Compositions and methods to selectively control invasive species

Abstract

Methods and compositions for controlling the growth of an invasive species by application of a composition comprised of a natural pesticide derived from a species to the invasive species, especially endocides.

Claims

1. A method of controlling a first invasive species comprising applying a composition comprising a natural pesticide to the first invasive species, wherein the natural pesticide is derived and/or isolated from Triadica sebifera, wherein the first invasive species is the same as or is within the same family as Triadica sebifera, and wherein the natural pesticide consists of an extract of Triadica sebifera.

2. The method of claim 1, wherein the growth, reproduction, and/or spread of the first invasive species is halted within 1 week or 1 month and/or is halted for at least 1 year.

3. The method of claim 1, wherein the composition comprising the natural pesticide is applied topically to the first invasive species; sprayed onto the first invasive species, spread around the first invasive species; and/or dissolved in water surrounding the first invasive species.

4. The method of claim 1, wherein the natural pesticide consists of an ethanol extract of Triadica sebifera or fraction thereof.

5. The method of claim 1, wherein the first invasive species is Triadica sebifera.

6. The method of claim 5, wherein the natural pesticide consists of an ethanol extract of Triadica sebifera or fraction thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1The scheme shows the discovery and development process and related experiments of endocides from invasive giant Salvinia (Salvinia molesta) as described herein (inactive: not able to inhibit the growth of giant Salvinia at all; active: able to fully inhibit the growth of giant Salvinia at high concentrations; very active: able to fully inhibit the growth of giant Salvinia at low concentrations).

(3) FIG. 2Salvinia species are rootless, free-floating aquatic ferns and they are well-known for their extremely water-repellent floating leaves. On the upper surfaces of the floating leaves of S. molesta, four multicellular glandular trichomes (called trichomes) have their apical cells connected to form egg-beater structures. The three short arrows point to the egg-beater structures of trichomes which were removed using a blade. The root-like submerged leaves are highly branched with multicellular trichomes on which sporocarps develop.

(4) FIG. 3Effects of dry plant matter of giant Salvinia (Salvinia molesta) (tea bag) on the growth of giant Salvinia in containers during the four weeks of greenhouse experiments (with bars presenting standard deviations).

(5) FIG. 4The photograph shows that giant Salvinia (Salvinia molesta) plants were totally eliminated by the dried plant matter of giant Salvinia (a bag of 400 g plant matter was placed in the bottom of each of the containers on the bottom row) by the end of the second week of experiments in the greenhouse in comparison with controls (containers on the top row); there was no new growth within the six months of observation.

(6) FIG. 5The photograph shows that the root-like submerged leaves of giant Salvinia (Salvinia molesta) were totally damaged before the floating leaves turning into brown and dead after the tea bag treatment.

(7) FIG. 6The photograph shows the wilted trichomes on the upper surfaces of the floating leaves of giant Salvinia (Salvinia molesta) caused by the water extract from tea bag (left) in comparison with those in the normal plant growth condition (right). The wilted trichomes released autotoxic compounds and enhanced the endocidal function of tea bag.

(8) FIG. 7The photograph shows that all giant Salvinia (Salvinia molesta) plants were eliminated within the 13 weeks after the treatment with water extract of the dried matter of giant Salvinia. The plants in the top row served as control without any treatment, and each plant has developed significant new growth. The plants in the middle row were treated with 0.01% water extract of giant Salvinia, and some plants have developed new growth. The plants in the bottom row were treated with 0.1% water extract of giant Salvinia, and all plants are dead and no new growth was developed from any plants during the eight months of observation. The three columns represent three replications of the experiment.

(9) FIG. 8The photograph showing growth performance of giant Salvinia (Salvinia molesta) plants two weeks after the treatments of water extract of the dried matter of giant Salvinia and its three fractions in comparison with those in the control group. Top row: plants cultured in water without any treatment (control); 2nd row: plants were treated with 0.1% MeOH fraction (all plants dead); 3rd row: plants were treated with 0.1% MeOH/CH.sub.2Cl.sub.2 (1:1) fraction; 4th row: plants were treated with 0.1% H.sub.2O fraction; and the bottom row: 0.1% water extract of giant Salvinia. The three columns represent three replications of the experiment.

(10) FIG. 9Structure of bioactive salviniside II as endocide isolated from giant Salvinia (Salvinia molesta).

(11) FIG. 10.sup.1H and .sup.13C-NMR Data () of salviniside II.

(12) FIG. 11Giant Salvinia (Salvinia molesta) can be totally eliminated by salviniside II isolated from giant Salvinia within two weeks (with bars presenting standard deviations).

(13) FIG. 12HPLC profiles of water extract from the dried matter of giant Salvinia (Salvinia molesta) on the preparation day (upper) and storage as the powder at RT for 26 weeks (bottom) using Agilent 1100 HPLC system (Eclipse XDB-C18 column (4.6150 mm, 3.5 M) at a flow rate of 0.6 mL/min, detection wavelength=254 nm, the extract concentration: 24 kg/L in fresh weight, and the injection volume: 3 L). The stored powder had approximately 70% of the total peak area of that in the newly prepared extract.

(14) FIG. 13HPLC profiles of 5% H.sub.2O fraction of water extract from the dried matter of giant Salvinia (Salvinia molesta) on the preparation day (upper) and stored in refrigerator (4 C.) for 26 weeks (bottom) using Agilent 1100 HPLC system (Eclipse XDB-C18 column (4.6150 mm, 3.5 M) at a flow rate of 0.6 mL/min, detection wavelength=254 nm, the extract concentration: 16 kg/L in fresh weight, and the injection volume: 50 L). The stored H.sub.2O fraction still had approximately 90% of the chemical contents of the newly prepared sample.

(15) FIG. 14The growth of giant Salvinia (Salvinia molesta) can be successfully controlled by the endocide treatments with or without surfactants. By the end of the third week, all plants treated with either tea bag of the dried matter, water extract of the dried matter with surfactants, or H.sub.2O fraction of the water extract from the dried matter with surfactants were dead and there was no new growth during the following four months of observation. By the end of the 4.sup.th week, over 90% or 95% of the plants treated with water extract and H.sub.2O fraction of the water extract were dead, respectively (with bars presenting standard deviations).

(16) FIG. 15The photograph shows that all giant Salvinia (Salvinia molesta) plants were eliminated within three weeks after the treatment using a tea bag of the dried matter of giant Salvinia (right) in comparison with control (left).

(17) FIG. 16The growth of giant Salvinia (Salvinia molesta) can be successfully controlled by the extract juice from the fresh matter of giant Salvinia by hydraulic press. After two treatments, the hydraulic extract juice with surfactants totally eliminated the giant Salvinia within four weeks and there was no new growth thereafter (with bars presenting standard deviations).

(18) FIG. 17The photograph shows that all giant Salvinia (Salvinia molesta) plants were eliminated within four weeks after the treatment with hydraulic extract juice from the fresh matter of giant Salvinia (right) in comparison with the control (left).

(19) FIG. 18The photograph shows that the trichomes on the upper surfaces of the floating leaves of giant Salvinia (Salvinia molesta) were significantly damaged by the hydraulic extract (right) in comparison with those in the control (left). The wilted trichomes caused by the hydraulic extract released autotoxic chemicals and enhanced the endocidal function of the hydraulic extract.

(20) FIG. 19The photograph shows that there was no plant survival or new growth in the cultured residues from the juice production from fresh plant matter of giant Salvinia (Salvinia molesta) within six weeks (white dots are living least duckweed (Lemna minuta)).

(21) FIG. 20The diagram shows that both water extract from the dried matter and hydraulic extract from the fresh matter of giant Salvinia with surfactants effectively inhibited the giant Salvinia after two treatments (with bars presenting standard deviations).

(22) FIG. 21HPLC profiles of water extract from the dried matter of giant Salvinia (Salvinia molesta) (upper) and hydraulic extract from fresh matter of giant Salvinia (bottom) by using an Agilent 1100 HPLC system coupled to an Agilent 1100 diode array detector, and an Eclipse XDB-C18 column (4.6150 mm, 3.5 M) at a flow rate of 0.6 mL/min. A gradient elution was performed by using H.sub.2O (A) and CH.sub.3CN (B) as mobile phases. Elution was performed according to the following conditions: 2% B at time 0, linear increase to 98% B in 22 min, and hold 98% B for 8 min. The column temperature was maintained at 23 C. The HPLC chromatogram was standardized on retention times and peak intensities of the peaks observed at a wavelength of 254 nm. The concentration of both water extracts and hydraulic extract were 2 kg fresh plant matter/L. The injection volume was 50 L for all analyses.

(23) FIG. 22HPLC profiles of water extract from the dried matter of giant Salvinia (Salvinia molesta) (upper) and hydraulic extract from the fresh matter of giant Salvinia (bottom) by using Agilent 1260 HPLC system coupled to an Agilent 1260 diode array detector, and an Acclaim HILIC-10 column (4.6150 mm, 3.0 M) at a flow rate of 0.6 mL/min. The mobile phase was CH.sub.3CN/H.sub.2O (65:35, v/v). The column temperature was maintained at 23 C. The HPLC chromatogram was standardized on retention times and peak intensities of the peaks observed at a wavelength of 230 nm. The concentration of both water extracts and hydraulic extract were 40 kg fresh plant matter/L. The injection volume was 20 for all analyses.

(24) FIG. 23The diagram shows that the hydraulic extracts are more stable when stored under refrigeration (4 C.) than that at RT. The total peak area of hydraulic extract stored at refrigeration (4 C.) for one, two, and 14 weeks contained 70%, 66%, and 60% of the total chemical contents in the newly prepared extract, respectively (with bars presenting standard deviations).

(25) FIG. 24HPLC profiles of hydraulic extract from the fresh matter of giant Salvinia (Salvinia molesta) on the preparation day (upper) and stored at refrigerator (4 C.) for 14 weeks (bottom) by using Agilent 1100 HPLC system (Eclipse XDB-C18 column (4.6150 mm, 3.5 M) at a flow rate of 0.6 mL/min, detection wavelength=254 nm, the extract concentration: 2 kg/L in fresh weight, and the injection volume: 50 L).

(26) FIG. 25Metal analysis of the dried plant matter, water extract powder, and its precipitated salts of giant Salvinia (Salvinia molesta) by Thermo Scientific ICAP 7200 ICP-OES.

(27) FIG. 26The diagram shows that the growth of the floating fern (Salvinia minima) plants can be totally controlled by the water extract of dried matter of giant Salvinia (Salvinia molesta) within six weeks (with bars presenting standard deviations).

(28) FIG. 27The diagram shows that the plants of Carolina mosquito fern (Azolla caroliana) was inhibited by the water extract at higher concentration (2.5% or higher, in dry weight) (with bars presenting standard deviations).

(29) FIG. 28The photograph shows that all giant Salvinia (Salvinia molesta) plants were eliminated within 13 weeks after the treatment with water extract of the dried matter of least duckweed (Lemna minuta). The plants in the top row served as control without any treatment, and each plant has developed significant new growth. The plants in the middle row were treated with 0.01% water extract of duckweed, and some plants have developed new growth. The plants in the bottom row were treated with 0.1% water extract of duckweed, and all plants are dead and no any new growth was developed from any plants during the eight months of observation. The three columns represent three replications of the experiment.

