WO2005039289A2

WO2005039289A2 - Herbicidal compositions

Info

Publication number: WO2005039289A2
Application number: PCT/US2004/035600
Authority: WO
Inventors: Jorge M. Vivanco; Frank R. Stermitz; Harsh Pal Bais
Original assignee: Colorado State University Research Foundation
Priority date: 2003-10-24
Filing date: 2004-10-25
Publication date: 2005-05-06
Also published as: CA2485780A1; WO2005039289A3

Abstract

The present invention provides herbicidal compositions based on exudates of the invasive plant Centaurea diffusa (diffuse knapweed). The extracts contain the quinoline derivative 8-hydroxyquinoline. Methods of isolating and making 8-hydroxyquinoline-containing compositions as well as methods of using 8-hydroxyquinoline and compositions containing it to control the growth of undesirable plants, microorganisms or prevent the germination of seeds are also provided.

Description

HERBICmAL COMPOSITIONS FIELD OF THE INVENTION The present invention resides in the field of herbicidal compositions and methods of making and using the same.

BACKGROUND OF THE INVENTION Many herbicides and pesticides in current use have human and animal toxicities requiring controlled application as well as clean up of contaminated land and ground water. These requirements greatly increase the actual cost of using these agricultural products beyond the costs associated with purchasing and applying the chemicals. One proposed solution to these problems has been the search for naturally-occurring chemicals with herbicidal or insecticidal properties without significant animal or human toxicities. This has led to the study of invasive plants which have the ability to spread over large areas by the secretion of allelochemicals to kill or inhibit the growth of surrounding plants. The few millimeters of soil immediately surrounding a plant root constitute a unique physical, biochemical and ecological environment. To a large extent, the rhizosphere is controlled by the root system through chemicals secreted into the surrounding soil. Root exudates include low molecular weight compounds such as amino acids, organic acids, sugars, phenolics and various secondary metabolites and high molecular weight compounds like mucilage and proteins. Through the exudation of a wide variety of compounds, roots may regulate the soil microbial community in their immediate vicinity, cope with herbivores, encourage beneficial symbioses, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species. Countering a challenge, roots may respond by secreting certain chemicals such as secondary metabolites, proteins and even volatiles. Root secretions may play symbiotic or defensive roles as a plant ultimately develops a positive or negative communication, depending on the other elements of its rhizosphere. An example of a negative communication is provided by Centaurea diffusa (diffuse knapweed). This noxious weed displaces other weeds and crops through root exudates. The past five decades of research in the field of knapweed allelopathy has witnessed minimal success in characterizing the responsible allelochemical(s). Thus, there is a long felt need for the identification of the chemicals responsible for the allelopathic chemical(s) from Centaurea diffusa that could be used as an effective, naturally-occurring herbicidal product.

