Endophytic microbial symbionts in plant prenatal care.

ENDOPHYTIC MICROBIAL SYMBOLS IN CARE PRIOR TO

GERMINATION OF PLANTS

FIELD OF THE INVENTION

The present description refers to fungal and bacterial endophytes of the plants that improve the vitality of the seed and / or the health of the plant, which confer general improvements in the agricultural traits of the plant, under normal and stress conditions. The description also refers to these isolated endophytes.

BACKGROUND OF THE INVENTION

Fungi and bacteria are ubiquitous microorganisms.

Endophyte is the term coined for the first time by Bary [1866] that defines those microbes that colonize plant tissues asymptomatically [Stone et al., 2000]. The existence of endophytes has been known for more than a century [Freeman 1904] and it seems that each individual host, among the 300,000 species of plants, hosts several hundred endophytes [Tan and Zou, 2001]. Endophytes are microbial organisms mostly associated in a symbiotic or mutualistic manner with living tissues of host plants. Many are able to confer tolerance of plants to abiotic stressors or can be used by the plant for defense against fungi and pathogenic bacteria [Singh et al. 2011]. Some of these microorganisms have proven useful for sectors of agriculture, forestry and horticulture, as well as plant production of medicinally important compounds.

Endophytes largely determine the genomic regulation of plant cells and the entire plant, which includes the life cycles of the plant: (I) events before and after seed germination (mycovitalism) [Vujanovic and Vujanovic 2007], (ii) absorption of nutrients from plants and mechanisms that promote growth (mycoheterotrophism) [Smith and Read 2008], and (iii) plant tolerance to environmental stress and systemic resistance induced against diseases and pests (mycosymbionicism) [Wallin 1927; Margulis, 1991]. These could fulfill a fundamental function in the production of plant biomass, CO2 sequestration, γ / or yield and, therefore, be fundamental actors in the regulation of the ecosphere, guaranteeing plant health and food safety. . In addition, they can be important sentinels (bioindicators) of environmental changes, as alterations in the structure and biomass of endophytic communities these can announce changes not only in nutrient pathways (N, P, K), energy transfer in food webs and biogeochemical cycles, but also in the tolerance to UV-B radiation, heat, drought or salt that influence the establishment and stability of the plant's total ecosystem. Despite its abundance and similar importance in all terrestrial ecosystems, almost nothing is known about the composition of endophytes in seeds or spermosphere, their interactions, or their common response to environmental changes.

While the spermosphere represents an area of ​​soil that surrounds a seed in germination that is microbiologically dynamic and rapidly changing soil [Nelson, 2004], the rhizosphere is a microbiologically active zone of the volume of soils surrounding the roots of the plant [Smith and Read 2008]. The rhizosphere supports mycoheterotrophy or a symbiotic relationship of mycorrhiza plant. The spermosphere, on the other hand, promotes mycovitality or a relationship of endophytic fungi with the seeds of the plant — which improves seed vigor, energy and germination uniformity that could be quite predictable. Fungal endophytes are different from mycorrhizae in that they can colonize not only roots, but also other plant organs including seeds [Vujanovic et al. 2000; Hubbard et al. 2011]. These belong to the multicellular edges Ascomycota and Basidiomycota and form symbiotic colonization structures different from those produced by the unicellular or cenocytic edge Glomeromycota, known as vesicular-arbuscular mycorrhiza symbiosis [Abdellatif et al. 2009]. Endophytic bacteria have also been found in practically all the plants studied, where they colonize an ecological niche similar to that of fungi, such as healthy internal tissues. Although the majority of bacterial endophytes seem to originate from the rhizosphere or philosopher; some can be transmitted through the seed [Ryan et al. 2008].

Seed germination is a vital phenotype for the survival and reproduction of plants, whether in optimal or stressful environmental conditions. Endophytic microbial colonization in the seed state is especially crucial due to the function of the seed as a generating organ in the regeneration and dispersion of flowering plants [Baskin and Baskin 2004] and the function of the associated mycobionts and bacteria of symbiotic manner (bactobionts) as potential drivers of seedling recruitment in natural habitats - unaltered, altered and contaminated - [Mühlmann and Peintner 2000; Adriaensen et al.

2006; White and Torres 2010]. Therefore, the development methods by which the appearance of seedlings can be improved and protected with the limitations of disease pressure, heat or drought are valuable. The use of endophytic symbionts is a promising method by which seed germination can be improved [Vujanovic et al 2000; Vujanovic and Vujanovic 2006; Vujanovic and Vujanovic 2007]. It was hypothesized that the resistance of the plant to stress can be conferred through a mecobionte-seed relationship known as mycovitality - a phenomenon that had been reserved for Orchidaceae [Vujanovic 2008] and through bactovitality, which refers to a form of bactosimbiosis, which uses different strains of endophytes with a variety of activities.

SUMMARY OF THE INVENTION

Endophytes can benefit plant hosts such as wheat, barley, legumes, cane, trees, shrubs or grass in various ways, including bactovitality, mycovitality and mycoheterotrophy, and improved tolerance to environmental stress, as demonstrated in the Present. Pre-germination care in agriculture, as demonstrated here with six endophytic strains, is more than just the vitality, health or vigor of germination or seed. This also determines what can be expected before and during the germination process, establishment of seedlings and the subsequent productivity or yield of the crop.

Several parameters of symbiotic efficacy (latency breakdown, germination, growth and yield) were evaluated using effective endophytic strain-harvest interactions from the Saskatchewan Microbe Collection and Database (SMCD) [Saskatchewan Microbial] Collection and Database] in in vitro, fitotron, greenhouse and field conditions.

The ability of bacterial endophytes to confer seed vitality was also evaluated. For fungal and bacterial endosymbionts, improved seed vitality can increase tolerance to abiotic and biotic stress in plants that have evolved beyond the seedling stage toward plant maturity through mycoheterotrophy.

Accordingly, the present description provides an isolated endophyte of the Streptomyces strain or culture thereof that is deposited with accession number 081111-06 of the International Depositary Authority of Canada (IDAC) [Laboratory] of National Microbiology, Public Health Agency of Canada. 1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2) or comprising the 16S rDNA sequence as shown in SEQ ID NO: 6; an endophyte isolated from the strain of the Paraconyothirium species or culture thereof that is deposited with the accession number 081111-03 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 5; an isolated endophyte of the Pseudeurotium species or culture thereof that is deposited with the accession number 081111-02 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 4; an isolated endophyte of the Penicillium species or culture thereof that is deposited with the accession number 081111-01 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 3; an isolated culture of the Cladosporium species that is deposited with the accession number 200312-06 of IDAC or that comprises the ITS rDNA sequence as shown in SEQ ID NO: 1; and / or an isolated endophyte of the Cladosporium species.o culture thereof that is deposited with the accession number 200312-05 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 2.

Also provided herein is a composition comprising an isolated endophyte or culture described herein or a combination or mixture thereof and a carrier.

A seed comprising an endophyte or culture described herein is also provided herein. In one embodiment, the seed is coated with the endophyte. In another embodiment, the seed is grown or planted near the endophyte so that the endophyte is able to colonize the seed.

The present description also provides methods to improve the health and performance of the plant under normal and stress conditions. Accordingly, a method is provided to improve the vitality of the seed, the health of the plant and / or the yield of the plant comprising inoculating a seed with an endophyte or crop described herein or a combination or mixture of these or with a composition described herein; and grow the seed in a first generation plant.

In one embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Streptomyces species, which is deposited with the accession number 081111-06 of IDAC or comprising the 16S rDNA sequence as it is shown in SEQ ID NO: 6. In one embodiment, the method increases the germination of the seed, decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed, increases the fresh weight of the seedlings, improves Rhizobium activity and nodulation frequency, and / or increases seedling yield. In another embodiment, the method comprises reducing the effects of stress, such as biotic stress, drought and / or heat, such as Fusarium infection.

In another embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this of the strain of the Paraconyothirium specimen, which is deposited with the accession number 081111-03 of IDAC or comprising the ITS rDNA sequence such as is shown in SEQ ID NO: 5. In one embodiment, the method increases the germination of the seed, decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed, increases the fresh weight of the seedlings and / o increases seedling yield. In another embodiment, the method comprises reducing the effects of stress, such as drought, heat and / or biotic stress, such as Fusarium infection.

In yet another embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this of the strain of the species Pseudeurotium, which is deposited with the accession number 081111-02 of IDAC or comprising the ITS rDNA sequence such as is shown in SEQ ID NO: 4. In one embodiment, the method decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed and / or increases the fresh weight of the seedlings. In another embodiment, the method comprises reducing the effects of stress, such as drought and / or heat stress.

In a further embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Penicillium species, which is deposited with the accession number 081111-01 of IDAC or comprising the ITS rDNA sequence such as is shown in SEQ ID NO: 3. In one embodiment, the method increases the germination of the seed, decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed and / or increases the yield of the seedlings . In another embodiment, the method comprises improving stratification, breaking latency and increasing resistance to stress by modulating the expression of hormonal ent-kaurenoic acid (KAO), outbreak growth repression (RSG) genes. ) by abscisic acid (ABA), gibberellic acid (GA), 14-3-3 or nitric acid (NO, for its acronym in English) and / or stress resistance genes using superoxide dismutase (SOD), manganese SOD (MnSOD), proline (Pro) or MYB genes, which reduces the effects of stress, such as biotic stress, by drought and / or heat, such as Fusarium infection.

In still a further embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Cladosporium species, which is deposited with the accession number 200312-06 of IDAC or, which comprises the ITS rDNA sequence. as shown in SEQ ID N0: l. In one embodiment, the method decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed and / or increases the fresh weight of the seedlings. In one embodiment, the method comprises reducing the effects of stress, such as drought and / or heat stress.

In yet another additional embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Cladosporium species, which is deposited with the accession number 200312-05 of IDAC or comprising the ITS rDNA sequence such As shown in SEQ ID NO: 2. In one embodiment, the method comprises reducing the effects of stress, such as drought and / or heat stress.

In one embodiment, the seed is coated with the endophyte, cultivated with endophyte or planted near the endophyte. In a particular embodiment, the seed planted near the endophyte is located at a distance of about 4 cm from the endophyte.

The plant can be any plant. In one embodiment, the plant is a cereal (wheat or barley), legumes (peas, lentils or chickpeas), flax, cane plants, coniferous tree (spruce or pine), broadleaf tree (willow or poplar), shrub (caragana or amarantáceas) or grass (fescue or wild rye).

In another aspect, a method is provided for improving the health of the plant and / or the yield of the plant comprising treating the plant propagation material or a plant with an endophyte or crop described herein or a combination or mixture of these. or with a composition described herein; and grow the plant propagation material in a first generation plant or allow the plant to grow.

In one embodiment, the plant propagation material is any generating / sexual (seed, bud or flower) and vegetative / asexual (stem, cut, root, bulb, rhizome, tuber, vegetative bud or leaf) that has the capacity of being grown in a new plant.

In one embodiment, the isolated endophyte or culture of this is an isolated endophyte of the Streptomyces strain or culture of this that is deposited with accession number 081111-06 of the International Depository Authority of Canada (IDAC, [by its acronym in English] National Microbiology Laboratory, Public Health Agency of Canada.

1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2) or comprising the 16S rDNA sequence as shown in SEQ ID NO: 6; an endophyte isolated from the strain of the Paraconyothirium species or culture thereof that is deposited with the accession number 081111-03 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 5; an isolated endophyte of the Pseudeurotium species or culture thereof that is deposited with the accession number 081111-02 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 4; an isolated endophyte of the Penicillium species or culture thereof that is deposited with accession number 081111-01 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID N0: 3; an isolated culture of the Cladosporium species that is deposited with the accession number 200312-06 of IDAC or that comprises the ITS rDNA sequence as shown in SEQ ID NO: 1 and / or an isolated endophyte of the Cladosporium species or culture thereof that is deposited with the accession number 200312-05 of IDAC or that comprises the ITS rDNA sequence as shown in SEQ ID NO: 2.

In one modality, the methods improve the development and shaking of the landscape. Accordingly, in one embodiment, a method is provided for reducing soil contamination comprising treating the plant propagation material or a plant with an endophyte or crop described herein or a combination or mixture thereof or with a composition described in the present; and grow the plant propagation material in a first generation plant or allow the plant to grow. In one embodiment, the soil contaminant is hydrocarbons, petroleum or other chemicals, salts or metals, such as lead, cadmium or radioisotopes.

In another embodiment, the methods reduce the effects of stress, such as biotic stress, heat and / or drought.

The plant can be any plant. In one embodiment, the plant is a cereal (wheat and barley), legumes (peas, lentils or chickpeas), flax, cane plants, coniferous tree (spruce or pine), broadleaf tree (willow or poplar), shrub (caragana or amarantáceas) or grass (fescue or wild rye).

Other features and advantages of this disclosure will be apparent from the detailed description that follows. It should be understood, however, that the detailed description and specific examples, despite indicating modalities of the description, are provided by way of illustration only, since several changes and modifications within the spirit and scope of the description will be apparent to the skilled in the art from this detailed description and the figures and legends of the respective figures.

BRIEF DESCRIPTION OF THE FIGURES

Next, the description will be described with respect to the figures, in which:

Figure 1 shows the phenotype appearance of the endophytic fungal strains SMCD 2204, 2004F, 2206, 2208, and 2210 and the bacterial strain SMCD 2215; after 10 days of growth in PDA at 21²C.

Figure 2 shows the phylogenic tree inferred by the method of joining neighbors of the Cladosporium SMCD 2204 and SMCD 2204F species as a function of the ITS rRNA. The numbers in the nodes indicate the bootstrap support values ​​for 1000 replicas; only values> 70% are provided. The bar indicates 0.01 nucleotide substitutions per site (nucleotide position).

Figure 3 shows the phylogenic tree inferred by the method of joining neighbors of the Penicillium SMCD 2206 species as a function of the ITS rRNA. The numbers in the nodes indicate the bootstrap support values ​​for 1000 replicas; only values> 70% are provided. The bar indicates 0.01 nucleotide substitutions per site (nucleotide position).

Figure 4 shows the phylogenic tree inferred by the method of joining neighbors of the Pseudeurotium SMCD 2208 species as a function of the ITS rRNA. The numbers in the nodes indicate the bootstrap support values ​​for 1000 replicas; only values> 70% are provided. The bar indicates 0.01 nucleotide substitutions per site (nucleotide position).

Figure 5 shows the phylogenic tree inferred by the neighbor binding method of the Coniothyrium SMCD 2210 strain as a function of the ITS rRNA. The numbers in the nodes indicate the bootstrap support values ​​for 1000 replicas; only values> 70% are provided. The bar indicates 0.05 nucleotide substitutions per site (nucleotide position).

Figure 6 shows the phylogenic tree inferred by the method of joining neighbors of the strain of the Streptomyces SMCD 2215 species as a function of the 16S rRNA. The numbers in the nodes indicate the bootstrap support values ​​for 1000 replicas; only values> 60% are provided. The bar indicates 0.05 nucleotide substitutions per site (nucleotide position).

Figure 7 shows the left compartments of divided plates (plant with microbial partner): appearance of the healthy wheat phenotype when the root is grown in contact with microbial mats; and the right compartments of divided plates (plant without microbial partner): massive formation of radical wheat hairs due to the plant-fungus association made in the left compartments of the separated plates.

Figure 8 (A) and (C) show the discontinuous colonization of SMCD2206 from wheat root tissue (epidermis and bark) compared to (B) and (D) showing the pathogenic uni-continuous / continuous cell colonization of Fusarium graminearum of the wheat root including the vascular cylinder.

Figure 9 shows the Ireg index - level of deviation (irregularity) in the cell form of the endophyte (SMCD).

Figure 10 shows the Idir index - level of direction changes when a host cell is colonized in a living plant.

Figure 11 shows endophytic hyphae in germinating wheat roots (A-SMCD 2204; B-SMCD 2206; C-SMCD 2210; and D-SMCD-2215) visualized with lactofucsin staining and fluorescence microscopy. Symbiotic organs / structures: (D) the bacterial endophyte SMCD 2215 formed mostly wavy intracellular filaments, while the endophytic fungus (Figures to the left) produced: intracellular loops and arbuscles of

SMCD 2204, intracellular vesicles of SMCD 2206, and SMCD 2110 intracellular nodes.

Figure 12 shows the appearance of wheat seedlings in symbiotic germination after 10 days on filter paper for moisture at 21 ^toC.

Figure 13 shows the length of leaves of germinating wheat seedlings after 10 days in filter paper for moisture at 21 ²C.

Figure 14 shows a method of inoculation in vitro (A). A 5mm agar plug², cut from the margin of the mother colony, was placed with the hypha down in the center of a 60 mm Petri dish containing a medium of potato and dextrose agar (PDA). Then, five sterilized seeds on the surface were placed at a distance equivalent to 48 hours of hyphal growth from the agar plug and germinated in the dark. The impact of the three methods of seed sterilization on the surface on the germination of the seed (B). Bars labeled with one or two asterisks (*) are significantly or very significantly different from those of the same endophyte grown under control conditions (p 0.05 or 0.01, respectively; ANOVA, followed by post-hoc test of LSD). Error bars represent the standard error of the average (SE).

Figure 15 shows the growth rates of autonomous endophytes SMCD 2204, 2206, 2208, 2210 and 2215 in vitro in potato and dextrose agar (PDA) under heat stress (36 ° C), drought stress (polyethylene glycol (PEG) ) 8000 to 8%) and control conditions for five days and simultaneous heat (36 ° C) and drought (PEG 8%) for six days. Bars labeled with one or two asterisks (*) are significantly or very significantly different from those of the same endophyte grown under control conditions (p 0.05 op 0.01, respectively; ANOVA, followed by post-hoc LSD test) . Error bars represent the standard error of the average (SE).

Figure 16 shows the germination percentage and fresh weight of the seedlings of the initial experiments in which the seeds were sterilized on the surface in 5% sodium hypochlorite for 3 min. Percentage of germination of wheat seeds in vitro after three days in potato and dextrose agar (PDA) under heat stress (36 ° C), drought stress (polyethylene glycol (PEG) 8000 to 8%) and control conditions (A, B and C) with the axis and normalized to a germination percentage obtained under the same conditions by means of seeds sterilized on the surface in 5% sodium hypochlorite for 1 min. Fresh weight of seedlings in vitro at seven days in PDA under heat stress, drought stress and control conditions (D, E and F). Bars labeled with one (*) or. two asterisks (**) are significantly or very significantly different from those of the endophyte control (p 0.05 op 0.01, respectively; ANOVA, followed by post-hoc LSD test). Error bars represent the standard error of the average (SE).

Figure 17 shows the percentage of germination with respect to the time of wheat seeds co-cultivated with the most effective endophytes to confer tolerance to abiotic stress (SMCD 2206, 2210 and 2215) compared to unstressed and non-colonized seeds (positive control) and stressed seeds not colonized (negative control). Germination energy (EG) is related to time, in days (x axis) in which 50% of germination (y axis) is reached. The symbols, x, o, Δ, and represent the positive control, seeds treated with SMCD 2206, seeds treated with SMCD 2210, seeds treated with SMCD 2215 and the negative control, respectively. The heat and drought treatments correspond to 36 ° C and 8000 polyethylene glycol (PEG) at 8%, respectively. Error bars represent the standard error of the average (SE). Note: The seeds used in the determination of EG were taken from the second round of experiments, and therefore were sterilized in 5% sodium hypochlorite for one minute, instead of three.

