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    • 专利摘要:
    compositions and methods for increasing biomass, iron concentration, and tolerance to pathogens in plants. methods for producing greater biomass in a plant, increasing a plant's tolerance to drought, producing a reduced concentration of lignin in a plant, producing a greater concentration of iron in a plant, or inhibiting fungal infection in a plant comprises administering bacillus subtilis fb17 to the plant, the seed of the plant, or the soil around the plant or seed, in an amount effective to produce greater biomass, increasing tolerance to dry, produce a reduced concentration of lignin, produce a higher concentration of iron, or inhibit infection by funds in the plant, compared to an untreated plant, respectively. agricultural vehicles and seed coatings comprising bacillus subtilis fb17 are provided. the biomass of a plant that has been administered bacillus subtilis fb17 can be converted into a biofuel or can be used as a food crop or for other uses.
    公开号:BR112012021952B1
    申请号:R112012021952
    申请日:2011-03-01
    公开日:2020-06-09
    发明作者:Janine Sherrier Darla；Bais Harsh；Lakshmanan Venkatachalam
    申请人:Univ Delaware；
    IPC主号:
    专利说明:

    Descriptive Report of the Invention Patent for METHOD TO IMPROVE A QUALITY OF A PLANT, AND COVERED PLANT SEED.
    REMISSIVE REFERENCE TO RELATED ORDERS
    This request claims priority to the application for serial number 61 / 309,134, filed on March 1, 2010, the application for serial number 61 / 414,108, filed on November 16, 2010, and application for no. Serial No. 61 / 416,039, filed on November 22, 2010, which are incorporated herein in their entirety by citation and for all purposes. GOVERNMENTAL FINANCING DECLARATION
    The research that gave rise to the described inventions was funded, in part, by the National Science Foundation (NSF) grant No. 0923806 and NSF grant No. IOS-0814477, with technical support from the USDA Experimental Field Station in Georgetown, DE, at University of Delaware. Accordingly, the United States government may have certain rights with respect to that invention.
    FIELD OF THE INVENTION
    The present invention relates, in general, to the use of plant growth-promoting rhizobacteria to improve various characteristics of plant growth, including to increase biomass, increase drought tolerance, reduce lignin content, increase germination of seeds, increase iron concentration, and increase tolerance to pathogens. In particular, the embodiments of the present invention relate to the administration of Bacillus subtilis FB17 to plants. The resulting plants can be used in the production of biofuels, food, or for other purposes.
    BACKGROUND OF THE INVENTION
    Food safety has always been a major global priority, and growing concern about the impact of crop production on the environment requires the development and use of new methods to increase productivity, while at the same time protecting the environment. Botanical biologists develop and put into practice strategies
    Petition 870190043849, of 05/09/2019, p. 10/31
    2/30 for the efficient production of crop plants in order to ensure the availability of essential raw materials for the growing world population. However, the development of biofuels and renewable technologies is added to the challenge as these have also become an increasingly important priority. Therefore, there is an increasing need for improved approaches to increase crop yields under different field conditions.
    The different types of geographic environment and different climates in the world present different types of challenges in the generation of greater biomass and potential yield of crop plants. Drought is one of the main factors that limit crop production in the world. Long-term drought or short-term drought during the growing season can severely limit or even eliminate crop production. Changes in global climate patterns have affected the frequency and intensity of drought, even in the main growing regions of the world.
    The availability of nutrients also limits crop production. Improving soil with nutrients is expensive and energy-consuming, and even when nutrients are available in sufficient quantities, crop plants are sometimes not efficient at absorbing nutrients. Poor absorption of essential nutrients results in low yield and food crops with low nutritional value. For example, rice (the seed of monocotyledonous plants Oryza sativa or Oryza glaberríma) is the most important staple food for more than two-thirds of the world's population, providing a significant proportion of the calories consumed. As rice is the main staple food for a large part of the global population, rice production with higher levels of iron can have a significant impact in reducing micronutrient malnutrition in the world, since iron deficiency is one of the main deficiencies of micronutrients in humans worldwide.
    Stress due to pathogens also limits productivity. Plants need to invest energy in order to survive the pathogen attack3 / 30 nos, and this energy diversion results in less yield. Plants also modify their composition in order to limit the progression of the disease, and these changes often make processing crops more difficult. Furthermore, some crop pathogens cannot be effectively limited by genetic diversity or chemical control, and have a significant impact on global crop production.
    The rice blast fungus (Magnaporthe grisea or Magnaporthe oryzaé) is a pathogenic plant fungus that causes a serious disease that affects rice. It causes significant annual losses from an economic point of view, contributing to an estimated 40% of crop yields. The rice blast fungus destroys an amount of rice that would feed millions of people around the world with each growing season. Since rice is an important staple food for much of the world, the effects of rice blast have a broad impact on human health and the environment. The deficit in rice directly contributes to human hunger. Rice blast contributes additionally to crop loss and requires the use of additional resources to compensate for the reduced yield. There is still a great need for strategies that improve various characteristics of plant growth in various growing conditions, such as tolerance to stress due to drought, tolerance to pressure from pathogens, availability of nutrients, and finally crop yield, so that greater amounts of foods with greater nutritional value may be available to the world population, and for other important benefits, such as the production of biofuels.
    SUMMARY OF THE INVENTION
    One embodiment of the present invention provides a method for producing greater biomass in a plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant seed, or the soil around the plant or plant seed, in an amount effective to produce greater biomass in the plant, compared to an untreated plant.
    Another method provides a method to increase a plant's drought tolerance, which comprises administering Bacillus subtilis FB17
    4/30 to the plant, plant seed, or soil around the plant or plant seed, in an amount effective to increase the plant's drought tolerance compared to an untreated plant.
    Another embodiment provides a method for producing a reduced concentration of lignin in a plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant seed, or the soil around the plant or the plant seed, in an amount effective to produce a reduced concentration of lignin in the plant compared to an untreated plant.
    Another method provides a method for increasing seed germination in plants, which comprises administering Bacillus subtilis FB17 to plants, plant seeds, or the soil around plants or plant seeds, in an effective amount to increase seed germination. plant seeds compared to untreated plants.
    Another modality provides a method for producing a higher concentration of iron in a plant, particularly a rice plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant's seed, or the soil around the plant or seed, in an amount 20 effective to produce a higher concentration of iron in the plant, compared to an untreated plant.
    Another modality provides a method for inhibiting the growth of a fungal plant pathogen and the infection of a plant, particularly a rice plant, by a fungal pathogen, particularly rice blast fungi, which comprises administering Bacillus subtilis FB17 to the plant. , to the seed of the plant, or to the soil around the plant or seed, in an amount effective to inhibit infection of the plant by the fungal pathogen.
