Bioactive compounds with pesticide activities derived from aged cultures of green microalgae

Abstract

Embodiments of the present disclosure pertain to a composition suitable for use in treating an agricultural material. In some embodiments, the composition includes a microalgae culture, an extract of the microalgae culture, compounds from the microalgae culture, or combinations thereof. Additional embodiments of the present disclosure pertain to methods of treating an agricultural material by applying a composition of the present disclosure to the agricultural material.

Description

TITLEBIOACTIVE COMPOUNDS WITH PESTICIDE ACTIVITIES DERIVED FROM AGED CULTURES OF GREEN MICROALGAECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 532,840, filed on August 15, 2023. The entirety of the aforementioned application is incorporated herein by reference.BACKGROUND

[0002] A need exists for the development of more effective and sustainable herbicides. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0003] In some embodiments, the present disclosure pertains to a composition suitable for use in treating an agricultural material. In some embodiments, the composition includes a microalgae culture, an extract of the microalgae culture, compounds from the microalgae culture, or combinations thereof. Additional embodiments of the present disclosure pertain to methods of treating an agricultural material by applying a composition of the present disclosure to the agricultural material.

[0004] The compositions of the present disclosure may be applied to various agricultural materials. For instance, in some embodiments, the agricultural material includes, without limitation, plants, seeds, plant parts, soil, root systems of plants or seeds, or combinations thereof. In some embodiments, the compositions of the present disclosure may be applied to a plant. In some embodiments, the compositions of the present disclosure may be applied to a seed. In some embodiments, the plant or seed includes, without limitation, crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, potato, grapes, varieties thereof, or combinations thereof.

[0005] The compositions of the present disclosure may have various activities. For instance, in some embodiments, the compositions of the present disclosure have at least one of pesticidal activities, herbicidal activities, anti-microbial activities, nematocidal activities, algicidal activities, or combinations thereof. In some embodiments, the compositions of the present disclosure have herbicidal activities. In some embodiments, the methods and compositions of the present disclosure may be used to prevent the growth of weeds along with a plant or seed.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 shows a fractionation process for obtaining a microalgae acid metabolite (AA)- fraction (AA-Fraction) using a combination of Strata X-AW (anion exchange) and Strata C18 (hydrophobicity-based) cartridges.

[0007] FIG. 2A shows the results of a disc diffusion assay to test AA-fractions. I. Viability Control: Demonstrates the baseline viability without treatment. II. Methanol Control: Using three different volumes of methanol (1 = 5 pL, 2 = 10 pL, 3 = 15 pL) to assess solvent effects. III. Raw Extract Bioassay: Testing the bioactivity of the raw extract at three volumes (1 = 5 pL, 2 = 10 pL, 3 = 15 pL). IV. Fractionation Bioassay: Bioactivity results after processing the raw extract through X-AW and C18 cartridges, testing AA (any acid), W2 (wash 2), and WA (weak acid) fractions.

[0008] FIG. 2B shows the nematocidal bioassay of an AA-Fraction against C. elegans. Methanol Control: Solvent control using 5 pL and 10 pL of methanol. Raw Extract Control: Bioassay testing of raw extract. AA-Fraction Bioassay: Testing 5 pL and 10 pL of AA-fraction for nematocidal activity. Viability Control: Ensures baseline viability of C. elegans without treatment.

[0009] FIG. 3 shows chromatographic peaks obtained from HPLC-MS analysis of the Raw Methanolic Extract and AA-Fraction. Chromatographic peaks with a signal-to-noise ratio greater than 3.0 and peak quality greater than 5.0. Normalized chromatographic area corresponding to the metabolome of the AA-Fraction (top), raw methanolic extract (middle), and methanol as a blank (bottom).

[0010] FIGS. 4A-4B show metabolomic data analysis of the AA-Fraction. FIG. 4A shows a metabolite set enrichment analysis (MSEA). The enrichment ratio represents the proportion of observed metabolites in the metabolite set relative to the expected number of metabolites. This ratio is calculated as the observed number of significant metabolites divided by the expected number based on the metabolite set size and the total number of metabolites analyzed. FIG. 4B shows a pathway analysis. Pathway impact measures the importance of a pathway within the overall metabolic network by integrating pathway topology analysis and MSEA. This comprehensive analysis highlights key pathways influenced by the metabolites identified in the AA-Fraction. Both analyses and plots were generated using MetaboAnalyst 6.0.

[0011] FIGS. 5A-5B show plots of the GO enrichment analysis of up-regulated genes in C. reinharcltii in response to 30 days of growth. Y-axis represents the top 20 enriched GO categories. X-axis represents the ratio of the genes enriched in each category. The diamond color represents enrichment significance, p < 0.05 was used as the threshold to select GO. The plot is created using the R package ggplot2.

[0012] FIG. 6 shows the inhibition of growth and germination in Lactuca sativa (upper panel) and Arabidopsis thaliana (lower panel) by the AA-Fraction. The upper panel displays the aerial parts of L. sativa seedlings grown in 24-well plates, while the lower panel shows A. thaliana seedlings grown under similar conditions. Control treatments include seed germination on Murashige and Skoog (MS) growth medium with water, as well as a solvent control (MeOH:methanol). The volume indicated in the images represents the amount of fraction used for the assay.

[0013] FIG. 7 shows jasmonic acid biosynthesis analysis at multiple time points (T14, T21, T30) through RNA-seq and Metabolomics Data. The figure illustrates the jasmonic acid biosynthesis pathway using RNA-seq data at three different time points: T14, T21, and T30, compared to the control (T07). It also includes metabolomics data obtained through HPLC-MS for the same time points. The BioCyc pathway tool was employed to create this figure, integrating output tables from edgeR (for transcriptomics data) and Compound Discoverer (for metabolomics data).DETAILED DESCRIPTION

[0001] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0002] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0003] Agriculture faces significant challenges in sustaining crop yields to feed a rapidly growing global population projected to reach 8.5 billion in 2030 and increase further to 9.7 billion in 2050. These challenges include soil degradation due to aggressive and non-sustainable practices, rapidly evolving pests and diseases that devastate complete fields, climate change comprising unpredictable weather events like rising temperatures and extreme water deficit, and herbicideresistant weeds that compete with crops. The use of agrochemicals, for example, has allowed farmers to control pests and diseases successfully.

[0004] However, the control of weeds, which compete with crops for soil nutrients and water and are one of the most significant sources of pests and diseases in the field, has become a very complex situation due to the appearance of weeds resistant to multiple herbicides. Overall, these herbicides exhibit only 25 distinct modes of action. Herbicides with only six modes of action that target essential cellular processes, such as cell division and the photosystem (PS) II, or specific enzymatic steps in the plant metabolisms, such as the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, acetolactate synthase (ALS), and acetyl CoA carboxylase (ACCase), dominate the market accounting for 75% of global herbicide usage.