Figure 18 shows the relationship between hydrothermal time (HTT) needed to reach 50% of. germination for heat and drought alone and 5% of germination for heat and drought combined (x axis) and the germination percentage achieved after seven days (y axis). Germination after seven days and HTT were based on the results of the second round of experiments. The symbols and A represent seeds exposed to heat (36 ° C), drought (polyethylene glycol (PEG) 8000 to 8%) or stress from both, heat and drought, respectively. The R squared values ​​associated with the trend lines are 0.96, 0.80, and 0.18 for seeds exposed to heat, drought or stress from both heat and drought, respectively. Note: The seeds used to determine the germination percentage at seven days HTT were taken from the second round of experiments, and therefore treated with 5% sodium hypochlorite for one minute, instead of three.

Figure 19 shows seeds treated or inoculated with SMCD strains, which demonstrates an improvement in all the parameters studied of the seed, including the effectiveness of the seed germination vigor (SGV).

.

Figure 20 shows the relationship between the values ​​of drought tolerance efficacy (DTE) for cultivated varieties of wheat (A) and barley (B) without (E-) and with (E +) Endophytes, depending on the average effect of symbiosis using all SMCD isolates, on the performance exposed to drought stress in a greenhouse.

Figure 21 shows (A) that endophytic inoculators (E +) (SMCD 2206, SMCD 2210, and SMCD 2215) improve grain yield in wheat genotypes compared to control treatment (E-) (yield in g / 3 rockers). (B) that endophytic inoculators (E +) (SMCD 2206, SMCD 2210, and SMCD 2215) improve grain yield in two-stroke barley genotypes (B_to) and six-stroke barley (Bt>) (grain yield: 3 plants / pot).

Figure 22 shows (A) six-stroke AC Metcalfe barley, from left to right: drought (E-), drought and SMCD 2206 (E +), Control (E-), Control and SMCD 2206 (E +); (B) Unity wheat cultivated variety, from left to right: drought (E-), drought and SMCD 2215 (E +), Control (E-), Control and SMCD 2215 (E +); (C) Verona wheat cultivated variety, from left to right: drought (E-), drought and SMCD 2215 (E +), Control (E-), Control and SMCD 2215 (E +); and (D) TEAL durum wheat, from left to right: drought (E-), drought and SMCD 2210 (Ε +), Control (Ε-), Control and SMCD 2210 (E +).

Figure 23 shows the dry stem weight of (A) chickpeas, (B) lentils, and (C) peas in symbiosis with endophytes SMCD (E +) under phytotron conditions under heat stress. Bars labeled with one (*) or two asterisks (**) are significantly or very significantly different from those of the control without stressed endophyte (p 0.05 op 0.01, respectively; ANOVA, followed by post-hoc LSD test ).

Figure 24 shows the dry weight of the pods of (A) chickpeas, (B) lentils, and (C) peas in symbiosis with endophytes SMCD (E +) under phytotron conditions under heat stress. Bars labeled with one (*) or two asterisks (**) are significantly or very significantly different from those of the control without stressed endophyte (p 0.05 op ñ 0.01, respectively; ANOVA, followed by post-hoc test of LSD).

Figure 25 shows the dry weight of the roots of (A) chickpeas, (B) lentils, and (C) peas in symbiosis with endophytes SMCD (E +) under phytotron conditions under heat stress. Bars labeled with one (*) or two asterisks (**) are significantly or very significantly different from those of the control without stressed endophyte (p 0.05 op 0.01, respectively; ANOVA, followed by post-hoc LSD test ).

Figure 26 shows the dry stem weight of (A) chickpeas, (B) peas, and (C) lentils under drought stress in a greenhouse. Bars labeled with one (*) or two asterisks (**) are significantly or very significantly different from those of the non-endophyte (E-) stressed control (p 0.05 op or 0.01, respectively; ANOVA, followed by test LSD post hoc).

Figure 27 shows the dry weight of the pods of (A) chickpeas, (B) peas, and (C) lentils in association with an endophyte (E +) under drought stress in a greenhouse. The bars labeled with one (*) or two asterisks (**) are significantly different from those of the control without endophyte (E-) stressed (p 0.05 op or 0.01, respectively; ANOVA, followed by post-hoc test of LSD).

Figure 2 8 shows the dry weight of the roots of (A) chickpeas, (B) peas, and (C) lentils under drought stress in the greenhouse. The bars labeled with one (*) or two asterisks (**) are significantly or very significantly different from those of the stress-free control (E-) under stress (p u 0.05 op £ 0.01, respectively; ANOVA, followed by post-hoc LSD test).

Figure 29 shows A. Vanguard chickpea plants with flowers that exhibit pods under drought stress in a greenhouse - the plant on the left is non-symbiotic (E-) and the plant on the right is symbiotic with the strain of SMCD 2215 ( E +); B and C, Vanguard chickpea plants that exhibit pods under drought stress in a greenhouse - (B) non-symbiotic and (C) symbiotic with SMCD 2215.

Figure 30 shows the nodulation of roots of pea varieties under heat stress in phytotron: Hendel (top) and Golden (bottom) inoculated (left) and not inoculated (right) with SMCD 2215. Note: all samples have been observed the natural infection with the Rhizobium species of pea seeds.

Figure 31 shows the considerable increase in seed germination energy of SMCD2206 and SMCD 2215 (^ 50%) in Glamis (lentils) as a function of time under heat stress and in vitro drought.

Figure 32 shows the considerable increase in seed germination energy of SMCD2206 and SMCD 2215 (^ 50%) in Hendel (peas) as a function of time under heat stress and in vitro drought.

Figure 33 shows that endophytic inoculators (SMCD 2206 and SMCD 2210) improve flax yield in drought conditions in a greenhouse. The different letters on the bars indicate statistically significant differences between the samples (p <0.05, Kruskal test26

Wallis).

Figure 34 shows that endophytic inoculators (SMCD 2206, SMCD 2210 and SMCD 2215) improve the performance of the cane in drought conditions in a greenhouse. The different letters on the bars indicate statistically significant differences between the samples (p <0.05, Kruskal-Wallis test).

Figure 35 shows the survival of in-vitro preinoculated wheat seeds (plaques in the upper rows) and preinoculated wheat seedlings in a greenhouse (pots in the bottom line) with endophytic SMCD 2206 - showing healthy plant growth, and with Fusarium avenaceum and Fusarium graminearum pathogenic - showing symptoms of disease and death of the plant.

Figure 36 shows Fusarium inoculators produced in wheat grain.

Figure 37 shows that post-onset fungal wilt has been prevented by the endophyte SMCD 2206 in the greenhouse.

Figure 38 shows the improvement of wheat biomass (aerial ad and root ef) in the presence of the endophyte SMCD 2206 compared to untreated plants, (a) control plant (E-), (b) inoculated plant ( E +), (c) plant with control flowers, (d) plant with inoculated flowers, (e) control plant (E-, left) compared to plant inoculated with SMCD 2206 (E +, right), and (f) fluorescence microscopy of wheat root colonization by SMCD 2206 (E +).

Figure 39 shows the aerial biomass of the plant / plant (left) and the biomass under 'ground (root) / plant (right) in control plants (E-) and inoculated with SMCD (E +) against F. graminearum and F Avenaceum The vertical error bars in the data points represent the standard error of the average.

Figure 40 shows the root length in control plants (Teal with CDC) without endophytes SMCD compared to plants inoculated with SMCD strains. The bars in the data points represent the standard error of the average.

Figure 41 shows the dry weight of the grains / plant (cultivated variety TEAL) using the dual preinoculation approach: a) Endophyte SMCD + Fusarium avenaceum (F.av), and b) Endophyte SMCD + Fusarium graminearum (F.gr). The vertical error bars in the data points represent the standard error of the average.

Figure 42 comparing the size of the TEAL spikes in the presence of the pathogen (negative control) and without the presence of the pathogen (positive control). Figure on the left - from left to right: i) plant + F.gr, ii) plant + F.av, and (iii) plant; Figure on the right - from left to right: i) plant; ii) plant + endophyte; iii) plant + endophyte + F. av; and iv) plant + endophyte + F.gr.

Figure 43 shows a symbiotic germination pattern of the specific seed of the strain representing mycovitality: Handel + 6% PEG - Control (A), Handel + 6% PEG + SMCD 2204 (B); Handel + 6% PEG + SMCD 2204F (C), Handel + 6% PEG + SMCD 2206 (D), Handel + 6% PEG + SMCD 2210 (E), Handel + 6% PEG + SMCD 2215 (F ) after 7 da 21²C in the dark.

Figure 44 shows the relative gene expressions of (A) SOD and (B) MnSOD in Handel exposed to PEG with and without endophytes.

Figure 45 shows the relative gene expression of proline in Handel exposed to PEG with and without endophytes.

Figure 46 shows the germination of wheat seeds in vi after three days in potato and dextrose agar (PDA). Cold stratification was imposed by keeping the seeds in a cold room of 4 ° C for 48 hours. For indirect treatment with endophytes and direct treatment with endophytes, the seeds germinated at approximately 4 cm distance and direct contact, respectively. A) Germination percentage compared to germination energy (50% germination). B) Efficiency of germination of wheat seeds subject to cold and biological stratification. The effectiveness was calculated by subtracting the germination percentage from the control of the treated seeds.

Figure 47 shows differential expression patterns of gibberellin genes (TaGA3ox2 and 14-3-3) and ABA (TaNCED2 and TaABA8 ΌΗ1) in the germination of wheat seeds germinating for three days under cold and biological stratification. Gene expression was calculated as 2 *^{Δ <? Τ}.

Figure 48 shows the relationship of expression levels (2^_ACT) of gibberellin genes (TaGA3ox2 and 14-3-3) and ABA (TaNCED2 and TaABAS'OHl) in the germination of wheat seeds germinating for three days under cold and biological stratification.

Figure 49 shows relative expression patterns of hormonal KAO and GSR regulator genes and resistance genes MYB 1 and MYB 2 in the germination of wheat seeds germinating for three days under cold and biological stratification. Gene expression was calculated as 2 ~ A^{c, r}.

Figure 50 shows the appearance of the radicle from a germinating wheat seed (A), inverted fluorescence (B) and fluorescence imaging of DAF-2DA fluorescence after reaction with NO in the

<td colspan="5"> 30</td>

<td>radicle cells</td><td>(C)</td><td>from AC Avonlea</td><td>in germination 5</td><td>min</td>

<td colspan="2">after treatment</td><td>[Nakatsubo et</td><td>to the. 1998] with</td><td>the</td>

<td>exudate from SMCD 2206</td><td colspan="2">fungal I dont know</td><td>observed reaction</td><td>from</td>

fluorescence in the cells of the control radicle. Bar = 25 μιη; Barrier = 50 pm.

Figure 51 shows the fluorescence intensity values ​​with DAF-2T 5 min after the treatment of the AC Avonlea wheat radicle in germination with the fungal SMCD 2206 exudate, the fungal exudate along with the NO scrubber CYTLE, and sterile water . The radicle segments were incubated for 30 min in 2 ml of detection buffer (10 mM Tris-Hcl, pH 7.4, 10 mM KC1) containing 15 μΜ of DAF-2DA (Sigma-Aldrich) with or without 2 - (4-carboxyphenyl) 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as a NO scrubber. The average fluorescence values ​​are reported as a ratio of the fluorescence intensity at 5 min with respect to the fluorescence intensity at time 0. The different letters indicate statistically significant differences between the samples (p &lt;0.05, Kruskal-Wallis test).

DETAILED DESCRIPTION OF THE INVENTION

Novel strains, compositions and seeds

The inventors of the present have isolated 6 novel endophyte strains that improve seed vitality and plant health and performance under normal and / or stress conditions. These endophytes have been deposited as follows: International Depositary Authority of Canada IDAC (original strains deposited - IDAC, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2; receipts and viability in Annex A) and Saskatchewan microbe collection and database - SMCD (copies of deposited strains) Strains:

(a) IDAC 081111-06 = SMCD2215;

(b) IDAC 081111-03 = SMCD2210;

(C) IDAC 081111-02 = SMCD2208;

(d) IDAC 081111-01 = SMCD2206;

(e) IDAC 200312-06 = SMCD 2204; and (f) IDAC 200312-05 = SMCD 2204F.

Accordingly, the present description provides an isolated endophyte of the Streptomyces species strain or culture thereof, which is deposited with accession number 081111-06 of IDAC or comprising the 16S rDNA sequence as shown in SEQ ID NO: 6; an endophyte isolated from the strain of the Paraconyothirium species or culture thereof that is deposited as 081111-03 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 5; an isolated endophyte of the Pseudeurotium species or culture thereof that is deposited as 081111-02 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 4; an isolated endophyte of the Pseudeurotium species or culture thereof that is deposited as 081111-01 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 3; an isolated culture of the Cladosporium species that is deposited as 200312-06 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 1; and / or an isolated endophyte of the Cladosporium species or culture thereof that is deposited as 200312-05 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 2; or combinations or mixtures of these.

The term endophyte, as used herein, refers to a fungal or bacterial organism that can live symbioticly in a plant and also referred to herein as endosymbiont. A fungal endophyte can live in the form of a spore, hypha or mycelia. A bacterial endophyte can be a cell or group of cells. The term endophyte, as used herein, includes the progeny of the strains mentioned herein.

. Also provided herein is a composition comprising an isolated endophyte or culture described herein or a combination or mixture thereof and a carrier.

Typical carriers include, but are not limited to, an inert material (which is not carbon-based) used to support and administer the active ingredient very dense to the target, and optionally adjuvants — compounds that promote and maintain the function of the active ingredient by UV radiation protection; ensure rain resistance on the target; retain moisture or protect against drying out; and / or promotes the spread and dispersion of biopesticide through standard agricultural equipment such as those described by Hynes and Boyetchko (2006, Soil Biology &amp; Biochemistry 38: 845-84).

In another embodiment, the composition comprises at least 2, at least 3, at least 4, at least 5 or 6 of the endophyte strains or cultures described herein.

A seed comprising an isolated endophyte or culture described herein or a combination or mixture thereof is also provided herein.

In one embodiment, the seed is inoculated by inoculation based on soil. In another embodiment, the seed is coated with the endophyte or culture thereof. In yet another embodiment, the seed is sprayed, injected, inoculated, grafted, coated or treated with the endophyte or culture thereof.

Methods,

Additionally, a method is provided to improve the vitality of the seed, the health of the plant and / or the yield of the plant comprising inoculating a seed with an endophyte or crop described herein or a combination or mixture thereof or with a composition described herein; and grow a first generation plant from the seed.

The phrase "inoculate a seed" as used herein refers to applying, infecting, coplanting or coating the seed with the endophyte. The techniques for inoculating the seed are known in the art, for example, as described by Hynes and Boyetchko (2006, Soil Biology &amp; Biochemistry 38: 845-84).

The term improve the vitality of the seed, as used herein, refers to the pre-germination care of the plant, which improves the ability of the seed to germinate and produce a plant under normal conditions and / or stress and include, non-taxatively, any one or more of the following: break dormancy, provide seed stratification, increase seed germination, modulate gene expression, decrease time to reach germination energy, protect against biotic stress, protect against abiotic stress, reduce the hydrothermal time needed for germination, increase the germination vigor of the seed, ³⁵increase the efficiency of seed germination, increase the uniformity of seed germination, improve the efficiency of heat / drought tolerance, increase seedling weight, and increase seedling yield. The efficacy of drought / heat tolerance (DTE / THE) is the opposite term (antonym) to susceptibility.

Germination energy is defined as 50% of germination, in relation to the amount of seeds tested. Seed germination vigor shows the difference between the total percentage of seeds treated in germination and untreated seeds in germination. Hydrothermal time postulates that an individual seed begins to germinate when the sum of the temperatures and the water potential accumulate sufficiently over a period of time that allows germination. The effectiveness of germination is defined as the total percentage of seeds treated in germination after a set period of time after planting in relation to the amount of seeds tested in a control without treatment. Biological stratification is defined as the dormancy of the seed that is released by a symbiote to promote germination. The uniformity of seed germination represents the maximum percentage of seed germination within a minimum incubation time.

The term improve the health and / or performance of the plant, as used herein, refers to general improvements in the agricultural traits of the plant (eg, health and productivity) of the resulting plant under normal conditions and / or stress and includes, non-taxatively, one or more of any of the following: agricultural traits of the plant RSG, KAO, ABA, GA, 14-3-3 or NO to improve the hormonal activities of the plant, agricultural traits of the plant MYB, Pro, SOD, or MnSOD to improve stress resistance, such such as abiotic and biotic stress, increase the weight of various tissues, such as root, stem, leaves and pods, increase the frequency of nodulation and Rhizobium activity and improve the characteristics of the seeds of the first or subsequent generation, which It includes, non-taxatively, one or more of any of the following: weight of the seed of subsequent generation and energy of germination of the subsequent generation.

The sets of primers and genes KAO, RSG, ABAs, GAs, 14-3-3 hormonal are as described by Zhang et al. [2007]. The sets of primers and stress resistance genes SOD, MnSOD, Pro and MYB are presented in Table 6 and

Table 9 (SEQ ID NO: 8-19).

The term "decrease or increase", as used herein refers to a decrease or increase in a characteristic of the resulting seed or plant treated with the endophyte as compared to a resulting seed or untreated plant. For example, a decrease in a characteristic can be at least 5%, 10%, 15%, 25%, 50%, 75%, 100% or 200% or less than the control without treatment and an increase can be at least 5 %, 10%, 15%, 25%, 50%, 75%, 100% or 200% or greater than the control without treatment.

In one embodiment, the plant is grown under conditions of biotic or abiotic stress.

The term abiotic stress, as used herein, refers to non-living stress that typically affects the vitality of the seed and the health of the plant and includes, non-taxatively, heat and drought stress. In one embodiment, abiotic stress is heat stress. In another embodiment, abiotic stress is drought stress, osmotic stress or saline stress. The term biotic stress, as used herein, refers to live stress that typically affects the vitality of the seed and the health of the plant and includes, non-taxatively, microbial infections of the plant .. In one modality , biotic stress is a Fusarium infection.

In one embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Streptomyces species, which is deposited with the accession number 081111-06 of IDAC or comprising the 16S rDNA sequence as it is shown in SEQ ID NO: 6. In one embodiment, the method increases the germination of the seed, to decrease the time to reach the germination energy, to reduce the hydrothermal time necessary for germination, to increase the vigor of the germination of the seed, to increase the fresh weight. of seedlings, to improve Rhizobium activity and nodulation frequency, and / or to increase seedling yield. In one embodiment, the method comprises reducing the effects of stress, such as biotic stress, drought and / or heat.

In another embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Paraconyothirium species, which is deposited with the accession number 081111-03 of IDAC or comprising the ITS rDNA sequence as it is shown in SEQ ID NO: 5. In one embodiment, the method increases the germination of the seed, decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed, increases the fresh weight of the seedlings and / o increases seedling yield. In another embodiment, the method comprises reducing the effects of stress, such as biotic stress, drought and / or heat.

In yet another embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this of the strain of the species Pseudeurotium, which is deposited with the accession number 081111-02 'of IDAC or comprising the ITS rDNA sequence such as shown in SEQ ID NO: 4. In one embodiment, the method decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed and / or increases the fresh weight of the seedlings. In another embodiment, the method comprises reducing the effects of stress, such as drought and / or heat stress.