SUMMARY OF THE INVENTION The present invention provides herbidical compounds and compositions and methods of using these chemicals to effectively control unwanted plants. The herbicidal compositions are based on exudates of C. diffusa and chemicals identified in those exudates. Specifically, herbicidal compositions are based on 8-hydroxyquinoline which has been identified as the active allopathic ingredient of C. diffusa exudates. Preferably, the herbicidal compositions of the present invention contain 8-hydroxyquinoline and additional ingredients such as extenders, thickners, dispersants, tackifiers, colorants, fertilizers, growth regulators, stabilizers, antifoams, preservatives, viscosity regulators, binders, surfactants or combinations thereof. These additional ingredients may represent between about 5% and about 95% of the composition. Typically, the 8-hydroxyquinoline is present in the herbicidal compositions at a concentration range of between about lOμg/ml and about 500μg/ml. However, the herbicidal compositions may be prepared as a concentrate for dilution. In this instance, the concentration of the 8-hydroxyquinoline in the composition may be greater than 500 μg/ml. The invention provides methods of controlling undesirable plant growth by contacting undesirable plants with a composition containing 8-hydroxyquinoline. In these methods, the 8-hydroxyquinoline is present in the composition in a concentration range of between about lOμg/ml and 500μg/ml. The method may include applying the composition to the media in which the undesirable plant is growing. Alternatively, the method may include mixing the composition into media in which the undesirable plant is growing. The application of the herbicidal composition may include preemergence application, postemergence application and/or seed dressing. The invention also provides methods of controlling growth of an undesired plant in crops of cultivated plants by contacting the undesired plant with a composition comprising 8-hydroxyquinoline. Similarly, the invention provides a method of inhibiting germination of a seed by contacting a seed with a composition comprising 8- hydroxyquinoline. In this method, the application may include contacting the media containing the seed such as soil. This may also include contacting or mixing of the composition with the media before the seed is contacted with the media. The present invention also provides a method of making the herbicidal compositions of the present invention by isolating a root extract of C. diffusa and combining the root exudate with a an ingredient such as extenders, thickners, dispersants, tackifiers, colorants, fertilizers, growth regulators, stabilizers, antifoams, preservatives, viscosity regulators, binders, surfactants or combinations thereof. In this method, the root extract may be extracted into a solvent such as methanol or hexane. Additionally, the solvent may be freeze dryed after the extraction. The present invention also provides a herbicidal composition made by the method of isolating a root extract of C. diffusa and extracting 8-hydroxyquinoline from the C. diffusa extract into a solvent followed by removing the solvent from the 8- hydroxyquinoline. The present invention also provides a method of inhibiting the growth of a microorganism by contacting a bacterium with an exudate of C. diffusa. This method may include applying a composition containing the extract or a chemical from the extract to a media prior to the microorganism contacting the media. In this method, the microorganism my include organisms such as Xanthomonas compestris, Pseudomonas syringae, Agrobacterium radiobacter, Erwinia carotovora, Aspergillus niger, Rhizoctonia solani, Phytopthora infestans and Fausarium Osysporum. DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of root exudates of C. diffusa (3 ml v v^"1) on (A) shoot and (B) root differentiation of 10-day-old -vztrσ-grown weeds and crop plants on the 14^th day after treatment. The data represent the percent inhibition relative to the untreated control in shooting and rooting efficiency response in various tested seedlings. (Values are Mean ± S.D, n=10). (C) shows the effect of root exudates of C. diffusa (3 ml v v^"1) on percent inhibition in germination of different weeds and crop plants on the 14^th day after treatment. The data represent the percent inhibition relative to the untreated control in shooting and rooting efficiency response in various tested seedlings. (Values are Mean ± S.D, n=10). Figure 2 shows the antibacterial activity of 8-hydroxyquinoline on different bacterial pathogens. Net bacterial growth was calculated by subtracting the OD₆₀₀ at the beginning from the OD₆₀o after 24 hours of incubation. Percent inhibition (%I) was calculated using net bacterial growth based on OD₆o₀ readings with the following formula: (untreated-treated/untreated) X (100). Values are mean ± S. D., (n=5). Figure 3 shows the concentrations of 8-hydroxyquinoline (chemical structure in inset) in natural soils supporting populations of Centaurea diffusa in native habitats (the Caucasus) and in invaded habitats (North America). Each bar represents one local population and error bars show one standard error. In a two-way ANOVA with continent and sites as fixed factors, F(continent) =88.05, df=l,68, PO.0001). Figure 4 (A) shows the proportional differences in the final biomasses of grass species from the native region (Caucasus) versus the invaded region (North America) of Centaurea diffusa treated with 8-hydroxyquinoline, a chemical exuded from the roots of the invasive weed. In an ANOVA testing the effects of 8-hydroxyquinoline and using the region of grass origin, and grass species as fixed factors F(8-hydroxyquinoline)=0.997, df=l,139, P=0.320; F(region of grass origin) =17.997, df=l,139, PO.0001 (grasses from the Caucasus were larger); F(8-hydroxyquinoline x region) =8.69, df=l,139, P=0.004. Data for Bothriochloa ischaemum are not presented because this species did not germinate abundantly in this experiment. (B) shows the total biomass of Centaurea diffusa invading experimental microcosms containing plant communities from either the native or invaded region of C. diffusa (the Caucasus or North America, respectively), planted in substrate with soil inocula from the native or invaded regions, and with soil from each region either sterilized or unsterilized. Error bars show one standard error. * designates significant effects of sterilization. In a three-way ANOVA with all factors fixed, F(region of plant origin) = 8.209, df = 1,51, P=0.006; F(region of soil

1.721, df = 1,51, P=0.197; F(soil sterilization) = 22.769, df = 1,51, PO.0001; F(soil origin x soil sterilization) = 4.863, df = 1,51, P=0.033; F(grass origin x soil sterilization) = 7.421, df = 1,51, P=0.010, no other signification interactions. Total grass biomass as covariate, F=1.243, df=l,51, P=0.271. Figure 5 demonstrates 8-Hydroxyquinoline concentrations in experimental microcosms containing plant communities from either the native or invaded region of C. diffusa (the Caucasus or North America, respectively), planted in substrate with soil inoculum from the native or invaded regions, with soil from each region either sterilized or unsterilized, and invaded with C. diffusa. Error bars show one standard error. * designate significant effects of sterilization. In a three-way ANOVA with all factors fixed, F(region of plant origin) = 0.002, df = 1,51, P=0.963; F(region of soil origin)= 73.206, df = 1,51, PO.001; F(soil sterilization) = 1.330, df = 1,51, P=0.255; F(soil origin x soil sterilization) =23.798, df = 1,51, PO.001; no other signification interactions. Total C. diffusa biomass as covariate, F= .134, df=l,51, P=0.048. Figure 6 shows the effect of low concentrations of 8-hydroxyquinoline (approximately 80 μg g^"1 soil) found in Eurasian soil on the rhizosphere of natural populations of two North American natives (Artemisia tridantata and Achillea millefolium) and C. diffusa. Plants were photographed and mortality counts were scored after two weeks of 8-hydroxyquinoline (approximately 80 μg g^"1 soil) addition in the native rhizosphere.