[0005] Importantly, there has not been an introduction of herbicides with new modes of action during the last three decades, which has complicated the control of weeds, especially those resistant to herbicides. Herbicide resistance, coupled with the destabilizing impacts of climate change on crop yields, underscores the urgent need for innovative herbicidal solutions. Identifying novel molecules with new modes of action is crucial to addressing these challenges and ensuring global food security.

[0006] As such, a need exists for the development of more effective and sustainable herbicides. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0007] In some embodiments, the present disclosure pertains to a composition suitable for use in treating an agricultural material. In some embodiments, the composition includes a microalgae culture, an extract of the microalgae culture, compounds from the microalgae culture, or combinations thereof. In some embodiments, the compositions of the present disclosure include a microalgae culture. In some embodiments, the compositions of the present disclosure include an extract from a microalgae culture. In some embodiments, the compositions of the present disclosure include compounds from a microalgae culture. Additional embodiments of the present disclosure pertain to methods of treating an agricultural material by applying a composition of the present disclosure to the agricultural material. As set forth in more detail herein the methods and compositions of the present disclosure can include numerous embodiments.

[0008] Microalgae cultures

[0009] Microalgae cultures, their extracts and their compounds may be derived from various sources. For instance, in some embodiments, the microalgae culture is derived from green microalgae. In some embodiments, the green microalgae are derived from strains belonging to the Chlorophyta phylum. In some embodiments, the microalgae culture is derived from genera belonging to Chlorella, Chlamydomonas, Chloroidium, or combinations thereof. In some embodiments, the microalgae culture is derived from Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella anitrata, Chlorella sp., Chloroidium saccharophilum, or combinations thereof. In some embodiments, the microalgae culture is derived from Chlamydomonas reinhardtii.

[0010] Microalgae culture extracts

[0011] In some embodiments, the compositions of the present disclosure include an extract of a microalgae culture. In some embodiments, the methods of the present disclosure may apply an extract of a microalgae culture to an agricultural material.

[0012] The methods and compositions of the present disclosure may utilize various microalgae culture extracts. For instance, in some embodiments, the extract of the microalgae culture includes solvent-extracted microalgae culture. In some embodiments, the solvent includes, without limitation, chloroform, hexane, methanol, ethanol, or combinations thereof. In some embodiments, the solvent includes methanol. In some embodiments, the extract of the microalgae culture is in crude form. In some embodiments, the extract of the microalgae culture is generated from early fractionation and purification processes. In some embodiments, the extract of the microalgae culture is a refined fraction.

[0013] Microalgae culture compounds

[0014] In some embodiments, the compositions of the present disclosure include compounds from a microalgae culture. In some embodiments, the methods of the present disclosure may apply compounds from a microalgae culture (or a combination of microalgae cultures) to an agricultural material.

[0015] The methods and compositions of the present disclosure may utilize various microalgae culture compounds. For instance, in some embodiments, the compounds from a microalgae culture include microalgae metabolites. In some embodiments, the compounds from a microalgae culture include microalgae lipid-based metabolites.

[0016] In some embodiments, compounds from a microalgae culture include, without limitation, benzenes, benzamides, amino acids, peptides, glycerides, oxylipins, fatty acids, polyunsaturated fatty acids, fatty acid conjugates, short-chain keto acids, sterols, isoprenoids, alpha-keto acids, phenylpyruvic acids, pyrrolidinyl pyridines, amines, carboxylic acids, dicarboxylic acids, hydroxycinnamic acids, analogues thereof, derivatives thereof, or combinations thereof.

[0017] In some embodiments, compounds from a microalgae culture include, without limitation, palmitoleic acid, coronaric acid, L-a-palmitin, 2,3-dihydroxypropyl stearate, coronaric acid, NP- 012766, 9(S)-HOTrE (9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid), 12-oxo phytodienoic acid, 13-HpODE (13-hydroperoxy-octadecadienoic acid), allelochemicals, isoquinoline alkaloids, aldehydes, cinnamic acids, Traumatin, Umbelliferone (7-hydroxycoumarin), Xestoaminol, 4- Linoleamide, palmitamide, (Z)-9-octadecenamide, palmitamide, cinnamic acid, (9Z)-9- octadecenamide, amide derivatives of oleic acid, derivatives thereof, or combinations thereof. In some embodiments, compounds from a microalgae culture include, without limitation, palmitoleic acid, L-a-palmitin, 2,3-dihydroxypropyl stearate, coronaric acid, NP-012766, 9(S)-HOTrE (9S- hydroxy-10E,12Z,15Z-octadecatrienoic acid), 12-oxo phytodienoic acid, and 13-HpODE (13- hydroperoxy-octadecadienoic acid), derivatives thereof, or combinations thereof.

[0018] Compounds from a microalgae culture may be in various forms. For instance, in some embodiments, compounds from a microalgae culture are in crude form. In some embodiments, compounds from a microalgae culture are in crude form in an extract of the microalgae culture. In some embodiments, compounds from a microalgae culture are in isolated form. In some embodiments, compounds from a microalgae culture are resuspended in a solvent, a combination of solvents, water, or combinations thereof. In some embodiments, compounds from a microalgae culture can act alone or in combination as compound cocktails. In some embodiments, compounds from a microalgae culture can be in the form of liquid, powder, pellets, granules, or combinations thereof.

[0019] Application of compositions to agricultural materials

[0020] Various methods may be utilized to apply the compositions of the present disclosure to agricultural materials. For instance, in some embodiments, the applying includes spraying a composition of the present disclosure onto an agricultural material. In some embodiments, the applying includes soaking an agricultural material (e.g., a plant or seed) with a composition of the present disclosure. In some embodiments applying includes in-furrow practices. In some embodiments, the applying occurs in a greenhouse or growth chamber prior to field transplantation.

[0021] In some embodiments, the applying includes applying a composition of the present disclosure onto a field that includes a plant or seed. In some embodiments, a composition of the present disclosure may then be incorporated into a soil. In some embodiments, the composition of the present disclosure can also be injected to the soil. In some embodiments, the applying occurs prior to associating the field with the plant or seed. In some embodiments, the applying occurs after associating the field with the plant or seed.

[0022] Agricultural materials

[0023] The compositions of the present disclosure may be applied to various agricultural materials. For instance, in some embodiments, the agricultural material includes, without limitation, plants, seeds, plant parts, soil, root systems of plants or seeds, or combinations thereof. In some embodiments, the compositions of the present disclosure may be applied to a plant. In some embodiments, the compositions of the present disclosure may be applied to a seed.

[0024] The compositions of the present disclosure may be applied to various plants or seeds. For instance, in some embodiments, the plant or seed includes, without limitation, crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palme ri, tobacco, tomato, lettuce, common beans, potato, grapes, varieties thereof, or combinations thereof.

[0025] Composition activities

[0026] The compositions of the present disclosure may have various activities. For instance, in some embodiments, the compositions of the present disclosure have at least one of pesticidal activities, herbicidal activities, anti-microbial activities, nematocidal activities, algicidal activities, or combinations thereof. In some embodiments, the compositions of the present disclosure have herbicidal activities.

