases, there can be a reduction in the standard chlorine, or with fatty alcohols derived from fats and oils to deviation in the flowering time of the plants in the population produce the desired type of oil or fat. Commodity plant prod of at least 5%, for example, at least 10%, at least 15%, at least ucts also include industrial compounds. Such as a wide variety 20%, at least 30%, at least 40%, at least 50%, at least 60% or of resins used in the formulation of adhesives, films, plastics, more, when compared with a population of reference agricul paints, coatings and foams. The above disclosure generally tural plants grown under the same conditions. describes the present application. A more complete under standing can be obtained by reference to the following spe Decreased Uniformity in Populations of Plants/Agricultural cific examples. These examples are described solely for the Fields purpose of illustration and are not intended to limit the scope of the disclosure. Changes inform and Substitution of equiva 0238. In certain circumstances, decreased uniformity in a lents are contemplated as circumstances might Suggest or population can be desirable. For example, plants within a render expedient. Although specific terms have been population that are not all at the same developmental stage employed herein, such terms are intended in a descriptive may not all be negatively affected by a biotic or an abiotic sense and not for purposes of limitation. stress event, and as a result, the population as a whole may 0240. The following non-limiting examples are illustra show a beneficial trait such as increased yield. As another tive of the present disclosure: example, a lack of uniformity may allow for the selection of plants/seeds with a trait that is not present in the other mem EXAMPLES bers of the population. Therefore, in another embodiment, there is a decreased uniformity with respect to a physiological 0241 Dormancy and germination depend on several pro parameter of the plants within the population. In some cases, cesses and factors. To ensure seedling establishment and Suc there can be a decreased uniformity in the height of the plants cess, it is important to control the underlying processes or when compared with a population of reference agricultural conditions. The role of plant genetics, hormones, and differ plants grown under the same conditions. For example, there ent seed tissues have been relatively well studied. The present can be an increase in the standard deviation in the height of the examples study the endophyte-plant seed relationship, tran plants in the population of at least 5%, for example, at least sitting into a root symbiotic stage towards plant maturation. 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more, when compared with a Example 1 population of reference agricultural plants grown under the same conditions. In other cases, there can be an increase Taxonomy and Physical Properties of the intracellular vesiculoid in the standard deviation in the flow Endophytes ering time of the plants in the population of at least 5%, for 0242. The endophytes used in the synthetic compositions example, at least 10%, at least 15%, at least 20%, at least 30%, described herein have been deposited as follows: Interna at least 40%, at least 50%, at least 60% or more, when com tional Depositary Authority of Canada—IDAC (original pared with a population of reference agricultural plants grown strains deposited IDAC, National Microbiology Labora under the same conditions. tory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2; receipts and viabil Commodity Plant Product ity in Appendix A) and Saskatchewan Microbial Collection 0239. The present invention provides a commodity plant and Database SMCD (copies of strains deposited) Interna product, as well as methods for producing a commodity plant tional Depository Authority of Canada—IDAC (original product, that is derived from a plant of the present invention. strains deposited) and Saskatchewan Microbial Collection As used herein, a "commodity plant product refers to any and Database SMCD (copies of strains deposited), (see composition or product that is comprised of material derived FIGS. 1-6 and Table 1). Strains: from a plant, seed, plant cell, or plant part of the present 0243 (a) IDAC 081111-06=SMCD 2215; invention. Commodity plant products may be sold to consum 0244 (b) IDAC 081111-03=SMCD 2210; ers and can be viable or nonviable. Nonviable commodity 0245 (c) IDAC 081111-02-SMCD 2208; products include but are not limited to nonviable seeds and 0246 (d) IDAC 081111-01=SMCD 2206; grains; processed seeds, seed parts, and plant parts; dehy 0247 (e) IDAC 200312-06=SMCD 2204; and drated plant tissue, frozen plant tissue, and processed plant 0248 (f) IDAC 200312-05=SMCD 2204F. tissue; seeds and plant parts processed for animal feed for 0249 SMCD 2215 strain was originally isolated as an terrestrial and/or aquatic animal consumption, oil, meal, endophytic bacterium of Phyalocephala sensu lato plant flour, flakes, bran, fiber, paper, tea, coffee, silage, crushed of endophytic SMCD fungus. Classification according to whole grain, and any other food for human or animal con Labeda et al. 2012. This phylogenetic study examines Sumption; and biomasses and fuel products; and raw material almost all described species (615 taxa) within the family in industry. Industrial uses of oils derived from the agricul Streptomycetaceae based on 16S rDNA gene sequences and tural plants described herein include ingredients for paints, illustrates the species diversity within this family, which is plastics, fibers, detergents, cosmetics, lubricants, and biodie observed to contain 130 statistically supported clades. The US 2016/017457.0 A1 Jun. 23, 2016 present 16S rDNA sequence data confirm that Streptomyces Example 2 sp. strain SMCD 2215 can be assigned to a separate unknown clade according to Labeda et al 2012 but separate species Symbiotic Microbe-Plant Association and Level of from Streptomyces lividans. Within a plant, it is capable of Compatibility forming intercellular hyphae-like filaments and intracellular 0256 The level of microbe-plant compatibility was individual spore-like cells. assessed using a slightly modified method of Abdellatif et al. 2009. In a bicompartmental agar 10 cm plate without nutri 0250 SMCD2204 is a filamentous, spore-forming facul ents (FIG. 7), the plants health and the formation of root tative endophyte. Within a plant, it is capable of forming hairs—the absorbants of water and minerals—were charac hyphal coils, microvesicles, microarbuscules, hyphal knots terized in co-culture, with and without microbial partners. In and intracellular or Hartig net-like structures. SMCD2204 is FIG. 7, the left compartment of each split plate shows a a fungus that is capable of being part of a plant-fungus sym culture with the microbial partner, and the right compartment biont that produces altered levels of phytohormones, and/or of each split plate shows a culture without the microbial altered levels of anti-oxidants, as compared to a plant that is partner. The experiment was repeated twice in three repli not in Symbiosis. This endophyte is also capable of being part Cates. of a plant-fungus symbiont that shows decreased aging and/or 0257. As shown in the left compartment of each split plate, senescence, and/or increased protection against pathogens, as healthy plant tissue formed even when the plant roots were compared to a plant or plant organ that is not in Symbiosis. grown directly on the dense microbial mats. The biomass of root hairs is enhanced to about twice as much compared to the 0251 SMCD2204F is filamentous, spore-forming facul right compartment of each split plate where the microbial tative endophyte that is capable of living within anotherendo partner is absent (see left compartments). phyte. It is capable of forming hyphal coils and intracellular 0258. The plant efficacy to establish symbiotic association or Hartig net-like structures within a plant. This endophyte is is dependent on the type of endophyte distribution within the also capable of being part of a plant-fungus symbiont that root endodermis. Typical endophytic root colonization is dis shows decreased aging and/or senescence, as compared to a continuous and partial with a lower number of occupied plant or plant organ that is not in Symbiosis. cells<50% (Table 2) compared to the colonization of fungal pathogens which is characterized by a uniform/continual (fre 0252 SMCD2206 is a filamentous, spore-forming facul quency: 60-80%) colonization of cells (FIG. 8). tative endophyte. Within a plant, it is capable of forming 0259 An endophyte's performance should not only be hyphal coils, microvesicles, microarbuscules, and hyphal assessed by measuring biomass production, because what knots. SMCD2204 is a fungus that is capable of being part of underlies the visibly increased yield is the endophyte's effi a plant-fungus symbiont that produces altered levels of phy ciency in colonizing the plant. This can be assessed by char tohormones, and/or altered levels of anti-oxidants, as com acterizing their association with plant cells, tissues, or organs pared to a plant that is not in Symbiosis. This endophyte is also (i.e. seed and radicles) using mathematical Indices which capable of being part of a plant-fungus symbiont that shows have been developed Abdellatif et al. 2009 and applied in decreased aging and/or senescence, and/or increased protec this study (FIG.9 and FIG. 10). tion against pathogens, as compared to a plant or plant organ 0260 These Indices are based on the following observa that is not in Symbiosis. tions: Endophytic symbionts show different radicle (root)- colonization patterns (regularity or level of deviation in endo 0253 SMCD2208 is a spore-forming facultative endo phyte cell form-Ireg and direction-Idir when colonizing phyte. living cell) compared to dead radicle-cell (which usually 0254 SMCD2210 is a facultative, spore-forming endo remain colonized by true saprophytes). phyte that is capable of living within another endophyte. 