(30) FIG. 29The diagram shows the new growth per plant of giant Salvinia (Salvinia molesta) by the end of the 13th week after treatment with water or ethanol extract of dried matter of least duckweed (Lemna minuta) in comparison with untreated plants (control) (with bars presenting standard deviations).

(31) FIG. 30The picture shows that all giant Salvinia (Salvinia molesta) plants were eliminated within 13 weeks after treatment with ethanol extract of the dried matter of least duckweed (Lemna minuta). The plants in the top row served as control without any extract treatment, and each plant has developed significant new growth. The plants in the middle row were treated with 0.01% ethanol extract of duckweed, and plants have developed new growth. The plants in the bottom row were treated with 0.1% ethanol extract of duckweed, and 12 plants are dead and only 3 plants have developed any new growth. However, all these 15 plants treated with 0.1% ethanol extract of least duckweed were dead by the end of 15 weeks of the experiment and no new growth occurred thereafter during the eight months of observation. The three columns represent three replications of the experiment.

(32) FIG. 31The photograph shows that some floating fern (Salvinia minima) plants were eliminated within the 13 weeks after the treatment with water extract of the dried matter of least duckweed (Lemna minuta). The plants in the top row served as control without any extract treatment. The plants in the middle row were treated with 0.01% ethanol extract of duckweed. The plants in the bottom row were treated with 0.1% ethanol extract of duckweed. The three columns represent three replications of the experiment.

(33) FIG. 32The diagram shows new growth per plant of floating fern (Salvinia minima) by the end of the 13th week after treatment with water or ethanol extract of the dried matter of least duckweed (Lemna minuta) in comparison with untreated plants (control) (with bars presenting standard deviations).

(34) FIG. 33The photograph shows that all floating fern (Salvinia minima) plants were eliminated by the end of 13 weeks after the treatment with 0.1% ethanol extract of the dried matter of duckweed (Lemna minuta). The plants in the top row served as control without any treatment, and there is significant new plant growth. The plants in the middle row were treated with 0.01% ethanol extract of duckweed, and new growth can be observed. The plants in the bottom row were treated with 0.1% ethanol extract of duckweed, and some plants are dead and minimal new growth can be observed. The three columns represent three replications of the experiment.

(35) FIG. 34The photograph shows that 3-week-old Brazilian pepper tree (Schinus terebinthifolius Raddi) seedlings had significant damage after the first treatment with 10% Brazilian pepper tree extract or Brazilian pepper tree extract with surfactants in comparison with those in control and surfactants treatment. By the end of the 4th week, all six pepper tree seedlings were dead following the pepper tree extract with surfactants treatment and five seedlings were dead and one had dead apical bud and young leaves after treated by the pepper tree extract.

(36) FIG. 35The photographs show that 3-week-old seedlings of Chinese tallow tree (Triadica sebifera (L.) Small) and Chinese privet (Ligustrum sinense Lour.) (family Oleaceae) under treatment with 10% Chinese tallow tree fruit extract. All six Chinese tallow tree seedlings treated by the ethanol extract of Chinese tallow tree fruits were significantly damaged (right on the upper row) in comparison with control (left on the upper row). However, the tollowtree extract did not cause any damage in Chinese privet seedlings (right on the bottom row) while the Chinese privet extract did not damage the tallow tree seedlings (left on the bottom row).

(37) FIG. 36The diagram shows the impacts of acetone extract and ethanol extract of the red imported fire ant (Solenopsis invicta Buren) on the workers of the red imported fire ant. The ethanol extract showed more significant toxicity against the fire ants than the acetone extract. During the 7 h of contact toxicity bioassays, an average of approximately 60%, 70%, or 80% of the 50 fire ants in contact with 1%, 5%, or 10% ethanol extract were dead, respectively.

(38) FIG. 37The diagram shows the impacts of ethanol extract of the red imported fire ants and formic acid on the workers of the red imported fire ant (Solenopsis invicta Buren). During the 90 min of contact toxicity bioassays, the combined applications of ethanol extract and formic acid had more significant effects than the use of either ethanol extract or formic acid alone (with bars presenting standard deviations).

(39) FIG. 38The diagram shows the impacts of ethanol extract of the red imported fire ant (Solenopsis invicta Buren) and formic acid on the injured workers of the red imported fire ants. During the 90 min of contact toxicity bioassays, the combined applications of ethanol extract and formic acid had more significant effects than the use of either ethanol extract or formic acid alone (with bars presenting standard deviations).

(40) FIG. 39The diagram shows the occurrence of formic acid (HCOOH) in chroroform (CHCl.sub.3) extract of the subterranean termite (Reticulitermes flavipes (Kollar)) by NMR analysis. .sup.1H-NMR experiments were performed on a JEOL ECS 400 spectrometer, with spectroscopic data referenced to the solvent used. According to standard formic acid .sup.1H-NMR spectrum, the unique singlet of the aldehyde proton should appear at .sub.H 8.02.

(41) FIG. 40The diagram shows the impacts of formic acid on the subterranean termite (Reticulitermes flavipes (Kollar)). Formic acid at higher concentrations (5 or 10%) killed all termites (including workers, soldiers, and reproductive) during the first hour of contact experiment. More than 40% of the termites were killed by the 1% formic acid during the 7 h of contact toxicity bioassays. However, formic acid at 0.1% concentration had no impact on the termites (with bars presenting standard deviations).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(42) Control of invasive species by its endogenous metabolites has never been proposed or used.

(43) The inventors recently discovered that some aquatic plants, trees, fire ants, or termites experienced slow autocide by their own toxins that were released into the environment by themselves or other individuals within the same species. It was known that some bioactive secondary metabolites may accumulate in glands (e.g., epidermal glandular trichomes of plants or exocrine glands of insects). These compounds usually do not interrupt normal growth of the producing species due to their non-bioavailability. However, it was discovered that external application of these bioactive agents to the parent species will inhibit and even eliminate the species. Further, the parent species may be more sensitive to its endogenous toxic metabolites than other species.

(44) As a proof of principle, the inventors found that endocides from giant Salvinia and extracts of least duckweed (Lemna minuta) (Lernnaceae) can eliminate existing giant Salvinia quickly and prohibit plant growth for at least six months in controlled conditions. The experiments showed that the effective materials used to extract bioactive agents to control Salvinia spp. include any ground dry biomass matters of Salvinia and Lemna. Experiments also found that the fresh biomass of Salvinia and Lemna, particularly chopped can also be useful to extract bioactive agents or direct application in control of Salvinia spp. Raw and unground dry plant matter can also be used in a similar manner, and it is believed that any other plant species containing the active agents as described herein could be useful in production of biocides to control Salvinia spp. This method is useful for controlling invasive aquatic plants, but this method also has potential in territorial plants and endophytic fungi. It is especially useful for controlling invasive plants with trichomes as the primary accumulation sites of bioactive compounds.

(45) Salvinia species are rootless, free-floating aquatic ferns and they are well-known for their extremely water-repellent floating leaves. On the upper surfaces of the floating leaves of S. molesta, four multicellular glandular trichomes have their apical cells connected to form egg-beater structures. The arrows point to the egg-beater structures of trichomes which were removed by blade. The root-like submerged leaves are highly branched with multicellular trichomes and on which sporocarps developed.

(46) Previous experiments found that the active agents of giant Salvinia are primarily accumulated in trichomes which are usually intact, and the plant can avoid poison by these toxins (Li et al., unpublished data). However, Salvinia spp. are well-known for their water-repellent floating leaves because of the leaves are densely covered with peculiar trichomes (Barthlott et al. 2009). On the upper surfaces of floating leaves of S. molesta, four multicellular trichomes have their apical cells connected to form egg-beater structures (FIG. 2). The root-like submergered leaves also have dense multicellular trichomes (FIG. 2). The basal cells of the trichomes in S. molesta are covered with wax (Barthlott et al. 2009). Therefore, the bioactive compounds in trichomes of the living plants are not released into the water because of this water-repellent feature. The removal or damage of the apical cells (egg-beater structures) of trichomes (FIG. 2) will not only release the chemicals in storage but also allow water to access the floating leaves to soak the bioactive agents out. This invention emphasizes that breaking the trichomes (e.g., mechanical treatments such as grinding, blending, squeezing, or heavily washing plant tissues) is necessary for effective extraction of the bioactive agents from giant Salvinia.

(47) Both water and organic solvents (e.g., ethanol) can be used to effectively extract the bioactive agents. The most effective solvent to dissolve these bioactive entities, fractions, or extracts to control Salvinia spp. is water, but organic solvents could also be useful in application. Plants respond to the treatment more quickly at higher temperatures than at lower temperatures (RT).

(48) The endocidal functions are not only in giant Salvinia, a fern species in the phylum Pteridophyta of the kingdom Plantae, but also discovered in Brazilian pepper tree and Chinese tallow tree, two tree species in the phylum Flowering Plants of the kingdom Plantae and the red imported fire ant and subterranean termite, two insect species from the phylum Arthropoda of the kingdom Animalia. The plant growth of Brazilian pepper tree seedlings had significant damage by the 10% ethanol extract of Brazilian pepper tree fruits after the first treatment (FIG. 34). Four weeks later, all six Brazilian pepper tree seedlings were killed by the extract with surfactants and five of the six seedlings treated by the extract alone were dead with one significantly injured. The seedlings of sweetgum (Liquidambar styraciflua L.) (family Altingiaceae) and Shumard oak (Quercus shumardii Buckland) (family Fagaceae) as well as poison ivy (Toxicodendron radicans (L.) Kuntze or Rhus toxicodendron L.) of the same family with Brazilian pepper tree were not impacted by the Brazilian pepper tree extract. Furthermore, the Brazilian pepper tree seedlings were no inhibited by either surfactants or the 10% ethanol extract of Chinese tallow tree (Triadica sebifera (L.) Small) fruits.

(49) The ethanol extract of the fruits of Chinese tallow tree (Triadica sebifera (L.) Small) (Eurporbiaceae) inhibited and even killed young seedlings of the tallow tree. However, the tollowtree extract did not cause any damage in the seedlings of Chinese privet (Ligustrum sinense Lour.) of the family Oleaceae. Further, the Chinese privet extract did not damage the tallow tree seedlings.

(50) Formic acid commonly occurs in ants (family Formicidae), termites (Isoptera), and some other insects. The organic acid is accumulated in exocrine glands of these insects and serves as defensive weapon fighting against attackers. The formic acid can be as concentrated as 60% of the secretion of ants, and workers can contain as much as 2 mg each (Morgan 2008). The acid is a known natural pesticide. Like their enemies, however, both the red imported fire ants and subterranean termites could not avoid toxicity of formic acid during external topical or fumigation application (FIGS. 37-40).

(51) This invention found that the red imported fire ants can be killed by the extracts of the fire ants (without formic acid), particularly ethanol extract (FIG. 36). However, the ethanol extract (primarily piperidine alkaloids) of the fire ants had selective activity against the ants and showed no effects on the subterranean termite. The fire ants and termites are in different superorders (Endopterygota and Exopterygota, respectively).

(52) Both formic acid and acetic acid have been used as herbicides to control weeds. This invention found that formic acid is more effective than acetic acid to control giant Salvinia. Formic acid at 0.05% concentration killed approximately 50% of the giant Salvinia plants within 24 h. It is more effective in Salvinia control when combined with giant Salvinia extracts.

