CYANOBACTERIUM TREATED DICOT PLANT SEEDS AND RELATED

METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international patent application claims the benefit of U.S. Provisional Patent Application No. 62/144,675, filed April 8, 2015 and incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing in text file (54886-154378-SEQ-LIST-ST25.txt; Size: 2,940 bytes (as measured in MS DOS); and Date of Creation: April 5, 2016) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND

[0003] Cyanobacteria are photosynthetic prokaryotes of the phylum Cyanobacteria. Although Cyanobacteria have been referred to as "blue-green algae," it is now recognized that Cyanobacteria should not be considered "algae" as they are prokaryotes rather than eukaryotes.

[0004] Treatment of various dicot plants with certain Cyanobacteria to improve dicot plant growth has been reported. Treatment of soil with a Nostoc strain soon after seed germination has been reported to improve maize growth and nitrogen uptake (Maqubela et al, Plant Soil Plant and Soil 315: 79-92, 2009). However, Maqubela et al. also report that the "high rate of application can not be recommended for field scale application as the amount of inoculum required would be unrealistically high" (Maqubela et al. at page 91). Treatment of rice paddy fields with a mixture of a mixture of Anabaena, Nostoc, Aulosira and Tolypothrix to improve rice yield has also been reported (Mishra and Pabbi, Resonance 9(6): 6-10, 20040). Although improved methods of producing the cyanobacterial inoculum are reported by Mishra and Pabbi, they report that "the effects of inoculation" are inconsistent and that the best results were obtained when the inocula are produced from local stocks and used with a low level of nitrogenous fertilizer (Mishra and Pabbi at page 9).

SUMMARY

[0005] Provided herein are treated dicot plant seeds that have been at least partially coated with a composition comprising: (i) at least one cyanobacterium species; and (ii) an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or a combination thereof. In certain embodiments, the dicot plant seed is selected from the group consisting of a soybean seed, a cotton seed, a Brassica sp. seed, a tomato seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed. In certain embodiments, the cyanobacterium species are characterized by having a gene encoding a 16S PvNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4. In certain embodiments, the cyanobacterium species are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l and/or SEQ ID NO:4. In certain embodiments, the cyanobacterium species is or are of the order Nostocales. In certain embodiments, the composition comprises one, two, three, or four cyanobacterium species that are members of the family Nostocaceae and/or at least one cyanobacterium species that is a member of the family Microchaetaceae. In certain embodiments, the member or members of the family Nostocaceae are selected from the group consisting of a Nostoc sp., an Aulosira sp., an Anabaena sp., and wherein the member the family Microchaetaceae is a Tolypothrix sp. In certain embodiments, the member or members of the family Nostocaceae are selected from the group consisting of a Nostoc commune UTEX B 1621 culture, an Aulosira bohemensis UTEX B 2947 culture, an Anabaena cylindrica UTEX B 1611 culture, isolate(s) therefrom, and a variant thereof and wherein the member or members of the family Microchaetaceae are selected from the group consisting of a Tolypothrix distorta UTEX 424 culture, isolate(s) therefrom, and a variant thereof. In certain embodiments, the member or members of the family Nostocaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3, and wherein the member or members of the family Microchaetaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO:4. In certain embodiments, the member or members of the family Nostocaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l and wherein the member or members of the family Microchaetaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO:4. In certain embodiments, the cyanobacteria are any one of: (i) Aulosira sp. and Tolypothrix sp.; (ii) Aulosira sp. and Anabaena sp.; (iii) Aulosira sp.; (iv) Aulosira sp., Anabaena sp., and Tolypothrix sp., or (v) Nostoc sp. In certain embodiments, the cyanobacteria are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of: (i) SEQ ID NO: l; (ii) SEQ ID NO: l and SEQ ID NO:2; (iii) SEQ ID NO: l and SEQ ID NO:4; or (iv) SEQ ID NO:3 and SEQ ID NO:4. In certain embodiments, the treated seed is a cotton, peanut, tomato, Brassica napus, or soybean seed and lateral root hair growth in a seedling obtained from the treated seed is increased in comparison to lateral root hair growth in a seedling obtained from an untreated control seed. In certain embodiments, the seed is a cotton, peanut, tomato, Brassica napus, or soybean seed and wherein biomass, yield, and/or nitrogen uptake of a plant obtained from the treated seed is increased in comparison to biomass, yield, nutrient uptake, and/or nitrogen uptake of a plant obtained from an untreated control seed. In certain embodiments, the seed is a cotton, peanut, tomato, Brassica napus, or soybean seed and at least one of root branching, lateral root growth, or vertical root growth in a seedling or plant obtained from the treated seed is increased in comparison to at least one of root branching, lateral root growth, or vertical root growth in a seedling or plant obtained from an untreated control seed. In certain embodiments, the seed is a cotton, peanut, tomato, Brassica napus, or soybean seed and wherein biomass, yield, and/or nutrient uptake of a plant obtained from the treated seed is increased in comparison to biomass, yield, and/or nutrient uptake of a plant obtained from an untreated control seed. In certain embodiments, the agriculturally acceptable adjuvant comprises an insecticide, a fungicide, a safener, and combinations thereof. In certain embodiments, the insecticide is selected from the group consisting of a carbamate, an organophosphate, a neonicotinoid, a pyrethroid, and combinations thereof. In certain embodiments, neonicotinoid insecticide is selected from the group consisting of thiamethoxam, imidacloprid, clothianidin, nitenpyram, nithiazine, thiacloprid, and combinations thereof. In certain embodiments, exogenous tackifier content is reduced in comparison to exogenous tackifier content of a standard microbial seed inoculant. In certain embodiments, the composition contains at least one element selected from the group consisting of iron, boron, manganese, zinc, molybdenum, copper, cobalt, salts thereof, and combinations thereof. In certain aspects of any of the aforementioned embodiments: (i) the cyanobacterium is not associated with the plant seed in nature; (ii) the composition comprises at least two cyanobacterium species that are not associated with the plant seed in nature; (iii) the composition comprises at least two cyanobacterium species that are not associated with one another in nature; or (iv) the composition comprises an exopolysaccharide (EPS). In certain aspects of any of the aforementioned embodiments, the composition has been lyophilized or has been dried upon the surface of the seed. In certain aspects of any of the aforementioned embodiments, the composition comprises about 1 milligram to about 500 milligrams per seed of the cyanobacterium species. Also provided are the use of the aforementioned treated seeds to: (i) improve biomass, yield, nitrogen uptake and/or nutrient uptake in a dicot plant including, but not limited to a soybean, a cotton, a Brassica sp., and a tomato plant; (ii) to improve plant growth in a dicot plant, including, but not limited to, improved growth in comparison to a dicot plant obtained from an untreated control seed where the plants are grown under conditions where nitrogen fertilizer is not used, a suboptimal amount of nitrogen fertilizer is used, or where less than 40, 60, 70, 80, 90, 100, 150 or 200 lbs of nitrogen fertilizer per acre is used. In certain embodiments, the seed is dormant before, during, and/or after treatment. [0006] Also provided are methods of improving plant growth in a dicot plant comprising exposing an seed to an effective amount of a composition comprising: (i) at least one cyanobacterium species; and (ii) an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or combination thereof and allowing the seed to germinate, wherein growth of a dicot plant obtained from the seed exposed to the composition is increased in comparison to growth of a dicot plant obtained from a control dicot plant seed that was not exposed to the composition. In certain embodiments, the dicot plant seed is selected from the group consisting of a soybean seed, a cotton seed, a Brassica sp. seed, a tomato seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed. In certain embodiments, the plant growth is improved under conditions where nitrogen is limiting. In certain embodiments, the exposing comprises applying the composition to a surface of the seed to obtain a seed that is at least partially coated with the composition. In certain embodiments, nitrogen fertilizer is not used. In certain embodiments, a suboptimal amount nitrogen fertilizer is used. In certain embodiments, less than about 40, 60, 80, 90, 100, 150, or 200 lb/acre of nitrogen fertilizer is used. In certain embodiments, the seed is dormant before, during, and/or after treatment.