    The additional arrangements provide agricultural vehicles and seed coatings, comprising Bacillus subtilis FB17. The biomass of a plant that has been administered Bacillus subtilis FB17 can be converted into a biofuel and the crop produced can be used
    5/30 safe as raw material for the production of products for humans or animals, or for other purposes.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1: Morphometric analysis (number of branches, number of leaves, height of shoots, weight of shoots, length of roots, weight of roots) of Brachypodium distachyon (Bd2-1) plants treated with B. subtilis FB17, compared to controls . This shows that inoculation with B. subtilis FB17 improves plant morphology.
    Figure 2: Biochemical analysis of Brachypodium distachyon plants treated with B. subtilis FB17, compared to controls, by measuring total chlorophyll and total carotenoids. This shows that inoculation with B. subtilis FB17 has a positive impact on the plants' ability to capture light energy.
    Figure 3: The amounts of B. subtilis FB17 and controls recovered from the soil and roots of Brachypodium distachyon. This shows the persistent association with the roots of plants inoculated with B. subtilis FB17.
    Figure 4: The total biomass gain in different species of plants treated with B. subtilis FB17. Significant increases (-28%) in both aerial and root biomass were observed in Z. mays (M017).
    Figure 5: Quantitative data showing the increase in root and shoot biomass in Z. mays Mo-17 plants whose seeds were treated with B. subtilis FB17.
    Figure 6: Quantitative data showing the increase in the number of leaves of cultivation plants for bioenergy, Brachypodium distachyon (genotype Bd2-1), whose seeds were treated with B. subtilis FB17.
    Figure 7: Quantitative data showing the increase in the biomass of roots and shoots in cultivation plants for bioenergy, Brachypodium distachyon (genotype Bd2-1), whose seeds were treated with B. subtilis FB17.
    6/30
    Figure 8: Quantitative data showing the increase in root and shoot biomass in Zinnia sp. Red Spider whose seeds were treated with B. subtilis FB17.
    Figure 9: Quantitative data showing the increase in the biomass of roots and shoots in Zinnia sp. Red Spider whose seeds were treated with B. subtilis FB17.
    Figure 10: The total chlorophyll content in plants treated with B. subtilis FB17. A significant increase in the total chlorophyll content was observed in tomatoes (14%), Z. mays CML10 (72%) and CML258 (87%) after treatment with FB17.
    Figure 11: The total carotenoid content in plants treated with B. subtilis FB17. A significant increase in the total carotenoid content was observed in soybeans (31%) and Z. mays MO17 (82%) after treatment with FB17.
    Figure 12: Quantitative data showing increased photosynthetic efficiency in Mo-17 plants, whose seeds were treated with β. subtilis FB17.
    Figure 13: Quantitative data showing the increased photosynthetic efficiency in cultivation plants for bioenergy, Brachypodium distachyon (genotype Bd2-1), whose seeds were treated with B. subtilis FB17.
    Figure 14: Quantitative data showing the increased photosynthetic efficiency in Zinnia sp. Red Spider whose seeds were treated with B. subtilis FB17.
    Figure 15: Quantitative data showing increased photosynthetic efficiency in exotic CML 10 and CML 258 corns, whose seeds were treated with B. subtilis FB17.
    Figure 16: The percentage of increased germination in seeds treated with B. subtilis FB17. A significant increase in the percentage of total germination content was observed in tomatoes (6%), Z. mays MO17 (2.1%) and CML258 (14%) after treatment with FB17. Notably, germination increased dramatically in the CML258 exotic maize strain.
    7/30
    Figure 17: The growth rate of Zea mays treated with B. subtilis FB17.
    Figure 18: Water storage capacity in plants treated with B. subtilis FB17. A significant increase in the total water retention and storage capacity was observed in tomatoes (2.1%) and Z mays MO17 (3.5%) after treatment with FB17.
    Figure 19: Drought tolerance in plants treated with B. subtilis FB17. A significant increase in the growth rate under drought treatment conditions (absence of water) was observed in MO17, that is, a 37.5% increase in relation to the control of uninoculated treatment with drought stress (absence of water) ) after treatment with FB17.
    Figure 20: Seed treatment with B. subtilis FB17 reduces the lignin content in corn. A significant reduction in the total lignin content was observed in Z. mays (MO17 = 46%; CML10 = 64% and CML58 = 49%) after treatment with FB17 without stress conditions.
    Figure 21: The increase in aerial biomass and roots in Oryza sativa (Nipponbare) treated with B. subtilis FB17. A significant increase in total biomass was observed in O. sativa (rice; cultivar Nipponbare) (greater than 200%) after treatment with FB17.
    Figure 22: The iron concentration observed in rice plants treated with Bacillus subtilis FB17, compared to untreated rice plants. These data show that inoculation with FB17 results in higher crop yield and higher concentrations of iron in rice grains.
    Figure 23: Summary of the effects of B. subtilis FB17 on different characteristics in various plant species.
    DETAILED DESCRIPTION OF THE INVENTION
    Applicants have found that a plant growth-promoting rhizobacterial strain (RPCP), Bacillus subtilis FB17, exhibits surprising effects when administered to plants. The B. subtilis FB17 strain was originally isolated from beet roots in North America (see Fali et al. 2004 System Appli. Microbiol. 27: 372-379, incorporated
    8/30 here per quote). This strain was isolated from the beet root due to its ability to form a superficial biofilm and promote dendrite growth.
    In particular, Bacillus subtilis FB17 provided a surprising increase in the biomass of phylogenetically diverse plants, as well as an increase in photosynthetic efficiency and an increase in growth rates in drought conditions. The administration of Bacillus subtilis FB17 to plants has also resulted in a decrease in lignin concentrations in plants, which can provide important benefits in the field of bioenergy, since lignin is one of the main obstacles in converting plant biomass into biofuel. In relation to rice plants, Bacillus subtilis FB17 has provided a surprising increase in the concentration of iron in rice and has also been shown to slow the growth of rice blast fungus, a fungal pathogen that destroys rice crops worldwide. The present invention provides methods to increase biomass, increase drought tolerance, reduce lignin content, increase seed germination, increase iron concentration, and increase tolerance to pathogens in various plants, particularly crop plants such as plants of corn, soy and rice. The present invention also provides agricultural vehicles and seed coatings, which comprise Bacillus subtilis FB17.