In a further embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Penicillium species, which is deposited with the accession number 081111-01 of IDAC or comprising the ITS rDNA sequence such as is shown in SEQ ID NO: 3. In one embodiment, the method increases the germination of the seed, decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed and / or increases the yield of the seedlings . In another embodiment, the method comprises reducing the effects of stress, such as biotic stress, drought and / or heat. In another embodiment, the method comprises improving stratification, breaking latency and increasing stress resistance by modulating the expression of the hormonal genes KAO, RSG, ABAs, GAs, 14-3-3 or NO and / or resistance genes to SOD, MnSOD, Pro or MYB stress, which reduces the effects of stress, such as biotic stress, drought and / or heat.

In still a further embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Cladosporium species, which is deposited with the accession number 200312-06 of IDAC or comprising the ITS rDNA sequence such as shown in SEQ ID NO: 1. In one embodiment, the method decreases the time to reach the germination energy, reduces the hydrothermal time necessary for germination, increases the vigor of the germination of the seed and / or increases the fresh weight of the seedlings. In one embodiment, the method comprises reducing the effects of stress, such as drought and / or heat stress.

In yet another additional embodiment, the method comprises inoculating the seed with an isolated endophyte or culture of this strain of the Cladosporium species, which is deposited with the accession number 200312-05 of IDAC or comprising the ITS rDNA sequence such As shown in SEQ ID NO: 2. In one embodiment, the method comprises reducing the effects of stress, such as drought and / or heat stress.

The term "plant", as used herein, refers to a member of the Plantae kingdom and includes all stages of the plant's life cycle, which includes, but is not limited to, the seeds. In one embodiment, the plant is a cereal (wheat and barley), legumes (peas, lentils or chickpea), flax or cane plant.

In one embodiment, the seed is coated with the endophyte, cultivated with endophyte or planted near the endophyte. In a particular embodiment, the seed planted near the endophyte is located at a distance of about 4 cm from the endophyte.

In another aspect, a method is provided to improve the health of the plant and / or the yield of the plant comprising treating the plant propagation material or a plant with an endophyte or crop described herein or with a combination or mixture of these or with a composition described herein; and cultivate the plant propagation material in a first generation plant or allow the plant to grow.

The term plant propagation material as used herein to any generator / sexual and vegetative / asexual part of the plant that has the ability to be grown in a new plant. In one embodiment, the propagation material of the plant is a generating seed, yolk or generating and vegetative flower stem, cut, root, bulb, rhizome, tuber, parts of vegetative leaves or vegetative bud).

In one embodiment, the isolated endophyte or culture of this is an isolated endophyte of the Streptomyces strain or culture of this that is deposited with accession number 081111-06 of the International Depository Authority of Canada (IDAC, [by its acronym in English] National Microbiology Laboratory, Public Health Agency of Canada. 1015 Arlington Street, Winnipeg, Manitoba, Canada, R3E 3R2) or comprising the 16S rDNA sequence as shown in SEQ ID NO: 6; an endophyte isolated from the strain of the Paraconyothirium species or culture thereof that is deposited with the accession number 081111-03 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 5; an isolated endophyte of the Pseudeurotium species or culture thereof that is deposited with the accession number 081111-02 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 4; an isolated endophyte of the Penicillium species or culture thereof that is deposited with the accession number 081111-01 of IDAC or comprising the ITS rDNA sequence as shown in SEQ ID NO: 3; an isolated culture of the Cladosporium species that is deposited with the accession number 200312-06 of IDAC or that comprises the ITS rDNA sequence as shown in SEQ ID NO: 1 and / or an isolated endophyte of the Cladosporium species or culture thereof that is deposited with the accession number 200312-05 of IDAC or that comprises the ITS rDNA sequence as shown in SEQ ID NO: 2.

In another embodiment, the methods reduce the effects of stress, such as biotic stress, heat and / or drought.

In one mode, the methods improve the development and recovery of the landscape.

Accordingly, in one embodiment, a method for phytoremediation or phytoremediation of a contaminated site is provided which comprises treating the plant propagation material or a plant with an endophyte or culture described herein or a combination or mixture thereof or with a composition described herein, and cultivate the plant propagation material in a first generation plant or allow the plant to grow, remedying or recovering the site in that way.

The term phytoremediation, as used herein, refers to the use of plants for the removal, reduction or neutralization of substances, wastes or hazardous material from the site to prevent and minimize any adverse effects on the environment. The term "recovery", as used herein, refers to the use of plants to convert the altered land back into its previous or other productive use.

In one embodiment, the site is land, just like in a landfill. In one embodiment, hazardous substances, wastes or materials comprise hydrocarbons, petroleum or other chemicals, salts or metals, such as lead, cadmium or radioisotopes.

The phrase "treat a plant propagation material or a plant," as used herein, refers to applying the endophyte or cultivation of this plant alone or with any solid or liquid carrier to the plant or plant propagation material. or a part of said plant. In one embodiment, the treatment includes foliar application or application to the soil of the endophyte or combination thereof with any solid or liquid carrier at all stages of plant growth.

The plant can be any plant. In one embodiment, the plant is a cereal (for example, wheat or barley), legumes (for example, peas, lentils or chickpeas), flax, cane plants, coniferous tree (for example, spruce or pine), tree of broad leaf (for example, willow or poplar), shrub (for example, caragana or amarantáceas) or grass (for example, fescue or wild rye).

The above description generally describes the present application. A more complete understanding can be obtained by reference to the specific examples that follow. These examples are described for illustrative purposes only and are not intended to limit the scope of the description. Changes in form and the replacement of equivalents are considered as circumstances that may suggest or become resources. Although specific terms have been used herein, it is intended that these terms have a descriptive sense and not for limiting purposes.

The following non-limiting examples illustrate the present invention:

EXAMPLES

EXAMPLES (1-14)

Latency and germination depend on several processes and factors. To ensure the establishment and success of seedlings, it is important to control the underlying processes or conditions. The function of plant genetics, hormones and different seed tissues have been studied relatively well. The present examples study the endogenous ito-vegetable seed relationship, which passes from a symbiotic stage of the root towards the maturation of the plant.

Example 1

Taxonomy

Canadian International Depositary Authority - IDAC (original deposited strains) and Saskatchewan microbe database - SMCD (copies of deposited strains) Strains: IDAC 081111-06 = SMCD 2215; IDAC 081111-03 = SMCD 2210; IDAC 081111-02 = SMCD 2208; IDAC 081111-01 = SMCD 2206; IDAC 200312-06 = SMCD 2204; IDAC 200312-05 = SMCD 2204F (Figures 1-6 and Table 1).

The strain of SMCD 2215 was originally isolated as a bacterium of the fungal endophytic SMCD plant Phyalocephala sensu lato. Classification according to Labeda et al. [2012]. This phylogenetic study studies almost all the described species (615 taxa) within the Streptomycetaceae family based on the 16S rDNA gene sequences and illustrates the diversity of the species within this family, which is observed to contain 130 statistically backed servants.

The present 16S rDNA sequence data confirm that the strain of the Streptomyces species of SMCD 2215 can be assigned to an independent unknown servant according to Labeda et al [2012] but independent species of Streptomyces lividans.

Example 2

Microbio-plant association and compatibility level

The level of microbe-plant compatibility was evaluated using a slightly modified method of Abdellatif et al. [2009]. In a plate of two compartments of 10 cm of agar without nutrients (Figure 7), the health of the plant and the formation of radical hairs - water and mineral absorbers - in coculture, with and without microbial partners, were characterized. In Figure 7, the left compartment of each divided plate shows a culture with its microbial partner, and the right compartment of each divided plate shows a culture without the microbial partner. The experiment was repeated twice in three replicas.

As shown in the left compartment of each divided plate, healthy plant tissue formed even when the roots of the plants were grown directly on dense microbial mats. The biomass of the radical hairs is improved to approximately twice more compared to the right compartment of each divided plate, where the microbial partner is absent (see the compartments on the left).

The effectiveness of the plant in establishing symbiotic association depends on the type of endophyte distribution within the root endodermis. The typical endophytic colonization of the roots is discontinuous and partial with a smaller number of occupied cells &lt;50% (Table 2) compared to the colonization of fungal pathogens characterized by a uniform / continuous cell colonization (frequency: 60-80 %) (Figure 8).

The performance of an endophyte should not only be evaluated by measuring biomass production, because what underlies the visibly increasing yield is the efficacy of the endophyte in plant colonization. This can be assessed by characterizing its association with plant cells, tissues or organs (ie seeds and radicles) using mathematical indexes that have been developed [Abdellatif et al. 2009] and applied in this study (Figure 9 and Figure 10).

These indices are based on the following observations: Endophytic symbionts show different patterns of radicle (root) colonization (regularity or level of deviation in the Treg form and the Ireg endophytic cell direction and the Tdir direction when colonizing a living cell ) compared to the dead cells of the radicle (which usually remain colonized by true saprophytes).

The high values ​​of the Ireg and Idir index determine mutualistic (beneficial) plant-symbiote relationships. In conclusion, the results show that the microbial-plant symbiotic association is characterized by a high level of compatibility between the two partners, leading to a balanced root colonization (&lt;50% of colonized cortex cells) and discontinuous by microbial endophytes measured using mathematical indices [Abdellatif et al. 2009]. This mutual association is also characterized by the direct effect of endophytic microbes on the healthy growth of the plant (bacto and mycodependence) when the plant is stimulated to use microbial partners as the sole source of nutrients or energy for growth. In addition, the improvement of root hair biomass by endophytes was observed and measured even in roots in distal compartments of the divided plaques where the microbial partners were absent, indicating a possible function to promote growth of the systemic plant of the endophytes.

Example 3

Symbiotic organs of endophytes in wheat

Each taxonomic group of endophytes establishes a single type of mycovitalism, which therefore forms different symbiotic organs. The characterization of mycovitalism was performed using the methodology of

Abdellatif et al. [2009], which consists of in vitro seed and microbe co-cultures that evaluates an early stage of the microbial-plant symbiotic association. The diversity of microbial symbiotic organs formed by SMCD 2204, 2206, 2210, and 2215 in germinating wheat is shown in Figure 11.

In summary, the results show differential types of symbiotic organs formed in the wheat root for each endophyte probably related to their different symbiotic functions. An abundance of balanced colonization, irregular colonization patterns, increased formation of the septum of the hyphae in living root cells, as well as the formation of arbuscles, knots, loops and vesicles - putative symbiotic functional organs - may indicate local specialization within the fungal endophytes to promote the mycovitality and mycoheterotrophy of the plant. Bactovitality is mainly characterized by wavy intercellular filaments of Streptomyces.

Symbiosis at the seed level resulted in an increase in germinating wheat after 10 days of joint inoculation (Figure 12 and Figure 13).

Example 4

Endophytes improve the germination of wheat seeds under heat and drought stress ⁵¹

Seed germination is a crucial stage of life for the survival of the plant and the timely establishment of seedlings especially in stressful environments. The hypothesis was raised that endophytes could improve the germination of wheat seeds under heat and drought stress. The germination hydrothermal time model (HTT) is a useful conceptual model for predicting germination time and energy (EG ) under a given set of conditions. HTT and EG are applied to determine if one or more compatible endophytes improve tolerance to heat or drought in wheat. The endophytes studied dramatically increased the germination percentage, improved the values ​​of EG and HTT, and decreased the susceptibility of wheat to heat and drought that was measured by the fresh weight of the seedlings. When they were colonized by the most effective endophyte, the values ​​of the parameters studied in wheat seeds exposed to heat stress resembled those of unstressed seeds.

Materials and methods

Hydrothermal germination time model and germination energy

The Hydrothermal Time Model (HTT) [Gummerson 1986] postulates that an individual seed begins to germinate when two conditions are met. First, the sum of daily temperatures, over a minimum cardinal value (Tmin), accumulated over a period of time, must pass a threshold value (θτ), measured in degree days. Second, the seed must accumulate sufficient water potential (Θη) per day-degree. Therefore, the ΗΤΤ (θ_Ητ) can be expressed as: © ht = (Θ_Η) (Θτ).

(Equation 1)

According to Kóchy and Tielborger [2007], θτ “(Taustrato - Tmin) t (Equation 2) where t represents the time elapsed in days, and

Θη Ψ substrate “ψπύ, η (Equation 3) in a constant environment assuming that Tsustrato is equal to or less than the optimum temperature for seed germination. In Equation 3, úsustrato and úmin represent the water potential of the substrate and the minimum water potential at which germination is possible, in MPa, respectively. Consistent with Bradford [2002], equations 2 and 3 can be substituted in equation 1 to provide:

© HT “(l | Fsustrato“ ψιηΐη) (Tsustrato ^— Tmin) t (Equation 4)

However, in the present study, the temperature exceeds the optimum temperature for wheat germination [reviewed by McMaster (2009)], which requires the consideration of a maximum temperature (T_max) on which germination cannot occur. Therefore, equation 2 was modified to:

⁼ [(Tsustrato "Tmin) (Tsustrato" Tmax |)] t (Equation 5) where Tmin Tsustrato Tmax. If equation 5 is replaced by 2 in equation 4, the following results:

Θητ ⁼ (ψ substrate “ψιηΐη) V [(Tsustrato“ Tmin) (| Tsustrato “Tmax |)] t (Equation 6) where Tmin Tsustrato - Tmax ·

Germination energy (EG) can be defined in several ways, which includes the percentage of seeds in germination after a set period of time after planting, in relation to the quantity of seeds studied [Rúa et al. 2002; Dong-dong et al. 2009], or 50% of germination achieved [Alien 1958]. In order to integrate the EG with the germination HTT model, the last definition was used, which means that the EG is equal to t in Equation 2.

Parameter Estimation

The estimation of Tmin and T_max for wheat was based on both the information available in the literature and those of the inventors here.

own observations

McMaster [2009] summarizes the data that originates from

Friend et al. [1962], Cao and Moss [1989], and Jame et al. [1998] indicating the existence of a curvilinear relationship between the speed of development of wheat and temperature. Since the germination and development of wheat do not occur below 0²C or above 40 ²C, to T_min and T_max was assigned the values ​​of 0 ²C and 40 ²C, respectively.

The ψπάη parameter was estimated in vitro by germinating wheat seed culture in potato and dextrose agar (PDA; Different) containing a range of polyethylene glycol (PEG) 8000 concentrations (Amresco Inc.). Water activity (a_w) PDA alone and PDA containing 8% PEG, 12% and 16% was measured using AquaLab 4TE, Series 4 Quick Start, Decagon Devices. Water activity was converted into water potential (ψ) using the adapted relationship of Bloom and Richard [2002]:

Ψ = [(RT) ln (a_w)] / V (Equation 7) where R is the universal gas constant (8.314 J mol^-1K '¹), T is the temperature in “K, and V is the partial molar volume of water (18 mL / mol). For conversion units, 1 J / mL = 1 MPa = 10 bar. The water potential is zero for a surface without water or saturated medium; All other values ​​are negative.

The water activities of PDA and PDA containing 8%, 12% and 16% PEG were 0.9974, 0.9890, 0.9863 and 0.9825, respectively. The values ​​are equivalent to -0.35, -1.51, -1.88, and -2.41 MPa, respectively and are consistent with those indicated in the literature [Leone et al. 1994].

Fungal and plant material

The plant material used was the cultivated variety of hard wheat Av Avonlea, which has low resistance to environmental stressors [SaskSeed guide 2008], The seeds used in the first round of experiments were produced by Paterson Grain in 2008, under field conditions , and it was not certified that it does not contain microbes. The seeds used in the second round of experiments were produced by Agriculture and Agri-Food Canada (AAFC) Seed lacrease Unit Research Farm in 2005 under greenhouse conditions, and it was certified that they had no microbes. Wheat seeds were surface sterilized with 95% ethanol for 10 s, rinsed in sterile distilled water for 10 s, submerged for 3 min (first round of experiments involving seeds of which it was not certified that they had no microbes) or 1 min (second round of experiments that used seeds that were not certified as having no microbes) in 5% sodium hypochlorite (Javex), they were rinsed three times in sterile distilled water and PDA for germination [Abdellatif et al. 2009]. A third method of seed sterilization was also studied, which involved a 3-hour exposure to chlorine gas (produced by combining 25 mL of 6% sodium hypochlorite with 1.0 mL of concentrated hydrochloric acid in one in a glass) in a closed plastic box placed in an extractor hood [Rivero et al. 2011]. The percentage of seed germination subject to each sterilization protocol and placed in PDA for three days is shown in Figure 14B. Only 3 min submersion in sodium hypochlorite resulted in a significant decrease in germination (p

0.01). Sterilization of the seed surface was intended to eliminate the microbes that could compete with the endophytes under investigation. In addition, the microbes present on the surface of the seeds could grow more than plaque and emerging seedlings, inhibiting plant growth. It was determined that all the seeds used in the study did not have microorganisms after sterilization, depending on the absence of unwanted microbial growth on the plate.

In this study, four mitosporic fungal isolates of endophytic Ascomycota (classified according to Kiffer and Morelet [2000]) were used: SMCD 2204, SMCD 2206, SMCD 2208 and SMCD 2210, plus the filamentous gram positive bacterial isolate of Actinomycetes SMCD 2215; compatible with Triticum turgidum L. [Abdellatif et al. 2009]. Endophytes were cultured in PDA for at least three days at room temperature in the dark before their experimental use.

Endophytes as autonomous organisms

Agar plugs (5 mm²) cut from the margin of the mother colony were placed in the center of a 90 mm Petri dish containing PDA alone or modified with 8% PEG (drought). The Petri dish was sealed with parafilm (Pechiney Plastic Packaging) to maintain sterility and placed in a countertop incubator (Precision Thermo Scientific, model 3522) at 23^toC, or under heat stress, 36 ²C, in the dark. The diameter of the colony was measured at 24,

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minimum of three replicas per isolation.

Endophytes ability to confer heat and drought tolerance on wheat

Each isolate is applied individually to wheat seeds before germination according to the method described in Abdellatif et al. [2010] and shown in Figure 14A. In summary, five sterilized seeds were placed on the surface at a distance equivalent to 48 hours of hyphae growth from a 5 mm agar plug², placed with the hypha down in the center of a 60 mm Petri dish. For slow-growing isolates, the agar plug of the endophyte colony was placed in the Petri dish one to four days before the introduction of the seeds. Seedlings germinated for a week under conditions of abiotic and control stress.

Drought stress was induced by PDA containing 8% PEG. Heat stress was induced in a dark countertop incubator; the temperature rose gradually from 2 ° C every 2 hours from 28 ° C to 36 ° C. In the initial round of experiments, the germination percentage at three days and the fresh weight at week were evaluated. Each experiment consisted of six Petri dishes and was repeated, independently, three times. In subsequent experiments, the germination percentage was evaluated every 24 hours for seven days. Each experiment consisted of 10 Petri dishes and was repeated twice (heat and drought stress combined) or three times (heat stress, drought stress and control conditions).

Stable internal colonization of wheat roots by the expected endophytes was confirmed by re-isolating the endophytic organism from roots that had been sterilized on the surface to remove external microbial growth using a modified procedure from Larran et al. [2002]. Root fragments (-0.5 cm) were sterilized on the surface in 95% ethanol for 10 s, rinsed in sterile distilled water for 10 s, immersed for 20 s in 5% sodium hypochlorite (Javex) , rinsed three times in sterile distilled water and placed on PDA in a 60 mm diameter Petri dish. The Petri dish was sealed with parafilm and incubated in the dark at room temperature for four to seven days prior to the exam.