DETAILED DESCRIPTION OF THE INVENTION The present invention is drawn to herbicidal compositions based on exudates and extracts of Centaurea diffusa and to methods of manufacturing and using these compositions. The herbicidal compositions preferably provide effective pre- and post- emergent herbicidal activity through the use of a naturally-occurring allelopathic chemical. Centaurea diffusa (C. diffusa or diffuse knapweed) is a member of a related group of invasive plants recognized as some of the most destructive plants in western North America. Since 1842, ecologists have known that knapweeds achieve their competitive advantage through the secretion of phytotoxic allelochemicals into the soil. The identity of these allelochemicals have been sought for almost fifty years. This long search concluded in 2001 with the discovery that (-)-catechin is the root-secreted compound responsible for Centaurea maculosά's (spotted knapweed) invasive behavior in the rhizosphere. Surprisingly, other members of the knapweed group of related plants secrete different allelochemicals to inhibit seed germination and root growth, and thereby displace other plants. The preset inventors have discovered that the roots of C. diffusa secrete 8-hydroxyquinoline, a chemical unrelated to (-)-catechin, to exert a phytotoxicity against other plants including C. maculosa. Interestingly, under natural conditions C. maculosa and C. diffusa do not share the same habitats and rarely displace each other, an observation presumably explained by the secretion of these different allelochemicals. Typical of the knapweed plants, C. diffusa is itself resistant to the effects of 8- hydroxyquinoline. Thus, one embodiment of the present invention is a herbicidal composition containing an exudate from the roots of C. diffusa or isolated 8-hydroxyquinoline or any agriculturally-acceptable salts thereof. The 8-hydroxyquinoline may be isolated from C. diffusa, synthesized, or purchased commercially. The herbicidal composition can be used to control, kill, suppress or inhibit the growth of susceptible plants. As used herein, the term "control" is inclusive of the actions of killing, inhibiting the growth, reproduction or proliferation, and removing, destroying or otherwise diminishing the occurrence and activity of plants and is applicable to any of the stated actions, or any combination thereof. In accordance with this invention it has been found that the growth of germinating seeds, emerging seedlings, maturing and established woody and herbaceous vegetation and aquatic plants can be controlled by exposing the emerging seedlings or above- or below-ground portions of maturing and established vegetation, or aquatic plants to an effective amount of the 8-hydroxyquinoline-containing compositions of the present invention. The 8-hydroxyquinoline compounds can be used individually, as admixtures with two or more compounds, or in an admixture with an adjuvant. These compositions are effective as pre- and post-emergent phytotoxicants or herbicides, e.g., the selective control of the growth of one or more monocotyledonous species and/or one or more dicotyledonous species in the presence of other monocotyledons and/or dicotyledons. Furthermore, these compounds are characterized by broad spectrum activity, i.e., they control the growth of a wide variety of plants including, but not limited to, ferns, conifer (pine fir and the like), aquatic, monocotyledons and dicotyledons. 8-Hydroxyquinoline is a commercially available compound, well-known as an analytical reagent for its metal chelating properties and described in the Merck Index as a fimgistat and antiseptic. Although a few simple 2- and 4-hydroxyquinolines are produced by microbes, 8-hydroxyquinoline has not previously been reported as a natural product. The quinoline ring system does occur as part of more complex plant alkaloid structures, in which anthranilic acid has been identified as a biosynthetic precursor. Although 8- hydroxyquinoline was phytotoxic against a variety of plant species, including C. maculosa, and repens, C. diffusa itself was resistant to 8-hydroxyquinoline. 8-Hydroxyquinoline also displayed strong antibacterial and antifungal activity against important plant pathogenic bacteria such as Xanthomonas compestris, Pseudomonas sy ngae Agrobacterium radiobacter, Erwinia carotovora, Erwinia amylovora and fungi viz., Aspergϊϊlus niger, Rhizoctonia solani, Phytopthora infestans and Fusarium oxysporum. Thus, it is possible that C. diffusa uses this allelochemical both as a phytotoxin and a protection against pathogens. The 8-hydroxyquinoline is preferably applied to the target plant as a liquid or a solid. The term "plant" as used herein means terrestrial plants and aquatic plants. The compositions of the present invention are suitable for all methods of application commonly used in agriculture, including preemergence application, postemergence application and seed dressing. For example, suitable application means include watering, spraying, atomizing, dusting and scattering. The 8-hydroxyquinoline compositions according to the invention can be applied before and after the plants have emerged, that is to say pre-emergence and post-emergence. They can also be incorporated into the soil before or during sowing. The active 8-hydroxyquinoline compounds or C. diffusa extracts can be converted to formulations customarily used in the agricultural industry such as solutions, emulsions, wettable powders, suspensions, powders, dusts, pastes, soluble powders, granules, suspension-emulsion concentrates, natural and synthetic materials impregnated with active components and microencapsulations in polymeric substances. These formulations are produced by methods commonly known to those of skill in the art, for example by mixing the active compounds with extenders, that is, liquid solvents, and/or solid carriers, optionally with the use of surfactants, that is emulsifiers and/or dispersants, and/or foam-formers. If the extender used is water, it is also possible to employ, for example, organic solvents as auxiliary solvents. Essentially, suitable liquid solvents include aromatics such as xylene, toluene or alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions, mineral and vegetable oils, alcohols such as butanol or glycol and also their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents such as dimethylformamide and dimethyl sulphoxide, and also water. Suitable solid carriers include, for example, ammonium