    One embodiment of the present invention provides a method for producing greater biomass in a plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant seed, or the soil around the plant or plant seed, in an amount effective to produce greater biomass in the plant, compared to an untreated plant. As used here, the term biomass in / of a plant refers to the total mass of matter in the plant. Unless otherwise specified, biomass comprises both the biomass above the soil surface (ie aerial biomass, including, but not limited to, the stalk, leaves and / or grains) and the biomass below the surface of the soil (ie the roots). The biomass of a plant that has been administered Bacillus subtilis FB17 can be
    9/30 measured using known methods. In one embodiment, the plant's biomass is measured according to the dry weight (PS) of the plant in grams.
    The biomass of a plant that has been administered Bacillus subtilis FB17 can be measured at a point in time between about 7 days and about 100 days, about 10 days and about 75 days, or about 15 days and about 35 days after administration of Bacillus subtilis FB17 to the plant. Alternatively, the biomass of a plant to which Bacillus subtilis FB17 has been administered can be measured at the time of harvesting the plant to harvest its grains or agricultural products, that is, at the time when the mature plant, such as a corn plant , soy or tomato, is harvested from the field. For example, a crop plant to which Bacillus subtilis FB17 was administered according to a method of the present invention produces a greater amount of total biomass above the soil surface and below the soil surface, measured in grams of dry weight, the amount about 1%, between about 5% and about 200%, between about 5% and about 100%, between about 7.5% and about 75%, between about 15% and about 60 %, or between about 30% and about 55%, higher compared to an untreated plant. In one embodiment, a method comprises administering Bacillus subtilis FB17 to the plant seed before sowing the seed in the soil in an effective amount so as to produce greater biomass in the plant from an amount of about 5% to about 100% greater in relation to an untreated plant, after administration of Bacillus subtilis FB17. For example, as illustrated in figure 4, an increase of about 28% in root and aerial biomass was observed in corn treated with Bacillus subtilis FB17, compared to untreated corn, 15 days after treatment.
    Another embodiment of the present invention provides a method for producing greater drought tolerance in a plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant seed, or the soil around the plant or plant seed, in an effective amount to produce a higher tolerance to drought in the plant, compared to an untreated plan10 / 30 ta. Drought is the absence of rain or irrigation for a period of time sufficient for depletion of soil moisture and damage to plants. Drought-induced stress results when the plant's water loss is greater than the plant's roots' ability to absorb water and when the water content in the plant is reduced to interfere with normal plant processes. A plant responds to a lack of water by interrupting its growth and reducing its photosynthesis and other plant processes in order to reduce water use. As used here, the term drought tolerance refers to the rate of plant growth per day in the absence of water, for example, grams per day of biomass increase in a plant inoculated with Bacillus subtilis FB17, compared to a plant not treated. For example, as illustrated in figure 19, the seeds of corn plants treated with Bacillus subtilis FB17 showed, in the absence of water, a growth rate per day of about 37.5% higher compared to untreated plants, 15 days after the treatment. In one embodiment, a method comprises administering Bacillus subtilis FB17 to the plant, to the soil around the plant, or to the seed of the plant before sowing in the soil, in an amount effective to produce greater drought tolerance in the plant by an amount at least about 10% higher compared to an untreated plant, after the administration of the said Bacillus subtilis FB17.
    Another embodiment of the present invention provides a method for producing a reduced concentration of lignin in a plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant seed, or the soil around the plant or plant seed, in an effective amount to produce a reduced concentration of lignin in the plant, compared to an untreated plant. The concentration of lignin can be measured using known methods. For example, as shown in figure 20, plants treated with Bacillus subtilis FB17 show reductions of about 46% to about 64% in the number of lignified cells compared to that observed in untreated plants. Lignin is an essential component of plants and is found in plant cell walls, as well as among plant cells. Lignin is one of the main obstacles to the conversion of plant biomass into biofuel. Cellulose, another plant component, is currently the main source for biofuels. While cellulose is easily fermented into alcohol, lignin cannot be converted using current fermentation processes and makes extracting fermentable sugars difficult. It would therefore be advantageous to produce plants with reduced concentrations of lignin. The present invention provides biofuels that are produced by converting any biomass from a plant (i.e., all plant biomass or any part of the plant biomass) to which Bacillus subtilis FB17 was administered according to any of the methods of the present invention in a biofuel. The biomass of a plant to which Bacillus subtilis FB17 has been administered can be converted into a biofuel by any known method, such as by fermenting the plant's sugar components.
    Another embodiment of the present invention provides a method for increasing the seed germination rate in plants, which comprises administering Bacillus subtilis FB17 to plants, plant seeds, or the soil around plants or plant seeds, in an effective amount to increase germination of plant seeds, compared to untreated plants. For example, as shown in figure 16, increases in percentages of total germination were observed in tomato and corn plants after administration of Bacillus subtilis FB17.
    Another modality provides a method for producing a higher concentration of iron in a plant, particularly a rice plant, which comprises administering Bacillus subtilis FB17 to the plant, the plant's seed, or the soil around the plant or seed, in an amount effective to produce a higher concentration of iron in the plant compared to an untreated plant. Iron deficiency is one of the main micronutrient deficiencies in humans in the world, and rice is the most important staple food for a large part of the world population; rice paddies produced according to the methods of the present invention can provide important nutritional benefits worldwide. The iron concentration in a plant that has been administered Bacillus subtilis FB17 can be measured using known methods, including inductively coupled plasma atomic emission spectrometry (ICP-AES; inductively coupled plasma-atomic emission spectroscopy), plasma mass spectrometry inductively coupled (ICP-MS; inductively coupled plasma mass spectroscopy) or other conventional methods. In one embodiment, the iron concentration in the plant is measured in milligrams of iron per kilogram of dry weight of the plant. As shown in figure 22, an increase of approximately 81% in the iron content was observed in rice plants treated with FB17 compared to untreated plants, measured in mg of iron per kg of dry weight of the plant.
    Rice plants suitable for use in the invention include Oryza sativa, Oryza glaberrima and all of its subspecies and cultivars. The iron concentration of a rice plant that has been administered Bacillus subtilis FB17 can be measured at the time of harvesting the rice to harvest its grains or agricultural products, that is, at the time when the ripe rice grains are harvested from a field . Alternatively, the iron concentration of a rice plant that has been administered Bacillus subtilis FB17 can be measured at a point in time that is, for example, about a week to about five months, preferably about three months, after administration of Bacillus subtilis FB17 to the rice plant. A rice plant to which Bacillus subtilis FB17 was administered according to a method of the present invention produces a greater amount of iron, measured, for example, in grams of iron per gram of dry weight of the rice plant's biomass above the surface of the rice. soil and below the surface of the soil.
