WO2022147314A1

WO2022147314A1 - Chlorella sp. accession no. ncma 202012055 and methods of use thereof to benefit plant growth

Info

Publication number: WO2022147314A1
Application number: PCT/US2021/065772
Authority: WO
Inventors: Chiliang Chen; Dana GEISLINGER; Rosaicela ROMAN; Christopher TEE; Yan Xu
Original assignee: Heliae Development, Llc
Priority date: 2020-12-30
Filing date: 2021-12-30
Publication date: 2022-07-07

Abstract

The present invention provides an isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 obtained by selective mutant propagation or a mutant thereof having all the identifying characteristics thereof. Also provided are compositions comprising the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof and an agriculturally acceptable carrier and methods of plant enhancement by applying the compositions to a plant, a plant part and/or a plant locus.

Description

CHLORELLA SP. ACCESSION NO. NCMA 202012055 AND METHODS

OF USE THEREOF TO BENEFIT PLANT GROWTH

TECHNICAL FIELD

The present invention relates generally to compositions and methods for stimulating and maintaining enhanced growth in plants. More particularly, the present invention relates to compositions comprising an isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 obtained by selective mutant propagation or a mutant thereof having all the identifying characteristics thereof.

BACKGROUND

It is a common practice in the agricultural field both for food production, ornamental shrubs and trees, and lawn grasses to accelerate growth by the application of chemical fertilizers, e.g., nitrates, phosphates, and potassium compounds, and also chemical materials such as pesticides, herbicides, and fungicides, etc., that can be toxic. Further, it is a present practice to overload the crops with these chemical materials and to repeatedly treat most crops multiple times in a growing season (typically four times, may be as many as eight times depending on the plant and location) because these water-soluble substances would wash off. The significant amount of runoff means that users must use more of these substances and apply more times, which increases both the monetary and labor cost. The runoff also results in these chemical materials finding their way into the soil and the ground water, and into rivers, lakes, ponds and ultimately the bays and oceans. While these chemicals do enhance the growth of desirable plants, the runoff has toxic effects. Thus, there is a need for environmentally friendly and sustainable means for enhancing plant growth.

Chlorella, a genus of single-celled green microalgae, is considered the most photosynthetically efficient organism in the world. Chlorella's chlorophyll content can reach levels as high as 8%; approximately 16 times more than most green foods. Chlorella conducts photosynthesis through the absorption of sunlight by chlorophyll A, chlorophyll B, and carotenoid pigments located in its chloroplast.

It has now been recognized that various characteristics including the quality, health, and/or color of plants can be improved through the application of effective amounts of biomass that has been obtained from the cell tissue of Chlorella species. In addition, application of Chlorella biomass to soil increases soil aggregation and water retention thereby providing a more productive growth medium for plants. There is a need to develop effective Chlor ella- based agricultural products to supplement or replace chemical soil amendments and enhance crop growth and yield in a sustainable manner.

SUMMARY

The present invention is directed to an isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 obtained by selective mutant propagation or a mutant thereof having all the identifying characteristics thereof.

In certain aspects, the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof grows efficiently under heterotrophic conditions comprising growth on at least one organic carbon source in the absence of a supply of light and/or carbon dioxide.

In other aspects, the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof has a maximum specific growth of at least 1.0 days and/or a productivity of at least 1.9 g/L/day when grown in a culture medium comprising glucose and sodium nitrate in a shake flask at 25°C. In one aspect, the maximum specific growth of at least 1.0 days and/or a productivity of at least 1.9 g/L/day are measured in a culture medium comprising about 15 g/L glucose and about 3.9 g/L sodium nitrate in a shake flask at 25°C.

In one aspect, application of the Chlorella sp. Accession No. NCMA 202012055 or mutant thereof to soil increases the culturable bacterial population in the soil compared to the soil without application thereof. In some aspects, the soil is loam soil, sandy loam soil, or sand soil.

In one aspect, application of the Chlorella sp. Accession No. NCMA 202012055 or mutant thereof to soil increases the water holding capacity of the soil compared to the soil without application thereof.

The present invention also provides a cell-free or inactivated preparation of the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof.

In other aspects, the present invention is directed to a composition comprising the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof and an agriculturally acceptable carrier.

In one aspect, the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof comprises whole cells, lysed cells, or a combination thereof. In another aspect, the composition is formulated as a solid, liquid or gel. In some aspects, the composition is a solid formulation selected from the group consisting of a powder, lyophilizate, pellet, and granule. In other aspects, the composition is a liquid formulation selected from the group consisting of an emulsion, colloid, suspension, and solution.

In yet other aspects, the composition further comprises at least one culture stabilizer selected from the group consisting of potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, or a combination thereof.

In some aspects, the present invention relates to a plant propagation material treated with a composition described herein in an amount of from 0.01 g to 10 kg per 100 kg of plant propagation material.

In certain aspects, the present invention provides a method of plant enhancement comprising the step of: applying to a plant, a plant part and/or a plant locus an effective amount of a composition comprising an isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055, a mutant thereof having all the identifying characteristics thereof, or a cell-free or inactivated preparation thereof to enhance at least one plant characteristic.

In some aspects, the plant characteristic is selected from the group consisting of seed germination rate, seed germination time, seedling emergence, seedling emergence time, seedling size, plant fresh weight, plant dry weight, utilization, fruit production, leaf production, leaf formation, leaf size, leaf area index, plant height, thatch height, plant health, plant resistance to salt stress plant resistance to heat stress, plant resistance to heavy metal stress, plant resistance to drought, maturation time, yield, root length, root mass, color, blossom end rot, softness, plant quality, fruit quality, flowering, sun bum, and any combination thereof. In one aspect, the plant characteristic is plant fresh weight, plant dry weight, or yield.

In other aspects, in the disclosed composition the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof comprises whole cells, lysed cells, or a combination thereof.

In certain aspects, the composition is applied as a soil drench, an in-furrow treatment, a foliar application, a side-dress application, a pivot irrigation application, a seed coating, or with a drip system. In other aspects, the composition is administered at a rate of 0. 1 -150 gallons per acre (0.935-1402.5 liters per hectare) to enhance the at least one plant characteristic.

In yet other aspects, the plant is a member of a plant family selected from: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae, Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae, Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae (Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae, Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae, Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae, Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae), Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae, Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae, Piperaceae, and Proteaceae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the growth as indicated by dry weight (g/L) measured over time of Chlorella sp. Accession No. NCMA 202012055 and several other Chlorella strains.

FIG. 2 depicts the maximum specific growth (days ) of Chlorella sp. Accession No. NCMA 202012055 and several other Chlorella strains.

FIG. 3 depicts the productivity (g/L/day) of Chlorella sp. Accession No. NCMA 202012055 and several other Chlorella strains.

FIG. 4 depicts the culturable bacterial populations obtained from soil samples (i.e., loam soil) from Granger, Iowa following treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae), compared to those obtained from the same soil left untreated.

FIG. 5 depicts the culturable bacterial populations obtained from soil samples (i.e., sandy loam soil) from Farmville, North Carolina following treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae), compared to those obtained from the same soil left untreated.

FIG. 6 depicts the culturable bacterial populations obtained from soil samples (i.e., sand soil) from Douglas, Georgia following treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae), compared to those obtained from the same soil left untreated.

FIG. 7 depicts the culturable bacterial populations obtained from soil samples (i.e., sandy soil) from Douglas, Georgia following treatment with Chlorella sp. Accession No. NCMA 202012055, PHYCOTERRA® (whole cell Chlorella microalgae), or PHYCOTERRA® ORGANIC (whole cell Chlorella microalgae), compared to those obtained from the same soil left untreated.