[0027] In some embodiments, the compositions of the present disclosure are capable of inhibiting the growth of photosynthetic organisms, non-photosynthetic organisms, nematodes, cyanobacteria (e.g., Synechoccocus elongatus, Nostoc sp., and / or Anabaena variabilis). enterobacteria (e.g., E. coli, Klebsiella sp., and / or Salmonella), phytopathogenic fungi, Chlorella sorokiniana, Arabidopsis thaliana, Amaranthus palmeri, Caenorhabditis elegans, Lactuca sativa, or combinations thereof. In some embodiments, the compositions of the present disclosure are capable of inhibiting the growth of Caenorhabditis elegans.

[0028] As such, the methods and compositions of the present disclosure can have numerous applications. For instance, in some embodiments, the methods and compositions of the present disclosure may be used to prevent the growth of weeds along with a plant or seed. In some embodiments, the methods and compositions of the present disclosure may be utilized to kill nematodes and prevent nematode egg hatching in the soil, either alone or in combination with a plant or seed. In some embodiments, the methods and compositions of the present disclosure may be utilized to prevent algae blooms onto the soil, either alone or in combination with a plant or seed.

[0029] In some embodiments, the compositions of the present disclosure (e.g., microalgae culture extracts) may be used directly to protect economically important crops from pests, diseases, and weeds. In some embodiments, the compositions of the present disclosure (e.g., microalgae culture extracts) may be used to control pests, diseases, and weeds by killing them or preventing their proliferation. In some embodiments, such tasks can be achieved by either spraying the compositions of the present disclosure or applying them directly to a plant (e.g., weed alone or in combination with crops), seeds (e.g., seeds of crops to offer some protection once germinated in the field), and / or soil (e.g., to directly attack pests, diseases, and weeds).

[0030] Additional Embodiments

[0031] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0032] Example 1. Harnessing green microalgae to discover novel bio-herbicides and bionematicides for sustainable agriculture

[0033] Applicant previously showed that extracts from green microalgae of the Chlamydomonas, Chlorella, and Chloroidium genera presented growth inhibitory effects on four target organisms: Chlorella sorokiniana, Arabidopsis thaliana, Amaranthus palmeri, and Caenorhabditis elegans. Brito-Bello et al., 2023, Bioactive Compounds with Pesticide Activities Derived from Aged Cultures of Green Microalgae, Biology (Basel) 12, 1 149 (herein referred to as “Applicant’ s previous work” and incorporated herein by reference). The established bioassays effectively showed an important degree of control of the green algae extracts over the growth of these four organisms. Notably, the methanolic extracts of one of the used strains, Chlamydomonas reinhardtii CC124, showed excellent control over a glyphosate-resistant ecotype of A. palmerii.

[0034] In this Example, Applicant builds on these findings by exploring the potential of metabolites from C. reinhardtii CC124- (hereafter CC124) as sources of new pesticide agents. Through metabolite profiling, fractionation techniques of the raw methanolic extract, and RNA sequencing (RNA-seq) studies, Applicant isolated and characterized compounds with potential pesticidal properties and to elucidate the pathways involved in their production, thereby providing new avenues for pesticide development.

[0035] Example 1,1. Cultivation of C. reinhardtii CC124

[0036] C. reinhardtii strain CC124 was sourced from the Chlamydomonas Resource Center. Following the procedure outlined by Applicant’ s previous work set forth above, CC124 was grown mixotrophically with constant illumination (50 pmol m-2s-1) at 28 °C in a Conviron A1000 growth chamber. Cultures were maintained in 50 mL glass flasks with 25 mL of Tris-acetate-phosphate (TAP) medium, shaken continuously at 120-130 rpm. TAP medium was supplemented with 15 g L-1Bacto agar for solid media.

[0037] To evaluate potential pesticidal activities, batch cultures of CC124 were cultivated as reported in Applicant’s previous work. Briefly, cultures were grown in one-liter glass bottles containing 900 mL of TAP medium under a 16:8 light / dark cycle with 50 pmol m-2s-1light at 28°C, with continuous air bubbling. Initial inoculation was done with 1% pre-culture to achieve an OD750 of 0.2, and the cultures were maintained for 30 days.

[0038] Example 1,2. Preparation of extracts of C. reinhardtii CC124

[0039] Following a method similar to Applicant’s previous study, batch cultures of C. reinhardtii CC124 were harvested at 30 days of growth by centrifugation at 4,000 rpm for 15 minutes. The collected biomass was flash-frozen in liquid nitrogen and then freeze-dried using a Labconco FreeZone 2.5 Liter -80°C Benchtop Freeze Dryer under 0.15 mbar vacuum pressure at -80°C for 48 hours. 250 mg aliquots were extracted from the dried biomass with 8 mL of methanol in glass vials. The mixture was sonicated for 30 minutes to enhance tissue disruption and filtered using Whatman No. 2 filter paper. The filtered extracts were dried in a fume hood for 30 hours. Once completely dried, the raw extracts (RE) were resuspended in 250 pL of methanol for use in various assays.

[0040] Example 1 ,3. Fractionation system

[0041] A fractionation procedure was developed to isolate the metabolites responsible for pesticide activity based on their polarity and acidic properties. The system utilized Solid-Phase Extraction principles, comprising Strata X-AW weak anion cartridges and Strata C18 cartridges (both from Phenomenex) containing 1 g of sorbents. The fractionation procedure using Sephadex cartridges followed the manufacturer’s protocol. Briefly, the cartridges were conditioned with 20 mL of methanol (MeOH). Strata X-AW cartridges were equilibrated with 20 mL of water at pH 6-7. RE samples, diluted 1:10 in MeOH and adjusted to pH 7, were loaded onto the cartridges. The cartridges were washed with 20 mL of 25 mM ammonium acetate buffer at pH 6-7, followed by a second wash with 20 mL of MeOH. Two fractions were obtained: acidic metabolites (AA) eluted with 5% NH4OH in MeOH, and weak acids (WA) eluted with 5% formic acid in MeOH. These fractions (AA and WA) were further fractionated using a Strata Cl 8 cartridge. The cartridge was conditioned and equilibrated with HPLC-grade water before loading the sample. A wash step was performed using 5% MeOH. Two sub-fractions were obtained: one eluted with 60% MeOH and the other with 100% MeOH. All fractions were processed under a vacuum pressure of 10 mmHg. The fractions obtained were tested using a disc-diffusion bioassay and in vitro seed germination, as previously described in Applicant’ s previous work.

[0042] Example L4. Assessment of algicidal effect of microalgae extracts

[0043] The algicidal effect of microalgae extracts was evaluated using the agar diffusion test method as outlined in Applicant’s previous work. Strain C. sorokiniana UTEX 3016 was used as the target organism, with TAP agar plates serving as the assay medium. Briefly, 5, 10, and 15 pL of microalgae extracts were applied to 8 mm diameter filter paper discs (Whatman No. 2) and dried in a fume hood. The dried discs were then placed onto freshly inoculated agar plates (50 x 15 mm, VWR) containing 200 pL of strain 3016 at an OD750 of 0.5, spread using sterile borosilicate beads. Culture viability controls were included. The plates were incubated for seven days under previously described conditions. The experiments were conducted independently two times, each with three technical replicates.