    For example, a rice plant that has been administered Bacillus subtilis FB17 according to a method of the present invention produces a greater amount of iron by dry weight of the rice plant's biomass above the soil surface and below the soil surface, being The
    13/30 amount at least about 5%, about 10% to about 200%, about 25% to about 150%, about 50% to about 100%, about 70% to about 90% , about 75% to about 85%, or about 80% higher compared to an untreated plant. For example, in one embodiment, a method comprising administering Bacillus subtilis FB17 to a rice seed before sowing the seed in the soil, in an amount effective to produce a greater concentration of iron in the plant, of an amount of at least about 25% higher compared to an untreated plant, after the administration of the said Bacillus subtilis FB17.
    Another embodiment of the present invention provides a method for inhibiting the infection of a plant by a fungal pathogen, which comprises administering Bacillus subtilis FB17 to the plant, the plant's seed, or the soil around the plant or seed, in an amount effective to inhibit plant infection by the fungal pathogen, compared to an untreated plant. Examples of plants include rice and barley plants, such as the rice cultivar Nipponbare. In a specific embodiment, the present invention provides methods for inhibiting infection of a rice plant by a fungal pathogen, particularly rice blast fungus, which comprises administering Bacillus subtilis FB17 to the rice plant, to the seed of the rice plant. rice, or to the soil around the rice plant or seed, in an amount effective to inhibit fungal pathogen infection of the rice plant, compared to an untreated rice plant. As used here, the term rice blast refers to the fungus Magnaporthe grisea or Magnaporthe oryzae, which is pathogenic to plants.
    Symptoms of rice blast include lesions or spots (which can, for example, be white or gray) produced on any part of the plant, particularly on the above-ground or aerial parts of the plant, such as the leaves. As used here, the term inhibit infection refers to the lower production of a fungal infection in the rice plant, measured as a reduction in the symptoms of the fungal infection, for example, by a smaller number of lesions in the aerial portions of the rice plant.
    14/30 rice, compared to an untreated plant, or a reduced size of some or all of the lesions. For example, in specific embodiments, Bacillus subtilis FB17 is administered to a rice plant, the seed of the rice plant, or to the soil around the rice plant or seed, in an amount effective to reduce the number of lesions on the rice plant, caused by rice blast fungus, in about 5% to about 100%, about 10% to about 80%, about 20% to about 60%, or about 25% about 45% compared to an untreated rice plant. Without being bound by any theory, Bacillus subtilis FB17 is believed to produce a volatile antifungal compound that attenuates or inhibits the growth of M. oryzae. In specific embodiments, in order to inhibit the growth of rice blast fungus and the infection of a rice plant, Bacillus subtilis FB17 is administered to a rice seed in an amount of about 1x10 7 CFU / seed a about 1x10 9 CFU / seed, more preferably about 1x10 8 CFU / seed, and the seed is subsequently planted in the soil.
    As used here, the term untreated plant refers to a plant of the same species and grown under basically the same conditions (for example, during the same period of time, with the same climate, and grown using the same methods using the same materials, with biomass, drought tolerance, lignin concentration, iron concentration, fungal infection and other characteristics being measured using the same methods) as a plant administered Bacillus subtilis FB17 according to a method of the present invention, except due to the fact that the untreated plant was not administered Bacillus subtilis FB17. As used here, a characteristic of a plant that has been administered Bacillus subtilis FB17, such as greater biomass, greater drought tolerance, lower lignin concentration, higher iron concentration, or less fungal infection, compared to a non-plant treated, refers to a higher biomass, greater drought tolerance, lower lignin concentration, higher iron concentration, or less fungal infection, measured at the same point in time, respectively.
    15/30
    In certain embodiments of the methods described here, Bacillus subtilis FB17 is administered to a seed in an amount of between about 1 ml / kg of an inoculum of Bacillus subtilis FB17 (i.e., 1 ml / kg of Bacillus subtilis FB17 grown over night in LB medium with an optical density (OD) of 0.5 at a wavelength of 600 nm measured using a Bio Rad SmartSpec spectrophotometer) and about 50 ml / kg, preferably about 5 ml / kg to about 25 ml / kg, more preferably from about 10 ml / kg to about 15 ml / kg, most preferably about 12.5 ml / kg. In alternative embodiments, Bacillus subtilis FB17 is administered to a seed in an amount of about 1x10 6 CFU / seed to about 1x10 9 CFU / seed, most preferably from about 1x10 7 CFU / seed to about 1x10 8 UFC / seed.
    The methods of the present invention can be used to treat various types of plants (as well as their seeds or soil around them) to increase biomass, increase drought tolerance, decrease lignin concentration, increase seed germination, increase iron concentration, and increase tolerance to pathogens. Plants can include monocots and dicots. In particular, plants can include crops such as corn, soybeans, tomatoes, rice or barley. Additional examples of plants that can be treated according to the methods of the present invention include Arabidopsis thaliana and Zinnia, as well as cultivation plants for bioenergy, that is, plants that are currently used or have the potential to be used as sources of bioenergy (for example, plants that are useful for the production of biofuels), such as Brachypodium distachyon.
    According to the invention, Bacillus subtilis FB17 can be administered to a plant by means of a known method, in which all or part of the plant is treated, such as by inoculating the roots, seeds or leaves. For example, Bacillus subtilis FB17 can be administered to the aerial parts of a plant, such as the leaves and stalk, to the roots of the plant, to the seed of the plant before sowing in the soil, or to the soil around the plant or seed of the plant. Administration methods include soaking, spraying, coating, injection or other methods known to those skilled in the art. As used herein, the term administration of Bacillus subtilis FB17 refers to a single administration, repeated administrations (i.e., more than one administration of Bacillus subtilis FB17), or to continuous administration. Bacillus subtilis FB17 can be administered at any point in the plant's life cycle (ie, before or after germination). For example, Bacillus subtilis FB17 can be administered to a plant seed before sowing the seed in the soil and before germination. Alternatively, Bacillus subtilis FB17 can be administered to the plant, the seed of the plant, or the soil around the plant after germination occurs. Once treated with Bacillus subtilis FB17, the seeds can be planted in the soil and grown using conventional methods to promote plant growth.