[0007] Also provided are methods of improving biomass, yield, nutrient uptake, and/or nitrogen uptake in a dicot plant comprising exposing a dicot seed to an effective amount of a composition comprising: (i) at least one cyanobacterium species; and (ii) an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or combination thereof and allowing the seed to germinate, wherein biomass, yield, nitrogen uptake and/or nutrient uptake in a dicot plant obtained from the dicot seed exposed to the composition is increased in comparison to biomass, yield, nitrogen uptake, and/or nutrient uptake in a dicot plant obtained from a control dicot seed that was not exposed to the composition. In certain embodiments, the dicot plant seed is selected from the group consisting of a soybean seed, a cotton seed, a Brassica sp. seed, a tomato seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed. In certain embodiments, the seed is dormant before, during, and/or after treatment.

[0008] Also provided are methods of obtaining an dicot plant seed that provides for improved growth in a dicot plant obtained from the seed comprising applying a composition comprising: (i) at least one cyanobacterium species; and (ii) an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or combination thereof to at least one surface of the seed to obtain a seed that is at least partially coated with the composition, wherein growth of a dicot plant obtained from the treated dicot plant seed is increased in comparison to growth in a dicot plant obtained from an untreated control dicot plant seed. In certain embodiments, the dicot plant seed is selected from the group consisting of a soybean seed, a cotton seed, a Brassica sp. seed, a tomato seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed. In certain embodiments, the seed is dormant before, during, and/or after treatment.

[0009] In certain aspects of any of the aforementioned methods or uses, the composition is lyophilized. In certain aspects of any of the aforementioned methods or uses, the composition is applied as a liquid or slurry. In certain aspects of any of the aforementioned methods or uses, the composition is dried upon the surface of the seed. In certain aspects of any of the aforementioned methods or uses, the composition comprises about 1, 2, or 4 milligrams to about 50, 100, or 500 milligrams per seed of the cyanobacterium species. In certain aspects of any of the aforementioned methods or uses, the cyanobacterium are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4. In certain aspects of any of the aforementioned methods or uses, the cyanobacterium are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l and/or SEQ ID NO:4. In certain aspects of any of the aforementioned methods or uses, the cyanobacterium species is or are of the order Nostocales. In certain aspects of any of the aforementioned methods or uses, the composition comprises one, two, three, or four cyanobacterium species that are members of the family Nostocaceae and/or at least one cyanobacterium species that is a member of the family Microchaetaceae. In certain aspects of the aforementioned method or use, the member or members of the family Nostocaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% sequence identity across the entire length of SEQ ID NO: l , SEQ ID NO:2, or SEQ ID NO:3, and wherein the member or members of the family Microchaetaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO:4. In certain aspects of the aforementioned method or use, the member or members of the family Nostocaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% sequence identity across the entire length of SEQ ID NO: l and wherein the member or members of the family Microchaetaceae are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% sequence identity across the entire length of SEQ ID NO:4. In certain aspects of the aforementioned method or use, the member or members of the family Nostocaceae are selected from the group consisting of a Nostoc sp., an Aulosira sp., an Anabaena sp., and wherein the member of the family Microchaetaceae is a Tolypothrix sp. In certain aspects of the aforementioned method or use, the member or members of the family Nostocaceae are selected from the group consisting of a Nostoc commune UTEX B 1621 culture, an Aulosira bohemensis UTEX B 2947 culture, an Anabaena cylindrical UTEX B 1611 culture, isolate(s) therefrom, and a variant thereof and wherein the member or members of the family Microchaetaceae are selected from the group consisting of a Tolypothrix distorta UTEX 424 culture, isolate(s) therefrom, and a variant thereof. In certain aspects of any of the aforementioned methods or uses, the cyanobacteria any one of: (i) Aulosira sp. and Tolypothrix sp.; (ii) Aulosira sp. and Anabaena sp.; (in) Aulosira sp.; (iv) Aulosira sp., Anabaena sp., and Tolypothrix sp.; or (v) Nostoc sp. In certain aspects of any of the aforementioned methods or uses, the cyanobacteria are characterized by having a gene encoding a 16S RNA that has at least 95%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% sequence identity across the entire length of: (i) SEQ ID NO: l; (ii) SEQ ID NO: l and SEQ ID NO:2; (iii) SEQ ID NO: l and SEQ ID NO:4; (iv) SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3; or (v) SEQ ID NO:4. In certain aspects of any of the aforementioned methods or uses, the cyanobacteria are any one of: (i) Aulosira bohemensis sp., and a Tolypothrix distorta.; (ii) Aulosira bohemensis; (iii) Aulosira bohemensis and Anabaena cylindrical; (iv) Aulosira bohemensis, Anabaena cylindrical, and Tolypothrix distorta; or (v) Nostoc commune. In certain aspects of any of the aforementioned methods or uses, the agriculturally acceptable adjuvant comprises an insecticide, a fungicide, a safener, and combinations thereof. In certain aspects of the aforementioned method or use, insecticide is selected from the group consisting of a carbamate, an organophosphate, a neonicotinoid, a pyrethroid, and combinations thereof. In certain aspects of the aforementioned method or use, the neonicotinoid insecticide is selected from the group consisting of thiamethoxam, imidacloprid, clothianidin, nitenpyram, nithiazine, thiacloprid, and combinations thereof. In certain aspects of any of the aforementioned methods or uses: (i) the cyanobacterium is not associated with the plant seed in nature; (ii) the composition comprises at least two cyanobacterium species that are not associated with the plant seed in nature; (iii) the composition comprises at least two cyanobacterium species that are not associated with one another in nature; or (iv) the composition comprises an exopolysaccharide (EPS). In certain embodiments, the seed is dormant before, during, and/or after treatment.