(53) This invention reports that bioactive compounds in giant Salvinia are primarily accumulated in glandular trichomes on the leaf surface. It is known that the surface of the fruits (drupes) of Brazilian pepper tree is also covered by glandular trichomes (Carmello-Guerreiro and Paoli 2002). Chinese tallow tree has glands on leaf stalks. Formic acid is a primary venom compound commonly accumulated in the abdominal exocrine glands of ants and termites. In all these five described distant-related species, glands act accumulation sites of bioactive compounds that play important roles in endocidal function of the species so that the bioactive compounds do not poison the producing species in normal growth. Thus, the endocide may have effects especially in the species with glands as accumulation sites of bioactive compounds.

(54) Furthermore, this invention disclosed that application of endocides at low concentrations (dosages) can control partial growth of an individual (e.g., a specific cell(s), tissue(s), or organ(s)) of the producing species rather than killing or eliminating the species.

(55) Application of endocides at low concentrations (dosages) may produce abnormal morphogenesis. In some embodiments, the endocides may induce mutations. In some embodiments, the induced mutations may be used to produce desirable genotypes.

(56) In some weeds and crops, autotoxicity reports are primarily on effects in the seed germination, growth, and yield of the producing plants and the resulting soil sickness and replanting problems. The reported autotoxic chemicals had weak activity against the parent species but they are more potent inhibitory effects on other non-closely-related species (e.g., species in other genera, families or orders) than on the producing plants. Therefore, it is impossible to develop a biocidal product to selectively control the producing invasive species. Further, it is commonly believed that a species can avoid self-toxicity by its endogenous toxic metabolites and the toxic compounds are primarily used for defense to protect itself from enemies. The present invention discovered that some invasive species with glands as accumulation site of their autotoxic chemicals cannot avoid autotoxicity by its own metabolite(s) when externally applied. More importantly, these parent species (and its closely-related species) are more sensitive to its endogenous toxic metabolites than the other tested species in different genera, families or orders. The mechanism of selective activity of the autotoxic chemicals to the producing species is not clear but it seems that these chemicals in glands are not available to the normal growth and development of the species.

(57) This discovery provides the foundation for development of a novel endocide product to selectively control an invasive species over the other species. It further suggests that how to avoid endogenous autotoxicity by the toxic metabolite(s) must be the priority for the producing species in the development and evolution. To protect itself from enemies by defensive metabolite(s) become possible only when the producing species can avoid endogenous autotoxicity.

A. Natural Pesticide Compositions

(58) 1. Endocides

(59) In some embodiments, the present invention relates to compositions containing endocides and methods of controlling the growth of invasive species using the same. An endocide (endogenous biocide) is a biocide derived from an endogenous bioactive agent (e.g., secondary metabolite) that does not cause apparent poison in normal growth of the producing species but will poison or inhibit and even eliminate the parent species when induced in producing species. It can selectively eliminate the parent species (and possibly its closely-related species) when externally applied. The dead tissues of species caused by an endocide will enhance the endocidal function to the species. The endocide can be developed as either a pure single entity or as a mixture of compounds (e.g., a fraction of extract, an extract, a dry or fresh matter of species). The endocides can be obtained in liquid or solid format from fresh or dried matter by any effective extraction methods (e.g., expression, distillation, solvent extraction, infusion, decoction, and percolation). For aquatic species, endocides are usually water soluble and can be dissolved in water in application. Endocides may have effects on the invasive species in all or some growth stages and all or some tissues.

(60) The endocide development process is summarized in FIG. 1. As an example, the inventors studied giant Salvinia plants. It was found that endocides derived from either dried or fresh plant matter of giant Salvinia can effectively control the invasive species in all growth stages (primary, secondary, and tertiary stages) (Examples 1-8). It was found that dry plant matter of giant Salvinia can fully eliminate giant Salvinia plants when adequate dried ground matter of giant Salvinia is placed in the water (FIG. 3).

(61) FIG. 3 illustrates that the minimum effective concentration to inhibit giant Salvinia is between 4 and 8 g of dried plant matter per liter of water. About 50% of giant Salvinia plants were killed at the concentration of 8 g/L. The Salvinia plants were fully eliminated at 16 g/L within two weeks in the greenhouse (FIGS. 3 and 4), and no new or living Salvinia plants were found within the six months of observation thereafter. This demonstrates that giant Salvinia contains some agents that are toxic to the parent plants and that these agents can fully eliminate and inhibit the growth of Salvinias at higher concentrations. The tea bag treatment killed the Salvinias bottom-up by damaging the root-like submerged leaves of giant Salvinia first (FIG. 5). Then the trichomes on the upper surfaces of the floating leaves of giant Salvinia were wilted following the damage of the submerged leaves (FIG. 6). The wilted trichomes released autotoxic compounds and thus enhanced the endocidal function. The bottom-up killing style of the tea bag is particularly effective in a shallow and still water body infested with multi-layers of Salvinia mat because the dead tissues of the bottom layer of Salvinias killed by the tea bag would enhance the endocidal function to eliminate the next layer of the Salvinia.

(62) In some cases, because higher effective concentration of dried ground plant Salvinia is needed to inhibit giant Salvinia, and some natural factors in lakes and other water systems may dilute the concentrations or prohibit the occurrence of higher concentrations, it may be necessary to place dried and ground matter of giant Salvinia in nylon or other net bags to have effective concentrations for the controlling target. In another cases, it was found that unground raw plant matter of giant Salvinia at 16 g/L was less effective than ground matter and took more than five times longer to kill the plants.

(63) To identify more effective agents from giant Salvinia to control the growth of the plant, experiments were conducted with a particular focus on water extracts of giant Salvinia (FIG. 1). It was confirmed that water extract of giant Salvinia can successfully eliminate giant Salvinia within 13 weeks at RT and prohibit the plant growth for at least eight months (FIG. 7), MeOH/CH.sub.2Cl.sub.2 and MeOH fractions eliminated all giant Salvinia plants within two weeks (FIG. 8), new compound salviniside II also eliminated the giant Salvinia plants (FIGS. 9-11), and the ethanol extract of giant Salvinia inhibited the growth of giant Salvinia effectively. In summary, while the bioactive MeOH/CH.sub.2Cl.sub.2 and MeOH fractions and salviniside II compound are more effective, each extract or fraction thereof can be produced as endocides to eliminate and prohibit giant Salvinia.

(64) The experiments showed that extraction temperature and solvent (water) volume significantly contributed to the quality of water extracts and its fractions. The higher extraction temperature (60 C.) and higher rate of solvent volume to dried plant matter weight (43.8:1, v/w) yielded an extraction rate of approximately 8.8% (Example 5), and the lower temperature (RT) and lower rate of solvent volume to dried plant matter weight (17.2:1, v/w) yielded a 6.25% extraction rate (Example 3). Also, H.sub.2O fraction from the Example 5 (FIG. 14) was much more effective than that from Example 3 (FIG. 8).

(65) The experiments indicate that extracts (juice) expressed from the fresh matter of giant Salvinia (e.g., by hydraulic press) can be directly used in control of giant Salvinia (FIGS. 16-20). The yield rate of extracted juice from the fresh matter of giant Salvinia may depend on the growth stage and moisture content of the harvested plant matter and approximately 50% of the plant matter in fresh weight. The extracts expressed from the fresh matter of giant Salvinia (0.28% or 0.40% in concentration) were even more effective than the concentrated water extracts (5%) from the dried matter of giant Salvinia in inhibition of the species (FIG. 20). The HPLC analysis showed that the hydraulic extracts had more complex chemical constituents in both detection conditions and higher contents of some compounds and even unique compounds may be responsible for the strong bioactivity of the hydraulic extract (FIGS. 21 and 22). Like aquatic herbicides, addition of a surfactant to the endocide may help it wet and penetrate the floating leaves with water-repellent trichomes and thereby result in more effective spray applications (FIGS. 14, 16, 17, and 20). In both experiments, all plant tissues of giant Salvinia after hydraulic extraction were dead and there was no new plant growth in the cultured plant residues from the hydraulic extraction within six weeks of the experiment (FIG. 19). The plant residues from the extraction of the fresh matter are safe to be discarded into the treated water bodies. Therefore, extracts from the fresh matter of giant Salvinia are desirable for use as endocides to eliminate and prohibit giant Salvinia. In its concentrated form, the extract juice from fresh matter may also be effective to control Salvinia. Because the degraded hydraulic extract after long-storage might be still useful in control of Salvinias, it is speculated that diluted several times with water, the newly produced extract juice from the fresh matter may also be effective to control Salvinias.

(66) According to the experiments, aqueous products of endocides may be better to be stored under refrigeration (4 C.) while powder samples can be stored at RT for relative stability. In general, the H.sub.2O fraction was more stable than the water extract or the hydraulic extract in storage. The extract powder stored at RT for 26 weeks had approximately 70% of the total peak area of that in the newly prepared extract (FIG. 12).

(67) The hydraulic extract stored under refrigeration (4 C.) is more stabilized than that stored at RT, and on the 14th week of the storage it contained approximately 60% of the total peak area of that in the newly-prepared extract, and it was still active in inhibition of giant Salvinia (FIGS. 23 and 24). After being stored at RT for 26 weeks, the H.sub.2O fraction powder preserved over 90% of its chemical contents, and its liquid form stored in a refrigerator (4 C.) still had approximately 90% of the total peak area of the newly prepared sample (FIG. 13).

(68) Salvinia spp. have the potential to hyperacumulate a wide range of heavy metals from water and have high levels of tolerance to the metals according to the literature. The ICP analysis showed that the concentrations of the main metals in the water extract powder are much lower than the toxic concentrations in the metal solutions to grow Salvinia spp. as reported (10 to 100 ppm). For example, the concentrations of cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), and molybdenum (Mo) in the dry extract powder are less than 1 ppm in dry weight, and the concentration of copper (Cu), iron (Fe), lead (Pb), and zinc (Zn) in the dry extract powder is 5.310525, 228.0387, 2.515505, and 166.7952, respectively (FIG. 25). The concentrations of these metals in the application of endocides were diluted by 20 to 1,000 times when using water extract at its spray concentration (0.1 to 5% in dry weight) in control of Salvinia. Thus, toxic metals are not the major active ingredients of Salvinia endocides, i.e., they are not responsible for inhibiting the survival and growth of giant Salvinia.

(69) The experiment showed that isolated salts account for 6.25% of the water extract from the dried matter of giant Salvinia in dry weight. Of the 5% water extract (the highest concentration used in the experiments), the concentration of the salts is 0.3125% (3.125 ppt). The experiment indicated that even the spray of the salts at even 5% concentration (50 ppt) of the salts had no significant impact on the growth of giant Salvinia. Therefore, the salts precipitated from the water extract are not the major active ingredients responsible for the bioactivity of water extracts (FIG. 25).

(70) The experiments indicated that both spraying and dissolving these agents were effective. A similar approach may be taken to identify endocides in all invasive plant species, including but not limited to Salvinia adnata, S. auriculata, S. biloba, S. cucullata, S. cyathiformis, S. hastata, S. herzogii, S. martynii, S. minima, S. molesta, S. natans, S. nymphellula, S. oblongifolia, S. radula, S. rotundifolia, S. sprucei, Azolla caroliniana, A. cristata, A. circinata, A. filiculoides, A. japonica, A mexicana, A. microphylla, A. nilotica, A. pinnata, A. rubra, Lemna minuta, or Eichharnia crassipes.