0261 High Ireg and Idir index values determine mutual Within a plant, it is capable of forming hyphal coils and istic (beneficial) plant-Symbiont relationships. In conclusion, microvesicles. SMCD2210 is a fungus that is capable of the results show that the symbiotic microbe-plant association being part of a plant-fungus symbiont that produces altered is characterised by a high level of compatibility between the levels of phytohormones, and/or altered levels of anti-oxi two partners, leading to an equilibrated (<50% of colonized dants, as compared to a plant that is not in Symbiosis. This cortex cells) and discontinuous root colonisation by the endophyte is also capable of being part of a plant-fungus microbial endophytes measured using mathematical indices symbiont that shows decreased aging and/or senescence, and/ Abdellatifetal. 2009. This mutualistic partnership is further or increased protection against pathogens, as compared to a characterised by the direct effect of endophytic microbes on plant or plant organ that is not in Symbiosis. plant healthy growth (bacto- and mycodependency) when the plant is challenged to use the microbial partners as the only 0255 SMCD2215 is also a facultative, spore-forming Source of nutrients or energy for growth. In addition, the endophyte that is capable of living within another endophyte. enhancement of the root hairs biomass by the endophytes was It is capable of forming hyphal coils and intracellular or observed and measured even in roots in distal compartments Hartig net-like structures within a plant. SMCD2215 is a of split plates where microbial partners were absent, indicat bacterium that is capable of being part of a plant-fungus ing a possible systemic plant growth promoting function of symbiont that produces altered levels of phytohormones, and/ the endophytes. or altered levels of anti-oxidants, as compared to a plant that is not in Symbiosis. This endophyte is also capable of being Example 3 part of a plant-fungus symbiont that shows decreased aging and/or senescence, and/or increased protection against patho Symbiotic Organs of Endophytes on Wheat gens, as compared to a plant or plant organ that is not in 0262 Each taxonomical group of endophytes establishes a symbiosis. unique type of mycoVitalism, consequently forming different US 2016/017457.0 A1 Jun. 23, 2016 26 symbiotic organs. Characterization of the mycoVitalism was Sured in degree days. Second, the seed must accumulate Suf done using Abdellatif et al. 2009 methodology, consisting ficient water potential (0) per degree-day. Thus, HTT (0) of in vitro seed and microbe co-cultures assessing an early can be expressed as: stage of the microbe-plant symbiotic association. The diver sity of microbial symbiotic organs formed by SMCD 2204. 0 (0(0). (Equation 1) 2206, 2210, and 2215 on wheat germinants is shown in FIG. 0268 According to Köchy and Tielbörger 2007, 11. 91 (Tsubstrate-Tain) (Equation 2) 0263. In summary, the results show differential types of symbiotic organs formed in wheat root by each endophyte with trepresenting the time elapsed in days, and likely related to their different symbiotic functions. An equili OHP substrate-Pain (Equation 3) brated colonization abundance, patchy colonization patterns, in a constant environment assuming that T is equal to increased hypha septation in living root cells, as well as or less than the optimal temperature for seed germination. In formation of arbuscules, knots, coils and vesicles—putative Equation3, and l, represent the Water potential of symbiotic functional organs—may indicate local specializa the Substrate and the minimum water potential at which ger tion within the fungal endophytes to promote plant mycoVi mination is possible, in MPa, respectively. Consistent with tality and mycoheterotrophy. Bactovitality is mostly charac Bradford 2002), equations 2 and 3 can be substituted into terized by Streptomyces intercellular curly filaments. equation 1 to yield: 0264 FIG. 66 shows symbiosomes formed in wheat root. The symbiosome is the new compartment that is formed in the OH T('substrate-Pain)(Tsubstrate-Tain) (Equation 4). plant cell when bacteria or fungi enter it. Symbiosomes can be 