BRIEF DESCRIPTION OF FIGURES

[0010] Figure 1 shows weight and weight measurements from the cotton greenhouse trial. The listed figures are an average of the two soil types, Crevasses and Loring. Addition of the cyanobacteria showed increases in height of 15.45% and weight of 24.73%, compared to an untreated control. [0011] Figure 2 shows differences in roots observed in the greenhouse trial. Roots from untreated control seeds had less root biomass, branching and depth than roots from seeds that had been treated with cyanobacterial Mixture.

DETAILED DESCRIPTION

[0012] Provided herein are dicot plant seeds treated with various cyanobacterial compositions that deliver improved dicot plant growth characteristics, methods of making the treated seeds, and methods of using the seeds. Such improved growth characteristics conferred by the seed treatments include, but are not limited to, improved nitrogen uptake, increased lateral root hair growth, and increased yield under nitrogen limiting growth conditions. Also provided are dicot plant seeds, methods of making, and methods of use where the seeds are treated with combinations of cyanobacterium species that can effect more pronounced improvements in the aforementioned growth characteristics when compared to untreated seeds or seeds treated with other combinations of cyanobacterium species.

[0013] Cyanobacteria used in the compositions provided herein include, but are not limited to, cyanobacteria in the order Nostocales. In certain embodiments, the composition comprises at least one, two, three, or four cyanobacterium species that are members of the family Nostocaceae and/or at least one cyanobacterium species that is a member of the family Microchaetaceae. In certain embodiments, the member or members of the family Nostocaceae are selected from the group consisting of a Nostoc sp., an Aulosira sp., an Anabaena sp., and the member the family Microchaetaceae is a Tolypothrix sp. Non-limiting examples of a Nostoc sp., an Aulosira sp., an Anabaena sp., and a Tolypothrix sp. and cultures comprising the same that can be used in certain embodiments of the compositions are provided in the following Table 1. Table 1. Cyanobacterium species

All strains with a "UTEX" designation are publically available through the University of Texas at Austin UTEX Culture Collection of Algae, 1 University Station A6700, Austin, TX, USA or via the internet on the world wide web site "utex.org."

[0014] Cyanobacterium that can be used in the compositions used to treat the seeds and related methods of making or use can also be identified by the sequence of the gene encoding the 16S RNA. In certain embodiments, the cyanobacteria that are used are characterized by having a gene encoding a 16S RNA that has at least about 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and or SEQ ID NO:4. Useful 16S RNA sequences for identifying cyanobacteria that can be used in the compositions are provided in Table 2. Table 2. Cyanobacterium 16S RNA sequences

6S consensus sequences were generated by harvesting liquid cyanobacterial cultures, amplifying the 16S ribosomal region by PCR, gel-purifying the 16S band, and sequencing the resulting PCR product. Sequences are made up of many reads that were combined to generate a consensus sequence. Lower-case letters in the published sequences denote locations where variation was found in the base at that position such that it reduced confidence to below the set threshold.

[0015] A cyanobacterium useful in certain compositions and methods provided herein can also be obtained by isolation from terrestrial sources including, but not limited to soil and plants, as well as aquatic sources. One useful method for isolating cyanobacteria that can be used involves nutrient saturated glass fiber filters in combination with a broad spectrum beta-lactam antibiotic to isolate cyanobacteria (Ferris and Hirsch, Appl. Environ. Microbio, 57(5): 1448-1452, 1991). Additional methods for obtaining cyanobacteria that use phototaxis and scored agar surfaces in the presence of certain antibiotics can also be used (Vaara et al. Appl. Environ. Microbiol. 38(5): 1011-4, 1979). Isolation methods including by not limited to those described by the aforementioned citations and references cited therein can also be used to obtain isolates from non-axenic cyanobacteria cultures to obtain cyanobacterium isolates that can be used in the methods and compositions provided herein. In certain embodiments, the isolates are obtained from a non-axenic Nostoc commune UTEX B 1621 culture, a non-axenic Aulosira bohemensis UTEX B 2947 culture, a non-axenic Anabaena cylindrica UTEX B 1611 culture, and a non-axenic Tolypothrix distorta UTEX 424 culture.

[0016] Variants of any of the aforementioned cyanobacteria can also be used in compositions and methods provided here. In certain embodiments, the cyanobacteria variants are variants that have been induced by mutagenesis and selected for one or more useful traits. Mutagenesis techniques include, but are not limited to use of alkylating agents, intercalating agents, transposons, and the like. Cyanobacteria variants can be screened and then selected for useful traits that include, but are not limited to, increased phototaxis, changes in genome copy number, increased protein production, or altered pigment expression. Variants can also be obtained where the cyanobacteria have been genetically transformed with a heterologous transgene. Methods for transforming cyanobacteria that have been disclosed (Chaurasia, et al., J Microbiol Methods. 73(2): 133- 41, 2008) can be used to obtain such variants.

[0017] Seeds treated with compositions provided herein can be used to improve a variety of dicot plant growth characteristics including, but not limited to, plant biomass, yield, lateral root growth, lateral root hair growth, root branching, lateral root growth, vertical root growth, growth under nitrogen limiting conditions, nitrogen uptake and/or nutrient uptake. A nutrient is defined as a mineral or inorganic compound that is absorbed from soil or other growth medium that is essential for plant growth or metabolism. Nutrients include nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, boron, chlorine, manganese, iron, zinc, copper, molybdenum, and nickel. In certain embodiments, the improvement in the dicot plant growth characteristic is in comparison to the growth characteristic of a dicot plant obtained from an untreated dicot plant seed. In certain embodiments, the improvement in the dicot plant growth characteristic is in comparison to the growth characteristic of a dicot plant that has been treated with a topical application of cyanobacteria. In certain embodiments, the improvement in the dicot plant growth characteristic is in comparison to the growth characteristic of a dicot plant that has been treated with a topical application of a cyanobacterial mixture of a Nostoc commune UTEX B 1621 culture, a Aulosira bohemensis UTEX B 2947 culture, a Anabaena cylindrica UTEX B 1611 culture, and a Tolypothrix distorta UTEX 424 culture. In certain embodiments, the improvement in the dicot plant growth characteristic is in comparison to the growth characteristic of a dicot plant obtained from a dicot plant seed treated with a cyanobacterial mixture of a Nostoc commune UTEX B 1621 culture, a Aulosira bohemensis UTEX B 2947 culture, a Anabaena cylindrica UTEX B 1611 culture, and a Tolypothrix distorta UTEX 424 culture. In certain embodiments, dicot seeds are treated with a composition having: (i) Aulosira sp. and Anabaena sp.; (ii) Aulosira sp.; or (iii) Aulosira sp. and Tolypothrix sp. In certain embodiments, dicot seeds are treated with a composition having: (i) Aulosira sp., Anabaena sp., and Tolypothrix sp., or (ii) Nostoc sp. In certain embodiments, dicot seeds are treated with a composition having Aulosira sp., Anabaena sp., Tolypothrix sp., and Nostoc sp. In certain embodiments, the Aulosira sp., Anabaena sp., Nostoc sp., and/or Tolypothrix sp. used in the composition can be characterized by having at least about 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4. In certain embodiments, the Aulosira sp., Anabaena sp., Nostoc sp., and/or Tolypothrix sp. used in the composition can be characterized by having at least about 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In certain embodiments, dicot seeds are treated with a composition having: (i) an Aulosira bohemensis sp. and a Tolypothrix distorta sp.; (ii) an Aulosira bohemensis sp.; (iii) an Aulosira bohemensis sp. and an Anabaena cylindrica sp.; (iv) an Aulosira bohemensis sp., an Anabaena cylindrica sp., and a Tolypothrix distorta sp.; or (v) a Nostoc commune sp. In certain embodiments, the cyanobacteria used in the composition can be characterized by having at least about 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4. Representative examples of cyanobacteria that can be used in the compositions and methods provided herein that are characterized by having at least about 95%, sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4 include, but are not limited to, one or more of the cyanobacteria provided in Table 3. Representative examples of cyanobacteria that can be used in the compositions and methods provided herein also include, but are not limited to, combinations of the cyanobacteria provided in Tables 1 and 3. In any of the aforementioned embodiments, the dicot plant or dicot seed can be a soybean seed, a cotton seed, a Brassica sp. seed, a tomato seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed.