(71) The water extract of dried matter of giant Salvinia can totally eliminate the plants of the floating fern (S. minima), a species within the same genus with giant Salvinia, at either 0.1% or 0.5% within six weeks (FIG. 26). Both water extracts and hydraulic extracts of giant Salvinia had significant impacts on the growth of Carolina mosquito fern (Azolla caroliniana), a fern species in a different family but the same order with the genus Salvinia. The hydraulic extract (0.4%) killed 100% of the Carolina mosquito fern plants by the end of the 15th day (FIG. 27). The water extract at higher concentrations (2.5, 5, or 7.5%) killed 100% of the Carolina mosquito fern plants within the 15 days of experiment (FIG. 27). At even 5% concentration, the water extract of the dried matter of giant Salvinia did not impact the growth of herbaceous flowering plants (e.g., least duckweed (Lemna minuta), Brazillan watermeal (Wolffia brasillensis Weddell), water hyacinth (Eichharnia crassipes), and hydrilla (Hydrilla verticillata (L.f.) Royle)), and native confers (e.g., bald cypress (Taxodium distichum (L.) Rich.) and loblolly pine (Pinus taeda L.)). The endocide of giant Salvinia has obvious selective activity against Salvinias (order Salviniales) especially the species of family Salviniaceae, but it has no impacts on seed plants (spermatophytes) including angiosperms (flowering plants) and gymnosperms.

(72) 2. Natural Pesticides Derived from Second Species

(73) Least duckweed (Lemna minuta) is a noxious invasive species itself. Its water extract successfully eliminated giant Salvinia and prohibited plant growth for at least eight months (see Example 14) (FIGS. 28 and 29). Ethanol extract of duckweed was also effective to control giant Salvinia (FIG. 30). Duckweed extracts were also able to control Salvinia minima (FIGS. 31-33).

B. Examples

(74) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Elimination and Prohibition of Giant Salvinia (Salvinia Molesta) by the Water-Extracted Dried Matters of Giant Salvinia

(75) General Experimental Procedures:

(76) Air-dried whole plants of giant Salvinia were ground to a coarse powder and placed in nylon net bags (called tea bags). Each of 12 containers had 400 g of living healthy plants of giant Salvinia in all growth stages (primary, secondary, and tertiary stages) in 25 L of tap water in the greenhouse (30 C. during the day time and 20 C. at night). Three containers had no treatment to serve as controls. The first treatment group had three containers and each had one bag of 100 g of giant Salvinia dried matter in the water. The second group of three containers had one bag of 200 g of giant Salvinia dried matter, and the last group of three containers had one bag of 400 g of giant Salvinia dried matter. There was no significant difference of pH values between the treatments and control during the experiments.

(77) Results:

(78) In the 400 g treatment group, more than 35% of plants were dead by the end of the first week and all plants were dead by the end of the second week and no plants survived or new growth occurred thereafter during the observation of six months (FIGS. 3 and 4). In this treatment, the root-like submerged leaves of giant Salvinia were totally damaged about a week later and then the floating leaves turned into brown and dead (FIG. 5). In contrast to those in the normal plant growth condition, the trichomes on the upper surfaces of the floating leaves of giant Salvinia were soon wilted following the response of submerged leaves to the tea bag treatment (FIG. 6). The wilted trichomes released autotoxic compounds and thus enhanced the endocidal function. It was also found that the 200 g treatment group inhibited almost 50% of the plants by the end of the fourth week of the experiment. However, no significant effect of the 100 g treatment was observed on Salvinia growth.

Example 2

Elimination and Prohibition of Giant Salvinia Plants by Water Extract of the Dried Matter of Giant Salvinia

(79) General Experimental Procedures:

(80) Air-dried whole plants (350 g) were ground to a coarse powder and percolated with H.sub.2O at RT to yield a 4 L aqueous solution (0.84%, g/mL). Of the aqueous solution, 119 mL was diluted to 900 mL 0.1% and 1,000 mL 0.01% solution (g/mL) for the following experiment. The experiment included 45 healthy and untreated living plants of giant Salvinia (in secondary growth stage, approximately 7 g in fresh weight each). The plants were cultured and tested in plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. Controls: 15 plants with five in each container (3 replications) were cultured with 300 mL of tap water; 0.01% water extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.01% of the water extract of giant Salvinia dissolved in tap water; and 0.1% water extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.1% of the water extracts of giant Salvinia dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of the 13th week, new growth biomass of the plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(81) Results:

(82) By the end of 13 weeks of treatment, each of the 15 plants in the control group had new growth, and 12 of the total 15 plants treated with 0.01% water extract of giant Salvinia had new growth (FIG. 7). All giant Salvinia plants treated with 0.1% water extract of giant Salvinia were dead and no new growth developed from any of the plants during the eight months of observation. pH values of the culture solution varied from 7.4 to 7.6 and there were no significant differences among control and treatments.

Example 3

Elimination and Prohibition of Giant Salvinia Plants by Fractions of Water Extract of the Dried Matter of Giant Salvinia

(83) General Experimental Procedures:

(84) Air-dried whole plants of giant Salvinia (1.4 kg) were ground to a coarse powder and extracted two times for 48 h with H.sub.2O (12 L2) at RT. The combined H.sub.2O extracts were concentrated to give extracts (88 g) under reduced pressure. The H.sub.2O extracts was applied on a column of silica gel (1,000 g) eluting with the mixture of MeOH/CH.sub.2Cl.sub.2 (1:1, v/v, 3 L), 3 L of 100% MeOH, and 1 L H.sub.2O to obtain three fractions, respectively (FIG. 1). The water extract and three fractions were all prepared as experimental solutions at the concentration of 0.1%. The total 45 healthy and untreated living plants of giant Salvinia (in secondary growth stage, approximately 10 g in fresh weight each) were cultured and tested in the plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. The five treatments are as follows. Controls: 9 plants with three in each container (3 replications) were cultured with 150 mL of tap water; water extract treatment: 9 plants with three in each container (3 replications) were sprayed with 150 mL 0.1% of the water extract of giant Salvinia dissolved in tap water; H.sub.2O fraction treatment: 9 plants with three in each container (3 replications) were sprayed with 150 mL 0.1% of H.sub.2O fraction dissolved in tap water; MeOH fraction treatment: 9 plants with three in each container (3 replications) were sprayed with 150 mL 0.1% of MeOH fraction dissolved in tap water; and MeOH/CH.sub.2Cl.sub.2 fraction treatment: 9 plants with three in each container (3 replications) were sprayed with 150 mL 0.1% of MeOH/CH.sub.2Cl.sub.2 (1:1) fraction dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 2nd week, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(85) Results:

(86) On the 3rd day after the treatment, all plants treated with MeOH/CH.sub.2Cl.sub.2 or MeOH fraction were dead in comparison to 100% survival of the plants in the control group. During the same day of observation, some leaves of the plants treated with water extract or H.sub.2O fraction started to turn into brown color. By the end of two weeks, all plants in the control group were still alive (FIG. 8). By then, in contrast, no plants survived or new growth in both MeOH/CH.sub.2Cl.sub.2 and MeOH treatments, almost all plants were dead with water extract, while the biomass of living plants under the H.sub.2O fraction treatment was less than 25% of the plants in the control group (FIG. 8). The experiment indicated that MeOH/CH.sub.2Cl.sub.2 and MeOH fractions were very active and can fully control giant Salvinia more quickly than its parent water extract and thus the following isolation of bioactive compounds was focused on these two fractions.

Example 4

Elimination and Prohibition of Giant Salvinia by Salviniside II, an Isolate from Water Extract of the Dried Matter of Giant Salvinia

(87) General Experimental Procedures:

(88) Continuous fractionation was based on the combined MeOH/CH.sub.2Cl.sub.2 (1:1, v/v) and 100% MeOH fractions which showed potent activity to inhibit growth of giant Salvinia in Example 3. The combined fractions were loaded on a pre-equilibrated open ODS column (60600 mm). The ODS column was eluted successively with 30%, 55%, 75% and 100% MeOH to yield four fractions F1, F2, F3, and F4, respectively. F3 (75% MeOH elute) was concentrated and separated by a preparative HPLC (MeOH/H.sub.2O, 35:65, 254 nm) to yield four compounds including active salviniside II and C3 and inactive C1 and C2 and active fraction F32 and inactive F31 (FIG. 1). NMR experiments were performed on a JEOL ECS-400 and a Bruker Avance 700 NMR instrument. NMR data were reported as (ppm) values and referenced to the solvent used. HRESIMS were acquired on an electrospray instrument (MDS Sciex Pulsar Qstar, Ontario, Canada). Octadecyl-functionalized silica gel, silica gel, Sephadex LH-20, and TLC plates were purchased from Aldrich Chemical Co. HPLC analysis was performed on a Hewlett Packard Series 1100 with a HP 1100 diode array detector using a Hypersil ODS column (1504.6 mm, 5 M, Supelco; flow rate, 1 mL/min; MeOH/H.sub.2O (v/v) linear gradient, 2:98-98:2 in 35 min). Preparative HPLC was performed with an Acuflow Series III pump connected with an Acutect 500 UV/VIS detector using an Econosil ODS column (25022 mm, 10 M, All tech). The total 27 healthy and untreated living plants of giant Salvinia (in secondary growth stage, approximately 10 g in fresh weight each) were cultured and tested in plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. The five treatments were as follows. Controls: 9 plants with three in each container (3 replications) were cultured with 150 mL of tap water; 0.01% salviniside II treatment: 9 plants with three in each container (3 replications) were sprayed with 150 mL 0.01% of salviniside II dissolved in tap water; and 0.1% salviniside II treatment: 9 plants with three in each container (3 replications) were sprayed with 150 mL 0.1% of salviniside II dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of two weeks, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(89) Results:

(90) The growth of giant Salvinia was totally inhibited by salviniside II (FIGS. 9 and 10) at 0.1% of concentration within two weeks in the lab at RT (FIG. 11). There was no new growth identified thereafter during the eight months of observation.