FIG. 8 depicts the percent water holding capacity of soil samples (i.e., sandy soil) from Douglas, Georgia following treatment with Chlorella sp. Accession No. NCMA 202012055, PHYCOTERRA® (whole cell Chlorella microalgae), or PHYCOTERRA® ORGANIC (whole cell Chlorella microalgae), compared to those obtained from the same soil left untreated. DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

The term “microalgae” as used herein refers to microscopic single cell organisms such as microalgae, cyanobacteria, algae, diatoms, dinoflagellates, freshwater organisms, marine organisms, or other similar single cell organisms capable of growth in phototrophic, mixotrophic, or heterotrophic culture conditions.

As used herein, the term “selective mutant propagation” refers to the selection of fast growing Chlorella sp. mutants in growth competition assays under heterotrophic conditions.

As used herein, a “biologically pure” strain is intended to mean the strain separated from materials with which it is normally associated in nature. A strain associated with other strains, or with compounds or materials that it is not normally found with in nature, is still defined as “biologically pure.” A monoculture of a particular strain is, of course, “biologically pure.” In different embodiments, a “biologically pure” culture has been purified at least 2x or 5x or lOx or 50x or lOOx or lOOOx or higher (to the extent considered feasible by a skilled person in the art) from the material with which it is normally associated in nature. As a nonlimiting example, if a culture is normally associated with soil, the organism can be biologically pure to an extent that its concentration in a given quantity of purified or partially purified material with which it is normally associated (e.g. soil) is at least 2x or 5x or lOx or 50x or lOOx or lOOOx or higher (to the extent considered feasible by a skilled person in the art) that in the original unpurified material.

The term “plant propagation material” is to be understood to denote all the generative parts of the plant such as seeds and vegetative plant material such as cuttings and tubers (e. g. potatoes), which can be used for the multiplication of the plant. This includes seeds, roots, fruits, tubers, bulbs, rhizomes, shoots, sprouts and other parts of plants, including seedlings and young plants, which are to be transplanted after germination or after emergence from soil.

Analysis of the DNA sequence of the parental strain of Chlorella sp. described herein was done in the NCBI 18s rDNA reference database at the Culture Collection of Algae at the University of Cologne (CCAC) and showed substantial similarity (i.e. , greater than 95%) with multiple known strains of Chlorella and Micractinium. Those of skill in the art will recognize that Chlorella and Micractinium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time within the Chlorella and Micractinium genera. As would be understood in the art, the reclassification of various taxa is not unusual, and occurs as developments in science are made. Any disclosure in the specification regarding the classification of exemplary species or strains should be viewed in light of such developments. While the exemplary microalgae strain is referred to in the instant specification as Chlorella, it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to the exemplary microalgae strain would reasonably be expected to produce similar results. Accordingly, any mention of Chlorella herein should be understood to include Micractinium species genetically and morphologically similar to species classified within the genus Chlorella as of the filing date.

By artificially controlling aspects of the microalgae culturing process such as the organic carbon feed (e.g., acetic acid, acetate), oxygen levels, pH, and light, the culturing process differs from the culturing process that microalgae experiences in nature. In addition to controlling various aspects of the culturing process, intervention by human operators or automated systems occurs during the culturing of microalgae through contamination control methods to prevent the microalgae from being overrun and outcompeted by contaminating organisms (e.g., fungi, bacteria). By intervening in the microalgae culturing process, the impact of the contaminating microorganisms can be mitigated by suppressing the proliferation of containing organism populations and the effect on the microalgal cells (e.g., lysing, infection, death, clumping). Thus, through artificial control of aspects of the culturing process and intervening in the culturing process with contamination control methods, the microalgae culture produced as a whole and used in the described inventive compositions differs from the culture that results from a microalgae culturing process that occurs in nature.

Certain publicly available strains described herein are identified by the term “UTEX” followed by a unique identifier containing letters and/or numbers. The term “UTEX” refers to the UTEX Culture Collection of Algae located at 205 W. 24th St., Biological Labs 218, The University of Texas at Austin (A6700), Austin, TX 78712 USA. The UTEX Culture Collection of Algae provides over 3,000 different strains of algae, representing more than 500 genera, to the public for a modest charge including the strains disclosed herein.

Methods of Culturing, Processing, and Formulating Chlorella

In certain aspects, the Chlorella strains disclosed herein are cultured heterotrophically, mixotrophically, and/or phototrophically. In a preferred embodiment, the Chlorella strains disclosed herein are cultured heterotrophically. As used herein, “heterotrophic” culturing conditions comprises supplying a culture of microorganisms with the at least one organic carbon source in the absence of a supply of light and/or carbon dioxide.

The Chlorella strains can be cultured on sources of organic carbon or combinations of organic carbon sources, such as: acetate, acetic acid, ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids, galactose, glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose, mannose, methanol, molasses, peptone, plant based hydrolysate, proline, propionic acid, ribose, saccharose, partial or complete hydrolysates of starch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea, industrial waste solutions, yeast extract, or combinations thereof.

In some embodiments, the Chlorella strains are cultured on a nitrogen source comprising monosodium glutamate (MSG), ammonia, ammonium (e.g., ammonium hydroxide, ammonium phosphate, ammonium acetate), urea, nitrates, glycine or a combination thereof. The methods of culturing the disclosed Chlorella strains include methods of mixing, organic carbon supply, nitrogen supply, lighting, culture media, nutrient stocks, culturing vessels, and optimization of the culture parameters such as but not limited to temperature, pH, dissolved oxygen, and dissolved carbon dioxide. The Chlorella culture can be harvested from the culturing vessel and/or concentrated by means known in the art, such as but not limited to, settling, centrifugation, filtration, and electro-dewatering before concentration and/or drying.

In some embodiments and Examples below, a microalgae composition may be referred to as PHYCOTERRA® or PHYCOTERRA® ST. The PHYCOTERRA® or PHYCOTERRA® ST Chlorella microalgae composition is a microalgae composition comprising Chlorella. The PHY COTERRA® product contains whole cell Chlorella biomass while the PHY COTERRA® ST contains lysed cell Chlorella biomass. The PHYCOTERRA® Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65°C - 75°C for between 90 - 150 minutes, adding potassium sorbate and phosphoric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The PHYCOTERRA® Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the PHYCOTERRA® Chlorella microalgae composition may comprise between approximately 0.3% potassium sorbate and between approximately 0.5%- 1.5% phosphoric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88.2%-89.2% water. It should be clearly understood, however, that other variations of the PHYCOTERRA® Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.

In some embodiments and Examples below, a microalgae composition may be an OMRI certified microalgae composition referred to as TERRENE®. The OMRI certified TERRENE® Chlorella microalgae composition is a microalgae composition comprising Chlorella. The OMRI certified TERRENE® Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65°C - 75°C for between 90 - 150 minutes, adding citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. The OMRI certified TERRENE® Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the OMRI certified TERRENE® Chlorella microalgae composition may comprise between approximately 0.5% - 2.0% citric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88%-89.5% water. It should be clearly understood, however, that other variations of the OMRI certified TERRENE® Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.

A composition comprising microalgae can be stabilized by heating and cooling in a pasteurization process. In certain aspects, the active ingredients of the microalgae based compositions maintain effectiveness in enhancing at least one characteristic of a plant after being subjected to the heating and cooling of a pasteurization process. In other embodiments, compositions with whole cells or processed cells (e.g., dried, lysed, extracted) of microalgae cells may not need to be stabilized by pasteurization. For example, microalgae cells that have been processed, such as by drying, lysing, and extraction, or extracts can include such low levels of bacteria that a composition can remain stable without being subjected to the heating and cooling of a pasteurization process.

In some embodiments, the composition is lysed. Lysing is a technique where the cell membrane of a cell is ruptured, which releases lysate, the fluid contents of lysed cells, from the cells. As an example, the lysing process can comprise anything suitable that ruptures a cell membrane. For example, a bead mill may be used for lysing, where feedstock biomass solids can be dispersed and wetted (e.g., placed into a liquid phase). In this example the bead mill can utilize ceramic, glass, or metal beats (e.g., of a suitable size for the desired result) disposed in a chamber, such as a rotating cylinder, to collide with and mechanically macerate the solid biomass in the mill, which can help rupture the cell walls (e.g., the hydrogen bonds that hold together a cell membrane). Accordingly, in this example, the whole biomass may be lysed with water at cooler temperatures, with the resulting lysate comprising lipids in the form of an oil, biomass cell contents and unbroken biomass solid (e.g., non-target portion of biomass), and water.