Statistic analysis

The growth rates of the colonies of autonomous endophytic organisms that are grown under heat or drought stress were compared with those of the same organism grown under control conditions by analysis of variance (ANOVA) followed by Fischer's post-hoc test of minimal significant difference (LSD). Germination percentage data underwent arcosine transformation before statistical analysis [McDonald 2009]. Statistical differences between germination percentage after three and seven days, and fresh weight at seven days were evaluated using a single ANOVA factor to compare all treatments. Subsequently, a post-hoc LSD test was used to assess the importance of differences between non-endophyte control and seeds treated with each mycobiont. The level of statistical importance associated with the differences between the EG and HTT needed to reach 50% of the germination of the control and colonized seeds with endophytes was studied by evaluating the EG for each of the three independent replicas of the experiment . The resulting data were subjected to an ANOVA and post-hoc LSD analysis. P values ​​less than 0.05 and 0.01 were considered significant and highly significant, respectively. Statistical tests were performed with SPSS Inc. 2011.

Results

Within each section, the results are organized according to the type of stress: heat, drought, heat and drought, or no stress. Within each stress, the results dealing with plant material are presented according to the measured characteristics of the seedling and / or germination: percentage of germination at three and seven days, fresh weight at seven days, EG and HTT .

Autonomous endophytes

The phenotypes of SMCD 2206, 2210 and 2215 were not altered by heat (36 ²C), while SMCD 2204 and 2208 did not grow to 36 ²C. Growth rates of the SMCD 2206 and 2210 colonies were reduced by 36 ^toC compared to unstressed conditions (p 0.01), while the growth rate of SMCD 2215 to 36 ²C increased (p 0.05) (Figure 15). To 36²C, SMCD 2215 was the fastest growing, followed in decreasing order by 2206 and 2210 (Figure 15).

The morphology of SMCD 2204, 2206, 2208 and 2215 was not significantly altered by the drought (8% PEG). However, when SMCD 2210 was exposed to drought, the organism lost its woolly appearance and instead acquired a shiny or silty appearance. The growth rates of the SMCD colonies 2204, 2206 and 2208 were reduced by drought (p 0.01, p 0.01, and p 0.05 respectively), while the growth rate of the other endophytes remained unchanged. (Figure 15). When drought stress was applied, SMCD 2204 grew at the highest speed, followed in decreasing order by 2206, 2210, 2208 and 2215 (Figure 15).

By stimulating SMCD 2204 and 2208 with 36 ²C heat and drought (8% PEG) simultaneously, these did not grow, while SMCD 2206, 2210 and 2215 grew at a significantly slower rate than in control conditions (p

0.01) (Figure 15). Under control conditions, SMCD 2204 was the fastest growing, followed in decreasing order by SMDC 2206, 2210, 2208 and 2215 (Figure 15).

Response of wheat colonized by endophytes to heat

To 36²C, colonization by SMCD 2206 and 2215 increased germination after three days (p 0.05 and p í 0.01, respectively; Figure 16A), while SMCD 2204, 2208 and 2210 did not alter this parameter (p&gt; 0, 1; Figure 16A). After seven days, 63% and 56% of the seeds germinated in coculture with SMCD 2204 and 2208, respectively. These values ​​were not statistically different (p&gt; 0.1) at the 59% germination achieved by the uncolonized control. In contrast, the SMCD endosymbionts 2206, 2210 and 2215 promoted germination after seven days (p &lt;0.01; Figure 17).

When they underwent .36 ²C, the fresh weight of wheat seedlings remained stable in coculture with SMCD 2204, 2206, 2208 and 2210, while SMCD 2215 significantly increased this parameter (p ¿0.01, respectively; Figure 16D).

EG for wheat seeds co-cultivated at 36 ²C with the fungal endophyte SMCD 2210 (p 0.05; Table 3, Figure 17) improved compared to the seeds without endophytes. However, SMCD 2204, 2206, 2208 and 2215 did not alter the EG (p&gt; 0.1; Table 3) with respect to the control. SMCD 2210 increased the EG to the greatest extent, followed by SMCD 2206 and 2215 (Table 3). SMCD 2210 reduced the time needed for 50% of the seeds to germinate to just two days.

When exposed to heat stress, the HTT required for germination was reduced for wheat seeds colonized by SMCD 2210 (p 0.05; Table 3), but did not occur with any of the other endophytes evaluated (p&gt; 0, 1; Table 3). Wheat seeds without endophytes needed 50 days^SC MPa more than the seeds colonized by SMCD 2210 (the most effective endophyte studied) to reach 50% germination (Table 3). There was a clear, linear negative correlation between the HTT required for 50% germination and the germination percentage after seven days under heat stress (Figure 18).

Response of wheat colonized by endophytes to drought

When they were subjected to drought stress for three days, a smaller percentage of wheat seeds germinated in co-cultivation with SMCD 2208, compared to seeds without endophytes (p.01; Figure 16B), while SMCD 2204, 2206, 2210 and 2215 did not alter this trait (p&gt; 0.1; Figure 16B). After seven days, treatment with SMCD 2206, 2210 and 2215 produced an increase in seed germination (p 0.01, p 0.05, and p 0.01, respectively;

Figure 17). On the contrary, 65 and 67% of the seeds co-cultivated with SMCD 2204 and 2208 had germinated after seven days. None of these values ​​differ statistically from 59% of the non-colonized seeds that germinated under the same conditions (p&gt; 0.1). In drought conditions, SMCD 2208 and 2210 decreased the fresh weight after seven days (p 0.05 and p 0.01, respectively; Figure 16E). None of the other mycobionts altered this parameter (p&gt; 0.1; Figure 16E).

The EG decreased for co-cultivated wheat seeds in drought conditions with all the endophytes studied, compared with the seeds without endophytes (0.05 &lt;p 0.1 for SMCD 2204 and 2208 and p 0.05 for 2206, 2210 and 2215 ; Table 3). SMCD 2206 improved the EG to the greatest extent, decreasing the time elapsed before 50% of germination was reached after 2.6 days (Table 3; Figure'17).

The HTT needed for germination was reduced for wheat seeds treated with all endophytes studied under drought stress (Table 3). While the uncolonized seeds needed 80 days^toC MPa to reach 50% of germination, the seeds colonized by the endophyte SMCD 2206 (the most effective endophyte studied) needed only days ²C MPa, which represents a reduction of 46 days ²C

MPa (Table 3). There was a visible linear negative correlation between the HTT required for 50% germination and the germination percentage at seven days under drought stress (Figure 18). However, the value of R² associated to this linear relationship was lower than the correlation found under heat stress. The intervals of HTT necessary to reach 50% of germination differ between heat and drought stress, with values ​​between 34 and 44 days^SC MPa and 80 and 94 days ^SC MPa, being unique for seeds exposed to drought and heat stress, respectively (Figure 18; Table 3). The germination percentage intervals after seven days are similar between the seeds exposed to drought and those subjected to heat, although the germination levels of the heat-stressed seeds cover a slightly longer interval (Figure 18).

Endophyte-colonized wheat response to a combination of drought and heat

Very few wheat seeds germinated when exposed to drought stress (8% PEG) and heat (36 ^toC) at the same time (Figure 17). Colonization by the endophytes SMCD 2210 and 2215 increased the germination percentage after seven days (p 0.01; Figure 17). On the other hand, SMCD 2204, 2206 and 2208 did not improve this feature (p

0.1). Seeds co-cultivated with SMCD 2215 (the most beneficial microorganism studied for this parameter) reached 24% of germination, four times the level reached by their counterparts without endophytes (Figure 17).

Because neither the colonized seeds nor those colonized by any of the endophytes reached 50% germination within seven days, the EG could not be determined and the HTT was calculated for 5% germination, instead of 50% . The time needed to reach 5% of germination varied from 24 to four days. None of the endophytes studied decreased the time needed to reach 5% of germination and HTT values ​​(P&gt; 0.1). In general, the HTT needed to reach 5% of germination varied from 11 to 43 days ²C MPa (HTT_praverage = 23.9) (Figure 18; Table 3).

The range of values ​​for HTT for the seeds subjected to both heat and drought stress were unique, compared to the HTT values ​​when only heat or drought was applied. There was a linear negative relationship between the necessary HTT and the germination percentage under the combination of heat and drought stress. However, the value of R² associated to this linear relationship was lower than the correlation found when heat stress or drought was applied individually (Figure 18).

Response of wheat colonized by endophytes to control conditions

Under unstressed conditions, SMCD 2215 significantly increased seed germination compared to uncolonized seeds after three days (p 0.01) (Figure 16C). SMCD 2206, 2208 and 2210 had a positive impact, while SMCD 2204 did not alter the germination percentage. Under unstressed conditions, SMCD

<td>2204, 2210 and 2215</td><td colspan="5">increased the fresh weight of</td><td>seedlings</td>

<td>of wheat after</td><td>of seven</td><td>days (p</td><td></td><td> 0,05</td><td>And p</td><td> £ 0,01,</td>

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no impact on fresh weight compared to uncolonized seedlings (Figure 16F).

Under control conditions, the parameters EG and HTT were slightly improved by the SMCD endosymbionts 2206 and 2215 (Table 3). Relatively little alteration in the parameters EG and HTT associated with unstressed wheat seeds in coculture with different isolates was measured.

Example 5

Endophytes improve the yield of wheat and barley genotypes under severe drought stress

Compendium: Due to climate change and population growth, the development of techniques that increase agricultural tolerance of crops in stressful environments is crucial. Inoculation with three symbiotic endophytes, indigenous to the Canadian prairies, increases the resistance of wheat and barley with respect to heat or drought, as well as grain yield and seed weight. The use of such fungal and bacterial endophytes in the field has the potential to increase the germination vigor of the seeds (SGV = difference between the total percentage of seeds in germination E- and seeds in germination E +) (Figure 19, Figure 20A and B), and to improve performance under conditions of stress propensity (Table 4; Figure 21 a, Ba and Bb). The evidence supports that SMCD strains increase seed vitality and plant vigor (Figure 22A-D). The general results show that the pre-germination care of the seeds using endophytic microbes, particularly SMCD strains, ensures the superior yield of wheat and barley genotype cultivation through physiological improvements.

Materials and methods

The seeds of the cultivated varieties of barley wheat were produced in experimental plots of the

University of Saskatchewan and Crop Science Field Laboratory (Saskatoon).

Visually, the healthy seeds were sterilized on the surface in 95% ethanol for 10, rinsed in sterile distilled water for 10, immersed for 1 min in sodium hypochlorite (Javex) and then rinsed three times in sterile distilled water.

The endophytic isolates used in this study were originally isolated from roots of Triticum turgidum L. durum wheat grown at field sites in Saskatchewan, Canada [Vujanovic 2007b]: SMCD 2204, 2206,

2208, 2210, 2215. All endophytic isolates can be grown on potato and dextrose agar (PDA; Di-fco) in the absence of a host plant. The isolates were grown in PDA for three days at room temperature 23²C) in the dark before experimental use.

The inoculations of the experiments were carried out in pots. Each of the endophytic isolates was applied to cereal seeds (wheat and barley) before germination according to the method described in Abdellatif et al. [2010]. In summary, five sterilized seeds on the surface were placed at a distance equivalent to 48 h of hyphae growth from a 5 mm agar plug², placed with the hypha down in the center of a 2 L plastic pot filled with 300 grams (dry weight) of Sunshine mix 4 potted soil with autoclavable field capacity. The seeds and the agar plug were then covered with a 3.5-4.0 cm layer of Sunshine 4 mixture. Five seeds were planted per pot and there were twelve pots per treatment. The pots containing plants were placed in a greenhouse for control and drought stress treatments. The pots were organized in a randomly selected block design.

Drought stress was induced from May to September, when the maximum night-day temperatures in the greenhouse ranged from 18 to 26 ²C. On sunny days, natural light provided irradiation, while cloudy or winter days with a shorter photoperiod, sunlight was. 10 supplemented by 1,000 watt high pressure sodium lamps, suspended from the ceiling about 2 m above the plants. In the first experiment, plants subjected to drought and control stress (well watered) were grown at 25% soil water content by weight and 100% water retention capacity, respectively. During the experiment, control plants were watered up to 100% water retention capacity three times a week, while plants subjected to drought stress were watered up to 100% water retention capacity weekly. This drought regime was adopted in order to mimic the natural drought cycle that may occur during the North American prairie growing season [Chipanshi et al. 2006].

The ripe ears were collected and the dried beans were weighed on a Mettler Toledo PG802-S scale in the laboratory.

Results and Discussion

Increased vigor of germination of wheat seeds (SGV)

Under in vitro control conditions, wheat seeds treated with SMCD (2204, 2206, 2208, 2210, 2215) germinated more consistently, more uniformly, and with a much higher SGE (seed germination efficiency). The SGV of the seeds inoculated with SMCD (E +) was 15% to 40% higher compared to the untreated seeds (E-) (Figure 19), which demonstrates the effectiveness of SMCD in controlling seed dormancy and improve the vigor of the seed. The positive effects of SMCD strains on the performance of wheat and barley genotypes under severe drought conditions were also demonstrated.

Barley genotypes generally show higher drought susceptibility (low DTE (drought tolerance efficacy) values) and lower yield than wheat (Table 4), possibly due to the most suitable greenhouse drought conditions in the greenhouse. the wheat. In particular, the two-run Kendall barley with CDC, without endophyte (E-), shows a high susceptibility to drought stress compared to other barley genotypes. However, endophytic (E +) treatments demonstrate a notable positive effect on the performance of all genotypes (Table 4). The conferred resistance ranges from the Kendall genotype with low-resistance CDC to the highly resistant New Dale, while the resistance conferred to wheat was consistently high.

During the maturity stage of wheat and barley, SMCD endophytes radically increase drought tolerance parameters of genotypes, such as DTE efficacy and yield. The application of SMCD in Avonlea, the cultivated wheat variety detected most susceptible to drought (DTE = 16.1), resulted in a large increase in yield (77%) in drought conditions compared to control or irrigation standard. Carberry benefited the most from endophytes under normal or control conditions, while Utmost VB with CDC and BW 423 had the same performance in both drought and control conditions.

In conclusion, the combination of drought-resistant genotypes with compatible microphytic symbionts SMCD 2206, SMCD 2210 and compatible endophytic SMCD 2215 maximizes the resistance of the plant to drought, an important aspect to ensure food safety. Without pretending to be limited to theory, this suggests that varieties of⁷³Wheat cultivation (Figure 19A) and barley (Figure 19b) more susceptible to drought (low DTE values) will obtain maximum benefit from symbiotic association when exposed to drought stress.

The only exception seems to be the six-run barley genotype, Legacy, which showed a very low DTE = 1.1. Although this responded positively to the presence of endophytes with a higher yield of 26.9% under control conditions, the yield was improved only by 5% in symbiosis under stress. Therefore, this crop variety was excluded from the barley model presented in Figure 19B.

Effect of individual strains of SMCD on the productivity of wheat and barley

Individual SMCD strains positively affect the average performance of the nucleus of each genotype, although the actual magnitude varies according to the genotype combination. Figure 21 presents the results obtained in drought conditions in the greenhouse (Figure 21: Atrigo; B_to-barley (two races), and Bb-barley (six races).

Early seed contact with compatible SMCD isolates is a prerequisite for crop protection against drought, which results in increased yield or grain production. SMCD 2206 generally confers the highest degree of improvement for most genotypes. However, the specificity of strain cultivation varieties ensures high improvements individually, for example, Wheat-PT580 and Barley-Copeland with CDC prefer SMCD 2210; while Trigo-BW423 and PT580, as well as Kendall with CDC show greater performance and drought resistance when inoculated with SMCD 2215.

The results highlight the importance of mycovitalism in wheat and barley seeds stimulated with stress, which helps growers in the elaboration of highly productive cultivated varieties capable of withstanding drought conditions significantly better than any variety grown alone (Figure 22 AD). After demonstrating the yield of SMCD strains in the fields, producers will have green symbiotic products to ensure crop and agricultural market performance

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Example 6

Heat stress experiment of legumes in phytotron

This experiment was carried out under phytotron conditions. All seed varieties were inoculated with endophytes (SMCD 2204F, SMCD 2206, SMCD 2210 and SMCD 2215) and without endophytes in pots containing the soil mixture.

Details on the methods used for inoculation with endophytes in the plant have been described above in Example 5. The pots containing the plants for heat stress were placed in a culture chamber.

Conviron PGR15 of phytotron (Controlled Environments Ltd.) using a randomized block design. A temperature of around 33 ° C was selected for heat stress.

The plants were exposed to this temperature for 8 hours, after which time the plants were exposed to a temperature of 21 ° C for 16 hours up to 10 days. After thermal shock, temperatures were changed at 16 ° C for 8 hours and 21 ° C for 16 hours.

Results

In summary, the results show that the efficacy of each endophyte studied to confer heat stress tolerance is related to the particular genotype of the host plant or variety (A-chickpea, B-lentil, and Carveja), and that the improvement in biomass it is associated with a particular organ of the plant, since each organ: goes ina 20 (Figure 23), stem (Figure 24) and root (Figure 25), is affected differently by heat stress.

SMCD 2215 mainly improved the stem and pod biomass in the peas, and the root biomass in the chickpeas. SMCD 2206 increased stem and pod biomass in lentils, and root biomass in chickpeas, peas and lentils. SMCD 2210 mainly improved the stem and pod biomass in the chickpeas, and the root biomass in the peas. SMCD 2204F improved the biomass of the pods in most of the crops studied (chickpeas, peas and lentils). The combination of endophyte-culture genotype with better yield (E +) showed an improvement of about 300% in the sheath, stem and root biomass compared to the heat-stressed control without endophytes (E-).

Stem: The following endophytes showed the best response to heat stress: Chickpea: Amit: SMCD 2210. Vanguard: SMCD 2204F; Pea: Golden: SMCD 2215. Handel: SMCD 2215; and Lentil: Glamis: SMCD 2206. Sedley: SMCD 2206.

Pods: The following endophytes showed the best response to heat stress: Chickpea: Amit: SMCD 2210. Vanguard: SMCD 2204F; Pea: Golden: SMCD 2204F. Handel: SMCD 2215; Lentil: Glamis: SMCD 2206. Sedley: SMCD 2204F.

Root: The following endophytes showed the best response to heat stress: Chickpea: Amit: SMCD 2215. Vanguard: SMCD 2206; SMCD 2215; Pea: Golden: SMCD 2210; SMCD2215. Handel: SMCD 2206; Lentil: Glamis: SMCD 2206;

Sedley: SMCD 2204F.

Example 7

Greenhouse Legume Drought Stress Experiment

In this study, six varieties of seeds were used [Amit, Vanguard (chickpeas), Golden, Handel (peas) and Glamis, Sedley (lentils)] and the endophytes SMCD 2204, SMCD 2204F, SMCD 2206, SMCD 2210 and SMCD 2215. These Experiments were carried out in the greenhouse. After sowing the miles and inoculating the endophytes, the pots were allowed to remain without water for 14 days to mimic severe drought as proposed by Charlton et al. [2008] and through the methodology and conditions indicated by Gan et al. [2004].

Results

In summary, the results show that each strain of SMCD has a positive effect on various agricultural parameters in the yield or production of pods (Figures 27), and the stem biomass (Figures 26) and the root (Figures 28) in chickpeas (A), peas (B), lentils (C) and under drought stress. In general, genotypes of cultures colonized by symbiotic endophytes (E +) became more resistant to drought with respect to heat stress. The level of efficacy of the endophytes studied to confer drought tolerance varied according to the particular organ of the ⁷⁸ plant: the performance of the pods in Glamis was greatly improved by means of SMCD 2204, in Vanguard by means of SMCD 2204F, in Sedley by means of SMCD 2206, in Golden by means of SMCD 2210, and in Handel by means of SMCD 2215 .