Table 3. Representative Cyanobacteria having 16S RNA Sequences with significant sequence identity to SEQ ID NO: l, 2, 3, and/or 4

Sequence Culture Identity NCBI Culture Collection

% Accession ¹ Collection ² Location

Nostoc azollae 0708 96% NC 014248.1 None N/A

American Manassas,

Anabaena variabilis Type Culture Virginia, ATCC 29413 96% NC 007413.1 Collection USA

Nodularia Culture

spumigena collection of Bratislava, CCY9414 96% CP007203.2 yeasts Slovakia

Nostoc sp. PCC Pasteur Culture

7107 95% NC 019676.1 Collection Paris, France

University of

Helisinki

Cyanobacterial

Culture Helsinki,

Anabaena sp. 90 95% NC 019427.1 Collection Finland

Isolates having at

least 95% 16S %

sequence identity Sequence

to Anabaena Identity to

cylindrica 16S SEQ SEQ ID

ID NO:2 NO:2

Anabaena

cylindrica PCC Pasteur Culture

7122 97% NC 019771.1 Collection Paris, France

Calothrix sp. PCC Pasteur Culture

7507 99% NC 019682.1 Collection Paris, France

Nostoc punctiforme Pasteur Culture

PCC 73102 97% NC 010628.1 Collection Paris, France

Nostoc sp. PCC Pasteur Culture

7120 96% NC 003272.1 Collection Paris, France Sequence Culture Identity NCBI Culture Collection

% Accession ¹ Collection ² Location

Cylindrospermum Pasteur Culture

stagnate PCC 7417 97% NC 019757.1 Collection Paris, France

Nostoc sp. PCC Pasteur Culture

7107 95% NC 019676.1 Collection Paris, France

American Manassas,

Anabaena variabilis Type Culture Virginia, ATCC 29413 96% NC 007413.1 Collection USA

Nodularia Culture

spumigena collection of Bratislava, CCY9414 96% NZ CP007203.1 yeasts Slovakia

University of

Helsinki

Cyanobacterial

Culture Helsinki,

Anabaena sp. 90 95% NC 019427.1 Collection Finland

Nostoc azollae 0708 96% NC 014248.1 None N/A

Nodularia Culture

spumigena collection of Bratislava, CCY9414 96% CP007203.2 yeasts Slovakia

Isolates having at

least 95% 16S %

sequence identity Sequence

to Nostoc Identity to

commune 16S SEQ SEQ ID

ID NO: 3 NO:3

Cylindrospermum Pasteur Culture

stagnale PCC 7417 97% NC 019757.1 Collection Paris, France

Calothrix sp. PCC Pasteur Culture

7507 99% NC 019682.1 Collection Paris, France

Nostoc sp. PCC Pasteur Culture

7107 95% NC 019676.1 Collection Paris, France Sequence Culture Identity NCBI Culture Collection

% Accession ¹ Collection ² Location

Nostoc sp. PCC Pasteur Culture

7120 96% NC 003272.1 Collection Paris, France

American Manassas,

Anabaena variabilis Type Culture Virginia, ATCC 29413 96% NC 007413.1 Collection USA

Anabaena

cylindrica PCC Pasteur Culture

7122 97% NC 019771.1 Collection Paris, France

Nostoc punctiforme Pasteur Culture

PCC 73102 97% NC 010628.1 Collection Paris, France

Isolates having at

least 95% 16S %

sequence identity Sequence

to Tolypothrix Identity to

distorta 16S SEQ SEQ ID

ID NO:4 NO:4

Nodularia Culture

spumigena collection of Bratislava, CCY9414 96% NZ CP007203.1 yeasts Slovakia

Calothrix sp. PCC Pasteur Culture

7507 99% NC 019682.1 Collection Paris, France

Nodularia Culture

spumigena collection of Bratislava, CCY9414 96% CP007203.2 yeasts Slovakia

Cylindrospermum Pasteur Culture

stagnale PCC 7417 97% NC 019757.1 Collection Paris, France

Anabaena

cylindrica PCC Pasteur Culture

7122 97% NC 019771.1 Collection Paris, France

Nostoc punctiforme Pasteur Culture

PCC 73102 97% NC 010628.1 Collection Paris, France Sequence Culture Identity NCBI Culture Collection

% Accession ¹ Collection ² Location

Nostoc sp. PCC Pasteur Culture

7120 96% NC 003272.1 Collection Paris, France

Sequences of the whole genomes or a selected chromosome of the indicated strains that contain the 16S RNA encoding sequences can be obtained from the National Center Biotechnology Information National Center for Biotechnology Information (NCBI) with the indicated accession numbers via the internet at the world wide web site "ncbi.nlm.nih.gov/nuccore." The NCBI is in the National Library of Medicine, Building 38A, Bethesda, MD 20894.

American Type Culture Collection (ATCC) isolates can be accessed via the internet at the World Wide Web site "atcc.org." The address for the ATCC is 10801 University Boulevard Manassas, VA 20110 USA.