In another aspect, the biomass is lysed using a shear mill. A shear mill utilizes a rotating impeller or high-speed rotor to create flow and shear of its contents. This causes the solid particles, such as biomass solid, to rupture due to shear stress.

In another aspect, the biomass is lysed using a pulsed electron field (PEF), high pressure homogenization, enzymes, and/or a chemical means (e.g., with a solvent).

In some embodiments, the inventive compositions are liquid formulations. Nonlimiting examples of liquid formulations include suspension concentrations and oil dispersions. In other embodiments, the inventive compositions are solid formulations. Non-limiting examples of liquid formulations include freeze-dried powders and spray-dried powders.

In a further aspect, the compositions can comprise a wetting agent or dispersant, a binder or adherent, an aqueous solvent and/or a non-aqueous co-solvent. The compositions provided herein can be formulated as a solid; as a powder, lyophilizate, pellet or granules; as a liquid or gel; or as an emulsion, colloid, suspension or solution.

In some embodiments, the composition can be heated to a temperature in the range of 50-130°C. In some embodiments, the composition can be heated to a temperature in the range of 55-65°C. In some embodiments, the composition can be heated to a temperature in the range of 58-62°C. In some embodiments, the composition can be heated to a temperature in the range of 50-60°C. In some embodiments, the composition can be heated to a temperature in the range of 60-90°C. In some embodiments, the composition can be heated to a temperature in the range of 70-80°C. In some embodiments, the composition can be heated to a temperature in the range of 100-150°C. In some embodiments, the composition can be heated to a temperature in the range of 120-130°C.

In some embodiments, the composition can be heated for a time period in the range of 1-150 minutes. In some embodiments, the composition can be heated for a time period in the range of 110-130 minutes. In some embodiments, the composition can be heated for a time period in the range of 90-100 minutes. In some embodiments, the composition can be heated for a time period in the range of 100-110 minutes. In some embodiments, the composition can be heated for a time period in the range of 110-120 minutes. In some embodiments, the composition can be heated for a time period in the range of 120-130 minutes. In some embodiments, the composition can be heated for a time period in the range of 130-140 minutes. In some embodiments, the composition can be heated for a time period in the range of 140-150 minutes. In some embodiments, the composition is heated for less than 15 min. In some embodiments, the composition is heated for less than 2 min.

After the step of heating or subj ecting the composition to high temperatures is complete, the compositions can be cooled at any rate to a temperature that is safe to work with. In one non-limiting embodiment, the composition can be cooled to a temperature in the range of 35- 45°C. In some embodiments, the composition can be cooled to a temperature in the range of 36-44°C. In some embodiments, the composition can be cooled to a temperature in the range of 37-43°C. In some embodiments, the composition can be cooled to a temperature in the range of 38-42°C. In some embodiments, the composition can be cooled to a temperature in the range of 39-41 °C. In further embodiments, the pasteurization process can be part of a continuous production process that also involves packaging, and thus the composition can be packaged (e.g., bottled) directly after the heating or high temperature stage without a cooling step.

In some embodiments, the composition can include 2.5-30% solids by weight of microalgae cells (i.e., 2.5-30 g of microalgae cells/100 mL of the composition). In some embodiments, the composition can include 2.5-5% solids by weight of microalgae cells (i.e., 2.5-5 g of microalgae cells/100 mL of the composition). In some embodiments, the composition can include 5-20% solids by weight of microalgae cells. In some embodiments, the composition can include 5-15% solids by weight of microalgae cells. In some embodiments, the composition can include 5-10% solids by weight of microalgae cells. In some embodiments, the composition can include 10-20% solids by weight of microalgae cells. In some embodiments, the composition can include 10-20% solids by weight of microalgae cells. In some embodiments, the composition can include 20-30% solids by weight of microalgae cells. In some embodiments, further dilution of the microalgae cells percent solids by weight can occur before application for low concentration applications of the composition.

In some embodiments, the composition can include less than 1% by weight of microalgae biomass or extracts (i. e. , less than 1 g of microalgae derived product/100 mL of the composition). In some embodiments, the composition can include less than 0.9% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.8% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.7% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.6% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.5% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.4% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.3% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.2% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.0001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.01% by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.0001-1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.0001-0.001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.001 -.01% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.01-0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.1-1% by weight of microalgae biomass or extracts.

In some embodiments, an application concentration of 0.1% of microalgae biomass or extract equates to 0.04 g of microalgae biomass or extract in 40 mL of a composition. While the desired application concentration to a plant can be 0.1% of microalgae biomass or extract, the composition can be packaged as a 10% concentration (0.4 mL in 40 mL of a composition). Thus, a desired application concentration of 0.1% would require 6,000 mL of the 10% microalgae biomass or extract in the 100 gallons of water applied to the assumption of 15,000 plants in an acre, which is equivalent to an application rate of about 1.585 gallons per acre. In some embodiments, a desired application concentration of 0.01% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.159 gallons per acre. In some embodiments, a desired application concentration of 0.001% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.016 gallons per acre. In some embodiments, a desired application concentration of 0.0001% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.002 gallons per acre.

In another non-limiting embodiment, correlating the application of the microalgae biomass or extract on a per plant basis using the assumption of 15,000 plants per acre, the composition application rate of 1 gallon per acre is equal to about 0.25 mL per plant = 0.025 g per plant = 25 mg of microalgae biomass or extract per plant. The water requirement assumption of 100 gallons per acre is equal to about 35 mL of water per plant. Therefore, 0.025 g of microalgae biomass or extract in 35 mL of water is equal to about 0.071 g of microalgae biomass or extract per 100 mL of composition equates to about a 0.07% application concentration. In some embodiments, the microalgae biomass or extract based composition can be applied at a rate in a range as low as about 0.001-10 gallons per acre, or as high as up to 150 gallons per acre.

In some embodiments, the applications are performed using a 10% solids solution by weight microalgae composition. For greenhouse trials, the concentrations vary and essentially refer to how much volume of the 10% solids solution are added in a given volume of water (e.g. 2.5% v/v - 5.0% v/v).

The present invention involves the use of a microalgae composition. Microalgae compositions, methods of preparing microalgae compositions, and methods of applying the microalgae compositions to plants are disclosed inWO 2017/218896 Al (Shinde etal.) entitled “Microalgae-Based Composition, and Methods of its Preparation and Application to Plants,” which is incorporated herein in full by reference. In one or more embodiments, the microalgae composition may comprise approximately 10%-10.5% w/w of Chlorella microalgae cells. In one or more embodiments, the microalgae composition may also comprise one of more stabilizers, such as potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, citric acid, or the like, or any combination thereof. For example, in one or more embodiments, the microalgae composition may comprise approximately 0.3% w/w of potassium sorbate or another similar compound to stabilize its pH and may further comprise approximately 0.5-1.5% w/w phosphoric acid or another similar compound to prevent the growth of contaminants. As a further example, in one or more embodiments where it is desired to use an OMRI (Organic Materials Review Institute) certified organic composition, the microalgae composition may comprise 1.0-2.0% w/w citric acid to stabilize its pH, and may not contain potassium sorbate or phosphoric acid. In one or more embodiments, the pH of the microalgae composition may be stabilized to between 3.0-4.0.