0269. However, in the present study, the temperature classified into two types: I and II. Both types are composed of exceeds the optimal temperature for the germination of wheat a perivesiculoid membrane and a partially fragmented outer reviewed by McMaster (2009), necessitating the consider vesiculoid membrane. Type I Symbiosomes are additionally ation of a maximum temperature (T) above which germi composed of an intercellular microvesiculoid compartment nation cannot occur. Thus, equation 2 was modified to: formed between two plant cell membranes, while type II symbiosomes are additionally composed of an intracellular or VI(Tsubstrate-Tain)(Tsubstrate-Tina)) (Equation 5) vesiculoid compartment. Both types can be seed in the form where T...sTfia satistrate sT. If equation 5 is Substituted for 2 of vesicles (A and B) and knots (C). in equation 4, the following results: 0265 Symbiosis at the seed level resulted in increased 0H1('substrate-pain)V(Tsubstrate- Tain)(Tsubstrate wheat germinants after 10 days of co-innoculation (FIG. 12 Tina.) It (Equation 6) and FIG. 13). where TsTsitbstrate sTina 0270 Energy of germination (EG) can be defined in sev Example 4 eral ways, including the percentage of seeds germinating after a settime period after planting, relative to the number of seeds Endophytes Improve Wheat Seed Germination tested Ruan et al. 2002: Dong-dong et al. 2009, or 50% of Under Heat and Drought Stress germination attained Allen 1958. In order to integrate EG 0266 Seed germination is a critical life stage for plant with the HTT model of germination the latter definition was Survival and timely seedling establishment especially in used, meaning that EG is equal to t in Equation 2. stressful environments. It was hypothesized that endophytes would improve wheat seed germination under heat and Estimation of Parameters drought stress. The hydrothermal time (HTT) model of ger 0271 The estimation ofT and T for wheat was based mination is a conceptual model useful for predicting the tim on both information available in the literature and the present ing and energy of germination (EG) under a given set of inventors’ own observations. McMaster 2009 summarizes conditions. The HTT and EG are applied to determine if one data originating from Friend et al. 1962, Cao and Moss or more compatible endophytes enhance heat or drought tol 1989, and Jame et al. indicating the existence of a curvilin erance in wheat. Endophytes tested dramatically increased ear relationship between wheat development rate and tem the percent of germination, improved EG and HTT values, perature. Since germination and development of wheat does and diminished wheat Susceptibility to heat and drought as not take place below 0°C. or above 40°C. T., and Twere measured by fresh weight of seedlings. When colonised by assigned the values of 0°C. and 40°C., respectively. the most effective endophyte, the values of the parameters 0272. The parameter up was estimated in vitro by ger tested in wheat seeds exposed to heat stress resembled those minating wheat seeds grown on potato dextrose agar (PDA: of unstressed seeds. Difico) media containing a range of polyethylene glycol (PEG) 8000 concentrations (Amresco Inc.). The water activ Materials and Methods ity (a) of PDA alone and PDA containing 8%, 12% and 16% PEG was measured using the AquaLab 4TE, Series 4 Quick Hydrothermal Time Model of Germination and Energy of Start, Decagon Devices. Water activity was converted to Germination water potential () using the relationship adapted from Bloom and Richard 2002: 0267. The hydrothermal time (HTT) model Gummerson 1986 postulates that an individual seed begins to germinate U=(RT)ln(a), V (Equation 7) when two conditions are met. First, the sum of daily tempera where R is the universal gas constant (8.314 J mol' K'), T tures, above a minimum cardinal value (T), accumulated is the temperature in K, and V is the partial molar volume of over a period of time, must pass a threshold value (0), mea water (18 mL/mol). For unit conversions, 1 J/mL=1 MPa = 10 US 2016/017457.0 A1 Jun. 23, 2016 27 bar. Water potential is Zero for a free water surface or a Endophytes Ability to Confer Heat and Drought Tolerance to saturated medium; all other values are negative. Wheat (0273. The water activities of PDA and PDA containing 0277 Each isolate was applied individually to wheat seeds 8%, 12% and 16% PEG were 0.9974, 0.9890, 0.9863, and prior to germination according to the method described in 0.9825, respectively. These values are equivalent to -0.35, Abdellatif et al. 2010 and shown in FIG. 14A. Briefly, five -1.51, -1.88, and -2.41 MPa, respectively and are consistent Surface-sterilized seeds were placed at a distance equivalent with those reported in the literature Leone et al. 