Culture Collection of Yeasts (CCY) isolates can be accessed via the internet at the world wide web site "ccy.sk." The address for the CCY is Institute of Chemistry, Slovak Academy of Sciences, Diibravska cesta 9, 845 38 Bratislava, Slovakia. Pasteur Culture collection of Cyanobacteria (PCC) isolates can be accessed via the internet at the http address "cyanobacteria.web.pasteur.fr/." The address for the PCC is Collections des Cyanobacteries, Institut Pasteur, 28, rue du Docteur Roux, 75724 PARIS Cedex 15, FRANCE.

[0018] In certain embodiments, at least one of root branching, lateral root growth, or vertical root growth in a seedling or plant obtained from a dicot seed treated with the compositions or methods provided herein is increased in comparison to at least one of root branching, lateral root growth, or vertical root growth in a seedling or plant obtained from an untreated control seed. In certain embodiments, the treated dicot seed is a cotton, peanut, tomato, Brassica napus, or soybean seed. Root branching is defined herein as the process in which lateral roots grow out from the taproot and other lateral roots for the purpose of providing plant stability and nutrient uptake. Lateral root growth is defined herein as the elongation of seminal roots or offshoots from the taproot in a direction perpendicular to or nearly perpendicular to the soil surface. Vertical root growth is defined herein as the elongation of the root, taproot, and seminal roots in a direction perpendicular to the soil surface.

[0019] Any method of measuring the growth characteristic can be employed. Typically, the biomass, yield, and the like can be assessed by determining the mass of plant material obtained, the mass of seeds obtained, or the number of seeds and a per plant or per unit area (e.g., per acre or hectare) basis. Root branching, vertical root growth, and lateral root growth can be measured by visual inspection to determine length and/or width or by imaging to quantify the length, width, density, volume or branching of roots and root systems.

[0020] Seeds treated with compositions provided herein can be used to improve dicot plant growth under nitrogen limiting conditions, to improve nutrient uptake, and/or to improve nitrogen uptake by dicot plants. As used herein, the phrases "nitrogen limiting conditions" or "a suboptimal amount of nitrogen fertilizer is used" refer to conditions where plant growth or yield can be increased upon supplementing the plant growth medium (e.g., soil, synthetic rooting mixes, hydroponic liquids, flooded fields, and the like) with exogenous nitrogen or by improving the availability of nitrogen already present in the plant growth medium. In certain embodiments where nitrogen fertilizer is not used or where a suboptimal amount of nitrogen fertilizer is used, compositions having: (i) Aulosira sp. and Anabaena sp.; (ii) Aulosira sp.; or (iii) Aulosira sp. and Tolypothrix sp. can be used. In certain embodiments where a suboptimal amount of nitrogen fertilizer is used, where less than an equivalent of 70 lbs/acre of nitrogen, or where between about 0, 5, 10, 15, 20, or 25 to 50, 60, 70, 80, 100, 120, 150, or 200 lbs/acre of nitrogen are used, compositions having: (i) Aulosira sp.; (ii) Aulosira sp. and Tolypothrix sp.; (iii) Aulosira sp., Anabaena sp., and Tolypothrix sp., or (iv) Nostoc sp. can be used. In such embodiments, the Aulosira sp., Anabaena sp., Nostoc sp., and/or Tolypothrix sp. used in the composition can be characterized by having at least about 95%, 97%, 98%>, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, respectively. In certain embodiments where nitrogen fertilizer is not used, where a suboptimal amount of nitrogen fertilizer is used, where less than an equivalent of 75 lbs/acre of nitrogen, or where between about 0, 5, 10, 15, 20, or 25 to 50, 60, or 70 lbs/acre of nitrogen are used, dicot plant seeds are treated with a composition having: (i) an Aulosira sp., and a Tolypothrix sp.; (ii) an Aulosira sp; (iii) an Aulosira sp. and an Anabaena sp.; (iv) an Aulosira sp., an Anabaena sp., and a Tolypothrix sp.; or (v) a Nostoc sp. In certain embodiments where nitrogen fertilizer is not used, where a suboptimal amount of nitrogen fertilizer is used, where less than an equivalent of 80, 90, 100, 125, 150, 175, 180, or 190 lbs/acre of nitrogen, or where between about 0, 5, 10, 15, 20, 25, 50 to 80, 90, 100, 125, 150, 175, 180, or 190 lbs/acre of nitrogen are used, dicot plant seeds are treated with a composition having: (i) Aulosira bohemensis and Tolypothrix distorta; (ii) Aulosira bohemensis; (iii) Aulosira bohemensis and Anabaena cylindrica; (iv) an Aulosira bohemensis, an Anabaena cylindrica sp., and a Tolypothrix distorta sp.; or (v) a Nostoc commune sp. In certain embodiments where nitrogen fertilizer is not used, where a suboptimal amount of nitrogen fertilizer is used, where less than an equivalent of 110 lbs/acre of nitrogen, or where between about 0, 5, 10, 15, 20, 25, 50 to 80, 90, 100, or 110 lbs/acre of nitrogen are used, seeds are treated with a composition having: (i) Aulosira bohemensis and Tolypothrix distorta; (ii) Aulosira bohemensis; (iii) Aulosira bohemensis and Anabaena cylindrica; (iv) an Aulosira bohemensis, an Anabaena cylindrica sp., and a Tolypothrix distorta sp.; or (v) a Nostoc commune sp. In certain embodiments, improvements in dicot plant growth or dicot plant yield obtained by seed treatments with cyanobacterial compositions provided herein under any of the aforementioned nitrogen limiting conditions can be at least about 3%, 4%, 9%, 10%, 15%, or 20%> in comparison to an untreated control dicot plant or can be about 3%, 4%, 5%, 10%, to about 15%, 18%, 20%, 30%, or 40% in comparison to an untreated control dicot plant. In certain embodiments, improvements in dicot plant growth or dicot plant yield under any of the aforementioned nitrogen limiting conditions with a cyanobacterial seed treatment provided herein can be at least about 3%, 4%, 9%, or 10%> in comparison to an untreated control dicot plant or can be about 3%, 4%, 5%, to about 10%, 15%, 18%, or 25% in comparison to a dicot plant subjected to a topical treatment of the cyanobacteria. In any of the aforementioned embodiments, the dicot plant or dicot seed can be a soybean seed, a cotton seed, a Brassica sp. seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed.