salts and ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals, such as highly dispersed silica, alumina and silicates. Suitable solid carriers for granules include, for example, crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite, and also synthetic granules of inorganic and organic meals, and granules of organic material such as sawdust, coconut shells, maize cobs and tobacco stalks; as emulsifiers and/or foam- formers for example nonionic and anionic emulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl polyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates and also protein hydrolysates. Suitable dispersants include, for example, lignin-sulphite waste liquors and methylcellulose. Tackifiers such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, as well as natural phosphohpids such as cephalins and lecithins, and synthetic phosphohpids, can be used in the formulations. Other additives can be mineral and vegetable oils. It is also possible to use colorants such as inorganic pigments, for example iron oxide, titanium oxide and Prussian Blue, and organic dyestuffs, such as alizarin dyestuffs, azo dyestuffs and metal phthalocyanine dyestuffs, and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. The formulations generally comprise between about 0.1% and about 95% by weight of active compound, preferably between about 0.5% and about 90%. The active compound according to the invention can be present in its commercially available formulations and in forms prepared from these formulations such as in a composition with other active compounds, such as insecticides, attractants, sterilizing agents, bactericides, acaricides, nematicides, fungicides, growth-regulating substances or other herbicides. The insecticides include, for example, phosphoric acid esters, carbamates, carboxylates, chlorinated hydrocarbons, phenylureas and substances produced by microorganisms. It is also possible to admix other known active compounds such as fertilizers and growth regulators with the herbicidal compositions of the present invention. The compositions may also contain inactive ingredients effecting the composition without imparting herbicidal activity on their own such as stabilizers, e.g. where appropriate epoxidised vegetable oils (epoxidised coconut oil, rapeseed oil, or soybean oil), antifoams, typically silicone oil, preservatives, viscosity regulators, binders, as well as other chemical agents including other herbidices such as imazamethabenz-methyl, sulfosulfuron, tribenuron-methyl, amidosulfuron, metosulam, flurtamone, 2,4-D, bromoxynil, dichlorprop-P, tribenuron(-methyl), diflufenican, glyphosate(-isopropyl- ammonium), metsulfuron-methyl, fluroxypyr, isoproturon, imazamox, diclofop-methyl, carfentrazone-ethyl, clodinafop-propargyl, thifensulfuron-methyl and mixtures thereof. The herbidical compositions of the present invention may contain any herbicidally-effective amount of 8-hydroxyquinoline or exudates of C. diffusa or salts thereof. Preferably, the liquid herbicidal compositions of the present invention contain between about lOμg/ml and about 500 μg/ml of the 8-hydroxyquinoline compounds of the present invention, more preferably the compositions contain between about 20μg/ml and about 200 μg/ml of the 8-hydroxyquinoline compounds, more preferably the compositions contain between about 50μg/ml and about 150 μg/ml of the 8- hydroxyquinoline compounds. Preferably, the solid herbicidal compositions of the present invention are applied to soil at a rate of between about 50 μg/gram of soil and about 500 μg/gram of soil, more preferably the compositions are applied at a rate of between about 100 μg/gram of soil and about 300 μg/gram of soil. Additionally, the 8-hydroxyquinoline compounds may be supplied in a concentrated form for dilution prior to application. In these forms, the 8- hydroxyquinoline compounds can be supplied in liquid compositions in concentrations exceeding 500 μg/ml up to the solubility of the 8-hydroxyquinoline in the desired solvent. In this embodiment of the present invention, the concentrate is preferably supplied with instructions for further dilution of the concentrate prior to use. Any of the additional components that may be added to the herbicidal preparations of the present invention may occupy between about 1% and about 99% of the composition. Preferably the additional components occupy between about 5% and about 95% of the composition. Further embodiments of the present invention include methods of controlling undesired plants by the application of 8-hydroxyquinoline or an exudate or extract of C. diffusa to the undesired plant. The 8-hydroxyquinoline and/or C. diffusa preparations described above may be applied directly to the plant targeted for control or applied to the area surrounding the plant including the habitat of the plant or the media in which the plant is growing. As used herein, the term media means any medium capable of sustaining plant growth including, but not limited to, soils, aqueous solutions, hydroponic systems, sterilized media, and nutrient-enriched or enhanced media. The herbicidal compositions of the present invention may be applied to the target vegetation prior to visible growth of the plant or after the plant has begun to grow. In a further embodiment of the present invention, the 8-hydroxyquinoline and/or C. diffusa preparations described above are used to inhibit germination of a seed. In this embodiment, the 8-hydroxyquinoline-containing composition may be applied to the seed or to media or containers in which the seeds are located. Thus, in this embodiment, the 8- hydroxyquinoline-containing composition may be preventively applied to the media or containers in which the seeds are located prior to the seeds becoming present in order to effectively inhibit the germination of the seeds should they later become present. Another embodiment of the present invention is a method for the selective control of weeds in crops of cultivated plants which includes treating the cultivated plants, the seeds or seedlings or the crop area thereof with a herbicidally effective amount of 8- hydroxyquinoline and/or an exudate of C. diffusa. The method is applicable to crop plants that are not affected by the 8-hydroxyquinoline-containing preparations of the present invention or can tolerate higher concentrations of the 8-hydroxyquinoline- containing preparations of the present invention than the vegetation targeted for elimination within the crop area.