    According to embodiments of the present invention, Bacillus subtilis FB17 can be administered to a plant, seed of a plant, or the soil, alone or in the form of a mixture with other materials. For example, Bacillus subtilis FB17 can be administered in a composition that basically consists of Bacillus subtilis FB17 in a culture medium without any additives or additional materials. Alternatively, Bacillus subtilis FB17 can be administered in a composition that comprises Bacillus subtilis FB17 in a culture medium, a vehicle, such as water, an aqueous solution, or a powder. The culture medium, vehicle, aqueous solution, or powder may contain additional additives, such as an insecticide or fungicide. Alternatively, Bacillus subtilis FB17 can be administered separately, with the other additives or materials being applied at different times. In certain embodiments, Bacillus subtilis FB17 is administered in a composition comprising Bacillus subtilis FB17 in an amount of between about 1 ml / kg (ie, 1 ml / kg of Bacillus subtilis FB17 grown overnight in the I_B with an optical density (OD) of 0.5 at a wavelength of 600 nm measured using a Bio Rad SmartSpec spectrophotometer) and about 50 ml / kg, preferably from about 5 ml / kg to about 25 ml / kg, most preferably from about 10 ml / kg to about 15 ml / kg, even more preferably about 12.5 ml / kg. In alternative embodiments, Bacillus subtilis FB17 is administered in a composition comprising Bacillus subtilis FB17 in an amount from about 1x10 6 UFC / seed to about 1x10 9 UFC / seed, most preferably about 1x10 7 UFC / seed at about 1x10 8 CFU / seed.
    The present invention further provides agricultural vehicles comprising Bacillus subtilis FB17, which can be applied to plants (e.g., roots), the soil around the plants, or to seeds before sowing, as well as seed coatings comprising Bacillus subtilis FB17 , which can be applied to plant seeds. The present invention also provides a plant seed, preferably a seed of a cultivation plant (for example, seed of a corn plant, a soybean plant, a rice plant, a tomato plant or a cultivation plant for bioenergy, such as Brachypodium distachyon), which is coated with Bacillus subtilis FB17, so that all or part of the seed has a coating or film comprising Bacillus subtilis FB17. The agricultural vehicle may comprise Bacillus subtilis FB17 in an amount of between about 1 ml / kg of an inoculum of Bacillus subtilis FB17 (i.e., 1 ml / kg of Bacillus subtilis FB17 grown overnight in LB medium with an optical density (OD) of 0.5 at a wavelength of 600 nm measured using a SmartSpec spectrophotometer from Bio Rad) and about 50 ml / kg, from about 5 ml / kg to about 25 ml / kg, from about 10 ml / kg to about 15 ml / kg, or about 12.5 ml / kg. The coating may comprise Bacillus subtilis FB17 in an amount of about 1x10 6 CFU / seed to about 1x10 8 CFU / seed, most preferably about 1x10 7 CFU / seed. The agricultural vehicle and seed coating can each consist basically of Bacillus subtilis FB17 in a culture medium without any additional additives or materials. Alternatively, the agricultural vehicle and the seed coating may each comprise Bacillus subtilis FB17 in a culture medium, such as water, an aqueous solution, or a powder. The culture medium,
    18/30 aqueous solution or powder may contain additional additives, such as an insecticide or fungicide.
    The present invention has basic and applied applications. In a broad sense, one could use the methods described here to increase biomass (for example, in alternative plant species used for biofuel or to influence the potential yield of crop plants) and to provide greater drought tolerance. In comparison to transgenic approaches, these methods are instantly applicable to any plant, without the time needed for gene identification, generation and characterization of transgenic strains, and do not involve the regulatory and social issues related to the use of transgenes. In comparison to the use of traditional agronomic practices (applications of chemical fertilizers and water), the methods described here are less intensive in terms of resources and labor for the grower and are safer for the environment. In addition, it is known that the application of chemical fertilizers increases the susceptibility of crops to diseases due to the induction of rapid but weak growth, while plants cultivated with this method are not more susceptible to diseases. In comparison to other rhizobacteria that are useful for seed treatment, FB17 requires low inoculants to produce advantageous results. Finally, these methods are compatible with organic farming practices, while other methods described above (for example, the application of chemical fertilizers) are not.
    A deposited B. subtilis FB17 strain has been available since before March 1, 2010, at the Delaware Biotechnology Institute, 15 Innovation way, Room No. 145, Newark, DE 19711. A deposit of the B. subtilis FB17 strain will also be available. held at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA. Access to this deposit at the ATCC will be available pending the application to the patent and trademark commissioner and persons with permission granted by the commissioner when requested. The deposit will be kept at the ATCC deposit center, which is a public deposit, for a period of 30
    19/30 years, or 5 years after the most recent application, or during the term of the patent, depending on which period is the longest, and will be replaced if it becomes non-viable during that period. The deposit will be available as required by international patent laws in countries in which counterparties to this application, or their offspring are deposited.
    In addition, the deposit of the object of the order will be stored and made available to the public in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms, that is, it will be stored with all necessary care so that it is kept viable and free from contaminants. for a period of at least five years after the most recent request to provide a sample of the filing, and in any event, for a period of at least thirty (30) years after the filing date or for the term of any patent that can present a revelation of the culture.
    The following examples are provided to describe the invention in greater detail and are intended to illustrate, but not limit, the invention. UD10-22, as used in some of the figures described below, refers to Bacillus subtilis FB17.
    EXAMPLES
    Example 1
    Brachypodium distachyon and corn plants were germinated and cultivated for 21 days. Once in 5 days (3 times), 5 ml of B. subtilis FB17 with OD of 0.5 was added per pot. As a control, 5 ml of E. coli OP50 with OD of 0.5 was added per pot. FB17 and OP50 had been grown overnight in LB medium and the optical density (OD) at a wavelength of 600 nm was measured using a SmartSpec spectrophotometer (Bio Rad). Ten days after the last treatment, the plants were analyzed. The controls described in all experiments here refer to plants that have not been treated with bacteria or that have been treated with E. coli OP50.
    Brachypodium distachyon (Bd2-1) and treated corn plants
    20/30 with B. subtilis FB17, treated with E. coli bacterial control, or that received simulated treatment, were grown in 4x4 inch pots under standard conditions (22 to 25 ° C, 60% humidity, 16 h photoperiod of light and 8 h of darkness) for 30 days after treatment. The aerial and root biomass of the B. distachyon energy crop increased with treatment with FB17. Figure 1 shows that the biomass of B. distachyon treated with FB17 increased at a statistical level. Figure 2 illustrates an increase in photosynthetic efficiency observed in B. distachyon treated with FB17. B. distachyon treated with FB17 contained more chlorophyll and total carotenoids compared to controls, demonstrating robust plant health. Figure 3 shows the quantities of FB17 recovered from the soil around the roots of B. distachyon. The figure shows that FB17 is more strongly associated with B. distachyon roots compared to E. coli, suggesting the true rhizobacterial nature of FB17.
    Corn plants also exhibited an increase in root and aerial biomass after growing for 30 days after treatment with B. subtilis FB17, treatment with E. coli bacterial control, or simulated treatment.