(91) Salviniside II is a new compound. It was obtained as colorless powders and showed an [M-1].sup.+ at m/z 473.0714 (calcd for 473.0720) in the HRESIMS, suggesting the molecular formula to be C.sub.22H.sub.18O.sub.12. The .sup.13C and .sup.1H-NMR spectrum (FIG. 10) displayed similarly two set of glycoside signals with two anomeric proton at 4.57 (1H, d, J=7.8 Hz, -H-1) and 5.14 (1H, d, J=3.7 Hz, -H-1), and two protonated carbon at .sub.C- 98.6 and .sub.C- 94.4 observed in the HSQC spectrum. The analysis of the COSY, HSQC spectra of salviniside II indicated it is an isomeric mixture of and -D-glucopyranosides (Choudhary et al. 2008; Ding et al. 1999; Narasimhulua et al. 2010). In addition, each signals of the aglycon part appeared essentially in duplicate owing to the formation of a mixture of and -anomers in the NMR spectrum. Five aromatic protons were found in the .sup.1H-NMR spectrum (FIG. 10), of which three signals at 7.39 (1H, dd, J=1.8 and 8.5 Hz, H-2), 6.84 (1H, d, J=8.5 Hz, H-3), and 7.45 (1H, d, J=1.8 Hz, H-6) were characteristic of a 1,2,4-trisubstituted phenyl moiety, the remaining two at 7.70 (1H, d, J=8.2 Hz, H-6) and 6.82 (1H, d, J=8.2 Hz, H-7) were assigned to a 1,2,3,4-tetra substituted phenyl group (Narasimhulua et al. 2010). A close inspection of the .sup.13C-NMR spectrum data, together with the MS data, clearly revealed that the 1,2,3,4-tetrasubstituted phenyl moiety should be a part of a 8-carbon benzofuran skeleton (Maurya et al. 2004; Wu et al. 2011). The .sup.13C NMR spectra of salviniside II displayed two ester carbonyl carbons at 168.1 and 168.7. As observed in the HMBC spectrum, two key HMBC correlation among H-6 at .sub.H 7.70 and Glc H-6 at .sub.H 3.94 with a carbonyl carbon at .sub.C 168.1 (C-11) established the linkage between the benzofuran unit and H-6 of Glc. Similarly, the other ester carbonyl carbon (.sub.C 168.7, C-10) was attached to H-4 of Glc position by the HMBC spectrum analysis. These long-range HMBC correlations of H-2 to C-1, C-6 and C-2, and of H-6 to C-1, C-2 and C-2 established the connectivity of the benzofuran skeleton with 1,2,4-trisubstituted phenyl moiety. The 1,1-ADEQUATE spectrum also confirmed the assignment. The relatively downfield shifted of C-3 at 110.3 and the remaining one degree of unsaturation established the linkage of the C-3 and C-10. The complete assignment of the protons and carbons was achieved by a combination of .sup.1H, .sup.13C, HSQC, .sup.1H-.sup.1H COSY, HMBC and 1,1-ADEQUATE spectral analyses. Thus, salviniside II was identified as 4,6-O-[3,5-dicarbonyl-8-hydroxy-2-(4,5-dihydroxy-phenyl)-1-benzofuran-2-yl]-D-glucopyranose.

Example 5

Elimination and Prohibition of Giant Salvinia Plants by Water Extract and H.SUB.2.O Fraction of the Dried Matter of Giant Salvinia with Surfactants

(92) General Experimental Procedures:

(93) Air-dried whole plants of giant Salvinia (8 kg) were ground to a coarse powder and extracted in a vacuum distillation system (Eden Labs, Seattle, USA) with H.sub.2O two times for 24 h each (200 L and 150 L, respectively) at 60 C. The combined H.sub.2O extracts were concentrated to give an extract (703 g) under reduced pressure. The water extract was mixed with 300 g silica gel and loaded into a column of silica gel (1,000 g). The column was then eluted with 4 L of MeOH and 4 L H.sub.2O. The water extract and H.sub.2O fraction were both prepared as experimental solutions at the concentration of 5%.

(94) The HPLC chromatographs of the newly prepared water extract and H.sub.2O fraction were also established by Agilent 1100 HPLC system coupled to an Agilent 1100 diode array detector, and an Eclipse XDB-C18 column (4.6150 mm, 3.5 M) at a flow rate of 0.6 mL/min. A gradient elution was performed by using H.sub.2O (A) and CH.sub.3CN (B) as mobile phases. Elution was performed according to the following conditions: 2% B at time 0, linear increase to 98% B in 22 min, and hold 98% B for 8 min. The column temperature was maintained at 23 C. The HPLC chromatogram was standardized on retention times and peak intensities of the peaks observed at a wavelength of 254 nm. The concentration of water extract was 24 kg fresh plant matter/L and the injection volume was 3 L for all analyses. The concentration of H.sub.2O fraction was 16 kg fresh plant matter/L. The injection volume was 50 L for all analyses.

(95) Part of each of water extract and H.sub.2O fraction was prepared separately as dry powder by SPD 2010 Integrated SpeedVac (Thermo Scientific, NC, USA) for 12 h and then was stored at RT for 26 weeks. In addition, both 5% water extract and 5% H.sub.2O fraction were stored at refrigerator (4 C.) for 26 weeks. To determine the stability of the water extract and H.sub.2O fraction during the storage, HPLC profiles of the powder and aqueous samples were established by Agilent 1100 at the condition described above.

(96) The experiment included 18 containers and each container had 750 g (in fresh weight) of healthy living Salvinia molesta in all growth stages (primary, secondary, and tertiary stages) cultured in 57 L of rain water. The plants were cultured and tested in the glass tank in the lab at RT. There were six treatments. Controls: each of the three containers had 750 g giant Salvinia plants cultured with rain water; tea bag treatment: each of the three containers with a nylon net bags of 800 g ground air-dried plant matter of giant Salvinia on the bottom (see Example 1); water extract treatment: plants in each of the three containers were sprayed twice (on the 1.sup.st and 18.sup.th day), each time with 100 mL 5% of the water extract of giant Salvinia dissolved in water; water extract with surfactants treatment: plants in each of the three containers were sprayed twice (on the 1.sup.st and 18.sup.th day), each time with 100 mL 5% of the water extract of giant Salvinia dissolved in water with 0.5 mL Inlet (polyalkoxylated and non-alkoxylated aliphatics and derivatives 90%, Helena Chemical Company, Collierville, Tenn., USA) and 0.25 mL Kinetin (proprietary blend of polyalkyleneoxide modified polydimethylsiloxane 99%, Helena Chemical Company, Collierville, Tenn., USA); H.sub.2O fraction treatment: plants in each of the three containers were sprayed twice (on the 1.sup.st and 18.sup.th day), each time with 100 mL 5% of H.sub.2O fraction of the water extract of giant Salvinia dissolved in water; and H.sub.2O fraction with surfactants treatment: plants in each of the three containers were sprayed twice (on the 1.sup.st and 18.sup.th day), each time with 100 mL 5% of H.sub.2O fraction of the water extract of giant Salvinia dissolved in water with 0.5 mL Inlet and 0.25 mL Kinetin. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 3.sup.rd or 4.sup.th week, biomass of living plants in each treatment was measured.

(97) Results:

(98) According to HPLC analyses, the powder samples stored at RT for 26 weeks were relatively stable. By the end of 26 weeks, the extract powder still had approximately 70% of the total peak area of that in the newly prepared extract (FIG. 12). By the end of 26 weeks, H.sub.2O fraction powder stored at RT preserved over 90% of its chemical contents and 5% H.sub.2O fraction liquid stored at refrigerator (4 C.) still had approximately 90% of the total peak area of the newly prepared sample (FIG. 13).

(99) The growth of giant Salvinia can be successfully controlled by the endocide treatments in comparison with control (FIG. 14). By the end of the third week, all plants treated with either tea bag (FIG. 15), water extract with surfactants, or H.sub.2O fraction of the water extract with surfactants were dead and there was no new growth during the following four months of observation. By the end of 4.sup.th week, over 90% or 95% of the plants treated with water extract and H.sub.2O fraction of the water extract were dead, respectively. Obviously, Surfactants made the Salvinia endocides more effective. Because the experiments were conducted in November and at RT, plants experienced relatively slow growth. The living biomass in the controls had lightly decreased because the mature tissues were dead and more young plants emerged during the four weeks of experiment.

Example 6

Elimination and Prohibition of Giant Salvinia Plants by Hydraulic Extract of the Fresh Matter of Giant Salvinia

(100) General Experimental Procedures:

(101) The total 58 kg fresh matter of giant Salvinia plants were chopped and shredded by food processor. Then the processed material was pressed by hydraulic press (Enerpac) at pressure of 27,579 kPa (4,000 psi) for 2 min to get 30 kg extract juice. To obtain the concentration of the extract juice, three 30 mL of juice samples were randomly collected and measured. Each of the three juice samples was centrifuged by accuSpin 3R Benchtop centrifuge (Fisher Scientific, PA, USA) at 3,000 rpm for 10 min. The upper liquid was first dried by SPD 2010 Integrated SpeedVac (Thermo Scientific, NC, USA) for 12 h. Then dried extract was measured. The average result of the three test samples was used to estimate the concentration of the extract juice. The concentration of hydraulic extract juices were 2 kg fresh plant matter/L. The spray treatment experiment included three plastic containers (20 gal, approximately equal to 75.7 L) with approximately 390 g (in fresh weight) of giant Salvinia living plants (in all growth stages: primary, secondary, and tertiary stages) in 50 L of tap water each in the greenhouse (30 C. during the day time and 20 C. at night). There were three treatments. Control: the plants in the first container were sprayed with 150 mL tap water only; extract juice treatment: the plants in the second container were sprayed with 150 mL of extract juice; and extract juice and surfactants treatment: the plants in the third container were sprayed with 150 mL extract juice with 0.375 mL Inlet (polyalkoxylated and non-alkoxylated aliphatics and derivatives 90%, Helena Chemical Company, Collierville, Tenn., USA) and 0.1875 mL Kinetin (proprietary blend of polyalkyleneoxide modified polydimethylsiloxane 99%, Helena Chemical Company, Collierville, Tenn., USA). The second same spay treatment was applied to each of these six contains a week later, respectively. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 6th week, the total biomass of living plants in each treatment was measured. To investigate the survival rate of giant Salvinia after hydraulic extraction, the 28 kg (in fresh weight) residual plant material of giant Salvinia expressed by hydraulic press was cultured in five containers with 50 L tap water each for six weeks.

(102) Results:

(103) The extraction rate of hydraulic extract from the fresh matter of giant Salvinia in this experiment was approximately 51.7%. Based on the three test samples, the estimated concentration of the experimental extract juice was 0.28% (in fresh weight). The spray experiment showed that the hydraulic extract juice effectively inhibited the giant Salvinia after two treatments (FIGS. 16 and 17). In comparison with those in the control treatment, the trichomes on the upper surfaces of the floating leaves of giant Salvinia treated by the hydraulic extract had significant damage (FIG. 18). The wilted trichomes released autotoxic chemicals to enhance the endocidal function of the hydraulic extract. With surfactants, hydraulic extract juice totally eliminated the giant Salvinia within four weeks and no new growth thereafter. The experiment indicates that the juice expressed from fresh matter of giant Salvinia (e.g., by hydraulic press) is effective enough and can be directly used in control of giant Salvinia without concentrating process. All plant tissues of giant Salvinia after hydraulic extraction were dead and there was no new plant growth in the cultured plant residues from the hydraulic extraction within six weeks of observation (FIG. 19).