1994. to 48-h hyphal growth from a 5 mm-agar plug, placed hyphal side down in the centre of a 60-mm Petri dish. For slow Plant and Fungal Material growing isolates, the agar plug of endophyte colony was placed in the Petridish one to four days prior to the introduc 0274 The plant material used was the durum wheat culti tion of the seeds. The seedlings were germinated for one week var AC Avonlea, which has low resistance to environmental under abiotic stress and control conditions. stressors SaskSeed guide 2008. The seeds used in the first 0278 Drought stress was induced using PDA containing round of experiments were produced by Paterson Grain in 8% PEG. Heat stress was induced in a bench-top incubator in 2008, under field conditions, and not certified to be free of darkness; the temperature was gradually raised by 2°C. every microbes. Seeds used in the second set of experiments were 2 h from 28°C. to 36°C. In the initial round of experiments, produced by the Agriculture and Agri-Food Canada (AAFC) percent germination at three days and fresh weight at one Seed Increase Unit Research Farm in 2006 undergreenhouse week was assessed. Each experiment consisted of six Petri conditions, and were certified to be free of microbes. Wheat plates and was repeated, independently, three times. In Sub seeds were surface-sterilized with 95% ethanol for 10 s. sequent experiments, percent germination was assessed every rinsed in sterile distilled water for 10s, submerged for either 24hrs for seven days. Each experiment consisted of 10 Petri 3 min (first round of experiments involving seeds not certified plates and was repeated either twice (heat and drought stress to be free of microbes) or 1 min (second round of experiments combined) or three times (heat stress, drought stress and using seeds certified to be microbe-free) in 5% sodium control conditions). hypochlorite (Javex), rinsed three times in sterile distilled 0279. The stable internal colonization of wheat roots by water and PDA for germination Abdellatif et al. 2009. A the intended endophytes was confirmed by re-isolation of the third seed sterilization method, involving a 3 hr exposure to endophytic organism from roots which had been Surface ster chlorine gas (produced by combining 25 mL 6% sodium ilized to remove an external microbial growth using a proce hypochlorite with 1.0 mL concentrated hydrochloric acid in a dure modified from Larranetal. 2002. Rootfragments (-0.5 beaker) in a closed plastic box placed in a fumehood Rivero cm) were surface sterilized in 95% ethanol for 10s, rinsed in et al. 2011 was also tested. The percent germination of seeds sterile distilled water for 10 s, submerged for 20 s in 5% subjected to each sterilization protocol and placedon PDA for sodium hypochlorite (Javex), rinsed three times in sterile three days is shown in FIG. 14B. Only the 3 min submersion distilled water and placed on PDA in a 60 mm diameter Petri in Sodium hypochlorite resulted in a significant decrease in dish. The Petridish was sealed with parafilm and incubated in germination (ps0.01). Seed surface sterilization was intended the dark at room temperature for four to seven days prior to eliminate microbes which could compete with the endo examination. phytes being investigated. In addition, microbes present on the Surface of the seeds could overgrow the plate and emerg Statistical Analysis ing seedling, inhibiting plant growth. All seeds used in the study were determined to be free from microorganisms after 0280. The colony growth rates of free-living endophytic sterilization, based on the absence of unintended microbial organisms grown underheat or drought stress were compared growth on the plate. to those of the same organism grown under control conditions using analysis of variance (ANOVA) followed by post-hoc 0275 Four endophytic Ascomycota mitosporic fungal iso Fischer's least significant difference (LSD) test. Percent ger lates (classified according to Kiffer and Morelet 2000): mination data was Subjected to arcsine transformation prior to SMCD 2204, SMCD 2206, SMCD 2208, and SMCD 2210, statistical analysis McDonald 2009. Statistical differences plus the Actinomycetes filamentous gram positive bacterial between percent germination after both three and seven days, isolate SMCD 2215; compatible with Triticum turgidium L. and fresh weight at seven days were assessed using a single Abdellatif et al. 2009 were used in this study. Endophytes factor ANOVA to compare all treatments. Subsequently, a were grown on PDA for at least three days at room tempera post-hoc LSD test was used to evaluate the significance of ture in darkness prior to experimental use. differences between the no endophyte control and seeds treated with each mycobiont. The level of statistical