[0044] Example 1,5. Assessment of the nematocidal effect of microalgae extracts

[0045] The nematocidal activity of microalgae extracts was evaluated following Applicant’s previous work. The ability of the extracts to inhibit the growth of Caenorhabditis elegans was tested by treating 10-15 LI larvae nematodes in S-basal medium with 2, 5, and 10 pL volumes of each algal extract. Negative controls included treatments with 2, 5, and 10 pL of the corresponding solvent. After 48 hours of treatment, the survival rate of LI larvae was determined by counting dead and alive worms using a stereomicroscope (Olympus, SZX10). The experiments were performed independently twice, with five technical replicates each.

[0046] Example 1,6. Metabolite profiling of microalgae extracts

[0047] Metabolite profiling was performed using a Vanquish Flex UHPLC system coupled with an Orbitrap Exploris 240 mass spectrometer (MS), following the methodology detailed in Applicant’s previous work. An XBridge PREMIER Peptide BEH C18 column (2.1 x 150 mm, 2.5 pm particle size) was utilized for separation. The system operated in positive ionization mode with an electrospray ionization (ESI) voltage of 3500V. Biomass extracts, prepared and filtered through a 0.25 pm PTFE syringe filter, were analyzed using the AcquireX workflow, which facilitated automated background exclusion list generation, data-dependent acquisition (DDA), and dynamic exclusion (DE).

[0048] The exclusion list, derived from a methanol matrix blank, was incorporated into the data acquisition methods. The automatic gain control (AGC) target was set to 2e5 for full MS and 5e4 for MS / MS. A pooled sample (20 pL from each strain) was used to generate the inclusion list and served as a quality control. HPLC-grade water with 0.1% formic acid (solvent A) and HPLC-grade methanol with 0.1 % formic acid (solvent B) were employed as solvents. The chromatographic run lasted 25 minutes with a gradient flow rate of 0.2 mL / min. Chromatographic and mass spectrometric conditions were consistent with those reported in Applicant’ s previous work.

[0049] Example 1,7, Data analysis

[0050] The chemical constituents of C. reinhardtii AA-fraction was analyzed using the method described in Applicant’s previous work. LC-MS RAW files were processed using Compound Discoverer (CD) v3.3 (ThermoFisher Scientific). The analysis adhered to the "Untargeted Metabolomics with statistics detect unknowns with ID using Online Database and mzLogic" workflow, utilizing databases such as BioCyc, Carotenoid Database, LipidMAPS, Chemical Biology Department of the Max Planck Institute of Molecular Physiology, Marine Drugs, Nature Chemical Biology, NIST, and Pesticide Common Names. This workflow included retention time alignment, compound identification via mzCloud and ChemSpider databases, and statistical analyses (ANOVA and Tukey comparisons). The compound table was filtered to include peaks with a quality rating above 5. For functional and over-representation analysis, the numeric mass (m / z), retention times (RT), and adjusted P-values of each metabolite were uploaded to the MetaboAnalyst server (http: / / www.metaboanalyst.ca / ) using version 6.0 and a mass tolerance of 5.0 ppm. Positive mode mass searches and enrichment analysis were performed using the Mummichog algorithm v2.0 (http: / / mummichog.org / ) with a P-value cutoff of l.OxlO"5, utilizing the C. reinhardtii library and the chemical structure main-class library for enrichment analysis.

[0051] Example 1,8. RNA isolation and RNA-seq

[0052] Total RNA was isolated from Chlamydomonas reinhardtii strain CC124 at four growth time points: 7 days (control), 14 days and 21 days (intermediate), and 30 days (nutrient limitation). The extraction was performed in two steps: initially using the TRIzol Reagent according to the manufacturer’s instructions (Invitrogen), followed by a secondary purification step with the Direct-zol RNA MiniPrep Plus kit (Zymo Research). RNA sequencing was then conducted by Novogene Bioinformatics Technology Co. (Beijing, China).

[0053] Example 1,9. RNA-seq data analysis

[0054] Transcript abundances from RNA-seq data were quantified using the pseudoalignment program Kallisto (vO.46.1). The C. reinhardtii v5.6 genome served as the reference for aligning the reads. To ensure the quality of the data, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) was employed for visualizing high-dimensional data in two dimensions, ensuring that samples clustered according to growth conditions. Differential gene expression (DGE) was analyzed by integrating Kallisto-derived transcript-level abundances into count-based statistical analyses using edgeR (v3.13) and DESeq2 through the tximport package. Comparisons were made between control (7 days), two intermediate time points (14 and 21 days after culture), and aged cultures (30 days). Genes with a log fold change (logFC) threshold of >1 or <-l and a false discovery rate (FDR) <0.01 were considered differentially expressed. Gene Ontology (GO) enrichment analysis was performed using the R package topGO to identify biological processes activated under stress conditions across different growth days. Data were categorized as upregulated if logFC > 0 and downregulated if logFC < 0, using the filtered dataset with FDR < 0.01.

[0055] Example 1.10, Metabolites of an acidic nature produced from CC124 exhibit algicidal activity

[0056] In Applicant’s previous work, Applicant observed that microalgae under nutrient limitation conditions produce metabolites that prevent germination of A. thaliana and A. palmeri seeds, inhibit the growth of the green algae C. sorokiniana, and kill the nematode C. elegans. These observations led Applicant to hypothesize that specific allelochemical molecules present in extracts of strain CC124 are responsible for these inhibitory effects.

[0057] To identify potential allelochemicals in CC124, the raw extract was subjected to a fractionation approach based on polarity and acidic properties (FIG. 1). This system utilized a weak anion exchange cartridge (Strata X-AW, Phenomenex) and a cartridge for the extraction of hydrophobic molecules (Strata C18, Phenomenex). The resulting fractions were tested using a paper disk-diffusion assay to determine their capacity to inhibit the growth of C. sorokiniana as the target organism, as described in Applicant’ s previous work.

[0058] To confirm the initial activity, Applicant tested the raw extract (RE) using different volumes; 5, 10, and 15 pL. A sharp halo of inhibition was observed at a volume of 10 pL of the RE, whereas no negative effects were observed for the treatment with methanol (MeOH) only (solvent control) (FIG. 2A). Applicant found that the fraction generated after running the RE through the fractionation system, corresponding to "Any Acid (AA)" in X-AW and elution in 100% MeOH in Cl 8, inhibited the growth of C. sorokiniana. Conversely, the fractions corresponding to "Weak Acids (WA)" (X-AW) / 100% MeOH (C18) and "Wash2(W2)" (X- AW) / 100% MeOH (Cl 8) did not show any inhibitory activity (FIG. 2A). These data suggest that the metabolites responsible for the growth inhibitory activity in C. sorokiniana are acids with pKa > 2, which are eluted in the "Any Acid" step and not in the "Weak Acid" step (FIG. 2A).