Example 7

Elimination and Prohibition of Giant Salvinia Plants by Water Extract from the Dried Matter and Hydraulic Extract of the Fresh Matter of Giant Salvinia with Surfactants

(104) General Experimental Procedures:

(105) The 5% water extract from the dried matter of giant Salvinia (called water extract) was prepared as described in the Example 5. The total 80 kg fresh matter of giant Salvinia plants were chopped and shredded by chipper shredder CS 3310 (Cub Cadet, Cleveland, Ohio, USA). Then the processed material was pressed by hydraulic press (Enerpac) at pressure of 27,579 kPa (4,000 psi) for 2 min to get 38 kg extract juice. To obtain the concentration of the hydraulic extract, three 30 mL of juice samples were randomly collected and measured. Each of the three juice samples was centrifuged by accuSpin 3R Benchtop centrifuge (Fisher Scientific, PA, USA) at 3,000 rpm for 10 min. The upper liquid was first dried by SPD 2010 Integrated SpeedVac (Thermo Scientific, NC, USA) for 12 h. Then dried extract was measured. The average result of the three test samples was used to estimate the concentration of the hydraulic extract (called hydraulic extract). The HPLC profiles of both water extract and hydraulic extract were established before the first treatments by using both of the following methods. (1) Method A: Agilent 1100 HPLC system coupled to an Agilent 1100 diode array detector, and an Eclipse XDB-C18 column (4.6150 mm, 3.5 M) at a flow rate of 0.6 mL/min. A gradient elution was performed by using H.sub.2O (A) and CH.sub.3CN (B) as mobile phases. Elution was performed according to the following conditions: 2% B at time 0, linear increase to 98% B in 22 min, and hold 98% B for 8 min. The column temperature was maintained at 23 C. The HPLC chromatogram was standardized on retention times and peak intensities of the peaks observed at a wavelength of 254 nm. The concentration of both water extract and hydraulic extract were 2 kg fresh plant matter/L. The injection volume was 50 L for all analyses. (2) Method B: Agilent 1260 HPLC system coupled to an Agilent 1260 diode array detector, and an Acclaim HILIC-10 column (4.6150 mm, 3.0 M) at a flow rate of 0.6 mL/min. The mobile phase was CH.sub.3CN/H.sub.2O (65:35, v/v). The column temperature was maintained at 23 C. The HPLC chromatogram was standardized on retention times and peak intensities of the peaks observed at a wavelength of 230 nm. The concentration of both water extract and hydraulic extract were 40 kg fresh plant matter/L. The injection volume was 20 L for all analyses. To determine the stability of hydraulic extract, HPLC profiles of the hydraulic extracts stored at refrigerator (4 C.) and RT was established separately by above Method A daily for the first week and on the 14.sup.th week before the third treatment in comparison with the profiles of the newly prepared extract for the first treatments. The spray treatment experiment included six 625 gal (approximately 2,366 L) containers with approximately 7,400 g (including approximately 7,000 g of plants in tertiary growth stage, approximately 300 g of plants in secondary growth stage, and approximately 100 g of plants in primary growth stage) of giant Salvinia living plant in 1,700 L of rain water each in the greenhouse (30 C.). There were three treatments. Control: the plants in each of the two containers were sprayed with 2.5 L rain water only; water extract with surfactants treatment: the plants in each of the two containers were sprayed with 2.5 L of 5% water extract with 6.25 mL Dyne-Amic (methyl esters of C16-C18 fatty acids, polyalkyleneoxide modified poly dimethylsiloxane, alkylphenol ethoxylate 99%, Helena Chemical Company), and 3.125 mL Kinetin (proprietary blend of polyalkyleneoxide modified polydimethylsiloxane 99%, Helena Chemical Company, Collierville, Tenn., USA); and hydraulic extract and surfactants treatment: the plants in each of two containers were sprayed with 2.5 L extract juice with 6.25 mL Dyne-Armic, and 3.125 mL Kinetin. The second spay treatment at the same dosage was applied to each of these six contains 10 days later, respectively. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 14th week, the total biomass of living plants in each treatment was measured. Then the third spay treatment at the dosage of 0.5 L (for water extract and extract juice, each with 1.25 mL Dyne-Amic, and 0.625 mL Kinetin was applied to each of these six contains, respectively. To investigate the survival rate of giant Salvinia after hydraulic extraction, the 42 kg (in fresh weight) residual plant material of giant Salvinia expressed by hydraulic press was cultured in six containers with 50 L tap water each for six weeks.

(106) Results:

(107) The extraction rate of hydraulic extract from the fresh matter of giant Salvinia in this experiment was approximately was 47.5%. Based on the three test samples in each experiment, the estimated concentration of the experimental hydraulic extract was 0.40% (in fresh weight). The hydraulic extract in this experiment had lower extraction rate of yield at higher concentration than that in the Example 6 because the plant matter used in this extraction had more dead tissues which holding less moisture. This experiment showed that both water extract and hydraulic extract with surfactants effectively inhibited the giant Salvinia after two treatments (FIG. 20). The hydraulic extract with surfactants totally eliminated the giant Salvinia within 15 weeks. The experiment indicates that hydraulic extract expressed from the fresh matter of giant Salvinia was effective enough without concentrating process and can be used directly in inhibition of the species (FIG. 20). All plant tissues of giant Salvinia after hydraulic extraction were dead and there was no new plant growth in the cultured plant residues from the hydraulic extraction within six weeks of observation.

(108) The experiment indicates that hydraulic extract expressed from the fresh matter of giant Salvinia at low concentration (0.40%) was more effective than the concentrated water extract (5%) from the dried matter of giant Salvinia in inhibition of giant Salvinia. HPLC profiles indicate that hydraulic extracts had more complex chemical constituents in both detection conditions and higher contents of some compounds and even unique compounds may be responsible for the strong bioactivity of the hydraulic extract (FIGS. 21 and 22).

(109) According to HPLC analyses, hydraulic extract degraded much more quickly at RT than that at refrigerator (4 C.). The total peak area of hydraulic extract stored at RT decreased to 35% by the end of the first week and to 30% by the end of the second week (vs. 70% and 66% stored at refrigerator (4 C.), respectively) in comparison with the newly prepared extract (FIG. 23). On the 14th week, hydraulic extract stored at refrigerator (4 C.) still contained approximately 60% of the total peak area of that in the newly prepared extract (first treatment) and still was active in inhibition of giant Salvinia during the third spray treatment (FIG. 24).

Example 8

Elimination and Prohibition of Giant Salvinia Plants by Hydraulic Extract of the Fresh Matter of Giant Salvinia in Combination with Formic Acid or Acetic Acid

(110) General Experimental Procedures:

(111) Formic acid (90.4%, certified ACS reagent grade, Fisher Scientific Company, Fair Lawn, N.J., USA) and acetic acid (99.7, ACS reagent grade, VWR International LLC, West Chester, Pa., USA) were prepared as solutions at the concentration of 0.01%, 0.1%, 1%, 5%, and 10% immediately before the bioassay experiments. The experimental hydraulic extract used in this example was prepared from the previous experiment (fresh matter of giant Salvinia (method see Example 7). The actual concentration of the extract during this experiment was approximately 0.28%. There were 37 treatments in this experiment. Each treatment included 9 living healthy plants of giant Salvinia in three Rubbermaid plastic containers (1415 cm, 0.68 L) (each container had two plants in secondary stage and one in tertiary growth stage, in total of 6 g in fresh weight). The plants in each container were sprayed with 12.5 mL of tap water, formic acid, acetic acid, or combination of hydraulic extract with formic or acetic acid. Control: the plants was sprayed with water only; nine formic acid treatments (0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 5%, and 10% concentrations); nine acetic acid treatments (0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 5%, and 10% concentrations); nine hydraulic extract treatments with various concentrations of formic acid (0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 5%, and 10%); and nine hydraulic extract treatments with various concentrations of acetic acid (0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 5%, and 10%). Plant growth and survival status were documented and photographed hourly for the first 5 h and then daily for seven days after the treatment.

(112) Results:

(113) Giant Salvinia is very sensitive to both formic acid and acetic acid. All plants treated with either formic or acetic acid at the 1% or higher concentrations were dead within 3 h of the treatment. However, at lower concentrations (<1%), formic acid was much more effective than acetic acid in control of giant Salvinia. For example, 0.05% formic acid was more effective than 0.25% acetic acid in giant Salvinia. Formic acid at the 0.01% concentration still effectively inhibited the growth of giant Salvinia. By combing hydraulic extract with 0.25% formic acid, all giant Salvinia plants were killed within 24 h. Treated by combing hydraulic extract with 0.05% formic acid, all giant Salvinia plants were dead within a week. Treated by combing hydraulic extract with 0.01% formic acid, more than 40% of the giant Salvinia plants were dead during the first week. The Salvinia plants responded to the hydraulic extract with the organic acids much quickly and severely than hydraulic extract, formic acid, or acetic acid alone.

Example 9

Metal Analysis of the Dried Matter of Giant Salvinia and its Water Extract and Extract Salts

(114) General Experimental Procedures:

(115) The dried matter of giant Salvinia and its water extract used this experiment were from Example 3. From the MeOH fraction of the 88 g water extract of the dried matter of giant Salvinia (see Example 3), 5 g salts were precipitated. 0.5 g of each of the ground dried plant matter of giant Salvinia, water extract powder, and the salts was pre-digested overnight with 5 mL nitric acid in Digi-tubes at RT. Then the Digi-tubes were incubated on DigiPREP HT High Temperature Digestion Systems (SCP SCIENCE) for 3 h at 100 C. Cool down and add 4 mL of hydrogen peroxide. Cook for 90 min at 95 C. The solution was diluted to a final volume of 50 mL for ICP analysis (Thermo Scientific iCap 7200 ICP-OES). The 5 ppt (0.5% in dry weight) and 50 ppt (5% in dry weight) solutions of the salts were prepared. 70 g living healthy plants of giant Salvinia in tertiary growth stage were cultured in each of nine plastic containers (2323 cm, 2.4 L). There were three treatments with three containers each. Control: the plants in each of three containers were sprayed with 12.5 mL tap water only; 5 ppt salts treatment: the plants in each of the three containers were sprayed with 12.5 mL 0.5% salt solutions; 50 ppt salts treatment: the plants in each of the three containers were sprayed with 12.5 mL 5% salt solutions. Plant growth and survival status were documented and photographed weekly for eight weeks after the treatment.

(116) Results:

(117) The ICP analysis showed that the concentrations of the main meals cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), molybdenum (Mo), and zinc (Zn), in the dry extract powder were 0.16047, 0.883666, not detected, 5.310525, 228.0387, 2.515505, 0.039819, and 166.7952 ppm (in dry weight) (FIG. 25). The salts precipitated from the water extract of the dried matter of giant Salvinia were mainly sodium (Na), potassium (K), calcium (Ca), and magnesium (Mg) salts according to the ICP analysis (FIG. 25). The 5 g precipitated salts accounts for 0.3333% of the 1.5 kg dry plant matter or 6.25% of the 88 g water extract in dry weight. Through eight weeks of observation, the plants treated with the salts at either 5 or 50 ppt did not have any significant damage. There was no significant biomass difference among the salts treatments and control. Obviously, the spray of the salts even at 5% did not inhibit the plant growth of giant Salvinia. In contrast, water extract of the dry plant of giant Salvinia can effectively control giant Salvinia as described in Examples 5 and 6. Thus, the salts were not the active ingredients of the water extract in responsible for inhibiting giant Salvinia.

Example 10

Elimination and Prohibition of Floating Fern (Salvinia Minima) Plants by Water Extract of the Dried Matter of Giant Salvinia

(118) General Experimental Procedures:

(119) The experiment included nine plastic containers (1415 cm, 0.68 L) and each container had 10 g (in fresh weight) of healthy living Salvinia minima cultured in tap water in the greenhouse (30 C. during the day time and 20 C. at night). There were three treatments. Controls: the floating fern plants in each of three containers were sprayed three times with the total of 90 mL tap water; 0.1% water extract treatment: the plants in each of three containers were sprayed three times with the total 90 mL 0.1% of water extract of giant Salvinia (the preparation method see Example 2); and 0.5% water extract treatment: the plants in each of three containers were sprayed three times with the total 90 mL 0.5% of water extract of giant Salvinia (the preparation method see Example 2). Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 1st, 2nd, 6th weeks, biomass of living plants in each container of all treatments was measured.