signifi Endophytes as Free-Living Organisms cance associated with differences between the EG and HTT required to reach 50% germination of endophyte-colonized (0276) Agar plugs (5 mm) cut from the margins of the and control seeds were assessed by evaluating the EG for each parent colony were placed in the centre of a 90-mm Petridish of the three independent replicates of the experiment. The containing either PDA alone or amended with 8% PEG resulting data were subjected to an ANOVA and post-hoc (drought). The Petri dish was sealed with parafilm (Pechiney LSD analysis. P-values less than 0.05 and 0.01 were consid Plastic Packaging) to maintainsterility and placed in a bench ered to be significant and highly significant, respectively. top incubator (Precision Thermo Scientific, model 3522) at either 23° C., or under heat stress, 36° C., in darkness. The Statistical tests were run with SPSS Inc. 2011. diameter of the colony was measured at 24, 48, 72, 96 h, and five and six days. The changes in diameter were used to Results calculate colony growth rate. The growth of a minimum of 0281. Within each section, the results are organised three replicates per isolate was measured. according to the type of stress: heat, drought, heat and US 2016/017457.0 A1 Jun. 23, 2016 28 drought, or no stress. Within each stress, the results dealing correlation between the HTT necessary for 50% germination with plant material are presented according to the germinant and the percent germination after seven days underheat stress and/or seedling traits measured: percent germination at three (FIG. 18). and seven days, fresh weight at seven days, EG and HTT. Response of Endophyte-Colonized Wheat to Drought Free-Living Endophytes 0289 When subjected to drought stress for three days, a 0282. The phenotypes of SMCD 2206, 2210 and 2215 diminished percentage of wheat seeds germinated in co-cul were not altered by heat (36° C.), while SMCD2204 and 2208 ture with SMCD 2208, compared to endophyte-free seeds did not grow at 36° C. The colony growth rates of SMCD (p<0.01; FIG. 16B), while SMCD 2204, 2206, 2210, and 2206 and 2210 were reduced by 36° C. as compared to non 2215 did not alter this trait (p>0.1; FIG. 16B). After seven stressed conditions (p 0.01), while the growth rate of SMCD days, treatment with SMCD 2206, 2210 and 2215 led to an 2215 at 36° C. was increased (p<0.05) (FIG. 15). At 36° C. increase in seed germination (ps0.01, ps0.05, and ps0.01, SMCD 2215 grew the most rapidly, followed in decreasing respectively; FIG. 17). In contrast, 65 and 67% of seeds order by 2206 and 2210 (FIG. 15). co-cultured with SMCD 2204 and 2208 had germinated after (0283. The morphology of SMCD 2204, 2206, 2208 and seven days. Neither of these values differed statistically from 2215 was not appreciably altered by drought (8% PEG). the 59% of uncolonized seeds that germinated under the same However, when SMCD 2210 was exposed to drought, this conditions (p-0.1). Under drought conditions, SMCD 2208 organism lost its “woolly' appearance and instead acquired a and 2210 decreased fresh weight after seven days (ps0.05 and “shiny’ or “slimy’ appearance. The colony growth rates of ps0.01. respectively; FIG. 16E). None of the other myco SMCD 2204, 2206, and 2208 were reduced by drought (p<0. bionts altered this parameter (p-0.1 FIG.16E). 01, ps0.01, and ps0.05 respectively), while the rate of colony 0290 The EG decreased for wheat seeds co-cultured in growth of all other endophytes remained unchanged (FIG. drought conditions with all endophytes tested, as compared to 15). When drought stress was applied, SMCD 2204 grew at endophyte-free seeds (0.05

Results 0331. This study hypothesizes that endophytes increase 0322. In summary, the results show that each SMCD strain the rapidity and uniformity of seed germination under opti positively affects several agricultural parameters on pod pro mal and stress conditions in-vitro. The aim was, firstly, to duction or yield (FIG. 27), and biomass of stem (FIG. 26) and measure the intrinsic symbiotic capacity of endophytes to root (FIG. 28) in chickpea (A), pea (B), lentil (C) and under trigger germination; and, secondly, to measure the efficiency drought stress. Overall, crop genotypes colonised by the sym of the compatible endophytes in conferring heat and drought biotic endophyte (E+) became more resistant to drought vs. resistance to pulses genotypes. heat stress. The level of efficacy of the tested endophytes in conferring drought tolerance varied with the particular plant Material and Methods organ: the pod yield was highly improved in Glamis by 0332 Two varieties of pulses, Glamis (lentil) and Handel SMCD 2204, in Vanguard by SMCD 2204F, in Sedley by (pea), were co-cultured with compatible SMCD 2206 and SMCD 2206, in Golden by SMCD 2210, and in Handel by SMCD 2215, fungal and bacterial symbiotic strains, respec SMCD 2215. tively. The endophytic strains ability to confer stress toler 0323 Stem: ance to Glamis (FIG. 31) and Handel (FIG. 32) genotypes 0324. The following endophytes showed the best response were tested during in-vitro seed germination modelling to drought stress: Chickpea: Amit: SMCD2204F, Vanguard: drought (6% PEG) and heat (33°C.) environments. SMCD 2206: Pea: Golden: SMCD 2204, Handel: SMCD 0333 Seeds were surface sterilized with 95% ethanol for 2204; SMCD 2210; SMCD 2215; Lentil: Glamis: SMCD 20s, rinsed twice insterile distilled water for 10s followed by 2204F: SMCD 2206. Sedley: SMCD 2204F: SMCD 2206. 