Stem: The following endophytes showed the best response to drought stress: Chickpea: Arnit: SMCD 2204F; Vanguard: SMCD 2206; Pea: Golden: SMCD 2204; Handel: SMCD 2204; SMCD 2210; SMCD 2215; Lentil: Glamis: SMCD 2204F; SMCD 2206. Sedley: SMCD 2204F; SMCD 2206.

Pods: The following endophytes showed the best response to drought stress: Chickpea: Arnit: SMCD 2204; SMCD 2210. Vanguard: SMCD 2204; SMCD 2206; SMCD 2215; Pea: Golden: SMCD 2210; SMCD2215. Handel: SMCD 2204F; SMCD 2206; SMCD 2215; Lentil: Glamis: SMCD 2204F; SMCD 2206. Sedley: SMCD 2210; SMCD2215.

Root: The following endophytes showed the best response to drought stress: Chickpea: Arnit: SMCD 2204; SMCD 2215. Vanguard: SMCD 2204F; SMCD 2206; Pea: Golden: SMCD 2204F; SMCD2215. Handel: SMCD 2204F; Lentil: Glamis: SMCD 2204F; SMCD 2206; SMCD 2210. Sedley: SMCD 2206; SMCD 2210.

Example 8

Streptomyces SMCD 2215 species increases Rhizobium activity and nodulation frequency in peas under heat stress

As recently observed for another species of Streptomyces, S. lydicus WYEC10 [Tokala et al. 2002], the novel species of Streptomyces SMCD2215 colonizes the roots of young pea seedlings from seeds produced from plants grown under control conditions. This specifically improves the flowering of the plant and the yield of the pods (Figure 29) and root nodulation by the Rhizobium species (Figure 30), which is a natural endophytic colonizer of pea seeds discovered in this study. (Table 5). Vegetative hyphae of the novel species of Streptomyces SMCD2215 colonize emerging nodule cells as discovered by culture plate amplification (PDA), fluorescence microscopy (Cari Zeiss Axioskop 2) and PCR (BioRad) [Schrey and Tarkka 2008]

Example 9

Endophytes confer tolerance to abiotic stress on legumes through improved seed viability

Legume crops refer to a group of more than sixty different grain vegetable crops grown worldwide. Vegetable crop seeds are important for human nutrition. The main limitations for legume production are biotic and abiotic stress such as drought, heat, cold and salinity. Recent research suggests that endophytic plant-microbe interactions are a fundamental determinant of plant adaptation.

This study hypothesizes that endophytes increase the speed and uniformity of seed germination under optimal conditions and in vitro stress. The objective was, first, to measure the intrinsic symbiotic ability of endophytes to trigger germination; and, secondly, to measure the efficacy of compatible endophytes for conferring heat and drought resistance to legume genotypes.

Materials and methods

Two varieties of legumes, Glamis (lentils) and Handel (peas), were combined with SMCD 2206 and SMCD 2215 compatible, fungal and bacterial symbiotic strains, respectively. The ability of endophyte strains to confer stress tolerance to the Golden genotypes (Figure 31) and Handel (Figure 32) were studied during germination of in vitro seeds that mimicked drought (6% PEG) and heat environments ( 33 ° C).

The seeds were sterilized on the surface with 95% ethanol for 20 seconds, rinsed twice in sterile distilled water for 10 seconds followed by 2 minutes in 3% sodium hypochlorite (Javex). Finally, the seeds were rinsed in sterile distilled water 4 times. The seeds were inoculated in PDA medium with and without endophytes in the dark at room temperature [Abdellatif et al. 2009]. Microbial organisms were grown in PDA for at least three days at room temperature in the dark before experimental use. The ability of endophytes to confer stress resistance to the plant was evaluated using germination energy, which is intended to capture the temporary nature of germination and is defined as the number of days needed to reach 50% of the seeds in germination.

Results

The present study demonstrates the differential ability of fungal or bacterial endophytes to confer legumes with resistance to drought and heat, which is specific to a combination of bacterial strain or fungal plant genotype-abiotic stress. This study used molecular and proteomic analysis to better understand the mechanism by which endophytes confer legumes symbiotic resistance to stress.

Strains of SMCD significantly increased the germination frequency of legume seeds under standard in vitro conditions (Figure 33). Under stress conditions, the two endophytes (SMCD 2206 and SMCD 2215) increased the frequency of germination compared to non-colonized seeds. The germination frequency was 70-100% in symbiotic treatments and 60-80% germination in the control, which means that the endophytes studied have the potential to increase the germination vigor of the seeds (SGV) by&gt; 15% . The highest germination frequency was observed (100%). in Glamis (lentils) associated with both SMCD 2206 and SMCD 2215 in conditions of drought stress vs. heat stress. When inoculated with SMCD strains, germination energy (&gt; 50% in germinating seeds) was reached in Glamis in 2 days in dry conditions and in 3 days in hot conditions. Similar results were obtained in Handel (peas), except that this genotype has a greater inherent ability to withstand thermal shock than Glamis (lentils).

Example 10

Endophytes improve the performance of flax genotypes and cane it under severe drought stress in greenhouse experiment

The objective of this study was to use three randomly selected isolates (SMCD 2206, SMCD 2210 and SMCD 2215 and extend the efficacy test to the production and yield of flax and cane under drought stress.

Materials and methods

The experimental design, the handling of flax seeds (Bethun and Sorel) and cane (1768S), the application of endophytic inoculators (SMCD 2206, SMCD 2210 and SMCD 2215), drought conditions, and performance evaluation are as detailed in Example 5 with minor modifications. In summary, control plants were watered up to 100% water retention capacity three times per week, while plants subjected to drought stress were watered up to 100% water retention capacity weekly. This drought regime was adopted in order to mimic the natural drought cycle that can occur during the Canadian prairie growing season in which no precipitation falls for seven consecutive days, or more.

Results and Discussion

Severe drying conditions jeopardized the performance of non-symbiotic flax, while endophytic inoculators SMCD 2206 and SMCD 2210 radically improved flax yield under these same conditions. In particular, in drought conditions, SMCD 2206 maintains a yield of almost 100% in Bethun while SMCD 2210 provides a 50% yield compared to the stress-free control in the greenhouse (Figure 34). In terms of pipe, improved performance was recorded in combination with SMCD 2210 (&gt; 100%), followed by SMCD 2206 (~ 50%) and SMCD 2215 (—30%) compared to the stress-free control (Figure 35).

The bioprotection capacity against

Fusarium avenaceum and F. graminearum in the greenhouse. The autoclaved seeds were infected by Fusaria inoculators at 25 ° C (Figure 36), and produced in plates from Abdellatif et al. [2009].

Earth was inoculated for darkness for 7 days at inoculated by endophytes petri as described by pots with twenty seeds that had Fusarium. The composition of the earth mixture was

55-65% Canadian Sphagnum peat moss, perlite and limestone mixed with sand. The standard greenhouse conditions were 8 days of light that were exchanged with a photoperiod regime of 16 hours (1000 lux) with a relative humidity of 70% and a constant temperature of 25 ° C ± 2 ° C.

Vegetable treatments were as follows:

Ti: Untreated plants (control)

T2: Plant + endophyte

T3: Plant + pathogen, Fusarium avenaceum

T4: Plant + pathogen, Fusarium graminearum

T5: Plant + fungal endophyte + Fusarium avenaceum T6: Plant + endophyte + Fusarium graminearum

Each treatment was repeated in three pots, and the seedlings were watered three times a week under control conditions. Root colonization by endophytes was studied using a fluorescence microscope to distinguish symbiotic endophyte-wheat relationships with respect to pathogens [Abdellatif et al. 2009].

Figures 37-40 show the positive effect of endophytes on the resistance of wheat seedlings after germination (Figure 37), foliage and root biomass (Figure 3 8 and Figure 39), and the flowering stage / anthesis and spikes (Figure 38, Figure 39 and Figure 40). All endophytes studied induced well-developed foliage compared to control, as well as well-developed flowers in the presence of endophytes.

To confirm the ability of endophytes to stimulate the growth of mature plants in the presence of Fusarium pathogens, the yield of ⁸⁶the flowering stage that showed the spikes as a more advanced stage of growth.

The histograms in Figure 41 illustrate the performance of endophytes to improve biomass or dry weight of wheat ears after double inoculation (endophyte SMCD and Fusarium pathogen).

Wheat yield in the presence of an endophyte and Fusarium significantly improves the use of all endophytic strains compared to treatment infected with F. graminearum and F. avenaceum but without an endophyte (E-) (Figure 41). Plants treated with the pathogen only show a significantly smaller spike size compared to control plants and plants with endophytes (E +) (Figure 42).

Example 11

Expression of the endophytic mediated abiotic stress resistance gene in legumes

Summary:

The beneficial effects of the genomic and proteomic mechanisms of plant endophytes on the resistance of the host plant to abiotic stressors are poorly understood. One of the contemporary theories suggests that symbiotic plants are protected against oxidative stress, produced by stress factors such as heat, drought and salinity, through the production of antioxidant molecules. The objective of this study is to better understand the defensive symbiosis of pea, chickpea and lentil genotypes by evaluating the gene expressions of Pro, SOD and MnSOD triggered by the association between host and endophyte genotypes. The results of this study demonstrated endophyte-mediated gene expression in plants inoculated with endophytes. These genes play an important role and provide protection to the host through improved stress tolerance to the abiotic stressors studied.

Materials and methods

For this analysis, leaves of 6 varieties of normal and low-stress seeds were collected (Amit, Vanguard [chickpea] (Figure 43), Golden, Handel [peas] and Glamis, Sedley [lentils]) with or without endophytes.

Real-time PCR was used to amplify genes such as proline (Pro), SOD and Mn SOD using primers as shown in SEQ ID NO: 8-15 (Table 6), it was found that stress proteins, in general , they perform special functions in the protection of the cytoplasm against dehydration and in the protection of plants by mitigating the toxicity produced by high concentrations of ions. PCR was carried out under the following conditions: 3 minutes at 95 ° C (enzyme activation), 40 cycles each 30 seconds at 95 ° C (denaturation) and 30 seconds at 60 ° C (hybridization / extension) . Finally, an analysis of the melting curve from 65 ° C to 95 ° C was performed in increments of 0.5 ° C, with a duration of 5 seconds each, to confirm the presence of a single product and the absence of primer dimers. The quantification is relative to the control gene by subtracting CT from the CT control gene of the gene of interest (ACT). The resulting difference in the cycle number is then divided by the normalized target value of the calibrator, and the value obtained (AACT) is the exponent of base 2 (due to the duplication function of the PCR) to generate expression levels relative.

Results

Different gene expressions were analyzed during drought. Table 6 shows the genes that were studied. Some of the results obtained from Handel variety when exposed to 6% PEG.

SOD and MnSOD

In general, SOD plays an important role in antioxidant defense mechanisms. In the present study, very high levels of SOD expression were observed in normal leaves (E-, control) exposed to 6% PEG, almost 200 times increase. Endophytes had a very important function in reducing this stress. Especially, SMCD 2215, followed by SMCD 2210, SMCD 2204 and SMCD 2206. These symbionts radically reduce stress with an observed increase in expression of only 9 and 24 times (Figure 44A).

MnSOD is one of the forms of SOD. Control sheets showed a 16-fold increase in gene expression, while SMCD 2215 suppressed stress and decreased change 16 times to 2 times, followed by SMCD 2206, SMCD 2210 and SMCD 2204 (Figure 44B) .

Proline

Proline is essential for primary metabolism. Proline biosynthesis is controlled by the activity of two P5CS genes in plants. This gene was evaluated in the Handel pea variety with endophytes in drought conditions. As expected, the P5CS gene regulated by increase and increased expression 5 times in leaves collected from plants exposed to PEG. While the cultivated seed leaves associated with SMCD 2206 expressed 2.8 times followed by SMCD 2215, in 3.4 times they expressed the proline encoding gene (Figure 45). These results confirmed that endophytes play an important role on stress resistance that modifies the proline gene expression compared to non-inoculated stressed plants.

Example 12

Patterns of gene expression in the wheat school in cold and biological stratification conditions

Summary;

Wheat is one of the main crops widely used in the world. However, world wheat production has decreased around 5.5% in the last two decades and a further decline has been forecast due to widespread global warming. Therefore, elucidating the conditions and techniques that improve seed germination is of great importance. Cold stratification is a method known long ago to release seed dormancy and promote germination. Biological stratification through fungal endophytes can also stimulate seed germination in many cereal crops. The coleorriza is one of the most active tissues in the seed and is also the first part that appears outside of germinating seeds. To assess the effectiveness of stratification methods, the percentage of germination of wheat seeds under cold and biological stratification conditions was evaluated and the level of expression of gibberellins and abscisic acid genes in the tailings was determined. Both cold and biological stratification treatments significantly improved (P &lt;0.05) the speed and efficiency of germination. The spatial distance between the fungal endophyte and the seeds is a determining factor of biological stratification since the seeds in direct contact with the fungal endophyte showed a higher percentage of germination (up to 86%). The high expression of the GA3ox2 gene in the wheat beetle was found throughout the germination period, which revealed constant production of the GA3 bioactive molecule. The expression of the 14-3-3 gene was lower in direct endophyte treatment. The expression of the abscisic acid-ABA biosynthesis gene, TaNCED2, was considerably high in cold stratification seeds that reflect the function of abscisic acid as a stress adaptation hormone. A high expression of the TaABA8ΌΗ1 gene was also found in the coleorhiza. Overall, this study provides molecular evidence of the importance of the coleorriza to germinate wheat seeds. By comparing the methods of cold and biological stratification, the germination of the seeds can be significantly improved by the application of fungal endophytes, and the spatial distance between the seeds and the endophyte is a driving factor towards mycovitality.

Materials and methods

Wheat seeds

In this study, seeds of the AC Avonlea hard wheat variety with low resistance to environmental stress conditions were used. These seeds were produced by Agriculture and Agri-Food Canada (AAFC) Seed Increase Unit Research Farm in 2006 under greenhouse conditions, and were recommended because they had no microbes. The seeds were kept in sterile ziploc bags and stored in a cold environment at 4 ° C until they were used again.

Comparison of seed sterilization protocols

Several methods for sterilizing the surface of wheat seeds have been proposed. Four broadly recognized seed sterilization methods were compared here to identify the best suitable protocol to effectively sterilize the seed surface without affecting the quality and vitality of the seed in this wheat variety. In the first method, the seeds were sterilized on the surface with 95% ethanol for 10 seconds, followed by a rinse in sterile distilled water three times for 1 minute [Zhang et al., 2007. BMC Genetics 8]. The second protocol was bleach sterilization, where the seeds were sterilized on the surface in 5% sodium hypochlorite for 3 minutes, followed by a thorough rinse with sterile distilled water three times for 1 minute. In the third protocol, the seeds were sterilized on the surface with 95% ethanol for 10 seconds, rinsed in sterile distilled water, and then immersed for 3 minutes in 5% sodium hypochlorite, rinsed three times in water distilled sterile and placed on potato and dextrose agar (PDA) for germination [Abdellatif et al., 2009]. The fourth method consisted of steam phase sterilization of the seeds with chlorine gas [Desfeux et al. , 2000]. In the gas hood chamber, a small glass with 20 ml of bleach is placed in a 5 liter snaptite box. Wheat seeds were placed in a 96-well plate and kept in the snaptite box. Then 3 ml of concentrated hydrochloric acid was added to the small beaker to create chlorine gas. The lid was kept closed for 4 hours to keep the seeds in contact with the chlorine gas. After sterilization, the 96-well plate was placed for 1 hour in a laminar flow hood to disperse the trace of chlorine gas. Then, the sterilized seeds were rinsed three times in sterile distilled water and placed on PDA plates. The comparison of these sterilization methods suggests that the chlorine gas sterilization protocol was the most effective method that shows 80% germination without contamination while the control seeds had the highest percentage of contamination (Table 7). Although bleach and ethyl methods inhibited contamination successfully, seed germination was significantly affected. Therefore, the chlorine gas protocol is a highly effective method for sterilization of wheat seeds and was selected to sterilize the seeds necessary for the experiments performed in this study.

Cold and biological stratification

For stratification a, the sterilized seeds on the surface were kept in wet filter paper in a cold environment of 4 ° C for 48 hours [Mukhopadhyay et al., 2004; Wu et al. , 2008]. After 2 days, the cold stratified seeds were brought to room temperature where they were quickly rinsed in sterile distilled water and placed on potato and dextrose agar (PDA) plates. For biological stratification, the sterilized seeds were incubated in the presence of SMCD 2206. The fungal endophyte was cultured in PDA at room temperature in the dark for at least three days before use. To assess this efficiency, wheat seeds were germinated in direct contact and at a certain distance from the fungal endophyte. An agar plug (5 mm was placed²) of the endophyte dissected from the margins of a mother colony in the center of a 90 cm Petri dish with PDA. Next, 10 sterilized seeds on the surface were placed on the periphery of the Petri dish that surrounds the fungal agar plug approximately 4 cm away. All Petri dishes were sealed with 5 layers of Parafilm® (Pechiny Plastic Packaging) to avoid any biological contamination and the diffusion of volatile / gaseous compounds. The direct contact impact of the fungal endophyte was elucidated by placing a 3 mm agar plug² between two adjacent wheat seeds sterilized on the surface and a 5 mm plug² in the center of the PDA plates. All treatments were carried out with three replicas of PDA plates with ten seeds sterilized on the surface in each plate. Petri dishes were incubated in a countertop incubator at room temperature (-20 ° C) in the dark. Incubation time was recorded and data collection and isolation of the coleorriza were carried out after 24, 48, and 72 hours.

Germination percentage

The appearance of the first radicles was carefully monitored. The germination percentage was calculated by estimating the number of germinated seeds in 10 wheat seeds in each dish with PDA. The germination rate of 50% was assumed as the germination energy. The effectiveness of germination in the different treatments was calculated using the following equation:

Efficacy =% of germination in a treatment -% of germination in the control [EC. one]

Germination rate was observed for all treated samples and replicas. For samples on day 2 and day 3, the germination rate was monitored from Day 1 to assess overall vitality. PDA plates were kept sealed throughout the data collection period.

Sausage Isolation

After observing the germination rate, the PDA plates were immediately transferred to a sterile biosecurity bell chamber for the isolation of the coleorriza. Wheat seeds were carefully dissected under a compound microscope and the coleorriza layers were excised using needle and sterile scalpel.

The isolated choles were stored in a sterilized microcentrifuge tube without RNase. The seeds of all the biological replicas of a treatment were combined to 30 buffers for and were approximately isolated to obtain plant material of optimum quantity for RNA extraction.

RNA extraction and cDNA synthesis

To avoid any degradation in the plant material, RNA extraction was carried out immediately after the isolation of the coleorriza on each day. Approximately 20 mg of cholera samples were taken for RNA extraction. Total RNA was extracted using the Aurum ™ total RNA mini kit according to the manufacturer's instructions (Bio-Rad Laboratories). RNA concentration was measured by Nanodrop spectrophotometry (Thermo Scientific). Immediately after RNA extraction, cDNA synthesis was performed using the iScript cDNA synthesis kit following the manufacturer's instructions (Bio-Rad Laboratories). A 600 ng aliquot of RNA was taken for cDNA synthesis. Reverse transcription was carried out at 42 ° C for 30 minutes with a final denaturation at 85 ° C for 5 minutes.