Compositions of the present invention may include formulation inerts added to compositions comprising cells, cell-free preparations or metabolites to improve efficacy, stability, and usability and/or to facilitate processing, packaging and end-use application. Such formulation inerts and ingredients may include carriers, stabilization agents, nutrients, or physical property modifying agents, which may be added individually or in combination. In some embodiments, the carriers may include liquid materials such as water, oil, and other organic or inorganic solvents and solid materials such as minerals, polymers, or polymer complexes derived biologically or by chemical synthesis. In some embodiments, the carrier is a binder or adhesive that facilitates adherence of the composition to a plant part, such as a seed or root. See, for example, Taylor, A. G., et al., “Concepts and Technologies of Selected Seed Treatments”, Annu. Rev. Phytopathol. 28: 321-339 (1990). The stabilization agents may include anti-caking agents, anti-oxidation agents, desiccants, protectants or preservatives. The nutrients may include carbon, nitrogen, and phosphors sources such as sugars, polysaccharides, oil, proteins, amino acids, fatty acids and phosphates. The physical property modifiers may include bulking agents, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersants, adjuvants, surfactants, antifreeze agents or colorants. In some embodiments, the composition comprising cells, cell-free preparation or metabolites can be used directly with or without water as the diluent without any other formulation preparation. In some embodiments, the formulation inerts are added after concentrating fermentation broth and during and/or after drying.

In some embodiments, the composition is a liquid and substantially includes of water. In some embodiments, the composition can include 70-99% water. In some embodiments, the composition can include 85-95% water. In some embodiments, the composition can include 70-75% water. In some embodiments, the composition can include 75-80% water. In some embodiments, the composition can include 80-85% water. In some embodiments, the composition can include 85-90% water. In some embodiments, the composition can include 90-95% water. In some embodiments, the composition can include 95-99% water. The liquid nature and high-water content of the composition facilitates administration of the composition in a variety of manners, such as but not limit to: flowing through an irrigation system, flowing through an above ground drip irrigation system, flowing through a buried drip irrigation system, flowing through a central pivot irrigation system, sprayers, sprinklers, and water cans.

In some embodiments, the composition can be used immediately after formulation, or can be stored in containers for later use. In some embodiments, the composition can be stored out of direct sunlight. In some embodiments, the composition can be refrigerated. In some embodiments, the composition can be stored at l-10°C. In some embodiments, the composition can be stored at 1-3°C. In some embodiments, the composition can be stored at 3-50°C. In some embodiments, the composition can be stored at 5-8°C. In some embodiments, the composition can be stored at 8-10°C.

Methods of Application and Application Rates for Plants

In some embodiments, administration of the composition to soil, a seed or plant can be in an amount effective to produce an enhanced characteristic in plants compared to a substantially identical population of untreated seeds or plants. Such enhanced characteristics can include accelerated seed germination, accelerated seedling emergence, improved seedling emergence, improved leaf formation, accelerated leaf formation, improved plant maturation, accelerated plant maturation, increased plant yield, increased plant growth, increased plant quality, increased plant health, increased fruit yield, increased fruit sweetness, increased fruit growth, and increased fruit quality. Non-limiting examples of such enhanced characteristics can include accelerated achievement of the hypocotyl stage, accelerated protrusion of a stem from the soil, accelerated achievement of the cotyledon stage, accelerated leaf formation, increased marketable plant weight, increased marketable plant yield, increased marketable fruit weight, increased production plant weight, increased production fruit weight, increased utilization (indicator of efficiency in the agricultural process based on ratio of marketable fruit to unmarketable fruit), increased chlorophyll content (indicator of plant health), increased plant weight (indicator of plant health), increased root weight (indicator of plant health), increased shoot weight (indicator of plant health), increased plant height, increased thatch height, increased resistance to salt stress, increased plant resistance to heat stress (temperature stress), increased plant resistance to heavy metal stress, increased plant resistance to drought, increased plant resistance to disease, improved color, reduced blossom end rot, and reduced sun bum. Such enhanced characteristics can occur individually in a plant, or in combinations of multiple enhanced characteristics. Additionally, the present invention is directed to a method of treating a plant, a plant part, such as a seed, root, rhizome, corm, bulb, or tuber, and/or a locus on which or near which the plant or the plant parts grow, such as soil, to enhance plant growth, the method comprising the step of applying to a plant, a plant part and/or a plant locus a composition comprising an isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof.

The compositions disclosed herein may be applied in any desired manner, such as in the form of a seed coating, soil drench, and/or directly in-furrow and/or as a foliar spray and applied either pre-emergence, post-emergence or both. In other words, the compositions can be applied to the seed, the plant or to the soil wherein the plant is growing or wherein it is desired to grow (plant's locus of growth).

In some embodiments, the microalgae based composition may be applied to soil, seeds, and plants in an in-furrow application. An application of the microalgae based composition infurrow requires a low amount of water and targets the application to a small part of the field. The application in-furrow also concentrates the application of the microalgae based composition at a place where the seedling radicles and roots will pick up the material in the composition or make use of captured nutrients, including phytohormones.

In some embodiments, the microalgae based composition may be applied to soil, seeds, and plants as a side dress application. One of the principals of plant nutrient applications is to concentrate the nutrients in an area close to the root zone so that the plant roots will encounter the nutrients as the plant grows. Side-dress applications use a “knife” that is inserted into the soil and delivers the nutrients around 2 inches along the row and about 2 inches or more deep. Side-dress applications are made when the plants are young and prior to flowering to support yield. Side-dress applications can only be made prior to planting in drilled crops, i.e. wheat and other grains, and alfalfa, but in row crops such as peppers, com, tomatoes they can be made after the plants have emerged.

In some embodiments, the microalgae based composition may be applied to soil, seeds, and plants through a drip system. Depending on the soil type, the relative concentrations of sand, silt and clay, and the root depth, the volume that is irrigated with a drip system may be about !4 of the total soil volume. The soil has an approximate weight of 4,000,000 lbs. per acre one foot deep. Because the roots grow where there is water, the plant nutrients in the microalgae based composition would be delivered to the root system where the nutrients will impact most or all of the roots. Experimental testing of different application rates to develop a rate curve would aid in determining the optimum rate application of a microalgae based composition in a drip system application.

In some embodiments, the microalgae based composition may be applied to soil, seeds, and plants through a pivot irrigation application. The quantity and frequency of water delivered over an area by a pivot irrigation system is dependent on the soil type and crop. Applications may be 0.5 inch or more and the exact demand for water can be quantitatively measured using soil moisture gauges. For crops such as alfalfa that are drilled in (very narrow row spacing), the roots occupy the entire soil area. Penetration of the soil by the microalgae based composition may vary with a pivot irrigation application, but would be effective as long as the application can target the root system of the plants. In some embodiments, the microalgae based composition may be applied in a broadcast application to plants with a high concentration of plants and roots, such as row crops.

In some embodiments, a composition can be administered before the seed is planted. In some embodiments, a composition can be administered at the time the seed is planted. In some embodiments, a composition can be applied by dip treatment of the roots. In some embodiments, a composition can be administered to plants that have emerged from the ground. In some embodiments, a liquid or dried composition can be applied to the soil before, during, or after the planting of a seed. In some embodiments a liquid or dried composition can be applied to the soil before or after a plant emerges from the soil.

In some embodiments, the volume or mass of the microalgae based composition applied to a seed, seedling, or plant may not increase or decrease during the growth cycle of the plant (i.e., the amount of the microalgae composition applied to the plant will not change as the plant grows larger). In some embodiments, the volume or mass of the microalgae based composition applied to a seed, seedling, or plant can increase during the growth cycle of the plant (i.e., applied on a mass or volume per plant mass basis to provide more of the microalgae composition as the plant grows larger). In some embodiments, the volume or mass of the microalgae based composition applied to a seed, seedling, or plant can decrease during the growth cycle of the plant (i.e., applied on a mass or volume per plant mass basis to provide more of the microalgae composition as the plant grows larger).

In one non-limiting embodiment, the administration of the composition may comprise contacting the foliage of the plant with an effective amount of the composition. In some embodiments, the composition may be sprayed on the foliage by a hand sprayer, a sprayer on an agriculture implement, or a sprinkler. In some embodiments, the composition can be applied to the soil. In certain aspects, the microalgae based composition is applied at 0.1-150 gallons per acre, 0.1-50 gallons per acre, or 0.1-10 gallons per acre.