[0059] Example 1,11. Metabolites of an acidic nature produced from CC124 exhibit nematocidal activity

[0060] The potential of the CC124 fraction to disrupt the growth of the model organism C. elegans was assessed, given its suitability as a model for investigating the control or eradication of pathogenic nematodes that infest crop fields. Using 96-well plates, 12-16 LI larvae were placed per well for testing the extracts. A viability control with no solvent and no treatment was included. Additionally, the potential toxic effect of the carrier solvent methanol (MeOH) was tested and showed no effect on the nematodes.

[0061] Interestingly, exposure to 5 and 10 pL of the AA fraction resulted in 100% mortality of the nematodes, evidenced by their straight shape and lack of movement upon light exposure (FIG. 2B). Similarly, 5 pL of the raw extract (RE) also killed C. elegans, consistent with Applicant’s previous findings (FIG. 2B). These results demonstrate that the fractionation system effectively isolates the allelochemicals responsible for these effects, specifically acids with pKa > 2. These findings highlight the potential of CC124 metabolites for controlling agricultural pests and pathogens, providing a basis for further research into their application in crop protection. The results indicate the potential presence of molecules with nematocidal properties, which could serve as a natural alternative to synthetic pesticides.

[0062] Example 1,12. Non-targeted metabolomics analysis of AA-fraction suggests lipid-based metabolites potentially involved in pesticidal activities

[0063] To gain deeper insights into the metabolites responsible for the algicidal and nematocidal activities observed in the AA-fraction, Applicant performed a non-targeted metabolomics analysis using UHPLC-MS. The metabolome of the A A fraction consisted of chromatographic peaks with a signal-to-noise ratio greater than 3.0 and peak quality greater than 5.0 in normalized chromatographic area (FIG. 3). The overall composition of the metabolome was determined through spectral searches against mzCloud MS / MS and additional databases. The metabolomic dataset for the AA fraction consisted of 1255 compounds, a reduction from the 4206 compounds identified in the RE in Applicant’s previous work, representing approximately 30% of the compounds previously identified. To further investigate the chemical classes of the profiled metabolites, the dataset was analyzed using the MetaboAnalyst server v6.0 and the Mummichog algorithm v2.0. The latest version of MetaboAnalyst allowed the use of libraries of known molecules from C. reinhardtii. Several enriched metabolite categories were identified in the AA fraction analyzed, including benzamides, amino acids, peptides, fatty acids, fatty acid conjugates, sterols, and isoprenoids (FIG. 4A).

[0064] To integrate metabolic pathway enrichment and topology analyses, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst were utilized. Metabolic pathways with impact values greater than 0.1 were analyzed, resulting in the identification of eight pathways (FIG. 4B). The pathway with the highest impact value was linoleic acid metabolism, followed by steroid biosynthesis, sphingolipid metabolism, ubiquinone and other terpenoidquinone biosynthesis, galactose metabolism, starch and sucrose metabolism, sesquiterpenoid and triterpenoid biosynthesis, and purine metabolism. Notably, some of these pathways are known to produce molecules that protect plants against various stresses or possess bioactivity. For example, linoleic acid metabolism is significant in the production of oxy lipins, which are involved in plant defense mechanisms and stress responses. In steroid biosynthesis, certain steroids play crucial roles in plant growth and defense, and some have been found to possess pesticidal properties.

[0065] Metabolites enriched in the AA-fraction include amino acids, peptides, alpha-keto acids and derivatives, amines, phenyl pyruvic acid derivatives, and pyrrolidinylyridincs, suggesting a significant role in stress responses. These compounds contribute to the observed bioactive properties by potentially producing or enhancing molecules with pesticidal effects. This capability can provide a competitive advantage through interspecific effects on survival, reproduction, and particularly the inhibition of the growth of competing species.

[0066] Some of the most abundant metabolites identified through UHPLC-MS were palmitoleic acid, L-a-palmitin, 2,3-dihydroxypropyl stearate, coronaric acid, NP-012766, 9(S)-HOTrE (9S- hydroxy-10E,12Z,15Z-octadecatrienoic acid), 12-oxo phytodienoic acid, and 13-HpODE (13- hydroperoxy-octadecadienoic acid) (Table 1).MolecularCompd Rt / min Formula m / z Identification1 12.148 C16H30O2 277.21611 Palmitoleic acid2 15.455 C19H38O4 313.27363 L-a-PALMITIN3 16.666 C21H42O4 341.30491 2,3-Dihydroxypropyl stearate4 12.756 C18H32O3 279.2318 NP-02052 (Coronaric acid)5 15.374 C31H46N4O7 625.30162 NP-012766 (CAS No. 1101810-02-9)6 12.714 C33H4 N3O6P 610.30217 15.21 C35H36N4O5 593.275498 12.169 C18H30O3 317.20872 9(S)-HOTrE9 12.295 C18H28O3 293.2111 12-oxo Phytodienoic acidC36H49N5O310 15.047 600.3894311 15.029 C29H40O6 485.2897812 12.885 C18H32O4 295.22673 (±)13-HpODETable 1. Most abundant metabolites in AA-Fraction.

[0067] These lipid-based metabolites encompass fatty acids, glycerides, and oxylipins, which are integral to various metabolic processes, including energy storage, structural integrity of cell membranes, and participation in signaling pathways. The identified oxylipins, such as coronaric acid, 9(S)-HOTrE, 12-oxo phytodienoic acid, and 13-HpODE, are specifically known for their roles in stress and defense responses in plants.

[0068] The metabolites identified in the UHPLC-MS analysis are predominantly lipid-based compounds that play crucial roles in metabolism, signaling, and stress responses. The presence of multiple oxylipins indicates a strong component of stress response and signaling pathways, vital for maintaining cellular homeostasis and responding to environmental changes.

[0069] Example 1.13. Transcriptomic insights into metabolite production and stress response in C. reinhardtii CC124

[0070] Transcriptomic analysis of the CC124 strain was performed to explore the metabolic pathways involved in producing metabolites contributing to the pesticide effect observed in fraction AA. The strain CC124 was cultured, and then the biomass was harvested at four different time points based on the growth inhibitory activity of methanolic extracts prepared from the same biomass. Therefore, RNA-seq was performed specifically at an early time point, seven days after culture (T07) when no pesticide effect was observed and used as control, two intermediate time points, 14 (T14) and 21 (T21) days after culture, and at a late time point, 30 days after culture (T30), when growth inhibitory effect was apparent.