Quantitative real-time PCR

The expression of the gibberellin and abscisic acid functional genes was estimated by relative quantification using real-time quantitative PCR (QRT-PCR). Several catabolic and biosynthetic genes were selected to evaluate their respective functions in cold and biological stratification. A 131 bp length fragment of wheat actin gwn was used as internal control [Nakamura et al., 2010]. QRT-PCR was performed using an MJ-Mini gradient thermal cycler (Bio-Rad Laboratories) following the manufacturer's instructions. The PCR condition was 1 cycle of 95 ° C for 1 minute and 40 cycles of 94 ° C for 2 0 seconds, 60 ° C for 3 0 seconds, and 72 ° C for 1 min. For real-time PCR, cDNA samples from the treatments were used and all reactions were carried out in three repetitions and two negative controls. Each 25 μΐ reaction contained 18 μΐ of green IQ supermix^TM SYBR® (Bio-Rad Laboratories), 10 pmol of suitable forward and reverse primers, 2.5 μΐ bovine serum albumin, and 25 ng cDNA template. Relative quantification was performed according to Zhang et al. [2007]. Expression levels were calculated using the cycle threshold value (Ct) determined according to a manually adjusted reference line. The difference between the Ct values ​​of the target gene and actin (Ct^objective; - Ct^actin) ae estimated as Ac t and then the expression level was calculated as 2^_ACt. The average values ​​of 2^_ACt They were used to assess the difference in expression between control and stratification treatments. To ensure the specificity and consistency of the amplicons, the analysis of the melting curve and agarose gel electrophoresis was performed after each QRT-PCR run.

Sequencing

Several actin amplicons and the GA and ABA genes were purified using the BioBasic PCR purification kit (Bio Basic Inc.). For each treatment, purified amplicons were sent to sequencing at the Plant Biotechnology Institute (NRC-PBI). The gene sequences were identified by analysis with basic local alignment search tools (BLAST) (http://blast.ncbi.nlm.nih.gov).

Statistic analysis

A variance analysis of the germination percentage and gene expression of data was performed using the IBM SPSS Statistics version 19 software. Differences between control and stratification treatments were examined with the Duncan post-hoc test.

Results and Discussion

Germination percentage and effectiveness

Cold stratification and biological stratification treatments significantly improve the germination rate and the three treatments have a higher percentage of germination than the control (Figure 46A; Table

100

8). The direct endophyte showed a higher germination percentage after each day and increased 60% from day 1 to day 3. During the entire germination period (3 days) it showed significantly higher germination (P &lt;0.05) than the others. Three treatments Only the biological stratification treatments produced more than 50% germination after Day 2. Interestingly, indirect endophyte treatments showed no germination after Day 1, but produced a remarkable 50% germination after Day 2. The cold stratification treatment showed no significant difference with the control after Day 1, and then steadily increased showing a significant difference after day 2 and day 3. The pattern of the increase in germination is also reflected in the values ​​of R². While the control showed a value of R² of 0.40, cold stratification and direct endophyte treatment showed 0.60 and 0.75 respectively. On the other hand, due to its 50% increase from Day 1 to Day 2, indirect endophyte treatment had the highest value of R² of 0.93, which is approximately 2.5 times greater than that of the control. Germination energy is a crucial parameter in determining the ability of seeds to break dormancy and begin germination. Germination energy

101 It is understood as the percentage of germination of the seeds after a certain time or the number of days needed to reach 50% germination. The direct endophyte showed the highest efficacy followed by the indirect endophyte and cold stratification (Figure 46B). As there was no germination in the seeds with indirect endophytes after Day 1, the effectiveness of germination was negative. In general, stratification treatments showed an extremely positive result when reaching 50% germination after 48 hours.

Stratification has an important ecological function in the release of primary latency and the improvement of seed germination [Bewley and Black 1982; Probert et al., 1989]. Relief of seed dormancy and improvement of germination through cold stratification have been achieved in many species, including herbs [Schutz and Rave 1999], mulberry [Koyuncu 2005], pine [Carpita et al., 1983], tobacco [Wu et al., 2008], rice [Mukhopadhyay et al. 2004], and apple [Bogatek and Lewak 1988]. Germination also increased by cold stratification in 33 species of annual herbs and it has been proposed that stratification is even capable of nullifying differences in seed germination between populations [Milberg and Andersson 1998]. Nevertheless,

102 Little information is available on the impact of cold stratification on the germination of wheat seeds. This study found that the effect of cold stratification requires an initial period and therefore the germination of seeds was not significantly different from the control on Day 1. However, it demonstrated considerable impact on germination from day 2 and the percentage Germination increased up to 20% above the control. The period of cold stratification time in this study was selected from previous reports that showed that a 48-hour period is effective for cold stratification in tobacco [Wu et al., 2008] and rice [Mukhopadhyay et al. 2004]. Previous studies have shown that the impact of cold stratification is proportional to its temporal duration [Baskin et al. 1992; Cavieres and Arroyo, 2000]. The findings support this and further extend the notion to conceive that a slightly longer stratification period (~ 4 days) for wheat may be necessary to achieve maximum geminability.

Several reports have demonstrated the improvement of seed germination through the application of fungal endophytes [Vujanovic 2007b; Hubbard et al. 2012; Vujanovic and 'Vujanovic 2007]. The present study supports the concept of mycovitalism, which is the increase in

103 vitality through fungal colonization. Fungal endophytes are known to produce volatile compounds that affect plant phenophases [Mitchell et al., 2009; Strobel et al., 2004]. Therefore, endophytes may be able to affect seed germination, even when they are not in direct contact with the seeds, and this attribute is particularly useful in field conditions. Here, the way in which physical distance can influence the germination of seeds under conditions of biological stratification was also tested. These findings suggest that seeds that are in direct contact with fungal endophytes are undoubtedly more beneficial than their counterparts. The direct endophyte produced a higher percentage and efficiency of seed germination on each day of the germination period. As in contact with the direct endophyte, the seeds placed 4 cm from the endophyte also germinated at a significantly higher rate than in the control. However, the germination percentage and effectiveness were effectively affected by distance and the seeds in indirect contact are between 14% and 27% less than those of direct contact germination. In addition, no germination activity was observed on Day 1 which was followed by a sharp increase (50%) on Day 2. Seed germination is a process

104 extremely complex and its underlying mechanisms are relatively less understood [Nonogaki et al., 2010]. Therefore, it is not clear how fungal endophytes facilitate release of dormancy and the beginning of seed germination. Given that fungi are capable of producing a wide range of substances that promote plant growth, it is possible that these substances are more effective nearby. Consequently, seeds with direct endophytes have a significantly higher germination rate than other treatments. In contrast, seeds with indirect endophytes showed high germination efficiency after 48 hours, this period may have allowed sufficient accumulation of growth promoting substances. There is a difference in the percentage of germination (6.6%) between control treatments and with indirect endophytes on Day 1, however, it is not substantial.

Level of expression of the gibberellin and abscisic acid genes in the tailor

The 2 GA3-oxidase and 14-3-3 genes were selected as the GA biosynthetic gene and negative regulator of the GA biosynthesis pathway respectively [Ji et al., 2011; Zhang et al. , 2007]. The NCED gene well known for its function in the ABA biosynthesis pathway while the ABA 8'- gene

105 Hydroxylase is involved in the catabolic pathway of ABA [Ji et al., 2011]. Quantitative real-time PCR analysis indicated that the differential value (Figure 47) and the expression ratio (Figure 48) of different functional genes varied significantly (P &lt;0.05) between treatments. Except for the 14-3-3 gene on Day 3, detectable expression was observed for all four genes on each day. On Day 1, all genes were regulated by decrease compared to the control. The expression of the GA biosynthesis gene, TaGA3ox2, was considerably higher in cold stratification treatment than in biological stratification. On the other hand, the expression of 14-3-3 did not vary significantly between cold and endophyte treatments although the expression of cold stratification was slightly greater than that of endophytes. The level of transcription of the ABA biosynthesis gene, TaNCED2, did not vary between control and cold stratification, and was regulated by significantly increased until endophyte treatments. The ABA 8'-hydroxylase gene, TaABA8'OHl, showed regulation by significant decrease in the three stratification treatments, with the lowest expression observed in cold stratification. The expression pattern did not vary between treatments with indirect endophytes and direct endophytes. On day 2, the expression of TaGA3ox2 is

106 regulation by decrease more significantly in all stratification treatments than in the control. Expression did not vary between cold stratification and indirect endophyte treatments, and the lowest expression was detected in coleorrizas with direct endophytes. No significant difference was observed for the transcription level of 14-3-3 between the four treatments, although the expression was somewhat higher by virtue of cold stratification. TaNCED2 gene expression was significantly lower in endophyte treatments than control and cold stratification. Similarly, the TaABA8ΌΗ1 gene demonstrated considerable regulation by decreasing stratification treatments with respect to control. The lowest expression was detected in the treatment with indirect endophyte. The transcription level of the TaGA3ox2 gene also varied significantly between treatments on Day 3. Cold stratification showed an expression ten times greater than the control, while the two treatments with endophytes did not vary significantly. In contrast, the TaNCED2 and TaABA8'0H1 genes were significantly regulated in all stratification treatments with the lowest expression in treatments with direct endophytes and indirect endophytes respectively. No detectable expression was observed

107 for the 14-3-3 gene on Day 3.

The ratio of GA and the expression of the ABA biosynthesis gene, TaGA3ox2: TaNCED2, shows no significant difference between treatments on Day 1, but increased steadily thereafter (Figure 48). The indirect endophyte exhibited higher value on Day 2, which is about 5-10 times greater than the other treatments; However, all three stratification treatments demonstrated similar values ​​on Day 3. In contrast, for the relationship of GA biosynthesis and catabolic genes (TaGA3ox2: 14-3-3), the direct endophyte showed the highest value on day 1, followed by indirect endophyte, cold stratification, and control, which is quite similar to its germination percentage. The ratio of the biosynthesis of GA and the ABA catabolic genes, TaGA3ox2: TaABAl, exhibited a similar pattern for all treatments in pia 1, however, the indirect endophyte was considerably higher than others on Day 2. Day 3 , cold stratification and indirect endophyte demonstrated similar level of expression and control was not appreciable. The relationship between ABA biosynthesis and catabolic genes (TaNCED2: TaABAl) did not vary between treatments during the entire germination period although cold stratification showed a slightly higher expression level on day 1.

108

The genes encoding the biosynthesis of GA and ABA and the catabolism enzymes show differential expression patterns based on the accumulation of transcription [Hedden and Phillips, 2000]. Expression patterns of GA3oxl genes have been studied to a large extent from plant species including Arabidopsis [Phillips et al., 1995], rice [Oikawa et al., 2004], and wheat [Zhang et al., 2007]. While other GA biosynthesis genes, such as GA-20ox are associated with growing vegetative tissues, and flowers, GA3ox (GA3ox2 or GA4H) is expressed exclusively during seed germination and supposedly has a crucial function [Phillips et al. ., nineteen ninety five; Yamaguchi et al., 1998; Hedden and Phillips, 2000]. Similar to the previous reports, this study also demonstrated a high expression of the GA3ox2 gene in the wheat rag throughout the germination period. Potentially, without intending to limit itself to any theory, this reflects the consistent production of the bioactive molecule of GA, GA3, in the wheat bulge during germination. On the other hand, the low expression of gene 14-3-3, a negative regulator of GA biosynthesis, was also detected in the backbone. With the growth and gradual increase of seedlings in the endogenous content of GA, the transcription level of 14-3-3

109 It also decreased and finally decreased after Day 2.

Interestingly, the control had the highest level of 14-3-3 followed by cold stratification, indirect endophyte, and direct endophyte, which was reflected in some way in its germinative capacity. These results were in agreement with the previous report by Zhang et al.

[2007], which showed GA biosynthesis and catabolic genes closely linked to GA content and outbreak growth.

Expression patterns of ABA pathway genes have been studied in a wide range of cereals and legumes including rice [Oliver et al., 2007], wheat [Ji et al., 2011; Nakamura et al. , 2010], beans [Qin and Zeevart, 1999]. The present results show that, except for control and cold stratification on day 1, the expression of the TaNCED2 gene did not vary between treatments.

Abscisic acid plays a fundamental role in the pathways of adaptation to stress in the plant [Nakamura et al., 2010]. Since the cold stratification seeds were kept at 4^SC for 48 hours before incubation at room temperature, the abscisic acid content may have been higher. On the other hand, the high expression of TaNCED2 in the control may have resulted in a greater synthesis of ABA and therefore in a rate of

110 slower germination. Recent reports suggest that ABA catabolism occurs mainly in the school [Millar et al., 2006; Okamoto et al., 2006]. In addition, Barrero et al. [2009] reported regulation by increase and the highest expression of ΑΒΑ8ΌΗ-1 in barley bark. As in these reports, here the pattern of high expression of TaABA8ΌΗ1 genes was found in the wheat horn. The ratio of the GA and ABA genes of biosynthesis was closely linked to the germination percentage. Although, TaGA3ox2: TaNCED2 did not vary markedly on Day 1, it was higher in the indirect endophyte on day 2 due to its significant increase. On the other hand, the three stratification treatments showed regulation by a considerable increase in TaGA3ox2: TaNCED2 on Day 3, which may have been reflected in its germination.

The underlying mechanisms of biological stratification are still relatively unknown but could reveal how plant-fungus interactions occur in the early stages of germination. The role of fungal endophytes as biopower enhancers is widely recognized [Arnold et al., 2001; Hubbard et al. 2011;

Saikkonen et al., 1998; Khan et. to the. 2012]. In this study, it has been shown that fungal endophytes can stimulate seed germination significantly, and

111 This mycovitality is proportional to the physical distance between the seed and the fungal endophyte. In addition, the effect of fungal endophyte-mediated biological stratification is considerably greater than that of cold pretreatment. Previous studies have shown that the onset of germination is proportional to the cold stratification time; [Cavieres and Arroyo, 2000b] taking this into account, the following study can be extended to the cold stratification period (&gt; 48 hours) to increase the germinability of the seeds in wheat. Although, cold stratification increased the level of transcription of the ABA biosynthesis gene, the fungal endophytes did not directly stimulate the expression of phytohormones genes in the choor. However, 'this study specifically evaluated. the expression of four genes in the coleorriza.

No study has compared germination patterns under 'cold and biological stratification, and elucidated the biosynthesis of GA and ABA and the expression of catabolic genes in the wheat coleorriza. The coleorriza has recently been demonstrated as a highly active component of the seed in germination [Barrero et al., 2009]. In accordance with this example, high expression of various functional genes was also demonstrated in the wheat seed rack in

112 germination. The germinability of the seed can be substantially improved through the application of fungal endophytes: 1) through indirect mycovitality or without the endophyte-seed contact in the distance studied (for example, the distance of 4 cm was used in this example) and 2) through direct mycovitality or once the endophyte reaches the seed.

Example 13

Effects of endophytic stratification on hormonal regulators (GSR and KAO) and MYB resistance genes

Stratification is the exposure of the seeds to cold and wet conditions in order to break dormancy or improve seed germination. Since stratification is currently limited to the role of abiotic factors, this study aims to make the definition more inclusive by recognizing the role of biotic factors using mycovitality or a fungus-seed symbiosis as a model. This recognizes the existence of both cold and biological stratifications. The germination of wheat seeds subjected to cold stratification at 4 ° C was compared with that of wheat seeds inoculated at room temperature. The seeds were inoculated with the endophytic strain SMCD2206. Changes in gene expression pattern were evaluated

113 seed growth promoters -regulators (GSR and KAO) and hormonal fit © gibberellins (GA); and acquired resistance genes (MYB) in abiotic conditions with respect to biotics, during the first breakage of seed dormancy and germination. Measurements were made in the cells of the cholester using QRT-PCR (as described in Example 12). The results indicate that the RSG and KAO genes (Figure 49), which encode the enzymes that promote GA biosynthesis, and the MYB resistance genes (Figure 49) are regulated in the inoculated seeds. Mycovitality, therefore, demonstrates a reprogramming effect on events before and after germination of wheat seeds towards a breakdown of dormancy and improved germination, effectively contributing to the pre-germination care of crop crops. cereals.

Materials and methods

RNA samples

This study is the continuation of Example 12. The same material (wheat and SMCD 2206) and in vitro methods were used, as well as RNA samples extracted to evaluate the expression of the GSR phytohormone and the KAO regulators and the MYB resistance gene by qRT-PCR.

114

Prior to the extraction of RNA from beginning, the tubes containing colony tissues were stored in liquid nitrogen immediately after they were isolated, the colossal tissues to preserve the cells and prevent denaturation of the RNA. The Aurum ™ total mini kit (Bio-Rad Laboratories) was used in the extraction of total RNA from plant tissues, and it was suggested that a minimum of 20 mg of plant tissues was suitable for each sample. The extraction stages were performed quickly and the entire process was kept on ice, since the RNAs easily denatured at room temperature. The recently extracted total RNA was directly loaded with premixed cDNA synthesis agents obtained from the iScript cDNA synthesis kit (Bio-Rad Laboratories). Reverse transcription was carried out at 42 ° C for 30 minutes with a final denaturation at 85 ° C for 5 minutes in a thermal cycler. The cDNA concentration was measured by Nanodrop spectroscopy (Thermo Scientific) and diluted or concentrated to 100 ng / μΐ.

Quantitative RT-PCR and statistical analysis

Quantitative real-time PCR (QRT-PCR) was performed in a MiniOpticon ™ real-time PCR detection system (Bio-Rad Laboratories) with SYQ® Green Supermixer kit IQ ™ Green (Bio-Rad Laboratories). In order to normalize the data of

115

QRT-PCR, the actin gene (131 bp fragment in length) a reference gene was selected and served as an internal control to avoid fluctuation bias of gene expression under conditions of low cDNA concentration [Zhang et al. 2007; Nicot 2005]. The KAO primer and and the RSG gene according to Zhang et al. [2007] were studied in this experiment, while the original primers were designed for MYB1 and MYB2 based on Triticum aestivum sequences available to the public at (http://compbio.dfci.harvard.edu/cgi-bin/tgi / geneprod_ search.pl) at the Computational Biology and Functional Genomics Laboratory (Harvard University). Newly designed MBY primers (Table 9):

the Myb2 mRNA transcription factor (158 bp) comprising the sequences as shown in SEQ ID NO: 16 and SEQ ID NO: 17 and the Mybl mRNA transcription factor (152 bp) comprising the sequences as shown in SEQ ID NO: 18 and SEQ ID NO: 19 (Table 9).

Samples of 100 ng / μ cDNA were further diluted to 10 ng / μΐ and 2 μΐ of cDNA was used for each 25 μΐ reaction. In addition, 12.5 μΐ of IQ ™ SYBR® green supermix, 8.5 μΐ of sterile milli-Q water, 1 μΐ of each forward and reverse primer (10 pmol) were included in the 25 μΐ reaction mixture. The thermocycle protocol was suggested

116 such as 95 ° C for 10 minutes and 40 cycles of 94 ° C for 20 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 min. All cDNA samples of the treatments were carried out in three repetitions and two negative controls in QRTPCR. Gene expression levels that refer to quantitative curves were carried out using the CFX Manager ™ software (Bio-Rad Laboratories). The quantification cycle value (Cq) from the recorded fluorescence measurements was manually adjusted with the reference line. Relative quantification is the statistical method chosen in this study [Gizinger 2002]. The gene of interest in relation to the endogenous control gene was used to compare with different treatments. The quantification (ACT) was performed with respect to the subtraction of the Cq value of the gene of interest from the Cq value of the control gene. ACT was further subtracted by the value of the calibrator and generated corresponding AACT values ​​that were transformed into log2 (duplication PCR function) to synthesize the relative gene expression levels [Jurado et al., 2010]. The amplified RSG, KAO and MYB genes were purified using the BioBasic PCR purification kit (Bio Basic Inc.) and submitted for sequencing at the Plant Biotechnology Institute (NRC-PBI). Gene sequences were identified by tool analysis.