The rate of application of the composition at the desired concentration can be expressed as a volume per area. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 10-50 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 10-15 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 15-20 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 20-25 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 25-30 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 30-35 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 35-40 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 40-45 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application can comprise a rate in the range of 45-50 gallons/acre.

In some embodiments, the rate of application of the composition in a soil or foliar application can comprise a rate in the range of 0.01-10 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 0.01-0.1 gallons/acre. In some embodiments, the rate of application of the composition in a soil or foliar application may comprise a rate in the range of 0.1-1.0 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 0.25-2 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 1-2 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 2-3 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 3-4 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 4-5 gallons/acre. In some embodiments, the rate of application of the composition in a foliar application may comprise a rate in the range of 5-10 gallons/acre.

In some embodiments, the v/v ratio of the composition can be between 0.001 %-50%. In some embodiments, the v/v ratio of the composition can be between 0.01-25%. In some embodiments, the v/v ratio of the composition can be between 0.03-10%. In some embodiments, the v/v ratio of the composition can be between 0.12-4%

In another non-limiting embodiment, the administration of the composition can include contacting the soil in the immediate vicinity of the planted seed with an effective amount of the composition. In some embodiments, the composition can be supplied to the soil by injection into a low volume irrigation system, such as but not limited to a drip irrigation system supplying water beneath the soil through perforated conduits or at the soil level by fluid conduits hanging above the ground or protruding from the ground. In some embodiments, the composition can be supplied to the soil by a soil drench method wherein the composition is poured on the soil.

The composition can be diluted to a lower concentration for an effective amount in a soil application by mixing a volume of the composition in a volume of water. The percent solids of microalgae sourced components resulting in the diluted composition can be calculated by the multiplying the original concentration in the composition by the ratio of the volume of the composition to the volume of water. Alternatively, the grams of microalgae sourced components in the diluted composition can be calculated by multiplying the original grams of microalgae sourced components per 100 mL by the ratio of the volume of the composition to the volume of water.

The rate of application of the composition at the desired concentration can be expressed as a volume per area. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 50-150 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 75- 125 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 50-75 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 75- 100 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 100-125 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 125-150 gallons/acre.

In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 10-50 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 10-20 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 20-30 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 30- 40 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 40-50 gallons/acre.

In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 0.01-10 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 0.01-0.1 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 0.1 -1.0 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 1- 2 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 2-3 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 3-4 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 4-5 gallons/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 5-10 gallons/acre.

In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 2-20 liters/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 3.7- 15 liters/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 2-5 liters/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 5-10 liters/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 10-15 liters/acre. In some embodiments, the rate of application of the composition in a soil application can include a rate in the range of 15-20 liters/acre.

Plants Benefitting from Application of the Compositions

Many plants can benefit from the application of compositions that provide a biostimulatory effect. Non-limiting examples of plant families that can benefit from such compositions include plants from the following: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae, Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae, Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae (Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae, Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae, Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae, Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae), Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae, Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae, Piperaceae, Proteaceae, and Cannabaceae.

The Solanaceae plant family includes a large number of agricultural crops, medicinal plants, spices, and ornamentals in its over 2,500 species. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Manoliopsida (class), Asteridae (subclass), and Solanales (order), the Solanaceae family includes, but is not limited to, potatoes, tomatoes, eggplants, various peppers, tobacco, and petunias. Plants in the Solanaceae can be found on all the continents, excluding Antarctica, and thus have a widespread importance in agriculture across the globe.

The Rosaceae plant family includes flowering plants, herbs, shrubs, and trees. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Magnoliopsida (class), Rosidae (subclass), and Rosales (order), the Rosaceae family includes, but is not limited to, almond, apple, apricot, blackberry, cherry, nectarine, peach, plum, raspberry, strawberry, and quince.

The Fabaceae plant family (also known as the Leguminosae) comprises the third largest plant family with over 18,000 species, including a number of important agricultural and food plants. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Manoliopsida (class), Rosidae (subclass), and Fabales (order), the Fabaceae family includes, but is not limited to, soybeans, beans, green beans, peas, chickpeas, alfalfa, peanuts, sweet peas, carob, and liquorice. Plants in the Fabaceae family can range in size and type, including but not limited to, trees, small annual herbs, shrubs, and vines, and typically develop legumes. Plants in the Fabaceae family can be found on all the continents, excluding Antarctica, and thus have a widespread importance in agriculture across the globe. Besides food, plants in the Fabaceae family can be used to produce natural gums, dyes, and ornamentals.

The Poaceae plant family supplies food, building materials, and feedstock for fuel processing. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Liliopsida (class), Commelinidae (subclass), and Cyperales (order), the Poaceae family includes, but is not limited to, flowering plants, grasses, and cereal crops such as barely, com, lemongrass, millet, oat, rye, rice, wheat, sugarcane, and sorghum. Types of turf grass found in Arizona include, but are not limited to, hybrid Bermuda grasses (e.g., 328 tifgm, 419 tifway, tif sport). The Vitaceae plant family includes flowering plants and vines. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Magnoliopsida (class), Rosidae (subclass), and Rhammales (order), the Vitaceae family includes, but is not limited to, grapes.

In certain aspects, any of a variety of plants may benefit from the workings of the composition according to the invention. In one embodiment, the plant is an ornamental plant, which includes flowering and non-flowering plants. In another embodiment, the plant is a consumable plant, which includes cereals, crops, fruit trees, herbs, medicinal plants and vegetables. In another embodiment, the plant is a member of the Alliaceae, Apiaceae, Asparagaceae, Asphodelaceae, Asteraceae, Araucariaceae, Begoniaceae, Brassicaceae, Bromeliaceae, Buxaceae, Chenopidiaceae, Cichorioideae, Chenopodiaceae, Coniferae, Cucurbitaceae, Fabaceae, Gentianaceae, Gramineaejridaceae, Leguminosae, Liliaceae, Malvaceae, Marantaceae, Marasmiaceae, Musaceae, Oleaceae, Orchidaceae, Paeoniaceae, Pleurotaceae, Pinaceae, Poaceae, Rosaceae, Rubiaceae, Rutaceae, Salicaceae, Solanaceae, Sterculiaceae, Taxaceae, Tuberacea, Vandeae, Vitacea or Xanthorrhoeaceae family, preferably of the Asteraceae, Begoniaceae, Brassicaceae, Chenopodiaceae, Cucurbitaceae, Gramineae, Leguminosae, Liliaceae, Malvaceae, Musaceae, Orchidaceae, Paeoniaceae, Rosaceae, Rubiaceae, Rutaceae, Salicaceae, Solanaceae, Sterculiaceae or Vandeae family, most preferably of the Begoniaceae, Brassicaceae, Orchidaceae, Paeoniaceae, Rosaceae or Solanaceae family. The plant may be a species of the genus Alchemilla, Allium, Aloe, Alstroemeria, Arabidopsis, Argyranthemum, Avena, Begonia, Brassica, Bromelia, Buxus, Calathea, Campanula, Capsicum, Cattleya, Cichorium, Citrus, Chamaecyparis, Chrysanthemum, Clematis, Cucumis, Cyclamen, Cydonia, Cymbidium, Cynodon, Dianthus, Dracaena, Eriobotrya, Euphorbia, Eustoma, Ficus, Fragaria, Fuchsia, Gaultheria, Gerbera, Glycine, Gypsophilia, Hedera, Helianthus, Hordeum, Hyacinthus, Hydrangea, Hippeastrum, Iris, Kalanchoe, Lactuca, Lathyrus, Lavendula, Lilium, Limonium, Malus, Mandevilla, Olea, Oryza, Osteospermum, Paeonia, Panicum, Pelargonium, Petunia, Phalaenopsis, Phaseolus, Pinus, Pisum, Platycodon, Prunus, Pyrus, Ranunculus, Rhododendron, Rosa, Rubus, Ruta, Secale, Skimmia, Solanum, Sorbus, Sorghum, Spathiphyllum, Trifolium, Triticum, Tulipa, Vanda, Vicia, Viola, Vitis, Zamioculcas or Zea. Preferably, the plant is a species of Arabidopsis, Begonia, Brassica, Fragaria, Paeonia, Phalaenopsis, Rosa, Solanum or Vanda.