[0071] Comparisons of the transcriptomic landscapes at T07 and T30, revealed significant enrichment in processes related to stress responses and metabolic pathways, suggesting the metabolites detected through UHPLC-MS were produced by the cell in response to the stress conditions. The arginine biosynthetic process exhibited the highest ratio of genes involved, indicating its potential importance in stress response and metabolic adaptation (FIG. 5A). Additionally, the starch catabolic process was upregulated, likely linked to nutrient starvation. Other upregulated stress response processes included response to freezing, homoiothermy, and response to nematodes, suggesting their involvement in protective mechanisms or stress signaling pathways. Conversely, several categories were downregulated under nutrient limitation, as expected. These included photosynthesis, chlorophyll biosynthetic process, photosystem II assembly, fatty acid beta-oxidation, carotenoid biosynthetic process, and protoporphyrinogen IX biosynthetic process (FIG. 5B). This downregulation is consistent with previous studies indicating that Chlamydomonas downregulates photosynthetic and related processes to conserve energy, reallocate resources, manage oxidative stress, and adapt metabolic pathways under adverse conditions.

[0072] In line with previous findings, the transcriptomic analysis of Chlamydomonas nivalis showed upregulation in nucleic acid metabolism (purine metabolism). This further supports the notion that algae accumulate lipids and other metabolites to endure environmental stresses, including nutrient starvation. This response is characterized by downregulation of the photosynthetic apparatus, reduction in cell division, and accumulation of starch or lipids as survival mechanisms.

[0073] Example 1,14. Inhibition of growth and germination in Lactuca sativa and A. thaliana by microalgae AA-fraction

[0074] The effects of the microalgae AA-fraction on the growth and germination of Lactuca sativa and A. thaliana are shown in FIG. 6. The growth-inhibition assays were conducted using various volumes of the AA-fraction (5, 10, and 15 pL per seed). In the case of L. sativa, even the smallest amount of AA-fraction tested (5 pL per seed) was sufficient to inhibit seed germination. The control treatments, which included seed germination on Murashige and Skoog (MS) growth medium with water and a methanol solvent control (MeOH), showed successful seed germination, indicating that the observed inhibition was specifically due to the AA-fraction. For A. thaliana, the germination assays showed that 5 pL and 10 pL of the AA-fraction per seed did not completely inhibit germination. Still, there was a noticeable reduction in the size of the seedlings. In contrast, using 15 pL of the AA-fraction resulted in the complete inhibition of A. thaliana seed germination. Increasing the volume of the AA-fraction further inhibited germination, demonstrating a dosedependent effect.

[0075] Similar to the results observed with L. sativa, the water and methanol controls exhibited normal growth and germination, underscoring the inhibitory effect of the AA-fraction. These results demonstrate that the fraction AA contains bioactive molecules that effectively present seed germination in very small amounts. Similar experiments with glyphosate-resistant A. palmeri and other herbicide-resistant weed seeds are in progress.

[0076] Example 1.15. Integration of metabolomics and transcriptomics to identify pathwaymetabolite relationships specific to the production of biocompounds

[0077] To perform the multi-omics analysis, two inputs were required: a list of the enriched metabolites obtained from the AA-fraction (including metabolite names and LogFC values) and a list of the differentially expressed genes (including gene names and LogFC values).

[0078] These inputs were obtained as described previously in Examples 1.7 and 1.9. Using these two lists, Applicant conducted an analysis using BioCyc (https: / / biocyc.org / ), specifically utilizing the Cellular Overview for C. reinhardtii. Applicant uploaded the datasets following the website's user guide for pathway tools v23 (Multi-Omics Viewer). This enabled Applicant to identify the pathways where genes and metabolites were differentially expressed or enriched.

[0079] Example 1.16. Discussion

[0080] Microalgae are recognized as cell biofactories, producing a diverse array of secondary metabolites. These include aldehydes, alkaloids, fatty acids, peptides, and carotenoids, which contribute to their significant biochemical and industrial potential. Numerous studies have highlighted the antioxidant and antibacterial activities of microalgae lipid extracts for health purposes, but few have focused on their pesticidal effects and the pathways involved in producing these compounds.

[0081] Here, Applicant demonstrated that green algae produce compounds with pesticidal activities with the potential to control seed germination and kill nematodes, thus expanding the potential applications of green algae beyond traditional purposes. Using a fractionation system based on polarity (Cl 8) and acidic properties (X-AW), Applicant fractionated the methanol raw extract (RE) of CC124, which possesses bioactivity, and produced a fraction that retain pesticidal activity (fraction AA) (FIG. 1). This approach aimed to reduce the number of metabolites responsible for the inhibitory activity and gain insights into the specific molecules responsible for the pesticidal activity.

[0082] Applicant identified that the fraction corresponding to Any Acid (X-AW) / 100% MeOH (Cl 8) (fraction AA) (FIGS. 2A-2B) contains the active metabolites with pesticidal effects, as shown in disc-diffusion, seeds and nematode assays (FIG. 2A). Applicant’s combined fractionation system allowed Applicant to purify a bioactive extract, reducing the number of putative compounds responsible for the inhibitory activity from 4206 compounds to 1255 compounds. UHPLC-MS analysis identified various lipid-based metabolites related to the pathways identified in MetaboAnlyst, such as linoleic acid metabolism (FIG. 4B), including palmitoleic acid, 12-oxo phytodienoic acid, and 9(S)-HOTrE (Table 1), which play critical roles in plant defense and stress responses. The presence of palmitoleic acid, known for its antimicrobial properties against Vibrio species, suggests a broad spectrum of bioactivity.

[0083] Secondary metabolites such as 2,3-dihydroxypropyl stearate from extracts of the tree Anthonotha cladantha have also demonstrated antibacterial effects, and Applicant found this compound in fraction AA. Several oxy lipins identified in Applicant’s study, including coronaric acid, 9(S)-HOTrE, 12-oxo phytodienoic acid, and 13-HpODE, act as crucial signaling molecules. Oxy lipins are biologically active oxygenated derivatives of polyunsaturated fatty acids, playing key roles in responding to environmental stresses.

[0084] For instance, 9(S)-HOTrE, produced by Leonotis nepetifolia, exhibits antifungal effects against Colletotrichum species. Another oxy lipin, 12-oxo phytodienoic acid, is produced in response to stress, such as herbivore attacks in com and wounding \n Arabidopsis. Similarly, hydroxylated unsaturated C18 oxylipins from the Mediterranean shrub Cestrum parqui were found to inhibit the germination and growth of Lactuca saliva, suggesting that these compounds may be responsible for the bioactivity observed.

[0085] These results align with the enrichment pathways identified in MetaboAnalyst, which are related to lipid-based metabolites, such as linoleic acid metabolism and sphingolipid metabolism. Certain fatty acids produced by algae have been found to exert inhibitory effects on a range of aquatic organisms, influencing interactions between species and contributing to the succession of phytoplankton in aquatic ecosystems. These fatty acids can serve as defense mechanisms against grazers. Overall, Applicant’s analysis suggests the fraction AA contains a cocktail of bioactive molecules of lipidic nature as responsible for the pesticidal activities, highlighting oxylipins as the potential molecules acting as bio-herbicides and bio-nematicides.