117 Basic Local Alignment Search (BLAST) (http://blast.ncbi.nlm.nih.gov). Similar or high-identity genes that correspond to different homologous organisms were assembled and aligned using the MEGA5 software (Molecular Evolutionary Genetic Analysis). A tree of phylogeny was performed with the statistical method of joining neighbors based on the aligned genes.

Example 14

Nitric oxide (NO) showed regulatory effect on mycovitalism during early seed germination events

Nitric oxide (NO) is a highly reactive signal molecule common to fungal, animal and plant systems. It is NOT also known as a signaling molecule involved in hormonal signaling of eukaryotic cells [Guo et al. 2003] and plant response to abiotic and biotic stress [Hayat et al. 2010]. Although there is evidence of NO accumulation, the increased activation of SOD and proline that contributes to the delay of O^2- and the accumulation of H2O2 in wheat leaves under saline stress, there is almost no information on fungal endophytes and there is interaction with seed germination (mycovitalism). At present, the occurrence of NO in the early stages of germination of AC Avonlea wheat seeds was investigated.

118 for three days - endophyte SMCD 2206 in PDA, focusing on the radicle's response to fungal diffusible molecules. It was NOT visualized in the radicle (early root organ) in germination cultures by fluorescence microscopy using the specific probe 4,5diaminofluoresce in diacetate; The evaluation was carried out after five minutes of exposure to fungal exudate, as sufficient to induce significant accumulation of NO [Calcagno et al. 2012]. Since, the exudate with SMCD 2206 induced an important production of NO in wheat root tissues; Without wishing to be bound by theory, it is possible that this production is regulated by a molecular dialogue found in the wheat symbiosis.

Materials and methods

The accumulation of NO in the root tissues was analyzed in germinating AC Avonlea wheat seeds (in vitro approach presented in Example 12) using the specific NO-permeable probe in the DAF-2DA cell according to Calcagno et al. [2012] which becomes its fluorescent triazole derivative DAF-2T after reaction with NO. The formation of DAF-2T was visualized by fluorescence microscopy (Cari Zeiss Axioscop 2). AC Avonlea in germination was evaluated at 5 min after treatment with the exudate of fungal SMCD 2206 after the procedure

119 proposed by Nakatsubo et al. [1998].

The specificity of this response to endophytic SMCD 22 06 was confirmed by the lack of response in non-inoculated radical cells. The analyzes were repeated in three independent biological replicas.

Results and Discussion

Seed treatment with fungal exudate may mimic - to some extent - the endophytic hyphae approach during the presymbiotic phase of the interaction, as suggested for the mycorrhiza AM in co-culture with Arabidopsis roots [Calcagno et al. 2010]. Fungal exudate could, therefore, be used with confidence to test whether diffusible fungal signals cause NO accumulation in host wheat tissues (Figure 51) during early germination events that improve mycovitality.

Cellular evidence, therefore, suggests that NO accumulation is a novel component in the signaling pathway that leads to mycosymbiosis related to wheat seed mycovitalism (Figure 51). This finding has both theoretical and practical values ​​in attempts to improve pre-germination care of the plant using endophytic symbionts.

Example 15

120

Study of the effects of endophytes on phytoremediation and phytoremediation

Phytoremediation is a promising environmental technique. It has been shown to be profitable for the recovery of hydrocarbons / petroleum, salts, heavy metals and soils contaminated with radioisotopes. In this study, the effects of conifers (Picea or Pinus) and deciduous trees (Salix or Populus), shrubs (Caragana or krascheninnikovia) and grasses (Festuca or Elymus) infected (E +) and uninfected (E-) will be investigated ) by endophytic organisms (through infection and colonization of plant propagation material, seeds or roots) (SMCD 2204, 2206, 2208, 2210 and 2215) in decomposition, transformation or degradation of petroleum hydrocarbons in soil contaminated with petroleum. The plants will be grown in pots with contaminated oil and uncontaminated soil. The plants will be inoculated and incubated for 6 months using the greenhouse method suggested by Soleimani et

<td>to the. (2010).</td><td>The flowerpots</td><td>no plants will be</td><td>used</td><td>how</td>

<td>control. To the</td><td>end of the</td><td>experiment is</td><td>will analyze</td><td>the</td>

<td>colonization</td><td>root plant</td><td>(Abdellatif et</td><td>to the. 2009),</td><td>the</td>

soil hydrophobicity (Chau 2012), total petroleum hydrocarbons (TPH), and polycyclic aromatic hydrocarbons (PAH) contained (Germida et al. 2010). I know

121 It will compare the difference between the root and shoot biomass of E + and E- plants and the photosynthesis of the leaf (Hubbard et al. 2012) with the elimination of PAH and TPH in the rhizosphere of plants. Unplanted pots will be used as a control to calculate the efficacy of symbiotic plants (E +) on the degradation of petroleum hydrocarbons (Soleimani et al. 2010). Infected plants will decompose, transform or degrade hydrocarbons and salts, capture and accumulate and clean or eliminate heavy metals and radioisotopes at the contaminated site, soil or environment.

While the present description has been described with reference to what is currently considered the examples, it should be understood that the description is not limited to the described examples. On the contrary, the description is intended to cover several modifications and equivalent provisions included within the spirit and scope of the appended claims.

All publications, patents and patent applications cited herein are fully incorporated by reference to the same extent to which they would be incorporated if it were indicated that each publication, patent or patent application was specifically incorporated herein in its totality by reference.

122

Table 1:

ITS rDNA 2204

CCTATAGCTGACTGCGGAGGGACATTACAAGTGACCCCGGTCTAACCACCGGGATG TTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGG CTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAA ATTAAAACTTTTAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGC GATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCC CCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTCGCTTGG TATTGGGCAACGCGGTCCGCCGCGTGCCTCAAATCGACCGGCTGGGTCTTCTGTCCCCTAA GCGTTGTGGAAACTATTCGCTAAAGGGTGTTCGGGAGGCTACGCCGTAAAACAACCCCATT TCTAAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGG AGGAAAAGAAACCAACAGGGATTGCCCCAGTAACGAA (SEQ ID NO: 1)

ITS rDNA&gt; 2204F

TCGATCTAGCTCATAGTGACTGCGGAGGGACATTACAAGTGACCCCGGTCTAACCA CCGGGATGTTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCG GGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTA ATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG CGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCAC ATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTC ACCACTCAAGCC TCGCTTGGTATTGGGAAACGCGGACCGACGCGTGCCTCAAATCGACCGGCAGGGTCTTCTG TCCCCTAAACGTTGTGAAAATTATTCGATAAAGGATGTTCCGTGCTACATTGTGAATAGAA CCGCATTTATAACATTGATTATAAACTAATTACGACTACATGGTAAGATAGATATATCAAG GAACTTCCTCTAAATGACCAAGAAACC (SEQ ID NO: 2)

123

ITS rDNA&gt; 2206

TCGACGGCGTATCCTAGTGACTGCGGAGGATCATTACCGAGTGAGGGCCCTCTGGG TCCAACCTCCCACCCGTGTTTAATTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCC GCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAA GATTGTAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTC CGGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAAATTCAGTGAAT CATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGC GTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTCCGATCCCGGGGGACG GGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCG CTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGA TCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA (SEQ ID NO: 3)

ITS rDNA&gt; 2208

TAACTGATTTGGCGGACTGGCGGAAGGACATTAAAGAGACGTTGCCCTTCGGGGTA TACCTCCCACCCTTTGTTTACCTTTTCCTTTGTTGCTTTGGCGGGCCCGTCCTCGGACCAC CGGTTTCGGCTGGTCAGTGCCCGCCAGAGGACCTAAAACTCTGTTTGTTCATATTGTCTGA GTACTATATAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAA CGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAA CGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTdATTACAACCCTC AAGCTCTGCTTGGTATTGGGCTCTGCCGGTCCCGGCAGGCCTTAAAATCATTGGCGGTGCC ATTCGGCTTCAAGCGTAGTAATTCTTCTCGCTTTGGAGACCCGGGTGCGTGCTTGCCATCA ACCCCCAATTTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA TCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGTCCCAATAACGAATTTATAAATAATA

124 (SEQ ID NO: 4)

ITS rDNA&gt; 2210

TCGAGAGTTCGGACTAAGTGCCTGATCCGAGGTCAAGACGGTAATGTTGCTTCGTG GACGCGGGCCACGCCCCCCCGCAGACGCAATTGTGCTGCGCGAGAGGAGGCAAGGACCGCT GCCAATGAATTTGGGGCGAGTCCGCGCGCGAAGGCGGGACAGACGCCCAACACCAAGCAGA GCTTGAGGGTGTAGATGACGCTCGAACAGGCATGCCCCATGGAATACCAAGGGGCGCAATG TGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACACTACTTATCGCATTTCGC TGCGTTCTTCATCGATGCCAGAGCCAAGAGATCCATTGTTGAAAGTTGTAACGATTGTTTG TATCAGAACAGGTAATGCTAGATGCAAAAAAGGTTTTGTTAAGTTCCAGCGGCAGGTTGCC CCGCCGAAGGAGAACGAAAGGTGCTCGTAAAAAAAGGATGCAGGAATGCGGCGCGTGAGGG TGTTACCCCTACCACCCGGGAGAGAACCCCCGAGGGCCGCGACCGCACCTGGTTGAGATGG ATAATGATCCTTCCGCAGGTTCACCTACGGAAACC (SEQ ID NO: 5)

RDNA 16S&gt; 2215

CCGGGGGCACTCCACTGCGTATGTGTGACGAGTAGACCGCTGCGCTTAGCTGAGGT CTGATGAAATGTAGAACACTTAACAAAAATATGCCCGGATGGATATACTTTTCAACGACAG GGCTGCGATTGGATGATCTCCTTTGAAACACAGAACTAGTCACGGCGACGAATACTCAACT TCGACCCCCCCCCTTTCTGGAGGCGCGTCTTAGTCCCCTCCTTGATGGAGCTGCCCCGTGC TCGGCGGCCGGAGTCGGCGGTGTTTTCCGCTGTACCTGAGACGCTGGACCAACTCCTTCGG GAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAG GGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACC TGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCG TTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGATTGTGAAAGC CCGAGGCTTAACCTCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTGTGGTAGGGGAGATC GGAATTCCTGGTGTAGCGCGCGCGCGCGCGCGCGCGCGG

125

GATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATA CCCTGGTAGTCCACGCCGTAAACGGTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGG TGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAA AGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAA GAACCTTACCAAGGCTTGACATACACCGGAAACATCCAGAGATGGGTGCCCCCTTGTGGTC GGCGTACAGGTCGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTAAGTCCCGC AACGAGCGCAACCTTGTTCTGGTGCTGCCAGCATGCCCTTCGGGTGATGGGÁCTTCACCAC GGAGACCGCGGCTCCACTCCGACGAGGTGGGGGACGACGTCAGTCATCATGCCCTAATGTC TGGCTG (SEQ ID NO: 6)

Table 2: Frequency of endophytic root colonization by SMCD evaluated in wheat germination 3D germination.

<td>Endophytes</td><td>SMCD2204</td><td>SMCD2206</td><td>SMCD2210</td><td>SMCD2215</td>

<td> %</td><td> 43</td><td> 40</td><td> 49</td><td> 48</td>

<td>colonization</td><td></td><td></td><td></td><td></td>

Table 3 - Germination energy (EG) and hydrothermal time (HTT) of wheat seeds grown in hot conditions (36 ° C), drought (potato and dextrose agar medium (PDA) plus polyethylene glycol (PEG) 8000 to 8 %), heat and drought, combined and in vitro control. Within a column, the data followed by an asterisk (*) are significantly different from control without endophyte

126 (ρ 0.05; ANOVA, followed by the post-hoc LSD test). Note: The seeds used in the determination of EG and HTT were taken from the second round of experiments, and therefore underwent sterilization in 5% sodium hypochlorite for one minute, instead of three; SMCD - Saskatchewan microbe collection and database.

<td></td><td colspan="2">Hot</td><td colspan="2">Drought</td><td colspan="3">Heat and drought</td><td colspan="2">Control</td>

<td>Endophyte</td><td>EG</td><td>HTT at</td><td>EG</td><td>HTT at 50</td><td>EG (days)</td><td></td><td>HTT at</td><td>EG (days)</td><td>HTT at 50</td>

<td></td><td>(days)</td><td>50% of</td><td>(days)</td><td>% of the</td><td></td><td></td><td>50% of</td><td></td><td>% of the</td>

<td></td><td></td><td>the</td><td></td><td>germinated</td><td></td><td></td><td>the</td><td></td><td>germinate</td>

<td></td><td></td><td>germinac</td><td></td><td>n (MPa</td><td></td><td></td><td>germinac</td><td></td><td>ón (MPa</td>

<td></td><td></td><td>ion (MPa</td><td></td><td>days ° C)</td><td></td><td></td><td>ion (MPa</td><td></td><td>days ° C)</td>

<td></td><td></td><td>days ° C)</td><td></td><td></td><td></td><td></td><td>days ° C)</td><td></td><td></td>

<td></td><td> 3,7 ±</td><td> 91 ± 7</td><td> 2,9 ±</td><td> 52 ± 5</td><td> 2,0 ± 0,8</td><td colspan="2"> 22 ± 8</td><td> 1,6 ± 0,2</td><td> 65 + 8</td>

<td>SMCD 2204</td><td> 0,3</td><td></td><td> 0,3</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 2,5 ±</td><td> 62 + 7</td><td> 1,9 ±</td><td> 34 ± 2 *</td><td> 2,0 ± 0,8</td><td colspan="2"> 22 ± 8</td><td> 1,5 ± 0,2</td><td> 61 ± 8</td>

<td>SMCD 2206</td><td> 0,3</td><td></td><td> 0,1 *</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 3,7 ±</td><td> 91 ± 7</td><td> 3,0 ±</td><td> 53 ± 5</td><td> 4,0 ± 1,0</td><td colspan="2"> 43 ± 10</td><td> 1,6 ± 0,2</td><td> 65 ± 8</td>

<td>SMCD 2208</td><td> 0,3</td><td></td><td> 0,3</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 1,8 _ +</td><td> 44 ± 5 ‘</td><td> 2,2 ±</td><td> 39 ± 3 *</td><td> 1,0 ± 0,5</td><td colspan="2"> 11 ± 5</td><td> 1,6 + 0,2</td><td> 65 ± 8</td>

<td>SMCD 2210</td><td> 0,2 *</td><td></td><td> 0,2 *</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 2,5 ±</td><td> 62 ± 7</td><td> 2,3 ±</td><td> 41 ± 3 *</td><td> 1,3 ± 0,2</td><td colspan="2"> 14 ± 2</td><td> 1,5 ± 0,2</td><td> 61 ±-8</td>

<td>SMCD 2215</td><td> 0,3</td><td></td><td> 0,2 *</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 3,8 ±</td><td> 94 ± 11</td><td> 4,5 ±</td><td> 80 + 8</td><td> 3,0 ± 1,5</td><td colspan="2"> 32 ± 15</td><td> 1,6 ± 0,2</td><td> 65 ± 8</td>

<td>No endo</td><td> 0,5</td><td></td><td> 0,5</td><td></td><td></td><td></td><td></td><td></td><td></td>

Table 4 - Endophytes increase the efficacy of drought tolerance (DTE) and yield on barley and wheat under stress.

127

<td>Culture</td><td>Genotype·</td><td></td><td colspan="3">Control condition</td><td colspan="3">Drought stress</td>

<td></td><td></td><td>OTE * (%)</td><td colspan="2">Average spike performance (3 plants / pot)</td><td>Increase %</td><td colspan="2">Average spike performance (3 plants / pot)</td><td>Increase %</td>

<td></td><td></td><td></td><td>E -</td><td>E +</td><td></td><td>E -</td><td>E +</td><td></td>

<td></td><td>AC Avonlea (Cont)</td><td> 16,1</td><td> 18, 27</td><td> 25, 52</td><td> 28,41</td><td> 2,94</td><td> 10,62</td><td> 72,32</td>

<td></td><td>PT control 580</td><td> 57,3</td><td> 23,42</td><td> 32,60</td><td> 28,16</td><td> 13,38</td><td> 21,53</td><td> 37,85</td>

<td></td><td>Utmost ve with CDC</td><td> 72,3</td><td> 20, 55</td><td> 35,4</td><td> 41,95</td><td> 16,67</td><td> 29,8</td><td> 44,06</td>

<td></td><td>Strongfield</td><td> 75,6</td><td> 13, 54</td><td> 16,77*</td><td> 19,26</td><td> 10,23</td><td> 14,98</td><td> 31,71</td>

<td>WHEAT</td><td>VB unit</td><td> 75,3</td><td> 20, 72</td><td> 26. 6</td><td> 22,11</td><td> 15,61</td><td> 23,2</td><td> 32,72</td>

<td></td><td>Teal with CDC</td><td> 76,9</td><td> 19, 51</td><td> 30,37</td><td> 35,76</td><td> 14,90</td><td> 25,1</td><td> 40,64</td>

<td></td><td>Carberry</td><td> 83,8</td><td> 17,31</td><td> 33,07</td><td> 47,66</td><td> 14,52</td><td> 22,9</td><td> 36,59</td>

<td></td><td>BW 423</td><td> 85,0</td><td> 13,26</td><td> 25,83</td><td> 48,66</td><td> 12,28</td><td> 21,41</td><td> 42,64</td>

<td></td><td>Veronna with CDC</td><td> 87,8</td><td> 15, 35</td><td> 22,58</td><td> 32,02</td><td> 13,49</td><td> 20,16</td><td> 33,09</td>

<td></td><td>Lillian</td><td> 87,8</td><td> 20, 50</td><td> 28,3</td><td> 27,56</td><td> 18,1</td><td> 23,6</td><td> 23,31</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Do racing barley</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Copeland with CDC</td><td> 4,9</td><td> 6,01</td><td> 10,78</td><td> 44,25</td><td> 2,91</td><td> 6,95</td><td> 58, 13</td>

<td></td><td>Kendall with CDC</td><td> 13,2</td><td> 9,93</td><td> 24, 19</td><td> 58,95</td><td> 0,32</td><td> 1,03</td><td> 68,93</td>

<td>Barley</td><td>AC Metcalfe</td><td> 43,2</td><td> 16, 5</td><td> 22,4*</td><td> 26,34</td><td> 7,3</td><td> 14,05</td><td> 48,04</td>

<td></td><td>New dale</td><td> 72,1</td><td> 9,55</td><td> 26,88</td><td> 64,47</td><td> 6, 89</td><td> 12,17</td><td> 43,39</td>

<td></td><td>Six-stroke barley ·</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Legacy</td><td> 1,1</td><td> 20, 42</td><td> 26,87»</td><td> 24,00</td><td> 2,26.</td><td> 2,38*</td><td> 5,04</td>

<td></td><td>Bold with CDC</td><td> 57,0</td><td> 9,16</td><td> 19, 9</td><td> 53,97</td><td> 5,22</td><td> 7,5</td><td> 30,40</td>

128 * Effectiveness of drought tolerance (DTE) = (performance under stress / performance without stress) xlOO; presented in increasing order within the Table. Genotypes with high DTE are considered drought resistant; while genotypes with low DTE are considered as susceptible to drought. Note: The effect of the absence (E-) or presence (E +) of endophytes on genotype performance was calculated as an average of the three strains studied SMCD 2206, SMCD 2210 and SMCD 2215.

Within the ranks, an average is not statistically significant apk 0.05.