In particular, the composition may be used to promote the growth of commercially important crops and plants, such as alfalfa, apples, bananas, begonias, bromeliads, cereals, cherries, citrus fruits, grapes, maize, melons, olives, onions, orchids, peaches, peonies, potatoes, rice, soybeans, sugar beets, spinach, strawberries, tomatoes or wheat.

The composition according to the invention may also be used for improving the growth or development of seeds, tubers or bulbs. The composition may be used as such or may be mixed with substrate or nutrition medium. It may be applied to the seeds, tubers or bulbs in any convenient way, including pouring, soaking and spraying. In one embodiment, the composition according to the invention is used to coat seeds, tubers or bulbs.

The effect of the application of the composition according to the invention is improved growth, such as improved root development, improved nutrient assimilation, improved efficiency of plant metabolism or increased photosynthesis. This may be apparent from improved yield, improved leaf formation, improved color formation, improved flowering, improved fruit formation, improved taste or improved health compared to a similar plant to which the liquid composition according to the invention has not been applied.

Improvements may be determined in any suitable way generally used by the person skilled in the art, for example by counting, weighing or measuring. Improvement in any one of these areas may be at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 300%, such as about 5% to 50%, about 5% to 100%, about 10% to 100%, about 20% to 50%, about 20% to 100% or about 100% to 200%.

Improved root development may be reflected in several ways, such as by more roots per plant, more roots per square area, accelerated root formation, earlier root formation, stronger roots, thicker roots, better functioning roots, more branched roots or a wider spread root network.

Improved yield may be reflected in several ways, such as by more plants per area, more branches per plant, more buds per plant, more bulbs per plant, more fruits per plant, more flowers per plant, more leaves per plant, more seedlings from seed, more seeds per plant, more shoots per plant, more spores per plant, more starch per plant, more tubers per plant, more weight per plant, higher dry matter content, more primary metabolites per plant or more secondary metabolites per plant.

Improved growth may be reflected in several ways, such as by earlier germination, accelerated germination, accelerated stem growth, a thicker stem, earlier fruit formation, accelerated fruit formation, earlier ripening of fruit or accelerated ripening of fruit. Improved leaf formation may be reflected in several ways, such as by more leaves per plant, more leaves per cm of stem, more buds per stem, larger leaves, broader leaves, thicker leaves, stronger leaves, better functioning leaves or earlier or accelerated leaf formation.

Improved color formation may be reflected in several ways, such as by earlier color formation, accelerated color formation, more diverse color formation, deeper color formation, more intense color or more stability of color.

Improved flowering may be reflected in several ways, such as by earlier flowering, accelerated flowering, larger flowers, more flowers, more open flowers, longer lasting flowers, longer open flowers, by flowers which are more diverse in color, by flowers having a desired color or by flowers with more stability of color.

Improved fruit formation may be reflected in several ways, such as by earlier fruit formation, accelerated fruit formation, longer period of bearing fruit, earlier ripening of fruit, accelerated ripening of fruit, more fruit, heavier fruit, larger fruit or tastier fruit.

Improved taste may be reflected in several ways, such as by less acidity, more sweetness, more flavor, more complex flavor profile, higher nutrient content or more juiciness.

Improved health may be reflected in several ways, such as by being more resistant to abiotic stress, being more resistant to biotic stress, being more resistant to chemical stress, being more resistant to physical stress, being more resistant to physiological stress, being more resistant to insect pests, being more resistant to fungal pests, growing more abundantly, flowering more abundantly, keeping leaves for a longer period or being more efficient in food uptake. In the present context, biotic stress factors include fungi and insects. Abiotic stress is the result of salinity, temperature, water or light conditions which are extreme to the plant under the given circumstances.

In one embodiment, the use of the composition according to the invention leads to harvesting more plants or plant parts per area, such as more barks, berries, branches, buds, bulbs, cut branches, cut flowers, flowers, fruits, leaves, roots, seeds, shoots, spores or tubers per plant per area. The use of the liquid composition according to the invention may lead to an increase in the yield of crops. The harvest may be more abundant, and harvesting may take place after a shorter period of time, in comparison with a situation in which the composition according to the invention is not applied.

In one embodiment, application of the liquid composition according to the invention leads to more kilos of flowers, fruits, grains or vegetables, such as apples, auberges, bananas, barley, bell peppers, blackberries, blue berries, cherries, chives, courgettes, cucumber, endive, garlic, grapes, leek, lettuce, maize, melons, oats, onions, oranges, pears, peppers, potatoes, pumpkins, radish, raspberries, rice, rye, strawberries, sweet peppers, tomatoes or wheat.

In another embodiment, the application of the method according to the invention leads to more kilos of barks, berries, branches, buds, flowers, fruits, leaves, roots or seeds from culinary or medicinal herbs, such as basil, chamomile, catnip, chives, coriander, dill, eucalyptus, fennel, jasmine, lavas, lavender, mint, oregano, parsley, rosemary, sage, thyme and thus to more aroma, flavor, fragrance, oil or taste in the same period of time or in a shorter period of time, in comparison to a situation in which the composition according to the invention has not been applied.

In another embodiment, the use of the liquid composition according to the invention leads to a higher yield of anti-oxidants, colorants, nutrients, polysaccharides, pigments or terpenes. In one embodiment, the sugar content of the plant cells is increased.

The period of comparison with a control plant or control situation may be any period, from several hours, several days or several weeks to several months or several years. The area of comparison may be any area, such as square meters or hectares or per pot.

BIOLOGICAL DEPOSIT OF CHLORELLA SP. ACCESSION NO. NCMA 202012055

A Biological Deposit of Chlorella sp. Accession No. NCMA 202012055 was made at the Provasoli-Guillard National Center for Marine Algae and Microbiota - Bigelow Laboratory for Ocean Sciences, (NCMA, 60 Bigelow Drive, East Boothbay, Maine 04544 U.S.A.) on December 16, 2020 under the provisions of the Budapest Treaty, and assigned by the International Depositary Authority the accession number 202012055. Upon issuance of a patent, all restrictions upon the Deposit will be irrevocably removed, and the Deposit is intended to meet the requirements of 37 CFR §§ 1.801-1.809. The Deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective, enforceable life of the patent, whichever is longer, and will be replaced if necessary during that period; and the requirements of 37 CFR §§ 1.801-1.809 are met.

The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

EXAMPLES Example 1. Identification of Chlorella sp. Accession No. NCMA 202012055 by Selective Mutant Propagation

Multiple pools of mutants were generated by UV mutagenesis of Chlorella sp. parental strains. The pools of mutants were subject to growth competition under heterotrophic conditions with a culture medium containing glucose in a closed system. Only those mutants with a selective advantage could outcompete the other mutants and propagate efficiently. After several rounds of growth competition under heterotrophic conditions, Chlorella sp. Accession No. NCMA 202012055 was isolated as one of the fastest growing mutants in the growth competition assays.

Example 2. Chlorella sp. Accession No. NCMA 202012055 Demonstrates Superior Heterotrophic Growth Compared to Other Chlorella Strains

Chlorella sp. Accession No. NCMA 202012055 was evaluated along with the Chlorella strains shown in Table 1 for heterotrophic growth in the same nutrient medium containing 15 g/L glucose and 3.9 g/L sodium nitrate (NaNOs) as the nitrogen source. Each strain was cultured in the nutrient medium in a shake flask at 25 °C until it reached a threshold growth level of at least 6 g/L dry weight.

Table 1. UTEX strains evaluated with Chlorella sp. Accession No. NCMA 202012055 for heterotrophic growth on glucose.