(120) Results:

(121) The growth of the floating fern plants treated with either 0.1% or 0.5% water extract of giant Salvinia were obviously inhibited during the first week (FIG. 26). By the end of the 6th week, all plants treated by water extract of giant Salvinia were dead and there was no new growth thereafter.

Example 11

Elimination and Prohibition of Carolina Mosquito Fern (Azolla caroliniana) Plants by Water Extract from the Dried Matter and Hydraulic Extract of the Fresh Matter of Giant Salvinia

(122) General Experimental Procedures:

(123) The experimental species included a fern species Carolina mosquito fern (Azolla caroliniana) (family Azollaceae). The experiments were conducted in the greenhouse (30 C. during the day time and 20 C. at night). Water extracts were prepared from the dried matter of giant Salvinia (the preparation method see Example 2) and the hydraulic extract was prepared from the fresh matter of giant Salvinia (see Example 7). The spray experiment started until the plants fully covered each of the 18 plastic containers (1415 cm, 0.68 L) with 400 mL tap water each. The plants were randomly classified into six groups: Control: each of the three containers was spayed three times with 10 mL of tap water each time; 0.4% hydraulic extract treatment: each of the three containers was spayed with 10 mL of 0.4% hydraulic extract; 0.1% water extract treatment: each of the three containers was spayed twice with 10 mL of 0.1% water extract; 2.5% water extract treatment: each of the three containers was spayed twice with 10 mL of 2.5% water extract; 5% water extract treatment: each of the three containers was spayed twice with 10 mL of 5% water extract; and 7.5% water extract treatment: each of the three containers was spayed twice with 10 mL of 7.5% water extract. By the end of the 5.sup.th, 10.sup.th, and 15.sup.th day, surface coverage of living plants in each container of all treatments was photographed and measured and EC.sub.50 (half maximal effective concentration) values were calculated by the PROBIT procedure of SPSS 13.0 for Windows.

(124) Results:

(125) Neither the hydraulic extract (0.4%) nor the water extract at 0.1% concentration showed any significant impacts on the plant growth of Carolina mosquito fern during the first five days of the experiment but both extracts significantly inhibited the plant growth thereafter. The hydraulic extract was able to kill 100% of the Carolina mosquito fern by the end of the 15th day (FIG. 27). The water extract at higher concentrations (2.5, 5, or 7.5%) significantly inhibited the growth of Carolina mosquito fern soon after the treatments. Both 2.5% and 5% water extracts killed 100% of the Carolina mosquito fern plants by the end of the experiment. The 7.5% water extract eliminated all treated plants within the five days of the experiment (FIG. 27). EC50 value of water extracts of the dried matter of giant Salvinia against Carolina mosquito fern is 4.42%, 1.32%, and 0.16% by the 5.sup.th, 10.sup.th, and 15.sup.th day, respectively.

Example 12

Impacts of Giant Salvinia Endocides on Other Selected Plant Species

(126) General Experimental Procedures:

(127) The experimental species included four herbaceous invasive aquatic species of angiosperms (flowering plants), namely, least duckweed (Lemna minuta) and Brazillan watermeal (Wolffia brasillensis Weddell) of the family Araceae, water hyacinth (Eichharnia crassipes) of the family Pontederiaceae and Hydrilla (Hydrilla verticillata (L.f.) Royle) of the family Hydrocharitaceae, and two native tree species of gymnosperms, namely baldcypress (Taxodium distichum (L.) Rich.) of the family Cupressaceae and loblolly pine (Pinus taeda L.) of the family Pinaceae. These species are often associated with giant Salvinia in the southeastern United States. The experiments were conducted in the greenhouse (30 C. during the day time and 20 C. at night). Water extracts were prepared from the dried matter of giant Salvinia (the preparation method see Example 2). Each species of least duckweed and Brazilian watermeal was separately cultured in nine plastic containers (1415 cm, 0.68 L) with 400 mL tap water each. The spray experiment started until the plants fully covered each of the containers. For each of these two species, the plants were randomly classified into three groups: Control: each of the three containers was spayed three times with 10 mL of tap water each time; treatment I: each of the three containers was spayed twice with 10 mL of 0.1% water extract each time and then a week later with 10 mL of 5% of water extract; and treatment II: each of the three containers was spayed twice with 10 mL of 0.5% water extract each time and then a week later with 10 mL of 5% water extract. Surface coverage of living plants in each container of all treatments was photographed and measured weekly for six weeks. Five plants of each species of water hyacinth and Hydrilla were cultured separately in container with 50 L tap water. For each species, three containers served as control and the plant leaves in each of the other three containers were sprayed twice with the total 100 mL of 5% water extract each time. Plant growth and survival status were documented and photographed weekly after the treatments for six weeks. Six 2-year old seedlings of each of bald cypress and loblolly pine in 2-gal pots were used in the experiment. For each species, three seedlings as controls received water only and the needles and stems in each of the other three plants were sprayed twice with the total 100 mL of 5% water extract each time. Plant growth and survival status were documented and photographed weekly after the treatments for six weeks.

(128) Results:

(129) During the six weeks of experiments, the growth of duckweed and watermeal had not been inhibited by 0.1%, 0.5% or 5% of water extract of giant Salvinia. By the end of the observation, the living plants of these three species in all treatments and controls stayed in full surface coverage on all containers. Similarly, there was no obvious leaf color change or damage observed in any treatments of the other two invasive flowering plants and two native confer species.

Example 13

Inhibition of Giant Salvinia Plants by Ethanol Extract of the Dried Matter of Giant Salvinia

(130) General Experimental Procedures:

(131) Air-dried whole plants (550 g) were ground to a coarse powder and percolated two times with 95% EtOH at RT (each 3 L, 24 h). The combined EtOH solution was concentrated to give ethanol extract (34.0 g) under reduced pressure. 1.0 g giant Salvinia ethanol extract was dissolved in 2 mL DMSO, and then diluted with water to yield 900 mL 0.1% and 1,000 mL 0.01% solutions (g/mL) for further experimental analysis. The experiment included 45 healthy and untreated living plants of giant Salvinia (approximately 7 g in fresh weight each). The plants were cultured and tested in the plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. Controls: 15 plants with five in each container (3 replications) were cultured with 300 mL of tap water; 0.01% ethanol extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.01% of the ethanol extracts of giant Salvinia dissolved in tap water; and 0.1% ethanol extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.1% of the ethanol extracts of giant Salvinia dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 13th week, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(132) Results:

(133) By the end of 13 weeks after the treatment, the plants in both Control and 0.01% ethanol extract treatment groups had new growth while about 60% of all giant Salvinia plants treated with 0.1% ethanol extract of the dried matter of giant Salvinia were dead and no significant growth on the other treated plants although they were still alive.

Example 14

Elimination and Prohibition of Giant Salvinia Plants by Water Extract of the Dried Matter of Least Duckweed (Lemna minuta)

(134) General Experimental Procedures:

(135) 180 g (dry weight) of air-dried whole plants of least duckweed (Lemna minuta) (Lernnaceae) was ground to a coarse powder and percolated three times with 95% EtOH (v/v) at RT (each 2 L, 24 h). After ethanol extraction, the residual plant matters were dried and then percolated two times with water at RT (each 2 L, 24 h). Combined water solution was concentrated in vacuo to give water extract (12.2 g). 1.0 g Duckweed water extract was dissolved in H.sub.2O to yield 900 mL 0.1% and 1,000 mL 0.01% solutions (g/mL) for the giant Salvinia inhibition experiment. The total 45 healthy and untreated living plants of giant Salvinia (approximately 7 g in fresh weight each) were cultured and tested in plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. The three treatments were as follows. Controls: 15 plants with five in each container (3 replications) were cultured with 300 mL of tap water; 0.01% duckweed water extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.01% of the duckweed extracts dissolved in tap water; and 0.1% duckweed water extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.1% of the duckweed extracts dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 13th week, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(136) Results:

(137) At the end of the 13th week of the experiment, each of the 15 giant Salvinia plants in the control group had developed significant new growth (FIG. 28). By the same time, in the Treatment I group (0.01% water extract of least duckweed), six giant Salvinia plants had developed significant new growth and four plants had somewhat new growth, while the rest five plants were dead and had no new growth. The total new plant growth of the Treatment I was about 40% of that of Control (FIG. 29). However, all 15 giant Salvinia plants in the Treatment II group (0.1% water extract of least duckweed) were dead and had no any new growth at all (FIGS. 28 and 29). Further, during the eight months of observation, no recovery or new growth of giant Salvinia were identified thereafter.

Example 15

Elimination and Prohibition of Giant Salvinia Plants by Ethanol Extract of the Dried Matter of Least Duckweed

(138) General Experimental Procedures:

(139) Air-dried whole plants (180 g) were ground to a coarse powder and percolated three times with 95% EtOH (v/v) at RT (each 2 L, 24 h). The combined EtOH solution was concentrated under reduced pressure to give ethanol extract (6 g). 1.0 g duckweed ethanol extract was dissolved in 2 mL DMSO, and then diluted with water to yield 900 mL 0.1% and 1000 mL 0.01% solutions (g/mL) for the inhibition experiment. The total 45 healthy and untreated living plants of giant Salvinia (approximately 7 g in fresh weight each) were cultured and tested in plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. The three treatments were as follows. Controls: 15 plants with five in each container (3 replications) were cultured with 300 mL of tap water; 0.01% duckweed ethanol extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.01% of the duckweed extracts dissolved in tap water; and 0.1% duckweed ethanol extract treatment: 15 plants with five in each container (3 replications) were sprayed with 300 mL 0.1% of the duckweed extracts dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 13th week, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(140) Results:

(141) Similar to those observed at the experiment 5, all 15 giant Salvinia plants in the control group had significant new growth at the end of the 13th week of the experiment (FIG. 30). By then the total new plant growth of the Treatment I group (0.01% ethanol extract of least duckweed) is less than 15% of that of Control (FIG. 29). During the same time, 12 giant Salvinia plants in the Treatment II group (0.1% ethanol extract of least duckweed) were dead and had no any new growth at all (FIGS. 29 and 30), and the rest three plants had dead mature tissues but developed somewhat new growth. However, the three new plants did not last long and eventually all 15 giant Salvinia plants were dead by the end of 15 weeks after the treatment and no new growth thereafter during the eight months of observation.