    Example 2
    Arabidopsis thaliana seeds were germinated and cultivated for 21 days. Once in 5 days (3 times), 5 ml of β. subtilis FB17 with OD of 0.5 were added per pot. As a control, 5 ml of E. coli OP50 with OD of 0.5 was added per pot. FB17 and OP50 had been grown overnight in LB medium and the optical density (OD) at a wavelength of 600 nm was measured using a SmartSpec spectrophotometer (Bio Rad). Ten days after the last treatment, the plants were subjected to drought (that is, no water was added) at 25 ° C with 40% humidity for 4 weeks. Thirty days after treatment, drought was assessed in terms of loss of the phenotype of delay in leaf senescence (stay-green phenotype) in untreated plants compared to plants treated with FB17, indicating that FB17 confers greater tolerance to drought in Arabidopsis.
    21/30
    EXAMPLE 3
    Seed treatment with B. subtilis FB17 promotes an increase in biomass in corn Mo17, CML258, CML10, Zinnia, and Brachypodium distachyon.
    To assess the effect of B. subtilis FB17 on the increase of biomass in maize (Mo17, CML258, CML10), soy (Will-82), tomatoes (Solanum lycopersicum), Zinnia and Brachypodium distachyon (an energy culture model), 50 seeds (n = 50) per plant species were treated with B. subtilis FB17 (about 1x10 7 CFU / seed or 12.5 ml / kg of Bacillus subtilis FB17 grown overnight in LB medium with an optical density (OD ) of 0.5 at a 600 nm wavelength measured using a Bio Rad SmartSpec spectrophotometer). After seed treatment, they were individually sown in pots (4x4 inches) with a mixture of soil for germination and biomass studies. Interestingly, the treatment of seeds with B. subtilis FB17 promoted the growth of roots and shoots in all crop species analyzed. Measurements were obtained 15 days after treatment.
    The plants whose seeds were treated promoted an increase in the biomass of the roots resulting in more dense root systems instead of increasing the length of the roots. A denser root system results from an increase in the lateral roots and hair in the roots providing a greater capacity for absorbing water and nutrients.
    Zea mays var. CML258 showed an increase in aerial biomass (g PS) of about 16% compared to the control. Zea mays var. CML10 showed an increase in aerial biomass (g PS) of about 9% compared to the control. Zea mays var. Mo-17 showed an increase in aerial biomass (g PS) of about 38% compared to the control. Brachypodium showed an increase in aerial biomass (g PS) of about 40% compared to the control. A significant increase of about 28% of the total root and aerial biomass was observed in Zea mays (MO17) in relation to the control. Figure 4 illustrates the total biomass gain in plants treated with B. subtilis FB17.
    22/30
    Figure 5 illustrates quantitative data showing the increase in root and shoot biomass in Mo-17 plants whose seeds were treated with B. subtilis FB17. Figure 6 illustrates quantitative data showing the increase in the number of leaves in cultivation plants for bioenergy Brachypodium distachyon (genotype Bd2-1) whose seeds were treated with B. subtilis FB17. Figure 7 illustrates quantitative data showing the increase in the biomass of shoots and roots in cultivation plants for bioenergy Brachypodium distachyon (genotype Bd2-1) whose seeds were treated with B. subtilis FB17. Figures 8 and 9 illustrate the quantitative data showing the increase in the biomass of shoots and roots in Zinnia sp. Red Spider whose seeds were treated with B. subtilis FB17.
    EXAMPLE 4
    Seed treatment with B. subtilis FB17 promotes photosynthetic efficiency in corn and tomatoes.
    To evaluate the effect of B. subtilis FB17 on photosynthetic efficiency in maize (Mo17, CML258, CML10), soy (Will-82), tomatoes (Solanum lycopersicum), Zinnia and Brachypodium (an energy culture model), 50 seeds ( n = 50) per plant species were treated with B. subtilis FB17 (12.5 ml / kg or 1X10 7 CFU / seed). After 15 to 32 days of treatment, the leaves were harvested and analyzed for total chlorophyll content. The results showed that tomato and corn plants (tomato and exotic corn lines CML258 and CML10) inoculated with B. subtilis FB17 showed a higher content of chlorophyll and carotenoid compared to untreated samples, as shown in figures 10 and 11 .
    The higher total chlorophyll values have the potential to promote greater vigor and biomass as observed in CML258 and CML10. The total chlorophyll content of tomatoes whose seeds were inoculated with B. subtilis FB17 showed an increase of about 14%. More significantly, the CML258 and CML10 exotic corn lines showed an increase of about 87% and about 72%, respectively.
    Although there are increases in the total chlorophyll content, this does not mean that the total carotenoid content will also show an increased value. Tomato and Zinnia showed a significant percentage reduction in total carotenoid when inoculated with B. subtilis FB17 and compared to untreated seeds. Corns CML258 and CML10 showed a significant percentage increase in total carotenoid, while soybeans, corn Mo17 and Brachypodium did not show statistically significant differences between treated and untreated seeds.
    Figure 12 illustrates quantitative data showing the increase in photosynthetic efficiency in Mo-17 plants whose seeds were treated with B. subtilis FB17. Figure 13 illustrates quantitative data showing the increase in photosynthetic efficiency in cultivation plants for bioenergy Brachypodium distachyon (genotype Bd2-1) whose seeds were treated with B. subtilis FB17. Figure 14 illustrates quantitative data showing the increase in photosynthetic efficiency in Zinnia sp. Red Spider whose seeds were treated with B. subtilis FB17. Figure 15 illustrates quantitative data showing the increase in photosynthetic efficiency in exotic CML258 and CML10 corns whose seeds were treated with B. subtilis FB17.
    EXAMPLE 5
    Seed treatment with B. subtilis FB17 promotes germination in tomato and corn plants.
    To assess the effect of B. subtilis FB17 on the percentage increase in germination in maize (Mo17, CML258, CML10), soybeans (Will-82), tomatoes (Solanum lycopersicum), Zinnia and Brachypodium (an energy culture model), 50 seeds (n = 50) per plant species were treated with B. subtilis FB17 (12.5 ml / kg or 1x10 7 CFU / seed). The final germination percentages were recorded 8 days after the date of sowing. The results showed that the treatment with B. subtilis FB17 promoted a statistically significant response in relation to germination in tomatoes and corn, as shown in figure 16.
    Tomato seeds and CML258 and CML10 exotic corn strains that were treated with B. subtilis FB17 showed a percentage increase in germination of 5.9% and 14%, respectively.
    Seed treatment with B. subtilis FB17 had
    24/30 positive and null in all evaluated culture species. There was no statistically negative response in terms of the percentage of germination with the application of treatments on the seeds.