[0086] Transcriptomic analysis of CC124 was conducted to gain insights into the metabolic pathways potentially involved in the production of metabolites with pesticidal activites. RNA sequencing at T07 (no pesticide effect) and T30 (inhibitory effect) revealed significant enrichment in stress response and metabolic pathways, which may be related to nutrient deficiency conditions (FIGS. 5A-5B). It was previously demonstrated that, when C. reinhardtii is cultivated in TAP medium under mixotrophic conditions similar to Applicant’s work, nutrient depletion begins as early as seven days post-inoculation, with phosphorus and nitrogen being the first to be depleted. The integration of metabolomics and transcriptomics data has revealed significant intersections in metabolic pathways and GO categories, highlighting potential coordinated regulation in the production of metabolites with pesticidal activities. Key metabolic processes, including purine metabolism, lipid metabolism, carbohydrate metabolism, and secondary metabolite biosynthesis, might be implicated in the alga defense mechanisms and response to environmental stresses.

[0087] Transcriptomic analysis revealed various biological processes related to the catabolism of amino acids, such as the purine-containing compound catabolic process, which is upregulated as a path of recycling nitrogen and other elements from purines, supporting continued cellular function during nutrient scarcity. This process involves breaking down amino acids to use as a nitrogen source, as previously reported in senescent plant tissues and is related to the GO category response to nitrogen compounds (FIGS. 5A-5B). The arginine biosynthetic process showed the highest ratio in the top upregulated GO categories.

[0088] Arginine, rich in nitrogen, is an important nitrogen storage molecule and plays a role in nitrogen assimilation regulation in gree algae, as it regulates the expression of key genes in the nitrogen assimilation pathway. Other upregulated stress response processes included responses to freezing, homoiothermy, nematodes, and fungi, suggesting their involvement in protective mechanisms and compound production. Conversely, processes related to photosynthesis, chlorophyll biosynthesis, photosystem II assembly, fatty acid beta-oxidation, carotenoid biosynthesis, and protoporphyrinogen IX biosynthesis were downregulated under nutrient limitation (FIGS. 5A-5B). This downregulation aligns with previous studies showing that Chlamydomonas reallocates resources and manages oxidative stress by reducing photosynthetic activity under adverse conditions.

[0089] Applicant’s analysis revealed a correlation between upregulated genes involved in long- chain fatty acid activation and the presence of bioactive metabolites, suggesting that these genes might be responsible for the accumulation of oxy lipins. This is particularly evident with Crel2.g512300.tl.2, annotated as lipoxygenase (LOX) (FIG. 7), a key enzyme in oxylipin biosynthesis. LOX enzymes catalyze the oxidation of linoleic acid and a-linolenic acid, leading to the production of oxylipins, such as 12-oxo-phytodienoic acid (OPDA), which are precursors in the jasmonic acid pathway. Jasmonic acid is crucial for regulating plant responses to environmental stimuli, including stress, development, and defense.

[0090] Applicant’s study identified (9Z, 13S, 15Z-12,13-epoxyoctadeca-9,l l,15-trienoate) (FIG. 7), a defense-related compound and precursor of jasmonic acid, which was reported to be more accumulated in response to nutrient deficiency stress in grapes. In the same pathway, Applicant identified the enzyme 12-oxophytodienoate reductase (Crel7.g727300.tL2) (FIG. 7), which was reported to increase in abundance after nitrogen removal from the growth media. Additionally, this enzyme was identified as a programmed cell death protein in C. reinhardtii. Little is known about the production of oxylipins in microalgae, with most studies focusing on the roles of oxylipins in human health, such as their antioxidant, anti-inflammatory, neuroprotective, or immunomodulatory activities.

[0091] These findings strongly suggest that lipid-based metabolites, specifically oxylipins, may regulate or are involved in the production of metabolites with pesticide activity. As shown in Table 1, each of the most abundant compounds has different retention times that can be separated using a preparative HPLC column combined with a fractionator.

[0092] Applicant’s findings demonstrate the potential of CC124 metabolites as bio-herbicides and bio-nematicides. The fractionation system effectively isolated the allelochemicals responsible for these activities, which include acids with pKa > 2. Metabolomics analysis revealed that lipid- based metabolites, particularly oxylipins, play crucial roles in these activities. These compounds are integral to stress response and signaling pathways, highlighting their potential application in sustainable agriculture. The RNA-seq analysis provided insights into the genetic regulation of these metabolites, identifying key upregulated pathways involved in stress responses and metabolic adaptations. The downregulation of photosynthetic processes under stress conditions aligns with the need to conserve energy and redirect metabolic resources to defense mechanisms.

[0093] In this Example, Applicant explored the pesticidal potential of metabolites produced by C. reinhardlii CC124 under nutrient limitation. Through a combination of fractionation-guided assays, non-targeted metabolomic profiling, and RNA sequencing, Applicant identified several key metabolites and pathways potentially involved in the algicidal, herbicidal and nematicidal activities observed. Applicant’s fractionation system effectively isolated bioactive compounds from the raw extract, specifically identifying lipid-based metabolites, such as oxy lipins, which play critical roles in stress response and signaling pathways. The identification of these metabolites underscores their potential as natural alternatives to synthetic pesticides, offering a sustainable approach to crop protection.

[0094] Transcriptomic analysis revealed significant upregulation in processes related to stress responses and metabolic adaptations, particularly in arginine biosynthesis and purine metabolism. These findings suggest a coordinated regulation of metabolic pathways in response to nutrient stress, highlighting the complex biochemical strategies employed by C. reinhardtii to survive and produce bioactive compounds. Overall, Applicant’s findings demonstrate the potential of C. reinhardtii CC124 metabolites as bio-herbicides and bio-nematicides. The integration of metabolomics and transcriptomics provides a comprehensive understanding of the metabolic and genetic mechanisms underlying the production of these bioactive compounds. Ongoing research focuses on further fractionation and characterization of these metabolites to fully elucidate their pesticidal properties and practical applications in sustainable agriculture.

[0095] Among the most abundant compounds identified by UHPLC in the AA-Fraction are the polar lipid L-a-PALMITIN, a derivative of palmitic acid (16:0), and the oxy lipin jasmonate, 12- oxo phytodienoic acid (Table 1). Previous studies have shown that L-a-PALMITIN exhibits no cytotoxic effects on human HaCaT (keratinocyte) cells and mouse RAW 264.7 (macrophage) cells at concentrations up to 100 pg / mL. See Frusciante et al. (2024). Phytochemical Composition, Anti-Inflammatory Property, and Anti- Atopic Effect of Chactomorpha linum Extract. Mar Drugs 22, 226. 10.3390 / md22050226. Additionally, 12-oxo phytodienoic acid has demonstrated cytoprotective effects against oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells. See Taki-Nakano et al. (2014). Cytoprotective effects of 12-oxo phytodienoic acid, a plant- derived oxylipin jasmonate, on oxidative stress-induced toxicity in human neuroblastoma SH- SY5Y cells. Biochimica et Biophysica Acta (BBA)-General Subjects 1840, 3413-3422. 10.1016 / j.bbagen.2014.09.003. These findings suggest that the metabolites of C. reinhardtii CC124, including L-a-PALMITIN and 12-oxo phytodienoic acid, are safe for humans and non-target organisms, supporting their potential application in sustainable agriculture.