EXAMPLES

EXAMPLE 1. Extraction, analysis and identification of the phytotoxic allelochemicals in root exudates from C. diffusa plants. Seeds of Centaurea diffusa were obtained from natural populations in Larimer and Routt Counties, CO. The seeds were washed in running tap water and were surface sterilized using sodium hypochlorite (0.3 % v v-1) for 10-15 min, followed by 3-4 washes in sterile distilled water. Surface sterilized seeds were inoculated on static MS21 (Murashige and Skoog, 1962) basal media in petri dishes for germination. The seeds were allowed to germinate for 10 days until roots and shoots emerged. The light intensity within the growth chamber was 4.4117 J m-2 s-1. Ten-day-old seedlings were transferred to 50 mL culture tubes with 5 mL of liquid MS basal media. Plant cultures were maintained on an orbital platform shaker set at 90 rpm. The root exudates were collected after 30 days from in vz'tro-grown C. diffusa plants, and were subsequently lyophilized, concentrated, and extracted using 5 ml of ethyl acetate. The extracts were vortexed and stored for 24 h at 4°C. The supernatant was transferred with a Pasteur pipette to a separate test tube, and 1 mL of ethyl acetate was added. The supernatant was further concentrated by freeze-drying, and the weighed powder was re-dissolved in 500 μL of absolute methanol for HPLC analyses. The roots of C. diffusa were also extracted for metabolic profiling. In vz^'tro-grown C. diffusa roots were also harvested after 30 days of growth cycle and 200 mg of fresh wet tissues were extracted in 2 mL of ethyl acetate for 24 hrs at 4°C. After the extracts were centrifuged at 10,000 rpm for 10 mins, supernatants were concentrated under vacuum and were re- suspended in 500 μL of ethyl acetate for HPLC analyses. Compounds in the root exudates and roots were chromatographed by gradient elution on a reverse phase 5 μm, C18 column (25 cm x 4.6 mm). Mobile phase Solution A consisted of double distilled water and Solution B (ethyl acetate). A multi-step gradient was used for all separations with an initial injection volume of 15 μL and a flow rate of 1 mL min-1. The multistep gradient was as follows: 0-5 min 5.0 % B, 5-10 min 20.0 % B, 15-20 min 20.0 % B, 20-40 min 80.0 % B, 40-60 min 100 % B, 60-70 min 100 % B, 70- 80 min 5.0 % B. Different peaks were collected for the bioassay against various other invasive weeds and crop plants. Various peak eluants were concentrated under vacuum at 30°C and further purified by injecting them back into HPLC under similar conditions and were collected at similar retentions. The eluant showing biological activity was dried under vacuum at 30°C resulting in two milligrams of an amorphous powder. The biological activity was detected in the whole fraction, but was missing in fractions collected before and after 41 min. The HPLC eluant passed through a UV detector with a flow rate of 0.25 mL min-1 was delivered into the electron spin mass spectrometer (ESI- MS). The mass spectrometer (MS) parameters were optimized to maintain a high gas temperature (200°C) and gas flow (50 psi). Ions were referred to both positive and negative splits. Scan ranges of 100 to 750 amu (milli absorbance units) were used for negative ions. A step size of 1 amu and dwell time of 1 ms was used during the analysis. The active eluant had m/z 145 (M+-1), for C9H7NO. The 1H and 13C NMR spectra of the HPLC-purified active exudate component from in vitro conditions were essentially identical to those of commercial 8- hydroxyquinoline and literature values for the compounds.