    EXAMPLE 6
    To evaluate the effect of B. subtilis FB17 on corn (Mo17, CML258, CML10), soy (Will-82), tomatoes (Solanum lycopersicum), Zinnia and Brachypodium (a model of energy culture), 50 seeds (n = 50 ) per plant species were treated with B. subtilis FB17 (1X10 7 CFU / seed or 12.5 ml / kg of Bacillus subtilis FB17 grown overnight in LB medium with an optical density (OD) of 0.5 in one 600 nm wavelength measured using a Bio Rad SmartSpec spectrophotometer). After seed treatment, they were individually sown in pots (4x4 inches). Measurements were performed after 15 days of treatment. Figure 17 illustrates the growth rate in Zea mays after treatment with B. subtilis FB17. Figure 18 illustrates the water storage capacity in plants treated with B. subtilis FB17. A significant increase in the total water retention and storage capacity was observed in tomatoes (2.1%) and Z. mays MO17 (3.5%) after treatment with FB17. Figure 19 illustrates drought tolerance in plants treated with B. subtilis FB17. A significant increase in the growth rate under drought treatment conditions was observed in MO17 (an increase of 37.5% over the treatment control that did not receive water), after treatment with FB17. Figure 20 illustrates that the treatment of seeds with B. subtilis FB17 reduces the lignin content in corn. A significant reduction in the total lignin content was observed in Z. mays (about 46% reduction in M017; about 64% reduction in CML10 and about 49% reduction in CML58) after treatment with FB17.
    EXAMPLE 7
    Figure 21 illustrates the increase in aerial biomass and roots in rice plants, Oryza sativa (Nipponbare), treated with B. subtilis FB17 60 days after inoculation. FB17 cultures were grown on LB overnight to produce an inoculum of 10 8 cells per ml. Plants of
    25/30 four-week rice grown by hydroponics (cultivar Nipponbare) was used for supplementation with FB17. Rice plants that were administered B. subtilis FB17 showed an increase of about 200% in biomass compared to untreated rice plants.
    EXAMPLE 8
    To evaluate whether Bacillus subtilis FB17 colonizes rice roots, rice plants (cultivar Nipponbare) were inoculated with Bacillus subtilis FB17 and the roots of rice plants were observed 96 hours after inoculation by confocal microscopy with laser scanning. The observations confirmed that beneficial rhizobacteria (Bacillus subtilis FB17) form a biofilm in plants. In particular, the data suggest that Bacillus subtilis FB17 efficiently colonizes rice roots after 96 hours of treatment, indicating that rice roots support colonization by beneficial microbes.
    To assess whether treatment of rice plants with rhizobacteria promotes any changes in stoma opening, applicants analyzed rice plants treated with rhizobacteria. The results showed that the treatment of rice with Bacillus subtilis FB17 rhizobacteria greatly reduced stomata opening in treated rice plants (Nipponbare cultivar). In the case of treatment with Bacillus subtilis FB17, guard cells were observed 1 week after the addition of Bacillus subtilis FB17. These results suggest that B. subtilis FB17 (Bacillus subtilis FB17) induces a general stomatal closure response in both monocotyledonous and dicotyledonous plants as observed in A. thaliana and rice.
    To assess whether Bacillus subtilis FB17 attenuates the growth of rice blast fungus, applicants exposed Magnaporthe oryzae to cultures of Bacillus subtilis FB17. Plates with qualitative compartments and quantitative data showed that Bacillus subtilis FB17 attenuated the growth of M. oryzae as shown by the decrease in radial growth in fungus cultures exposed to Bacillus subtilis FB17. A comparison with the controls (TY and LB) shows how much the pathogen would have grown26 / 30 in the absence of treatment. As shown in Table 1, Bacillus subtilis FB17 restricted the growth of M. oryzae by about 25%, in vitro. These results suggest that B. subtilis FB17 produces a volatile antifungal compound that can attenuate or inhibit the growth of M. oryzae.
    Table 1
    Treatment Average diameter of the fungus colonies (cm) Percentage growth of the fungus in relation to the control treatment Control ΤΎ 3,175 100 LB Control 3,098 100 Bacillus subtilis FB17 2,342 75.59
    Bacillus subtilis FB17 induced systemic resistance in rice and barley against Magnaporthe oryzae. The rice and barley plants that were exposed to M. oryzae show a decrease in the formation of lesions in the leaves of rice and barley cotyledons, respectively, in plants treated with FB17 compared to controls, as shown in table 2 (infected was defined as a leaf with at least one diamond-shaped lesion typical of blast).
    Table 2
    No. of infected leaves Total number of injuries None / Mo 3 23 FB17 / Mo 5 13
    EXAMPLE 9
    To assess whether Bacillus subtilis FB17 increases rice fortification with iron, applicants analyzed the total iron content in leaves, roots and grains of rice in plants supplemented with Bacillus subtilis FB17, using inductively coupled plasma atomic emission spectrometry (ICP -AES). The results showed that the supplementation of Bacillus subtilis FB17 in rice helps to mobilize iron in the plants, that is, iron, which is an essential element, is actively captured by the plant in which it is used for the growth and development of the plant. As shown in figure 22, an 81% increase in iron content was observed in rice plants treated with FB17 compared to untreated control,
    27/30 as measured in mg of iron per kg of dry weight of the plant (UD1022, as shown in figure 1, refers to Bacillus subtilis FB17). Thus, the administration of Bacillus subtilis FB17 to plants, particularly rice plants, can greatly increase the nutritional value of foods by increasing the 5 iron concentrations in foods.
    Figure 23 summarizes the effects of B. subtilis FB17 on different characteristics in the various plant species described above.
    Although the present invention has been described with reference to specific embodiments, it should be understood that the invention of the claimed form should not be limited to such specific embodiments. In fact, various modifications and variations of the described compositions and methods of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims.
    28/30
    Applicant's file reference number or International application Agent No.: UOD-306WO To be determined
    INDICATIONS REGARDING A DEPOSITED MICROORGANISM (PCT Rule 3bis)
    A. The indications made below refer to the microorganism mentioned in the specification on page _________ 5_________, line 22-25 ________.
    B. DEPOSIT IDENTIFICATION Other deposits are identified on an additional sheet
    Depositary institution name
    American Type Culture Collection (ATCC)
    Address of the depositary institution: (including postal code and country)
    10801 University Boulevard
    Manassas, Virginia 20110-2209
    United States of America
    Deposit date Access number to be determined to be determined
    C. Additional information (leave blank if not applicable) This information is continued on an additional sheet Microorganism deposit is a culture of Bacillus subtitlis FBI7
    D. Designated States for which nominations are made:
    E. SEPARATE SUPPLY OF INDICATIONS (leave blank if not applicable)
    The nominations listed below will be submitted to the International Office later (please specify the general nature of the nominations, for example, deposit accession number)
    Deposit Access Number
    For use by the receiving office only
    Ã] This sheet was received with the international application / Catherine Williams /
    Authorized Responsible:
    Catherine Williams
    Form PCT / RO / 134 (July 1998, reprinted January 2004)
    For International Office use only | ____ I This sheet was received by the international office
    Authorized Responsible:
    29/30
    Applicant's file reference number or International application Agent No.: UOD-306WO PCT / US2011 / 026683
    INDICATIONS CONCERNING A DEPOSITED MICROORGANISM (PCTRule 13 /> A)
    A. The indications made below refer to the microorganism mentioned in the specification on page _________ 5_________, line 22-25 ________.