[0096] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

WHAT IS CLAIMED IS:

1. A method of treating an agricultural material, said method comprising: applying a composition to the agricultural material, wherein the composition comprises a microalgae culture, an extract of the microalgae culture, compounds from the microalgae culture, or combinations thereof.

2. The method of claim 1, wherein the composition has at least one of pesticidal activities, herbicidal activities, anti-microbial activities, nematocidal activities, algicidal activities, or combinations thereof.

3. The method of claim 1, wherein the composition is capable of inhibiting the growth of photosynthetic organisms, non-photo synthetic organisms, nematodes, cyanobacteria, enterobacteria, phytopathogenic fungi, Chlorella sorokiniana, Arabidopsis t Indiana, Amarant hits palmeri, Caenorhabditis elegans, Lactuca sativa, or combinations thereof.

4. The method of claim 1, wherein the agricultural material is selected from the group consisting of plants, seeds, plant parts, soil, root systems of plants or seeds, or combinations thereof.

5. The method of claim 1, wherein the agricultural material comprises a plant or seed.

6. The method of claim 5, wherein the composition is used to prevent the growth of weeds along with the plant or seed.

7. The method of claim 1, wherein the composition comprises the microalgae culture.

8. The method of claim 1, wherein the microalgae culture is derived from green microalgae.

9. The method of claim 8, wherein the green microalgae are derived from strains belonging to the Chlorophyta phylum.

10. The method of claim 1, wherein the microalgae culture is derived from genera belonging to Chlorella. Chlamydomonas, Chloroidium, or combinations thereof.

11. The method of claim 1, wherein the microalgae culture is derived from Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella anitrata, Chloroidium saccharophilmn, Chlorella sp., or combinations thereof.

12. The method of claim 1, wherein the microalgae culture is derived from Chlamydomonas reinhardtii.

13. The method of claim 1, wherein the composition comprises an extract of the microalgae culture.

14. The method of claim 13, wherein the extract of the microalgae culture comprises solvent- extracted microalgae culture.

15. The method of claim 14, wherein the solvent is selected from the group consisting of chloroform, hexane, methanol, ethanol, or combinations thereof.

16. The method of claim 13, wherein the extract of the microalgae culture is in crude form.

17. The method of claim 1, wherein the composition comprises compounds from the microalgae culture.

18. The method of claim 17, wherein the compounds are selected from the group consisting of benzenes, benzamides, amino acids, peptides, glycerides, oxylipins, fatty acids, polyunsaturated fatty acids, fatty acid conjugates, short-chain keto acids, sterols, isoprenoids, alpha-keto acids, phenylpyruvic acids, pyrrolidinyl pyridines, amines, carboxylic acids, dicarboxylic acids, hydroxycinnamic acids, analogues thereof, derivatives thereof, or combinations thereof.

19. The method of claim 17, wherein the compounds are selected from the group consisting of palmitoleic acid, coronaric acid, L-a-palmitin, 2,3-dihydroxypropyl stearate, coronaric acid, NP- 012766, 9(S)-HOTrE (9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid), 12-oxo phytodienoic acid, and 13-HpODE (13-hydroperoxy-octadecadienoic acid), allelochemicals, isoquinoline alkaloids, aldehydes, cinnamic acids, Traumatin, Umbelliferone (7 -hydroxy coumarin), Xestoaminol, 4-Linoleamide, palmitamide, (Z)-9-octadecenamide, palmitamide, cinnamic acid, (9Z)-9-octadecenamide, amide derivatives of oleic acid, derivatives thereof, or combinations thereof.

20. The method of claim 17, wherein the compounds from the microalgae culture are in crude form in the extract of the microalgae culture.

21. The method of claim 1, wherein the applying comprises spraying the composition onto the agricultural material.

22. The method of claim 1, wherein the applying comprises applying the composition onto a field that comprises the agricultural material.

23. The method of claim 1, wherein the agricultural material comprises a plant or seed selected from the group consisting of crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, potato, grapes, varieties thereof, or combinations thereof.

24. A composition suitable for use in treating an agricultural material, wherein the composition comprises a microalgae culture, an extract of the microalgae culture, compounds from the microalgae culture, or combinations thereof.

25. The composition of claim 24, wherein the composition has at least one of pesticidal activities, herbicidal activities, anti-microbial activities, nematocidal activities, algicidal activities, or combinations thereof.

26. The composition of claim 24, wherein the composition is suitable for use in preventing the growth of weeds along with a plant or seed.

27. The composition of claim 24, wherein the composition comprises the microalgae culture.

28. The composition of claim 24, wherein the microalgae culture is derived from green microalgae.

29. The composition of claim 28, wherein the green microalgae are derived from strains belonging to the Chlorophyta phylum.

30. The composition of claim 24, wherein the microalgae culture is derived from genera belonging to Chlorella. Chlamydomonas, Chloroidium, Chlorella sp., or combinations thereof.

31. The composition of claim 24, wherein the microalgae culture is derived from Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella anitrata, Chloroidium saccharophilum, Chlorella sp. , or combinations thereof.

32. The composition of claim 24, wherein the microalgae culture is derived from Chlamydomonas reinhardtii .

33. The composition of claim 24, wherein the composition comprises an extract of the microalgae culture.

34. The composition of claim 24, wherein the extract of the microalgae culture comprises solvent-extracted microalgae culture.

35. The composition of claim 24, wherein the solvent is selected from the group consisting of chloroform, hexane, methanol, ethanol, or combinations thereof.

36. The composition of claim 24, wherein the extract of the microalgae culture is in crude form.

37. The composition of claim 24, wherein the composition comprises compounds from the microalgae culture.

38. The composition of claim 37, wherein the compounds are selected from the group consisting of benzenes, benzamides, amino acids, peptides, glycerides, oxylipins, fatty acids, polyunsaturated fatty acids, fatty acid conjugates, short-chain keto acids, sterols, isoprenoids, alpha-keto acids, phenylpyruvic acids, pyrrolidinyl pyridines, amines, carboxylic acids, dicarboxylic acids, hydroxycinnamic acids, analogues thereof, derivatives thereof, or combinations thereof.

39. The composition of claim 37, wherein the compounds are selected from the group consisting of palmitoleic acid, coronaric acid, L-a-palmitin, 2,3-dihydroxypropyl stearate, coronaric acid NP-012766, 9(S)-HOTrE (9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid), 12-oxo phytodienoic acid, and 13-HpODE (13-hydroperoxy-octadecadienoic acid), allelochemicals, isoquinoline alkaloids, aldehydes, cinnamic acids, Traumatin, Umbelliferone (7- hydroxy coumarin), Xestoaminol, 4-Linoleamide, palmitamide, (Z)-9-octadecenamide, palmitamide, cinnamic acid, (9Z)-9-octadecenamide, amide derivatives of oleic acid, derivatives thereof, or combinations thereof.

40. The composition of claim 24, wherein the compounds from the microalgae culture are in crude form in the extract of the microalgae culture.

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