EXAMPLE 2. The phytotoxicity of root exudates from C. diffusa. Ten-day-old seedlings of C. maculosa, C. diffusa, Festuca idahoensis, Koeleria micrantha, A. repens, L. esculentum, T. aestivum, and A. thaliana were placed on MS basal medium in petri dishes after initial surface sterilization. Petri dishes were kept under a 16 hr light and 8 hr dark photoperiod in an incubator. Root exudates collected from 30- days old cultures of C. diffusa were subjected to autoclaving at 120°C for 30 min at 15 lb pressure and were then administered in different concentrations (1-3 ml v v-1) over the surface sterilized seedlings to analyze their phytotoxic effects. Autoclaving was performed to denature macromolecules and to narrow down the effect to a secondary metabolite. Arabidopsis was used to assess the phytotoxicity minimum inhibitory concentration (MIC) of 8-hydroxyquinoline. Ten plants per species were used for analysis of mortality rates. Root exudates were also tested against C. diffusa itself. All species except C. diffusa showed 80-100% mortality 14 days after addition of root exudates from C. diffusa. Plants showed wilting symptoms prior to senescence with reduced shoot and root differentiation (Figure l , b). All species except C. diffusa showed reduced germination in response to C. diffusa root exudates (Figure lc). Although 8-hydroxyquinoline was phytotoxic to all other plant species tested, including C. maculosa, C. diffusa itself was resistant to 8-hydroxyquinoline (Supplementary Figure 6).

EXAMPLE 3. The antimicrobial activity of the phytotoxic chemicals ofC. diffusa. This example demonstrates the antimicrobial activity of the phytotoxic chemicals of C. diffusa. Both fungal and bacterial isolates from a broad phylogenetic range were tested for inhibition of growth against 8-hydroxyquinoline. Inhibition of hyphal growth in Aspergillus niger, Rhizoctonia solani, Phytophthora infestans and Fusariurn oxysporum was tested by a linear growth assay. Fungal isolates were maintained on PDA in the dark at 24°C. 8-Hydroxyquinoline was applied to sterile filter discs and allowed to air dry before being arranged in a circle on a 35 mm petri dish. A four-mm plug of fungal hyphae was placed in the center of the petri dish and inhibition was observed on a daily basis for 7 days. Each fungal isolate was tested against 8-hydroxyquinoline and concentrations (10-250 μg mL-1) in two separate replicates. Bacterial assays were performed in 96-well, sterile, flat bottom microtiter plates. Bacterial suspension cultures of Xanthomonas compestris, Pseudomonas syringae pv. tomato DC3000, Agrobacterium radiobacter, Erwinia carotovora and Erwinia amylovora were grown overnight at 37°C to OD600= 0.2. Test wells contained 5 μL of the tested bacteria in combination with varying amounts (10-250 μg mL-1) of standard 8-hydroxyquinoline. Control wells contained 5 μL (about 7.5 x 10⁵ cells) of bacteria alone with the highest volume of methanol used. The plates were covered with sterile lids and placed in polystyrene boxes lined with moistened filter paper to maintain high humidity, and incubated at 37°C. The absorbance of each well was determined at OD600 nm with an Opsys MR microtiter plate reader. Net bacterial growth was calculated by subtracting the initial OD600 from the OD600 after 24 h of incubation. Percent inhibition (1%) was calculated using net bacterial growth based on OD600 readings with the following formula: (untreated- treated/untreated) x 100. 8-Hydroxyquinoline displayed strong antibacterial and antifungal activity against important plant pathogenic bacteria including Xanthomonas compestris, Pseudomonas syringae Agrobacterium radiobacter, Erwinia carotovora, Erwinia amylovora (Figure 2) and fungi Aspergillus niger, Rhizoctonia solani, Phytophthora infestans and Fusarium oxysporum. Suggesting that C. diffusa uses this allelochemical both as a phytotoxin and antimicrobial.