[0021] Nitrogen uptake by plants obtained from seeds treated with the cyanobacterial compositions provided herein can be improved under nitrogen limiting conditions and under conditions where nitrogen is not limiting. Nitrogen uptake can be determined by a variety of methods that include, but are not limited to, isotopic and non- isotopic methods that have been described (Sandrock et a. Hort. Sci. 40(3):665, 2005; Norman et al. Soil Sci. Soc. Am. J. 56: 1521-1527. 1992). In certain embodiments, improvements in nitrogen uptake by dicot plants obtained by seed treatments with cyanobacterial compositions provided herein under any of the aforementioned nitrogen limiting conditions can be at least about 3%, 4%, 5%, 10%, 20%, 40%, 50% or 60% in comparison to an untreated control dicot plant or can be about 3%, 4%, 5%, 10%, to about 15%), 18%), 20%), 40%), 60%o, or 70% in comparison to an untreated control dicot plant. In certain embodiments, improvements in nitrogen uptake under any of the aforementioned nitrogen limiting conditions with a cyanobacterial seed treatment provided herein can be at least about 3%, 4%, 9%, or 10% in comparison to an untreated control dicot plant or can be about 3%, 4%, 5%, to about 10%, 15%, 20%, 30%, or 40% in comparison to a dicot plant subjected to a topical treatment of the cyanobacteria. In any of the aforementioned embodiments, the dicot plant or dicot seed can be a soybean seed, a cotton seed, a Brassica sp. seed, a peanut seed, a biofuel crop seed, or a forage crop seed. In certain embodiments, the Brassica sp. seed is a Brassica napus, Brassica oleracea, or other Brassica sp. seed. In certain embodiments, the Brassica napus seed is a canola seed. In certain embodiments, the forage crop seed is an alfalfa, clover, vetch, or other forage crop seed. In certain embodiments, the biofuel crop seed is a Camelina, Jatropha, sunflower, flax, or other biofuel crop seed.

[0022] Compositions used to treat dicot plant seeds can comprise (i) at least one cyanobacterium species; and (ii) an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or a combination thereof. In certain embodiments, the composition will provide about 1, 2, 4, or 5 milligrams to about 50, 100, 200, 300, 400, or 500 milligrams per seed of the cyanobacterium species. In certain embodiments where more than a single cyanobacterium species is used, a total of about 1, 2, 4, or 5 milligrams to about 50, 100, 200, 300, 400, or 500 milligrams per seed of the collected group of cyanobacterium species is provided. Ratios of the cyanobacteria within the composition can be varied. In certain embodiments, equal parts of each cyanobacterium species can be provided in the composition. In certain embodiments, a 3: 1 : 1 : 1 mixture by mass of Nostoc sp., Aulosira sp., Anabaena sp., and Tolypothrix sp., respectively, can be used. In certain embodiments, ratios with a range of about 1 : 1, 0.5: 1 to 0.3: 1, 0.2: 1 or about 1 :0.5, to 1 :0.3, or 1 :0.2 of (i) an Aulosira sp., and a Tolypothrix sp.; or (ii) an Aulosira sp. and an Anabaena sp. can be used. In certain embodiments, ratios of about 1 : 1 : 1 of an Aulosira sp., an Anabaena sp., and a Tolypothrix sp. can be used. In certain embodiments, any of the aforementioned ratios are used to provide a total of about, 2, 4, or 5 milligrams to about 50, 100, 200, 300, 400, or 500 milligrams per seed of the collected group of cyanobacterium species.

[0023] Agriculturally acceptable adjuvants used in the compositions can include, but are not limited to, one or more insecticides, fungicides, safeners, and combinations thereof. Such adjuvants can be selected for an absence of bacteriocidal, bacteriostatic, or bacterio-inhibitory activities that would reduce the effectiveness of the cyanobacteria provided in the composition. In certain embodiments, the adjuvants can include one or more of a carbamate, an organophosphate, a neonicotinoid, or a pyrethroid insecticide. In certain embodiments, the fungicide can include one or more of an azole, strobilurin, or metalaxyl compounds. Useful azole fungicides include, but are not limited to, difenoconazole, prothioconazole, and tebuconazole compounds. Useful strobilurin fungicides include, but are not limited to, kresoxim-methyl, azoxystrobin, trifloxystrobin, fluoxastrobin, picoxystrobin, pyraclostrobin, dimoxystrobin, pyribencarb, metominostrobin, orysastrobin, enestrobin, pyraoxystrobin and pyrametostrobin compounds. Useful metalaxyl fungicides include, but are not limited to, metalaxyl and mefenoxam.

[0024] Agriculturally acceptable excipients used in the compositions can include, but are not limited to, one or more bulking agents, binding agents, colorants, emulsifiers, oils, tackifiers, trace elements, and /or extending agents. Useful bulking agents include, but are not limited to, peat, wood flour, calcium carbonate, lime, diatomaceous earth, forms of clay such as bentonite and kaolin, and combinations thereof. Useful binding agents can be water soluble polymers and/or waxes. Binding agents that are used can include, but are not limited to, polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone, polyurethane, methyl cellulose, carboxymethyl cellulose, hydroxylpropyl cellulose, sodium alginate, polyurethane, polyacrylate, casein, gelatin, pullulan, polyacrylamide, polyethylene oxide, polystyrene, styrene acrylic copolymers, styrene butadiene polymers, poly(N-vinylacetamide), and combinations thereof. Waxes used as binders can include, but are not limited to, waxes such as carnauba wax, paraffin wax, polyethylene wax, bees wax, and polypropylene wax. Emulsifiers can be either ionic or non-ionic agents. Emulsifiers that can be used include, but are not limited to, lecithin, polysorbates, polyethylene glycols, derivatives thereof, and the like. Oils that can be used include, but are not limited to, silicon, animal, plant, or mineral oils and mixtures thereof. In certain embodiments, and exogenous tackifier can be added to the composition. However, content of an exogenous tackifier can be reduced in certain embodiments and in comparison to exogenous tackifier content of a standard microbial seed inoculant. In certain embodiments, the composition can also contains at least one element selected from the group consisting of iron, boron, manganese, zinc, molybdenum, copper, cobalt, salts thereof, and combinations thereof. Extenders are materials that provide for improvements in the viability of cyanobacteria on the seed either pre or post planting. Useful extenders include, but are not limited to, compounds such as trehalose, sucrose, glycerol, sorbitol, and combinations thereof. Liquid seed treatment inoculums containing various microorganisms and extenders comprising one or more of trehalose, sucrose, glycerol or sorbitol at about 5% to about 50% by weight/volume and related methods where a partially desiccated liquid inoculant product for application to seeds is prepared are described in US Patent No. 8,020,343 and can be adapted to use with the cyanobacteria compositions provided herein. US Patent No. 8,020,343 is incorporated herein by reference in its entirety. Other liquid seed treatment compositions that can be adapted for use with the cyanobacteria compositions provide herein can comprise sucrose, sorbitol, or a combination thereof at about 5% to 60% weight/volume, mineral oil or silicon oil at about 0.15%) to about 3% weight, and an emulsifying agent selected from the group consisting of lecithin and polysorbate and are described in US Patent No. 8,551,913. US Patent No. 8,551,913 is incorporated herein by reference in its entirety. Exopolysaccharides (EPS) produced by the cyanobacteria or by other bacteria can also be used as agriculturally acceptable excipients in the compositions provided herein. In certain embodiments, the EPS can be provided in the composition by simply adding fermentation broths, filtrates, supernatants, purified fractions, partially purified fractions, and the like that are obtained from cyanobacterial or other bacterial cultures. Without seeking to be limited by theory, it is believed that such EPS can improve water retention and/or desiccation tolerance of cyanobacteria in the compositions provided herein by slowing the desiccation process. It has been reported that bacterial EPS can help bacteria adapt to variable hydration conditions (Or et al. Advances in Water Resources, 30 (2007), pp. 1505-1527; Redmile-Gordon et al., Soil Biology & Biochemistry 72 (2014) 163el71). In certain embodiments, exopolysaccharides obtained from sources other than cyanobacterial or other bacterial cultures that include, but are not limited to, fungal or yeast sources can be used either alone or in combination with the aforementioned cyanobacterial or bacterial cultures as agriculturally acceptable excipients in the compositions provided herein. Furthermore, the aforementioned reductions in content of exogenous tackifier can be achieved in certain embodiments by using such EPS in the as agriculturally acceptable excipients in the compositions provided herein. Seed coating equipment and associated techniques used to coat the seeds include, but are not limited to, drum coaters, fluidized beds, rotary coaters, side vended pan, tumble mixers, and spouted beds.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of inconsistencies between the present disclosure and the issued patents, applications, and references that are cited herein, the present disclosure will prevail.