As shown in FIG. 1, Chlorella sp. Accession No. NCMA 202012055 reached the threshold growth level of 6 g/L dry weight by Day 3, whereas the other Chlorella strains did not reach this threshold growth level until later.

Using the growth curves, growth parameters were calculated for each of the strains. For productivity, the following equation was used.

Max DW - DWto tMaxDW — t0 The following equation was used for Maximum Specific Growth. ln(Max DW) - In(Wto) tMaxDW — t0

In these equations, MaxDW is the maximum dry weight achieved, DWto is the dry weight on the day the cultures were inoculated, tMaxDW is the number of days at which the maximum dry weight was reached, and tO is the initial day on which the cultures were inoculated.

Chlorella sp. Accession No. NCMA 202012055 demonstrated the greatest maximum specific growth among the strains (see FIG. 2). In addition, Chlorella sp. Accession No. NCMA 202012055 had the highest productivity among any of the strains (see FIG. 3). The ability of Chlorella sp. Accession No. NCMA 202012055 to grow more efficiently under heterotrophic conditions than the other strains indicates that this strain is better adapted to growth in a fermenter, which is generally preferred for controlled, large scale production of algal biomass.

Example 3. Chlorella sp. Accession No. NCMA 202012055 Increases Culturable Bacterial Populations and Water Holding Capacity in Various Soil Types

In a first set of experiments, soil samples representing three different soil textures determined by the USDA NRCS Soil Texture Calculator were collected from different regions in the United States: 1) Loam soil from Granger, Iowa; 2) Sandy loam soil from Farmville, North Carolina; and 3) Sand soil from Douglas, Georgia. The soil samples had slightly different moisture contents with the soil from Farmville, North Carolina and Douglas, Georgia having slightly more moisture than the soil from Granger, Iowa. The following treatments were applied to each type of soil: conventional PHYCOTERRA® (whole cell Chlorella microalgae) (i.e., PHYCOTERRA® #1); PHYCOTERRA® (whole cell Chlorella microalgae) cultured with an alternative carbon source (i.e., PHYCOTERRA® #2); PHYCOTERRA® ORGANIC (whole cell Chlorella microalgae); Chlorella sp. AccessionNo. NCMA 202012055 cultured with a first nitrogen source (i.e., NCMA 202012055 #1); and Chlorella sp. Accession No. NCMA 202012055 cultured with a second nitrogen source (i.e., NCMA 202012055 #2).

The treated soil samples were diluted via a series of 10-fold dilutions and were plated onto Petri dishes containing an agar-based medium 8 days post-treatment. The resulting culturable bacterial populations/colonies were counted after an additional 7-day incubation period. Untreated soil samples were plated and counted, according to the same protocol, for comparison.

The treatments generally resulted in significant increases in culturable bacterial populations in the soil samples compared to the untreated control soil samples, with Chlorella sp. Accession No. NCMA 202012055 having the most pronounced effect on the loam soil from Granger, Iowa (see FIGs. 4-6).

In a second set of experiments, sand soil from Douglas, Georgia was used to determine the impact of various treatments on percent (%) water holding capacity and culturable microbial growth in the soil. The following treatments were applied to samples of the soil: Chlorella sp. Accession No. NCMA 202012055; PHYCOTERRA® (whole cell Chlorella microalgae); and PHYCOTERRA® ORGANIC (whole cell Chlorella microalgae).

The treated soil samples were diluted via a series of 10-fold dilutions and were plated onto Petri dishes containing an agar-based medium 7 days post-treatment. The resulting culturable bacterial populations/colonies were counted after an additional 6-day incubation period. Untreated soil samples were plated and counted, according to the same protocol, for comparison.

The treatments resulted in significant increases in culturable bacterial populations in the soil samples compared to the untreated control soil samples, with Chlorella sp. Accession No. NCMA 202012055 having the most pronounced effect, followed by PHYCOTERRA® ORGANIC (whole cell Chlorella microalgae) and PHYCOTERRA® (whole cell Chlorella microalgae) treatments (see FIG. 7).

At 17 days post-treatment, the soil samples were dried to evaluate their respective percent water holding capacities. Post-drying, 25 mL of water was added to each sample and and completely eluted. The weight of each sample was taken pre- and post-elution to determine the percentage of moisture retained. Compared to the untreated control samples, the water holding capacity of the samples treated with Chlorella sp. Accession No. NCMA 202012055 increased by an average of approximiately 6.79%; the water holding capacity of the samples treated with PHYCOTERRA® (whole cell Chlorella microalgae) increased by an average of approximately 6.66%; and the water holding capacity of the samples treated with PHYCOTERRA® ORGANIC (whole cell Chlorella microalgae) increased by an average of approximately 7.44% (see FIG. 8).

Without wishing to be bound by any theory, the revitalization of the native microbiome in the soil by Chlorella sp. Accession No. NCMA 202012055 can contribute to a healthier growth substrate for crops by helping to build soil structure and to retain water more efficiently. Example 4. Chlorella sp. Accession No. NCMA 202012055 Increases Lettuce Shoot and Root Biomass in the Greenhouse

In a first set of experiments, Chlorella sp. Accession No. NCMA 202012055 was applied as a drench to Romaine lettuce (Valley Heart variety) plants at a concentration of 5% v/v at seeding. The Chlorella sp. Accession No. NCMA 202012055 were either washed cells with spent culture medium removed or unwashed cells with spent culture medium remaining in the drench. In both cases, a similar concentration of cells was applied to the plants. PHYCOTERRA® (whole cell Chlorella microalgae) was applied as a drench at the same concentration (i.e., 5% v/v) as a positive control. Control Romaine lettuce (Valley Heart variety) plants were seeded and grown without any treatment for comparison. All lettuce plants were grown for five (5) weeks in a greenhouse following a standard fertilizer program and afterwards evaluated for shoot and root biomasses.

As shown in Table 2, both the washed and unwashed Chlorella sp. Accession No. NCMA 202012055 produced increased shoot and root biomass compared to untreated control plants. Surprisingly, the increases in plant growth observed with Chlorella sp. Accession No. NCMA 202012055 exceeded those observed with the commercial product, PHYCOTERRA® (whole cell Chlorella microalgae).

Table 2. Increases in Romaine lettuce (Valley Heart variety) shoot and root biomass after treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae) compared to untreated control plants.

In a second set of experiments, Chlorella sp. Accession No. NCMA 202012055 was applied as a drench to Romaine lettuce (Valley Heart variety) plants at a concentration of 5% v/v at seeding. The Chlorella sp. Accession No. NCMA 202012055 were either whole cells or lysed cells. As in the previous experiment, PHYCOTERRA® (whole cell Chlorella microalgae) was applied at the same concentration (i.e., 5% v/v) as a positive control, and untreated plants were seeded and grown for comparison. All plants were grown in a greenhouse following a standard fertilizer program.

As shown in Table 3, both the whole cell and lysed cell Chlorella sp. Accession No. NCMA 202012055 produced increased shoot and root biomass compared to untreated control plants. Surprisingly, the increases in plant growth observed with Chlorella sp. Accession No. NCMA 202012055 exceeded those observed with the commercial product, PHYCOTERRA® (whole cell Chlorella microalgae).

Table 3. Increases in Romaine lettuce (Valley Heart variety) shoot and root biomass after treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae) compared to untreated control plants.

In a third set of experiments, Chlorella sp. Accession No. NCMA 202012055 was applied as a drench to Romaine lettuce (Valley Heart variety) plants at concentrations of 1%, 2.5% and 5% v/v at seeding. The Chlorella sp. Accession No. NCMA 202012055 were either whole cells or lysed cells. PHYCOTERRA® (whole cell Chlorella microalgae) was applied as a drench at the same concentrations (i.e., 1%, 2.5% and 5% v/v) as a positive control. Control lettuce plants were seeded and grown without any treatment for comparison. All lettuce plants were grown for several weeks in a greenhouse following a standard fertilizer program and afterwards evaluated for shoot biomasses.