    B. DEPOSIT IDENTIFICATION Other deposits are identified on an additional sheet 1 1
    Depositary institution name
    American Type Culture Collection (ATCC)
    Address of the depositary institution: (including postal code and country)
    10801 University Boulevard
    Manassas, Virginia 20110-2209
    United States of America
    Deposit date Access number April 26, 2011 PTA-11857
    C. Additional Information (leave blank if not applicable) This information is continued on an additional sheet
    Microorganism deposit is a culture of Bacillus subtitlis FBI7
    D. Designated States for which nominations are made:
    E, SEPARATE SUPPLY OF INDICATIONS (leave blank if not applicable)
    The nominations listed below will be submitted to the International Office later (please specify the general nature of the nominations, for example, deposit accession number)
    For use by the receiving office only
    with the international order
    Authorized Responsible:
    Catherine Williams
    For International Office use only
    I I This sheet was received by the international office
    Authorized Responsible:
    Form PCT / RO / 134 (July 1998, reprinted January 2004)
    30/30
    BUDAPEST TREATY
    ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PROCESSING PATENTS
    INTERNATIONAL FORM
    RECEIPT IN CASE OF ORIGINAL DEPOSIT ISSUED UNDER RULE 7 1 AND STATEMENT OF FEASIBILITY ISSUED UNDER RULE 10.2
    The American Type Culture Collection (ATCC®) received your seed / lineage (s) / strain (s) deposit in association with filing a patent application for a patent. The following information is provided to satisfy the requirements of the Patent Registry.
    Mr. Harsh Bais
    University of Delaware
    Innovation Way
    Newark, DE 19711
    Deposited in the Name of: University of Delaware
    Date of receipt of seeds / lineage (s) by ATCC®: April 26, 2011
    Ref, from Ident. by the Depositor: ATCC® Patent Deposit Designation: Amount received:
    Bacillus subtilis: UD1022 PTA-11857 25 vials
    ATCC® understands that:
    1. The deposit of these seeds / strain (s) does not grant ATCC® a license, express or implied, to infringe the patent and our release of these seeds / strain (s) to others does not grant them a license, express or implied , to infringe the patent.
    2. If the deposit needs to expire or be destroyed during the effective term of the patent, it must be your responsibility to replace it with viable material: It must also be your responsibility to provide a sufficient amount for distribution during the deposit term. ATCC® will distribute and maintain the material for 30 years or 5 years after the most recent deposit application, whichever is longer. The United States of America and many other countries are signatories to the Budapest Treaty.
    Prior to granting a US Patent, ATCC® agrees to pay a one-time service fee, not to distribute these seeds / strain (s) or any information relating to them or their deposit except as instructed by the depositor or registration of relevant patent. After relevant patent grants, we are responsible for releasing the seeds / strain (s) and these will become available for distribution to the public without any restrictions. We will inform you of the requests for the seeds / strain (s) for 30 years from the date of deposit.
    The deposit was tested on May 10, 2011 and until that date, the seeds / strain (s) were viable International Depositing Authority: American Type Culture Collection (ATCC®), Manassas VA, USA
    Signature of the person authorized to represent ATCC®:
    digital signature of Latha Ramakrishaman
    Latha Ramakrishaman CN: cn = Latha Ramakrishman, o = ATCC ou = IPLS Department e-mail: lramakrishnan@atcc.org, c = US date: 10/05/2011 15:30:45 -04'00 '—--- ---------------------------- May 10, 2011
    Patent Depositor at ATCC® j) minutes
    权利要求:
    Claims (10)
    [1]
    1. Method for improving the quality of a plant, characterized by the fact that it comprises administering a minimum quantity of 1 x 106 CFU / seed of Bacillus subtilis FB17 (ATCC PTA-11857) to the plant's seed and subsequently planting the seed in the soil, wherein said administration results in (a) greater biomass in the plant, compared to an untreated plant;
    (b) greater tolerance to aridity in the plant, compared to an untreated plant;
    (c) inhibition of plant infection by a fungal pathogen, as compared to an untreated plant; and / or (d) increased tolerance for plant pathogens.
    [2]
    2. Method according to claim 1, characterized by the fact that the plant is a crop plant for bioenergy.
    [3]
    3. Method according to claim 2, characterized by the fact that the plant is Brachypodium distachyon.
    [4]
    4. Method according to claim 1 (a), characterized in that it comprises administering Bacillus subtilis FB17 to produce a greater biomass in the plant of about 5% to about 100% compared to an untreated plant.
    [5]
    5. Method according to claim 1, characterized in that the plant is selected from the group consisting of a corn plant, a soy plant, a rice plant, a tomato plant, a Zinnia plant, and barley .
    [6]
    6. Method according to claim 1 (c), characterized by the fact that the plant is a rice plant and the fungal pathogen is the rice blast fungus.
    [7]
    7. Method according to claim 6, characterized by the fact that it comprises administering Bacillus subtilis FB17 to the seed of the rice plant before planting.
    [8]
    Method according to claim 1 or 7, characterized
    Petition 870200008705, of 1/17/2020, p. 6/12
    2/2 because it comprises administering Bacillus subtilis FB17 to the seed in an amount above about 1x108 CFU / seed.
    [9]
    9. Method according to claim 6, characterized in that the symptoms of rice blast are reduced by about 5%
    5 to about 100% compared to an untreated plant.
    [10]
    10. Plant seed, characterized by the fact that it comprises a plant seed coating comprising Bacillus subtilis FB17, in which the amount of Bacillus subtilis FB17 is between about 1 x 10 6 CFU / seed and about 1 x 10 9 CFU /seed.
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    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-02-12| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2019-10-22| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-06-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/03/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    2021-08-17| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2579 DE 09/06/2020 QUANTO AO INVENTOR. |
    优先权:
    申请号 | 申请日 | 专利标题
    US30913410P| true| 2010-03-01|2010-03-01|
    US41410810P| true| 2010-11-16|2010-11-16|
    US41603910P| true| 2010-11-22|2010-11-22|
    PCT/US2011/026683|WO2011109395A2|2010-03-01|2011-03-01|Compositions and methods for increasing biomass, iron concentration, and tolerance to pathogens in plants|
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