[0026] The following examples are offered by way of illustration and not by way of limitation. Examples

EXAMPLE 1. Microbial biomass production

[0027] Nostoc commune UTEX B 1621, Anabaena cylindrica UTEX B 1611, Aulosira bohemensis UTEX B 2947, and Tolypothrix distorta UTEX 424 will be maintained under constant shaded cool white fluorescent light on sterile BGl l plates. BGl l media has been described (Rippka Archiv fur Mikrobiologie 87, Issue 1, pp 93-98, 1972) and is commercially available (Sigma-Aldrich, St. Louis, MO). Plates will be re- streaked onto fresh plates as colonies appear using a microbiological loop under sterile conditions. For amplification, biomass from plates will be transferred into several autoclaved 250 ml Erlenmeyer flasks containing 100 ml sterile BG11 by swishing a sterile microbiological loop containing microbial biomass (obtained from the plates) in the liquid media. The Erlenmeyer flasks will be stoppered with autoclaved foam stoppers.

[0028] The 250 ml Erlenmeyer flasks containing monocultures will be shaken at 120 rpm on an orbital shaker in constant cool white fluorescent light at 22 °C until cultures reach translucent green cell density. The entirety of four 250 ml Erlenmeyer flasks with the same species of cyanobacteria, containing 100 ml of BGl l each, will then be transferred to autoclaved tubular 4 L flasks containing 3.5 L sterile BG11. The 4 L bubble flasks will be mixed by aeration through an autoclaved bubble tube connected to an aquarium pump. A filter made of autoclaved cotton fibers will be placed in the tubing line to maintain sterility. An autoclaved foam stopper with a hole for the air-in port will be used to seal the 4 L flasks. The culture will be supplied with constant cool white fluorescent light at 22 °C.

[0029] Upon reaching translucent green cell density, 2 L culture from the 4 L bubble flasks will be transferred to a 20 L carboys containing 14 L sterile BGl l (for a total culture volume of 16 L). The 4 L bubble flask will be replenished with 2 L sterile BG11 to allow for additional 20 L inoculations as necessary. Monocultures growing in 20 L carboys will be diluted by half with fresh sterile BGl l as necessary to maintain dense, translucent green cultures. As necessary, upon dilution with fresh BGl l, excess culture will be transferred to plastic 20 L carboys that are washed with antibacterial soap and sterilized with ethanol. These cultures will also be supplied with constant light at 22 °C. If necessary to generate appropriate biomass, 20 L carboys will be used to inoculate 100 L flat panel photobioreactors containing 80 L sterile BG11.

[0030] Harvesting of cyanobacterial monocultures will begin by settling monocultures growing in 20 L carboys or 100 L photobioreactors for 2-4 hours (total settling time depended on time it takes individual containers to settle). After settling, clear supernatant will be siphoned off using autoclaved plastic tubing. A total of 1-6 L of each monoculture per 20 L carboy will remain after settling. Settled cultures will then be pulse blended in an Oster blender for less ~5 seconds. Cultures will then be centrifuged at 6,000 x g for 10 minutes in either a 6 x 50 ml benchtop centrifuge or a 6 x 1 L floor centrifuge. Centrifuge size will be chosen based upon total biomass required for subsequent plant treatments. Each vessel will be decanted after centrifugation, and biomass pellets will be combined into a sterilized beaker. Total wet weight biomass will be determined by measuring the weight of the beaker before and after the addition of biomass. Biomass cyanobacterial mixtures will be prepared by mixing biomass paste of each monoculture by weight with the other members of the cyanobacterial mixture to give the prescribed ratio. Biomass paste will then be diluted to a working volume with sterile Millipore water, giving a known working biomass concentration.

EXAMPLE 2. Dicot Seed Coating

[0031] A total of 4 mg cyanobacteria will be applied per seed to dicot seeds (e.g. soybean, cotton, tomato, Brassica sp., a peanut, biofuel crop, or a forage crop seeds) in batch applications. A control will be also created with no cyanobacteria applied. Cyanobacterial biomass will be prepared by harvesting excess liquid cyanobacterial monoculture, centrifuging, and removing the supernatant. The culture will then be centrifuged in tubes for 10 min at 6000 x g. The biomass pellets will then be diluted with a small amount of Nanopure™ water (Thermo Fischer Scientific, Inc. USA), harvested and combined into one tube. The cyanobacterial culture will then be homogenized by blending on a pulse setting in a kitchen blender for less than five seconds. As little blending as possible will be used to prevent unnecessary cell damage. After homogenization, the biomass will be spun for 10 min at 6000 x g to remove any remaining liquid. Cyanobacterial biomass will be added to individual tubes as a paste. Biomass will be calculated by subtracting the weight of the tube and paste from the weight of the tube only (determined at beginning of experiment). The 0 and 4 mg per seed application rates will then be diluted with Nanopure™ water to a known total volume before coating. Six replicates of each application rate will be prepared. Four will be used in seed coating and two will serve as backups in case there are unforeseen issues with coating.

[0032] Dicot seeds will be coated with 4 mg per seed of cyanobacteria (prepared as above) using Hege™ 11 liquid seed treater. Coating will be performed in 3 applications. Between coatings, seeds will be allowed to dry on a cookie sheet at room temperature for four hours. Drying between applications will be performed to prevent early germination.

EXAMPLE 3. Germination and root hair formation

[0033] Dicot seeds (e.g. soybean, cotton, tomato, Brassica sp., a tomato, a peanut, a biofuel crop, or a forage crop seeds) will be germinated and images of the roots will be captured daily for one week. Changes in the formation of lateral root hairs will be measured.