2 minin 3% sodium hypochlorite (Javex). Finally, seeds were 0325 Pods: rinsed insterile distilled water 4 times. Seeds were inoculated 0326. The following endophytes showed the best response on PDA media with and without endophytes in the dark at to drought stress: Chickpea: Amit: SMCD 2204; SMCD room temperature Abdellatif et al. 2009. Microbial organ 2210. Vanguard: SMCD 2204; SMCD 2206: SMCD 2215; isms were grown on PDA for at least three days at room Pea: Golden: SMCD 2210; SMCD2215. Handel: SMCD temperature in darkness prior to experimental use. The endo 2204F: SMCD 2206: SMCD 2215; Lentil: Glamis: SMCD phytic ability to confer plant stress resistance was assessed 2204F: SMCD 2206. Sedley: SMCD 2210; SMCD2215. using the energy of germination, which is meant to capture the 0327 Root: temporal nature of germination and which is defined as the 0328. The following endophytes showed the best response number of days required to reach 50% of germinating seeds. to drought stress: Chickpea: Amit: SMCD 2204; SMCD 2215. Vanguard: SMCD 2204F: SMCD 2206: Pea: Golden: Results SMCD 2204F: SMCD2215. Handel: SMCD 2204F; Lentil: Glamis: SMCD 2204F: SMCD 2206: SMCD 2210. Sedley: 0334. The present study demonstrates the differential SMCD 2206: SMCD 2210. capacity of fungal or bacterial endophytes to confer drought and heat resistance in pulses specific to a fungal or bacterial Example 8 strain-plant genotype-abiotic stress combination. This study used molecular and proteomic analyses to better understand Streptomyces sp. SMCD 2215 Increases Rhizobium the mechanism by which endophytes confer symbiotic stress Activity and Nodulation Frequency in Peas Under resistance to pulses. Heat Stress 0335 SMCD strains significantly increased the frequency 0329. As was recently observed for another Streptomyces of pulse seed germination understandard in-vitro conditions (FIGS. 31 and 32). Under stressful conditions, both endo species, S. lydicus WYEC10 Tokala et al. 2002), the Strep phytes (SMCD 2206 and SMCD 2215) increased the fre tomyces sp. nov. SMCD2215 colonizes the roots of young pea quency of germination when compared to non-colonized seedlings from seeds produced from plants grown under con seeds. Frequency of germination was from 70-100% in sym trol conditions. It specifically enhances plant flowering and biotic treatments and 60-80% germination in the control, pod yield (FIG. 29), and root nodulation by Rhizobium sp. meaning that the tested endophytes have the potential to (FIG. 30), a native endophytic colonizer of pea seeds discov increase seed germination vigour (SGV) by >15%. The high ered in this study (Table 5). Vegetative hyphae of Streptomy est frequency of germination (100%) was observed in Glamis ces sp. nov. SMCD2215 colonize the cells of emerging nod (lentil) associated with both SMCD 2206 and SMCD 2215 ules as discovered by culture plate (PDA), fluorescence under drought stress vs. heat stress. When co-inoculated with microscopy (Carl Zeiss Axioskop 2) and PCR (BioRad) SMCD strains, the energy of germination (>50% germinating amplification methods Schrey and Tarkka 2008 seeds) in Glamis was achieved in 2 days under drought and in Example 9 3 days under heat conditions. Similar results were achieved in Handel (pea), except that this genotype has inherently a Endophytes Confer Abiotic Stress Tolerance to higher ability to support heat shock than Glamis (lentil). Pulses Via Enhanced Seed Viability Example 10 0330 Pulse crops refer to a group of more than sixty different grain legume crops grown around the world. The Endophytes Enhance Yield of Flax and Canola seeds of pulse crops are important to human nutrition. The Genotypes Under Severe Drought Stress in chief constraints to pulse production are biotic and abiotic Greenhouse Experiment stresses such as drought, heat, cold and Salinity. Recent research Suggests that endophytic microbe-plant interactions 0336. The aim of this study was to use three randomly are an instrumental determinant of plant adaptation. selected isolates (SMCD2206, SMCD 2210 and SMCD2215