EXAMPLE 4. Soil differences in the toxicity ofC. diffusa exudates to different plants. To further investigate the potential role of 8-hydroxyquinoline in the invasion process, biogeographical differences in its abundance and effects were explored in more realistic ecological experiments. Soil was collected directly beneath 5-10 individual Centaurea diffusa plants in each of four populations in The Republic of Georgia (Lisi Lake, near Tblisi: N41°44.389' E044°44.229"; Ksani: N 41°54.687' E044°34.065'; Okzolkara N41°40.892' E044°46.299'; and Rustavi N 41°34.540' E044°57.445*) and from 6-20 individuals in each of three populations in the northwestern United States (The Dalles, OR N45°37.433' W121°12.841; Spokane, WA N47°36.996' W117°30.331; and Superior, MT N47°l 1.770' W114°52.960'. These soils were measured directly for 8- hydroxyquinoline concentrations using HPLC as described above. Seeds of Festuca idahoensis, Koeleria micrantha, Pseudoroegneria spicata, and Stipa comata were collected in Missoula County, Montana, and seeds of Agropyron cristatum, Melica transsilvanica, Phleum nuniculatum, and Bothriochloa ischaemum were collected near Tbilisi, Georgia from plants intermixed with Centaurea diffusa. C. diffusa seeds were collected in Upper Kittitas County, Washington. Individual plants were grown from these seeds in 100 cm³ tubes for six months and then transplanted into 525 cm³ pots containing a 80:20 silica sand : Montana field soil mixture. Twenty days after the transplant 250 μL of 5 mg mL-1 of 8-hydroxyquinoline and ethyl acetate were applied around the base of each grass and the same concentration was added again 42 days after the first one. The grasses were harvested 105 days after transplanting. Microcosms using soil and grass species described above were constructed. Twenty-liter buckets were filled with 20/30 grit sand, except for the top 5 cm, which was filled with a 1 : 1 combination of sand and soil. For each soil type both a sterilized and a non-sterilized treatment was included. Sterilization was a triple-autoclave procedure, where four liters of soil were autoclaved for two hours, then left to cool for 24 hours, and then autoclaved and cooled two more times. Grasses endemic to each country were grown in native soil and foreign soil, with a sterilized and non-sterilized treatment for each, for a total of 51 replications. Treatment combinations did not have equal replication because the quantity of soil collected was inadequate in some cases. The larger grass species, Agropyron and Pseudoroegneria, were planted in the center of the buckets, with two individuals of each of the other three species from the regional communities planted in a circle around them, for a total of seven grasses in each community. The grasses were allowed to establish for 136 days under aseptic conditions for sterilized soil treatments before the communities were seeded with 50 seeds of C. diffusa. This establishment period allowed for the development of 50-75% cover of aboveground grass tissue. After seeding, the invasions were allowed to proceed for another 186 days under aseptic conditions for sterilized soil treatments before harvesting the total biomass of all plants. Nutrients were applied every three weeks, with each community receiving 400 ml each week. The total biomass of all C. diffusa plants was analyzed using the region of grass origin, the region of soil origin, and the sterilization of the soil as fixed factors in a three way ANOVA with the total grass biomass as a covariate. The concentration of 8-hydroxyquinoline in the mesocosms was measured and compared among treatments using a three-way ANOVA using C. diffusa biomass as a covariate. To compare the susceptibility of North American natives grown under natural conditions against 8-hydroxyquinoline, two North American natives' monocultures (Artemisia tridentata and Achillea millefolium) in C. diffusa-iavaded regions were identified at Walker Ranch, Boulder County, CO. 8-Hydroxyquinoline dissolved in methanol was added at the low concentration (about 80 μg g-1 soil) found in Eurasian soil to the rhizosphere of natural populations of A. tridentata and A. millefolium). Controls received only methanol. The 8-hydroxyquinoline-treated natives along with the untreated controls were allowed to grow for 14 more days, and were subsequently photographed and analyzed for biomass and mortality counts. To evaluate the proximal localization of 8-hydroxyquinoline in C. diffusa's habitat, soil samplings was performed both horizontally (10-15 cm) and vertically in depth (10-40 cm) around the rosette and was further extracted and subjected to HPLC analysis as described in Example 1. The concentration of 8-hydroxyquinoline was over three times higher in soils supporting C. diffusa in North America than in similar soils in Eurasia (Figure 3). This difference may have been due to greater exudation from North American plants, longer persistence in North American soils due to slower microbial breakdown, or higher densities of C. diffusa in North American plant communities. However, these regional differences in 8-hydroxyquinoline were not the only possible explanation for C. diffusa 's exceptional success in North America. North American plant species were much more susceptible than Eurasian species to identical concentrations of 8-hydroxyquinoline added to field soil (Figure 4a), supporting the hypothesis that the success of some exotic invasive plants may be due to the use of competitive mechanisms that do not occur in the natural communities they invade and which disrupt inherent and co-evolved interactions among long-associated native species. The mechanism by which whole communities resist C. diffusa was tested and the antibacterial and antifungal activity of 8-hydroxyquinoline was tested in more realistic conditions by establishing microcosms in which North American and Eurasian plant communities were constructed in both North American and Eurasian soils. The regional source of the plant community was a highly significant factor in the resistance to C. diffusa (Figure 4b, F(region of plant origin) = 8.209, df = 1 ,51 , P=0.006), with North American plant communities showing far less resistance than Eurasian communities. Importantly, sterilization of North American soils resulted in much stronger suppression of C. diffusa growth than sterilization of Eurasian soils, suggesting that differences in the microbial communities in Eurasia and North America may also play a major role in C. diffusa invasions (Figure 4b, F(soil origin x soil sterilization) = 4.863, df = 1,51, P=0.033). One important difference in the function of the Eurasian and North American microbial communities may be how they break down or even utilize 8-hydroxyquinoline. Even when the biomass of C. diffusa was incorporated as a covariate (to account for large differences in the biomass of C. diffusa in different treatments), Eurasian soils accumulated much less 8-hydroxyquinoline than North American soils (Figure 5). More importantly, sterilization of Eurasian soils resulted in a 134% increase in 8-hydroxyquinoline concentrations, whereas sterilization of North American soils resulted in only a 41% increase (F(soil origin x soil sterilization) =23.798, df = 1,51, PO.001). These results suggest that microbes in Eurasian soils supporting populations of C. diffusa may be better adapted to 8-hydroxyquinoline and perhaps even use the exudate as an organic carbon source. To address the effects of root-secreted 8-hydroxyquinoline on naturally growing North American natives in fully realistic field conditions, a low concentration of 8- hydroxyquinoline (about 80 μg g^"1 soil, the concentration found in the soil of Eurasian C. diffusa communities) was added to the rhizosphere of populations of two North American natives growing naturally in the field (Artemisia tridentata and Achillea millefolium). Similar to the findings of the greenhouse experiments, the field results clearly indicated that North American plant species were susceptible (about 90 % mortality) to low concentrations of 8- hydroxyquinoline Figure 6). However, this concentration of 8-hydroxyquinoline did not affect Eurasian species under greenhouse conditions (Figure 4a). These results have several important implications for community ecology. First, they suggest that interactions among plant species and plants and microbes may drive natural selection. Second, the indication that plant species may be selected to tolerate specific characteristics of their neighbors suggests that natural plant communities may be less individualistic than generally thought. Third, thses results imply that North American species may evolve resistance over time to the unfamiliar root exudates of invaders. Finally, they imply that natural biological communities may evolve in some functional manner and that some exotic invasive plants may use aggressive mechanisms that are not present in the natural communities they invade to disrupt inherent, co-evolved functions. The foregoing examples have been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