EXAMPLE 4: Greenhouse testing of Cyanobacterium treated seed

[0034] A greenhouse trial will be performed with plants grown from treated and untreated control dicot seeds (e.g. peanut, soybean, cotton, tomato, Brassica sp., a tomato, a biofuel crop, or a forage crop seeds). Mixture A will be Nostoc commune UTEX B1621 alone. Mixture B will be a 3: 1 : 1 : 1 mixture of Nostoc commune UTEX B 1621, Anabaena cylindrica UTEX B 1611, Aulosira bohemensis UTEX B 2947, and Tolypothrix distorta UTEX 424. Mixture C will be a 1 : 1 : 1 mixture of Aulosira bohemensis UTEX B 2947, Anabaena cylindrica UTEX B 1611, and Tolypothrix distorta UTEX 424. All cyanobacterial mixtures will be applied at a rate of 4 mg cyanobacterial mixture per seed. During the trial, seeds coated with Mixture A, Mixture B, Mixture C, and a control with no seed coating (Table 2) will be grown for 8-16 weeks in a greenhouse. Plants will be grown in plastic pots in potting soil. They will be watered in four stepwise chemical nitrogen regimes which represent the typical amounts applied in commercial agricultural operations. Physiological parameters that include, but are not limited to: chlorophyll content, biomass, stem diameter and leaf formation will be measured. It is anticipated that seeds coated with Mixture A, Mixture B, or Mixture C will provide dicot plants with improved growth characteristics when compared to dicot plants obtained from control seeds with no seed coating.

EXAMPLE 5. Cyanobacterial treatment of dicot plants by treatment of fields or by seed treatment in field tests

[0035] A mixture of Nostoc commune UTEX B 1621, Aulosira bohemensis UTEX B 2947, Anabaena cylindrica UTEX B 1611, and Tolypothrix distorta UTEX 424 respectively will be maintained and harvested essentially as described in Example 1. The cyanobacterial mixture will be diluted to a working concentration that allows the biomass to be treated as a liquid. Seed coating will be carried out in a Hege™ 11 Seed Coater. A desired coating rate of 500 g cyanobacterial mixture per acre will be achieved. This cyanobacterial mixture will be applied in three separate aliquots, and seeds will be allowed to dry before applying additional cyanobacterial mixture. After all coating runs, seeds will be spread out into a single layer and allowed to dry overnight at room temperature in a well-ventilated area.

[0036] Dicot seeds {e.g. soybean, cotton, tomato, Brassica sp., a tomato, peanut, a biofuel crop, or a forage crop seeds) treated as described above will then be planted in fields and grown in 0, low (-75), and high (-150) lbs/acre nitrogen. Upon reaching full maturity, dicot plants will be harvested and yield and total nitrogen uptake will be measured. Total above ground biomass will be collected from a i m section of the first bordered row. Following maturity, plots will be harvested. Statistical analysis will be conducted using a standard ANOVA and means were separated using Fisher's protected LSD at the a=0.05 level where appropriate.

EXAMPLE 7: Greenhouse testing of Cyanobacterium treated seed

[0037] A greenhouse trial was performed with plants grown from treated and untreated control cotton seeds. The Mixture was a 2: 1 ratio of Aulosira bohemensis UTEX B 2947 and Tolypothrix distorta UTEX 424. It was applied at a rate of 4 mg cyanobacterial mixture per seed. The cotton cultivar Stoneville 4946 GLB2 was used, as it is currently the most commonly used cultivar in the United States.

[0038] The study was implemented as a randomized complete block design with a factorial arrangement of two soils (Crevasses and Loring) and four N treatments (Nitrogen-Treatment). The four N-Treatments were: 1) 0 Cyanobacteria, 0 N fertilizer (control); 2) 500g/acre dosage of cyanobacteria, 0 synthetic N fertilizer; 3) 0 Cyanobacteria, urea to meet half of crop's N requirement to simulate the pre plant N application rate; and 4) no Cyanobacteria, urea to meet all of the crop's N requirement.

[0039] Four different fertilization levels were tested in the trial. Control seeds, with no coating and no additional fertilizer; seeds coated with Mixture; uncoated seeds with nitrogen fertilizer (46-0-0 NPK) equal to a 90 lbs per acre; and uncoated seeds with nitrogen fertilizer (46-0-0 NPK) equal to 45 lbs per acre. These were grown for 98 days in a greenhouse. Two types of soil were tested, Crevasses and Loring. Soil samples were dug up from research plots at the University of Arkansas, sieved and added to plastic pots. Soil pH, organic matter, NO ₃ -N, and Mehlich-3 extractable nutrients were measured by standard methods. Soil texture was measured by the hydrometer method.

[0040] Three cotton seeds were planted in each pot and thinned to one seed per pot five days after emergence. Supplemental lights were provided for 12 hours from 7:00 AM to 7:00 pm. The greenhouse temperature was maintained at no lower than 68°F.

[0041] Analysis of variance (ANOVA) was performed to evaluate the effect of soil type, N-Treatment and their interaction on early cotton growth and development parameters by using the SAS Package. When appropriate, means were separated by the least significant difference (LSD) method and interpreted as significant when <0.10. Results are found in Figure 1.

[0042] A noticeable difference in cotton growth and development between the two soils where the plants grew faster in Loring soil as compared to the Crevasses was observed, which was confirmed by statistical analysis of the data. Soil type and N- Treatment both significantly (P < 0.1) influenced the total number of nodes, monopodial (vegetative) nodes, sympodial (fruiting nodes), average internode length (the distance between the two successive nodes), final plant height, and final dry biomass. However, the interaction of soil type by N-Treatment significantly influenced only dry biomass per plant. Plant height and dry weight per pot of the cotton grown in Loring soil were 70% and 140% more than the plants in the Crevasses soil.

[0043] Visual evidence of root stimulation was observed. Seeds which had been treated with Mixture showed significantly more branching and deeper, thicker roots. Results are shown in in Figure 2.

[0044] In Crevasses soil, cotton fertilized with the full N-rate produced significantly higher biomass than cotton treated with cyano bacteria or cotton that did not receive any N. However, there was no significant difference in biomass production among the all treatments that received less than the full N rate. Biomass production rate in Crevasses may have been limited by the low soil pH. Cotton fertilized with 0.5 X N-rate produced numerically higher biomass than the cyano treated cotton which produced numerically higher biomass than the cotton that did not receive any cyano bacteria. In Loring soil cotton fertilized with the full N rate produced the highest dry biomass and cotton that did not receive any N or cyano bacteria produced the lowest biomass dry weight. The amount of biomass per single plant produced by the cyano treated bacteria was 25% more and significantly higher than the cotton that did not receive any N fertilizer or cyanobacteria.