Table 5 - Maximum Rhizobium sequence identity against the GenBank database

<td>Access</td><td>Description</td><td>Score Max</td><td>Score Total</td><td>Coverage of the search</td><td>E value</td><td>Ident Max</td>

<td>EF549401.1</td><td>16S ribosomal DNA gene of Rhizobium CCBAU 83431 species, partial sequence</td><td> 1007</td><td> 1007</td><td> 46 %</td><td> 0,0</td><td> 99 %</td>

Natural nodulation rhizobium in interaction with

Streptomyces SMCD2215

16S F (Golden) Rhizobium species

129

GGAAGGGGGGCGGCTTACCATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACG

GGTGAGTAACGCGTGGGAATCTACCCTTGACTACGGAATAACGCAGGGAAACTTGTGCTAA TACCGTATGTGTCCTTCGGGAGAAAGATTTATCGGTCAAGGATGAGCCCGCGTTGGATTAG CTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATC AGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTG GACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTA AAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGT GCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGCCGTAAAGCG CACGTAGGCGGATCGATCAGTCAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGTCTTT GATACTGTCGATCTGGAGAACTTCCTGCTCGAGTGATTTACCCACATGGCGAGCACCGGCA CCCCGTTTCGACATGCAAAAAATGATGCCCAGGCTTATGTTTGÁCCTGGCTGCTACGGCTC TCTTCGGCGTGGACCCCGGCCTCCTATCCCCGGAGATGCCACCCATGGACGCCGCAGTCTC CATGGATATATCATGGAGGTGGGTTTTCTCCGACTCATGATGCCGGCTTCTTGCTGGAAGT TGATGAAGCAACTAAACATCAGCCCTGAGAGAAAGCTTCGCATGCCGCGCAGGGTGCTCCG AGTGTTCGTCTGGAGATGATGAAATAGACGAAGATCATCTCATGTCATGTTGGTAACGACG AGAACAAGATGGTGTGGATTTTGTGTCTTCCATCCTCCATGACCCTGACGATGCTGATGAT GACGTGGTTCATGCTATGATGACTCGATACTGGTCGCTGCAAGCGGATACAGTTGGGACCT ACCGCTAACATGGTTCTTTCTACAACCTCCCCCCAAACCGCATAGGATCGTGGTCAATCAT TCGGCACGAACCTCTTCCCCCATTGCCTCCAACTAGTTTATCGCTCTAGAGTTGGGGAGCC CTGTGTGACCTTTCGTACGCGA 7TQG

Table 6 - Set of SOD, MnSOD and Pro primers used to evaluate pea expression genes [Handel] exposed to stress by still with PEG / osmotic using qpcr

130

<td>Gene name</td><td>Primer</td><td>Reference</td>

<td>internal control with PP2A</td><td>CCACATTACCTGTATCGGATGACA (F) (SEQ ID NO: 8) GAGCCCAGAACAGGAGCTAACA (R) (SEQ ID NO: 9)</td><td>Die et. Al, Plant (2010) 232: 145-153</td>

<td>saline with MnSOD and by drought</td><td>gcagaaaaaccctatcctccgtgct (F) (SEQ ID NO: 10) gctccaaagctccgtagtcg (R) (SEQ ID NO: 11)</td><td>Wong Vega et.ál., Plant Mol. Biol. 17 (6), 1271- 1274 (1991)</td>

<td>SOD pea</td><td>ctgtactcgctgttggggtg (F) (SEQ ID NO: 12) gcatggatatggaagccgtg (R) (SEQ ID NO: 13)</td><td>Nakamura et.ál., Plant Biotechnol 20, 247-253 (2003).</td>

<td>Proline (Pro)</td><td>aatggccgaaagcattgcca (F) (SEQ ID NO: 14) aaggacggtgatgccgatggactc (R) (SEQ ID NO: 15)</td><td>Williamson, CL and Slocum, RD, Plant Physiol. 100, 1464-1470 (1992).</td>

Table 7

Evaluation of the effectiveness of seed sterilization methods.

The seeds were germinated in potato and dextrose agar for 4 days at room temperature (20 ° C). Each petri dish had 10 seed of

131 wheat

<td>Sterilization type</td><td>from</td><td colspan="2">Potato and dextrose aerar (PDA)</td>

<td colspan="2"></td><td>Pollution</td><td>Germination</td>

<td colspan="2">Control</td><td> 50 %</td><td> 80 %</td>

<td colspan="2">50% bleach</td><td> 0</td><td> 50 %</td>

<td>Ethyl alcohol</td><td>to the</td><td> 0</td><td> 70 %</td>

<td> 95 %</td><td></td><td></td><td></td>

<td>50% bleach</td><td> +</td><td> 0</td><td> 50 %</td>

<td>Ethyl alcohol</td><td>to the</td><td></td><td></td>

<td> 95 %</td><td></td><td></td><td></td>

<td colspan="2">Chlorine gas</td><td> 0</td><td> 80 %</td>

Table 8 - Average germination of wheat seeds under cold and biological stratification treatments

<td>Day</td><td>Control</td><td>Cold stratification</td><td>Indirect endophyte</td><td>Endophyte-direct</td>

<td> 1</td><td> 6,66 ± 6,66^</td><td> 16,6 ± 3,33^ab</td><td> 0,00 ± 0,00^to</td><td> 26,6 ± 12,02^b</td>

<td> 2</td><td> 16,6 ± 8,81®</td><td> 40,0 + 11,5^P«</td><td> 50,0 ± 5,77«</td><td> 66,6 ± 8,81«</td>

<td> 3</td><td>33.3 ± 12.0 IV</td><td> 53,3 + 8)81^</td><td>73.3 ± 3.33 zz</td><td> 86,9 ± 7,24 ^z</td>

* The Duncan test was performed to study the significant difference between treatments (Control,

132 cold stratification, indirect endophyte, and direct endophyte) on day 1 (a, b, c), day 2 (p, q), and day 3 (x, y, z)

Different letters indicate significant difference P &lt;0.05.

Table 9

Myb2 mRNA transcription factor (158 bp)

TaMyb2 1F acatcaagcgcggcaacttca (SEQ ID NO: 16)

TaMyb2 IR gagccgcttcttgaggtgggtgt (SEQ ID NO: 17)

Mybl mRNA transcription factor (152 bp)

TaMybl 1F ccagggaggacggacaacga (SEQ ID NO: 18)

TaMybl IR ctctgcgccgtctcgaagga (SEQ ID NO: 19)

133

REFERENCES

Abdellatif et al. 2009. Mycological Research, 113: 782791.

Abdellatif et al. 2010. Can JPlant Pathol, 32: 468-480.

Adriaensen et al. 2006. Mycorrhiza, 16: 553-558.

Agius et al. 2006. PNAS, 103: 11796-11801.

Ali et al. 1994. Annals of Applied Biology, 125: 367375.

Alien 1958. Forest Chron, 34: 266-298.

Armas et al. 2004. Ecology, 85: 2682-2686.

Arnold et al. 2001. Mycological Research, 105: 15021507.

Bacon and White 2000. In: Bácon CW and White JFJ (Eds), Microbial endophytes. Marcel Dekker Inc; New York, NY, USA. 237-263.

Bae et al. 2009. J Exp Bot 60: 3279-3295.

Baird et al. 2010. Mycorrhiza. 20: 541-549.

Barrero et al. 2009. Plant Physiology, 150: 1006-1021.

Baskin et al. 1992. International Journal of Plant Sciences, 153: 239-243.

Baskin and Baskin 2004. Sci. Res., 14: 1-16. ·

Bewley and Black 1982. Physiology and Biochemistry of

Seeds 2. Viability, Dormancy, and Environmental Control.

134

Springer-Verlag, Berlin.

Bloom and Richard 2002. ASAE document No. 027010. ASAE, St. Joseph, MI.

Bogatek and Lewak 1988. Physiology Plantarum, 73: 406411.

Boyko and Kovalchuk 2008. Environmental and Molecular Mutagenesis, 49: 61-72.

Bradford 2002. In: J. Kigel J, Galili G (eds), Seed Develop and Germin. Marcel Dekker Inc, New York, p. 351396.

Calcagno et al. 2012. Mycorrhiza, 22: 259-69.

Cao and Moss. 1989. Crop Sci, 29: 1018-1021.

Carpita et al. 1983. Physiology Plantarum 59: 601-606.

Cavieres and Arroyo, 2000. Plant Ecology 149: 1-8.

Cavieres and Arroyo, 2000b. Botanical Gayana 64: 40-45.

Charlton et al. 2008. Metabolomics, 4: 312-327.

Chau et al. 2012. Fungal Biology, 116: 1212-1218.

Chipanshi et al. 2006. Clim Res, 30: 175-187.

Choi y Sano, 2007. Molecular Genetics and Genomics, 277: 589-600.

Davitt et ál. 2010. New Phytol, 188: 824-834.

de Bary 1866. Vol. II. Hofmeister's Handboo k of

Physiological Botany. Leipzig, Germany

.Defeux et al., 2000. Plant Physiology, 123: 895-904.

135'

Dong-dong et al. 2009. J Zhejiang Univ-Sci B, 9: 964968.

Farquhar and Richards 1984. Australian Journal Plant Physiology 11: 539-552.

Farquhar et al. 1989 In: Jones HG, Flowers TJ and Jones MB (Eds) Plants under stress. Cambridge University Press, Cambridge, p 47-69.

Farquhar et al. 1989b. Annual Review of Plant Physiology and Plant Molecular Biology. 40: 503-537.

Finnegan et al. 1998. Plant Molecular Biology 95: 58245829.

Freeman 1904. Philosophical Transactions of the Royal Society London (Biology) 196: 1-27.

Friend et al. 1962. Can J Bot, 40: 1299-1311.

Gan et al. 2004. Can. J Plant Sci, 84: 697-704.

Germida et al. 2010. Scale evaluation in the field of battery recovery in an old oil tank in Bruderheim, Alberta. Withgreso Mundial de la Ciencia del Suelo, Soil Solutions for a Changing World, 1-6 agosto de 2010. Brisbane, Australia. Disponible en línea en: http://www.iuss.org/19th%20WCSS/Symposium/pdf/0694.pdf

Gizinger 2002. Experimental Hematology, 30: 503-512.

Gornall et ál. 2010. Phil. Trans. R. Soc. B, 365, 29732989.

136

Grant et al. 2009. Tree Physiology, 29: 1-17.

Guimnerson 1986. J Exp Bot, 37: 729-741.

Gundel et ál. 2010. Evol Appl, 3: 538-546.

Guo et ál. 2003. Science, 302: 100-103.

Hayat et ál. 2010. Nitric Oxide in Plant Physiology, Issue 58, Willey-VCH Verlag, Alemania.

Hedden y Phillips, 2000. Trends in Plant Science, 5: 523-530.

Hubbard et ál. 2011. En: Wheat: Genetics, Crops and Food Production. Nova Science Publishers Hauppauge, NY, EUA. p. 333-345.

Hubbard et ál. 2012. Botany, 90(2): 137-149.

Jame et ál. 1998. Agrie Forest Meteorol 92: 241-249.

Ji et ál., 2011. Plant Physiology, 156: 647-662.

Johannes et ál. 2009. Píos Genetics 5: el000530.

Johannes et ál. 2011. Genetics, 188: 215-227.

Johnson et ál. 1990. Crop Science, 30: 338-343.

Jost et ál. 2001. Nucleic Acids Research, 29: 4452-4461.

Jumpponen y Trappe 1998. New Phytologist, 140: 295-310.

Jurado et ál., 2010. Food Microbiology, 27: 50-57.

Kane 2011. Environmental and Experimental Botany, 71: 337-344.

Kang et ál. 2008. International Journal of Sustainable Development and World Ecology, 15: 440-447.

137

Karavata &amp; Manetas 1999. Photosynthetica, 36: 41-49.

Khan et ál. 2010. Pakistán Journal of Botany, 42: 259267.

Khan et ál. 2012. BMC Microbiol, 12; 12:3.

Kiffer y Morelet 2000. Science Publisher Inc, Enfield, NH, Plymouth.

Kóchy y Tielborger 2007. Basic Appl Ecol 8: 171-182.

Koyuncu 2005. Acta Biológica Cracoviensia Series

Botánica, 47: 23-26.

Labeda et ál., 2012. Antonie van Leeuwenhoek, 101:73104.

Lang-Mladek et ál. 2010. Molecular Plant, 3: 594-602.

Larran et ál. 2002. World Journal of Microbial Biotechnology, 18: 683-686.

Leone et ál. 1994. Physiol Plantarum, 92: 21-30.

Li et ál. 2008. Ecological Research, 23: 927-930·.

Li et ál. 2011. Agronomy Journal, 103: 1619-1628.

Lu et ál. ‘2007. Plant Biology, 49: 1599-1607.

Lucht et ál. 2002. Nature Genetics, 30: 311-314.

Madsood et ál. 2005. Engineering Applications of

Artificial Intelligence, 18: 115-125.

Margulis, 1991. En Symbiosis as a Source of Evolutionary

Innovation, L. Margulis y R. Fester, ed. The MIT Press: Cambridge, pp. 1-14.

138

Márquez et ál. 2007. Science, 315: 513-515.

McCormick MC, Siegel (eds.) 1999. Prenatal Care: Effectiveness and implementation. Cambridge University Press uk .

McDonald 2009. Handbook of biological statistics. 2.^aed. Sparky House Publishing, Baltimore, Maryland·

McMaster 2009. En: Carver BF (ed), Wheat, science and trade, Wiley-Blackwell, lowa, EUA, p. 31-55.

Milberg y Andersson 1998. Plant Ecology, 134: 225-234.

Millar et ál., 2006. Plant Journal, 45: 942-954.

Miransari et ál. 2011. Applied Microbiology and

Biotechnology, 92: 875-885.

Mitchell et ál., 2009. Microbiology-SGM, 156: 270-277.

Mühlmann y Peintner 2000. Mycorrhiza, 18: 171-180.

Mukhopadhyay et ál., 2004. PNAS, 101: 6309-6314.

Nakatsubo et ál. 1998. FEBS Lett 427:263-266.

Nakamura et ál., 2010. Euphytica, 171: 111-120.

Nelson, 2004. Annu Rev Phytopathol, 42: 271-309.

Nicot 2005. Environmental and Experimental Botany, 56: 2907-2914.

Nonogaki et ál., 2010. Plant Science, 179: 574-581.

Oikawa et ál., 2004. Plant Molecular Biology, 55: 687700.

Okamoto et ál., 2006. Plant Physiology, 141:97-107.

139

Oliver et ál., 2007. Plant and Cell Physiology, 48: 1319-1330.

Penterman et ál. 2007. PNAS, 104: 6752-6757.

Phillips et ál., 1995. Plant Physiology, 108: 1049-1057.

Probert et ál., 1989. Environmental and Experimental Botany, 40: 293-301.

Qin y Zeevart, 1999. PNAS, 96: 15354-15361.

Reynolds et ál. 2007 Journal of Experimental Botany, 58: 177-186.

Richards et ál. 2002. Crop Science, 42: 111-121.

Ries et ál. 2000. Nature, 406: 98-101.

Rivero et ál. 2011. Internacional Conference on

Arabidopsis Research. June 22-25, Madison EUA.

Rúan et ál. 2002. Seed Sci Technol, 30: 61-67.

Ryan et ál. 2008. FEMS Microbiol Lett, 278: 1-9.

Saikkonen et ál., 1998. Annual Review of Ecology and Systematics, 29: 319-343.

Saze 2008. Seminars in Cell and Developmental Biology, 19: 527-536.

Schrey y Tarkka 2008. Antonie van Leeuwenhoek, 94:11-19.

Schutz y Rave 1999. Ecology, 144: 215-230.

Semenov y Shewry 2011. Scientific Reports, 1: 66-71.

Sinclair et ál. 1984. BioScience, 34: 36-40.

Singh et al. 2011. Plant Signal Behav, 6: 175-191.

140

Smith y Read 2008. Mycorrhizal symbiosis, tercera Edición. Elsevier Ltd. Mycorrhizas in acholorophyllous plants (mycoheterotrophs). Capítulo 13: 458-507.

Solaiman et ál. 2010. Australian Journal of Soil

Research, 48: 546-554.

Soleimani et ál. 2010. Chemosphere, 81: 1084-1090.

Stone et ál., 2000. En: Bacon, C. W. y White, J. F. eds., Microbial Endophytes, Marcel Dekker: Nueva York Capítulo 1: 3-29.

Strobel et ál., 2 004. Journal of Natural Products, 67: 257-268.

Sun et ál. 2010. Journal of Plant Physiolog, 167: 10091017 .

Tan y Zou, 2001. Nat Prod Rep, 18: 448-45.

Tokala et ál. 2002. Appl Environ Microbiol, 68:21612171.

Vaughn et ál. 2007. PloS Biology, 5: 1617-1629.

Verhoeven et ál. 2010. New Phytologis, 185: 1108-1118.

Vujanovic et al. 2000. Annals of Botany, 86: 79-86.

Vujanovic y Brisson 2002. Mycological Progress. 1: 147154.

Vujanovic y Vujanovic 2006. Floriculture, Ornamental and

Plant Biotech, 63: 563-569.

Vujanovic y Vujanovic 2007. Symbiosis, 44: 93-99.

141

Vujanovic 2007b. Can J Plant Pathol, 29: 451-451.

Vujanovic 2008. 19.^a Conferencia Internacional sobre

Arabidopsis. Research Proceedings- ICAR13, Julio 23-27, Montreal, QC, Canadá.

Waller et ál. 2005. PNAS, 102: 13386-13391.

Wallin 1927. Symbionticism and the Origin of Species. Londres: Bailliére, Tindall y Cox.

Wang et ál. 2011. Journal of Experimental Botany, 62: 1951-1960.

Whalley et ál. 2006. Plant and Soil, 280: 279-290.

White y Torres 2010. Physiol. Plant, 138: 440-446.

Wu et ál. 2008. Plant Physiology, 148: 1953-1963.

Wu y Sardo 2010. Lichtfouse E. (Ed.), Sociology, Organic Farming, Climate Change and Soil Science. Sustainable Agriculture Reviews. 3: DOI 10.1007/978-90-481-3333-8_3.

Yamaguchi et ál. 1998. Plant Cell 10: 2115-2126

Yang et ál., 2002. Planta, 215: 645-652.

Zadoks et ál. 1974. Weed Research, 14:415-421.

<td>Zhang</td><td>et</td><td>ál.,</td><td> 2007.</td><td>BMC Genet,</td><td> 2007, 8:</td><td> 40.</td>

<td>Zhang</td><td>et</td><td>ál.</td><td> 2010.</td><td>Journal of</td><td>Cereal</td><td>Science, 52: 263-</td>

<td> 269.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Zhang</td><td>et</td><td>ál</td><td> . 2011</td><td>African</td><td>Journal</td><td>of Microbiology</td>

Research, 5: 5540-5547.

Zhao et ál. 2007. Journal of Plant Nutrition, 30: 947142

963.Zhong et ál. 2009. African Journal of Biotechnology, 8:

6201-620.

Zhu et ál. 2007. Current B.iology, 17: 54-59.

Foresight. The future of food and farming: challenges and choices for global sustainability. Final Project Report.

Londres: The Government Office for Science, UK, 2011.

IPCC Climate Change 2007. En: Solomon S, Qin D, Manning

M, Chen Z, Marquis M, Averyt KB, Tignor M y Miller HL (Eds) .

Cambridge University Press, Cambridge, UK.

Saskatchewan Ministry of Agriculture 2008. Varieties of

Grain Crops. SaskSeed guide. Regina, SK, Canadá.