What is claimed is: 1. A herbicidal composition comprising an exudate of C. diffusa.

2. The herbicidal composition of Claim 1, wherein the exudate comprises 8- hydroxyquinoline.

3. A herbicidal composition comprising 8-hydroxyquinoline and an ingredient selected from the group consisting of extenders, thickners, dispersants, tackifiers, colorants, fertilizers, growth regulators, stabilizers, antifoams, preservatives, viscosity regulators, binders, surfactants and combinations thereof.

4. The herbicidal composition of Claim 3, wherein the 8-hydroxyquinoline is present in a concentration range of between about lOμg/ml and about 500μg/ml.

5. The herbicidal composition of Claim 3, prepared for further dilution wherein the 8-hydroxyquinoline is present in a concentration of greater than 500 μg/ml.

6. The herbicidal composition of Claim 3, wherein the at least one ingredient comprises between about 5% and about 95% of the composition.

7. A method of controlling undesirable plant growth comprising the step of contacting an undesirable plant with a composition comprising 8-hydroxyquinoline.

8. The method of Claim 7, wherein the 8-hydroxyquinoline is present in the composition in a concentration range of between about lOμg/ml and 500μg/ml.

9. The method of Claim 7, wherein the contacting comprises applying the composition to the media in which the undesirable plant is growing.

10. The method of Claim 9, wherein the applying comprises mixing the composition into media in which the undesirable plant is growing.

11. The method of Claim 7, wherein the contacting comprises a treatment selected from the group consisting of preemergence application, postemergence application and seed dressing.

12. The method of Claim 7, wherein the contacting step comprises applying the composition comprising 8-hydroxyquinoline to the habitat of the undesirable plant.

13. A method of controlling growth of an undesired plant in crops of cultivated plants comprising the step of contacting the undesired plant with a composition comprising 8-hydroxyquinoline.

14. A method of inhibiting germination of a seed comprising the step of contacting a seed with a composition comprising 8-hydroxyquinoline.

15. The method of Claim 14, wherein the contacting comprises applying the composition to a media containing the seed.

16. The method of Claim 15, wherein the media containing the seed is soil.

17. The method of Claim 14, wherein the contacting comprises applying the composition to a media prior to contacting the media with the seed.

18. A method of making a herbicidal composition comprising: isolating a root extract of C. diffusa and combining the root exudate with a an ingredient selected from the group consisting of extenders, thickners, dispersants, tackifiers, colorants, fertilizers, growth regulators, stabilizers, antifoams, preservatives, viscosity regulators, binders, surfactants and combinations thereof.

19. The method of Claim 18, wherein the isolating step comprises extracting 8-hydroxyquinoline from the root extract into a solvent.

20. The method of Claim 19, wherein the solvent is selected from the group consisting of methanol and hexane.

21. The method of Claim 19, comprising the additional step of freeze drying the solvent after the extracting step.

22. A composition produced by the process comprising: a. isolating a root extract of C. diffusa; b. extracting 8-hydroxyquinoline from the C. diffusa extract into a solvent; and, c. removing the solvent from the 8-hydroxyquinoline.

23. A method of inhibiting the growth of a microorganism comprising the step of contacting a bacterium with a composition comprising an exudate of C. diffusa.

24. The method of Claim 23, wherein the contacting comprises applying the composition to a media prior to the microorganism contacting the media.

25. The method of Claim 23, wherein the microorganism is selected from the group consisting of Xanthomonas compestris, Pseudomonas syringae, Agrobacterium radiobacter, Erwinia carotovora, Aspergillus niger, Rhizoctonia solani, Phytopthora infestans and Fausarium Osyspomm.