As shown in Table 4, both the whole cell and lysed cell Chlorella sp. Accession No. NCMA 202012055 and the PHYCOTERRA® (whole cell Chlorella microalgae) produced increased shoot biomass (at all concentrations) compared to untreated control plants. With one exception, i.e., lysed cell at 1% v/v application, the increases in plant growth observed with Chlorella sp. Accession No. NCMA 202012055 exceeded those observed with the commercial product, PHYCOTERRA® (whole cell Chlorella microalgae). Table 4. Increases in lettuce shoot biomass after treatment with Chlorella sp. Accession No. NCMA 202012055 (lysed or whole) or PHYCOTERRA® (whole cell Chlorella microalgae) compared to untreated control (UTC) plants.

Example 5. Chlorella sp. Accession No. NCMA 202012055 Increases Cauliflower Shoot

Biomass in the Greenhouse

In a set of experiments, Chlorella sp. Accession No. NCMA 202012055 was applied as a drench to cauliflower plants at concentrations of 1%, 2.5% and 5% v/v at seeding. The Chlorella sp. Accession No. NCMA 202012055 were either whole cells or lysed cells. PHYCOTERRA® (whole cell Chlorella microalgae) was applied as a drench at the same concentrations (i.e., 1%, 2.5% and 5% v/v) as a positive control. Control cauliflower plants were seeded and grown without any treatment for comparison. All cauliflower plants were grown for several weeks in a greenhouse following a standard fertilizer program and afterwards evaluated for shoot biomasses. As shown in Table 5, both the whole cell and lysed cell Chlorella sp. Accession No.

NCMA 202012055 produced increased shoot biomass (at all concentrations) compared to untreated control plants. The increases in plant growth observed with Chlorella sp. Accession No. NCMA 202012055 exceeded those observed with the commercial product, PHYCOTERRA® (whole cell Chlorella microalgae).

Table 5. Increases in cauliflower shoot biomass after treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae) compared to untreated control (UTC) plants.

Example 6. Chlorella sp. Accession No. NCMA 202012055 Increases Yield in Lettuce Field Trials A field trial was conducted in Yuma, Arizona with Romaine lettuce. Chlorella sp.

Accession No. NCMA 202012055 and PHYCOTERRA® (whole cell Chlorella microalgae) - each at approximately 10% solid biomass concentration - were applied to the lettuce plots at 1.5 gallons per acre per season. At the conclusion of the growing season, the yields (Ib/acre) were determined for the treated plots. Chlorella sp. Accession No. NCMA 202012055 had a 46% increase in yield compared to PHY COTERRA® (whole cell Chlorella microalgae) confirming the surprising improvement in performance of Chlorella sp. Accession No. NCMA 202012055 over PHYCOTERRA® (whole cell Chlorella microalgae) observed in the greenhouse.

Table 6. Increases in Romaine lettuce yield after treatment with Chlorella sp. Accession No.

NCMA 202012055 compared to PHYCOTERRA® (whole cell Chlorella microalgae).

Example 7. Chlorella sp. Accession No. NCMA 202012055 Increases Yield in Spinach Field Trials

A field trial was conducted with Revere spinach in Yuma, Arizona where the plants grew in clay soil having 0.8% organic matter. Chlorella sp. Accession No. NCMA 202012055 and PHYCOTERRA® (whole cell Chlorella microalgae) - each at approximately 10% solid biomass concentration - were sprayed over the spinach beds at 1 quart per acre at planting. At the conclusion of the growing season, the yields (Ib/acre) for the treated plots were determined and compared to the grower standard.

Chlorella sp. Accession No. NCMA 202012055 had a 6% increase in yield compared to the grower standerd, and PHYCOTERRA® (whole cell Chlorella microalgae) had an 18% increase in yield compared to the grower standerd.

Table 7. Increases in Revere spinach yield after treatment with Chlorella sp. Accession No. NCMA 202012055 or PHYCOTERRA® (whole cell Chlorella microalgae) compared to grower standard.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. An isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 obtained by selective mutant propagation or a mutant thereof having all the identifying characteristics thereof.

2. The isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof of Claim 1, wherein the Chlorella sp. Accession No. NCMA 202012055 or mutant thereof grows efficiently under heterotrophic conditions comprising growth on at least one organic carbon source in the absence of a supply of light and/or carbon dioxide.

3. The isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof of Claim 1 or 2, having a maximum specific growth of at least 1.0 days and/or a productivity of at least 1.9 g/L/day when grown in a culture medium comprising glucose and sodium nitrate in a shake flask at 25°C.

4. The isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof of any one of Claims 1 to 3, wherein application of the Chlorella sp. Accession No. NCMA 202012055 or mutant thereof to soil increases the culturable bacterial population in the soil compared to the soil without application thereof.

5. The isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof of any one of Claims 1 to 3, wherein application of the Chlorella sp. Accession No. NCMA 202012055 or mutant thereof to soil increases the water holding capacity of the soil compared to the soil without application thereof.

6. The isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof of Claim 4 or 5, wherein the soil is loam soil, sandy loam soil, or sand soil.

7. A cell-free or inactivated preparation of the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof of any one of Claims 1 to 6.

34

8. A composition comprising the isolated biologically pure culture of Chlor ella sp. Accession No. NCMA 202012055 or mutant thereof of any one of Claims 1 to 7 and an agriculturally acceptable carrier.

9. The composition of Claim 8, wherein the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof comprises whole cells, lysed cells, or a combination thereof.

10. The composition of Claim 8 or 9, wherein the composition is formulated as a solid, liquid, or gel.

11. The composition of Claim 10, wherein the composition is a solid formulation selected from the group consisting of a powder, lyophilizate, pellet, and granule.

12. The composition of Claim 10, wherein the composition is a liquid formulation selected from the group consisting of an emulsion, colloid, suspension, and solution.

13. The composition of any one of Claims 8 to 12, further comprising at least one culture stabilizer selected from the group consisting of potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, or a combination thereof.

14. A plant propagation material treated with the composition of any one of Claims 8 to 13 in an amount of from 0.01 g to 10 kg per 100 kg of plant propagation material.

15. A method of plant enhancement comprising the step of: applying to a plant, a plant part and/or a plant locus an effective amount of a composition comprising an isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055, a mutant thereof having all the identifying characteristics thereof, or a cell- free or inactivated preparation thereof to enhance at least one plant characteristic.

16. The method of Claim 15, wherein the plant characteristic is selected from the group consisting of seed germination rate, seed germination time, seedling emergence, seedling emergence time, seedling size, plant fresh weight, plant dry weight, utilization, fruit

35 production, leaf production, leaf formation, leaf size, leaf area index, plant height, thatch height, plant health, plant resistance to salt stress, plant resistance to heat stress, plant resistance to heavy metal stress, plant resistance to drought, maturation time, yield, root length, root mass, color, blossom end rot, softness, plant quality, fruit quality, flowering, sun bum, and any combination thereof.

17. The method of Claim 16, wherein the plant characteristic is plant fresh weight, plant dry weight, or yield.

18. The method of any one of Claims 15 to 17, wherein the isolated biologically pure culture of Chlorella sp. Accession No. NCMA 202012055 or mutant thereof comprises whole cells, lysed cells, or a combination thereof.

19. The method of any one of Claims 15 to 18, wherein the composition is applied as a soil drench, an in-furrow treatment, a foliar application, a side-dress application, a pivot irrigation application, a seed coating, or with a drip system.

20. The method of any one of Claims 15 to 19, wherein the composition is administered at a rate of 0.1-150 gallons per acre (0.935-1402.5 liters per hectare) to enhance the at least one plant characteristic.

21. The method of any one of Claims 15 to 20, wherein the plant is a member of a plant family selected from: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae, Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae, Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae (Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae, Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae, Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae, Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae), Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae, Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae, Piperaceae, and Proteaceae.