Example 16

Elimination and Prohibition of Floating Fern (Salvinia Minima) Plants by Water Extract of the Dried Matter of Least Duckweed

(142) General Experimental Procedures:

(143) The water extract of least duckweed used in this experiment was the same as used in Example 6. 1.0 g duckweed water extract was dissolved in H.sub.2O to yield 900 mL 0.1% and 1,000 mL 0.01% solutions (g/mL) for floating fern inhibition experiment. The total 90 healthy and untreated living plants of floating fern Salvinia were cultured and tested in plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. The three treatments were as follows. Controls: 30 plants with 10 in each container (3 replications) were cultured with 300 mL of tap water; 0.01% duckweed water extract treatment: 30 plants with 10 in each container (3 replications) were sprayed with 300 mL 0.01% of the duckweed extracts dissolved in tap water; and 0.1% duckweed extract treatment: 30 plants with 10 in each container (3 replications) were sprayed with 300 mL 0.1% of the duckweed extracts dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 13th week, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(144) Results:

(145) At the end of the 13th week of the experiment, each of the 30 floating fern plants in the control group had developed significant new growth (FIG. 31). The floating fern plants had no response with the treatment at 0.01% water extract of least duckweed during the first 13 weeks of experiment (FIGS. 31 and 32). By the same time, the plants treated with 0.1% water extract of least duckweed had only about 35% of growth as observed in the control (FIGS. 31 and 32). However, plants treated with 0.01% or 0.1% water extract of least duckweed were all dead by 20 weeks while untreated plants had new growth.

Example 17

Elimination and Prohibition of Floating Fern (Salvinia minima) Plants by Ethanol Extract of the Dried Matter of Least Duckweed

(146) General Experimental Procedures:

(147) The ethanol extract of least duckweed used in this experiment was the same as used in the Experiment 7. 1.0 g duckweed ethanol extract was dissolved in 2 mL DMSO, and then diluted with water to yield 900 mL 0.1% and 1000 mL 0.01% solutions (g/mL) for the inhibition experiment. The total 90 healthy and untreated living plants of floating fern Salvinia were cultured and tested in plastic containers (1415 cm, 0.68 L) in the NCPC Lab at RT. The three treatments were as follows. Controls: 30 plants with 10 in each container (3 replications) were cultured with 300 mL of tap water; 0.01% duckweed ethanol 0.01% duckweed extract treatment extract treatment: 30 plants with 10 in each container (3 replications) were sprayed with 300 mL 0.01% of the duckweed extracts dissolved in tap water; and 0.1% duckweed ethanol extract treatment: 30 plants with 10 in each container (3 replications) were sprayed with 300 mL 0.1% of the duckweed extracts dissolved in tap water. Plant growth and survival status were documented and photographed weekly after the treatments. By the end of 13th week, new growth biomass of plants in each treatment was measured. The biomass of new growth was a primary factor to measure the inhibition of each treatment on the target plant.

(148) Results:

(149) At the end of the 13th week of the experiment, each of the 30 floating fern plants in the control group had developed significant new growth (FIG. 33). More than 80% of the floating fern plants were dead after the treatment at 0.01% ethanol extract of least duckweed and all plants treated with 0.1% level were dead within the 13 weeks (FIGS. 32 and 33). No new growth was observed thereafter during the eight months of observation.

Example 18

Elimination and Prohibition of Brazilian Pepper Tree (Schinus terebinthifolius Raddi) Seedlings by the Ethanol Extract of Brazilian Pepper Tree Fruits

(150) General Experimental Procedures:

(151) The fruits of Brazilian pepper tree (S. terebinthifolius) (200 g, in dry weight) and Chinese tallow tree (Triadica sebifera (L.) Small) (Eurphorbiaceae) (260 g, in dry weight) were ground to coarse powders and extracted two times with 70% ethanol at RT (each 2 L, 48 h). After evaporated under reduced pressure, 25.4 g ethanol extract of Brazilian pepper tree and 27 g ethanol extract of Chinese tallow tree (both in dry weight) were obtained, respectively. Each extract was dissolved in water and then was prepared as experimental solutions at the concentration of 10%, respectively. The spray treatment experiment included 36 Brazilian pepper tree seedlings (three weeks old) in pots cultured in the greenhouse (30 C.). There were six treatments. Control: six seedlings were sprayed twice with a total 3 mL of tap water each time; surfactant treatment: six seedlings were sprayed twice with a total 3 mL of tap water with 7.5 uL Inlet and 3.75 uL Kinetin each time; Brazilian pepper tree extracts treatment: six seedlings were sprayed twice with total a 3 mL of 10% pepper tree fruit extracts each time; Brazilian pepper extracts with surfactants treatment: six seedlings were sprayed twice with a total 3 mL of 10% pepper tree fruit extracts with 7.5 uL Inlet and 3.75 uL Kinetin each time; Chinese tallow tree extracts treatment: six seedlings were sprayed twice with total 3 mL of 10% tallow tree fruit extracts each time; and Chinese tallow tree extracts with surfactants treatment: six seedlings were sprayed twice with total a 3 mL of 10% tallow extracts with 7.5 uL Inlet and 3.75 uL Kinetin each time. In addition, newly-spread leaves of six seedlings of each of the three native species in North America, namely poison ivy (Toxicodendron radicans (L.) Kuntze or Rhus toxicodendron L.) (family Anacardiaceae), sweetgum (Liquidambar styraciflua L.) (family Altingiaceae), and Shumard oak (Quercus shumardii Buckland) (family Fagaceae) were sprayed twice with a total 3 mL of 10% pepper tree fruit extracts each time, respectively. Plant growth and survival status were documented and photographed daily after the treatments.

(152) Results:

(153) All Brazilian pepper tree seedlings treated with Brazilian pepper tree extract or the extract with surfactants had significant damages three days after the first treatments (FIG. 34). Four weeks later, all six pepper tree seedlings were killed by the pepper tree extract with surfactants while five seedlings were killed and one was significantly damaged with dead apical bud and young leaves by the pepper tree extract (FIG. 34). In contrast, the Brazilian pepper tree plants had no damages in the control, surfactants, 10% ethanol extract of Chinese tallow tree fruits, or 10% ethanol extract of Chinese tallow tree fruits with surfactants treatment. None of the poison ivy, sweetgum, or Shumard oak seedlings had any damages by the Brazilian pepper tree extract.

Example 19

Elimination and Prohibition of Chinese Tallow Tree (Triadica sebifera (L.) Seedlings by the Ethanol Extract of Chinese Tallow Tree Fruits

(154) General Experimental Procedures:

(155) The fruits of Chinese tallow tree (Triadica sebifera (L.) Small) (Eurphorbiaceae) (260 g, in dry weight) and Chinese privet (Ligustrum sinense Lour.) (family Oleaceae) (400 g, in dry weight) were ground to coarse powders and extracted two times with 70% ethanol at RT (each 2 L, 48 h), respectively. After evaporated under reduced pressure 27 g ethanol extract of Chinese tallow tree fruits and 25.3 g ethanol extract of Chinese privet fruits (both in dry weight) were obtained, respectively. Each extract was dissolved in water and then was prepared as experimental solutions at the concentration of 10%. The spray treatment experiment included 18 Chinese tallow tree seedlings (three weeks old) and 12 Chinese privet seedlings under the parent trees in the field. There were four treatments. Control: six seedlings of each species were sprayed with 3 mL of tap water; Chinese tallow tree extract treatment on Chinese tallow tree seedlings: six tallow tree seedlings were sprayed with 3 mL of 10% tallow tree fruit extract; Chinese tallow tree extract treatment on Chinese privet seedlings: six privet seedlings were sprayed with 3 mL of 10% tallow tree fruit extract; and Chinese privet extract treatment on Chinese tallow tree seedlings: six tallow tree seedlings were sprayed with 3 mL of 10% Chinese privet fruit extract. Plant growth and survival status were documented and photographed daily after the treatments.

(156) Results:

(157) All six Chinese tallow tree seedlings treated by the ethanol extract of Chinese tallow tree fruits were significantly damaged (FIG. 35). During the one week of experiments, two tallow tree seedlings were dead and four were severely injured. However, the tollowtree extract did not cause any damage in Chinese privet seedlings and the Chinese privet extract did not damage the tallow tree seedlings.

Example 20

Elimination and Prohibition of the Red Imported Fire Ant (Solenopsis invicta Buren) by the Fire Ant Extracts and Formic and Acetic Acids

(158) General Experimental Procedures:

(159) Preparation of the fire ant extracts: (1) Acetone extract: 97 g (in fresh weight) of the red imported fire ant workers were extracted two times with acetone at RT (each 250 mL, 24 h). After evaporated under reduced pressure, the combined extractions yielded 2.13 g (in dry weight) acetone extract. (2) Ethanol extract: 110 g (in fresh weight) of the whole worker bodies of the red imported fire ant (Solenopsis invicta Buren) (family Furmicidae) were extracted two times with 95% EtOH at RT (each 250 mL, 24 h). After evaporated under reduced pressure, the combined extractions yielded 2.72 g (in dry weight) ethanol extract. Both extracts were dissolved in water and then prepared as experimental solutions at the concentration of 0.1%, 1%, 5%, and 10%, respectively. Preparation of the formic and acetic acids: Formic acid (88%, Sigma Aldrich) and acetic acid (99.7, ACS reagent grade, VWR International LLC) were prepared as solutions at the concentration of 0.1%, 1%, and 5% immediately before the bioassay experiments. Detection of formic acid by NMR analysis: Approximately 100 fire ant workers were anesthetized by CO.sub.2 and dissolved in 1 mL deuterated CHCl.sub.3 (chroroform). The mixture was put under ultrasound for 30 min extraction. The fire ants were then removed and the extract was dried with anhydrous magnesium sulfate. 500 L of the extract was transferred to NMR tube for detection. .sup.1H-NMR experiments were performed on a JEOL ECS 400 spectrometer, with spectroscopic data referenced to the solvent used. According to standard formic acid .sup.1H-NMR spectrum, the unique singlet of the aldehyde proton should appear at .sub.H 8.02. Contact Toxicity Assays: (1) The impacts of acetone and ethanol extracts of the red imported fire ants on the fire ants: the experiment includes nine treatments and each treatment had the total 150 workers of the red imported fire ant with 50 ants in each of the three 100 mL beakers (as three replications). The ants in each beaker were topically sprayed with a total 1 mL of 0.1%, 1%, 5%, or 10% solutions of either acetone or ethanol extract, respectively. The control group was sprayed with 1 mL pure water. The beakers in all treatments were covered by cloth to prevent the escape of the fire ants. The surviving number of the fire ants in each treatment was counted by 1 h interval for 7 h. (2) The impacts of formic and acetic acids on the fire ants: the experiment had seven treatments and each treatment had the total 150 workers of the red imported fire ant with 50 ants in each of the three 100 mL beakers (as three replications). The ants in each beaker were topically sprayed with a total 1 mL of 0.1%, 1%, or 5% solutions of either formic or acetic acids, respectively. The control group was sprayed with 1 mL pure water. The beakers in all treatments were covered by cloth to prevent the escape of fire ants. The surviving number of the fire ants in each treatment was counted by 1 h interval for 7 h. For extracts and acids, LD.sub.50 (the dose required to kill half of the exposed fire ants) and LD.sub.90 (the dose required to kill 90% of the exposed fire ants) were calculated by the PROBIT procedure of SPSS 13.0 for Windows. (3) The impacts of combined application of ethanol extract and formic acid on the fire ants: The experiment had the total 720 workers of the red imported fire ants with 30 ants in each of the 24 Petri Dishes (85 mm in diameter). Control: the ants in three dishes had no treatment; Water treatment: the ants in each of the three dishes were in contact with 1 mL water for 10 sec; 2.5% ethanol extract treatment: the ants in each of the three dishes were in contacted with 1 mL of 2.5% ethanol extract for 10 sec; 2.5% formic acid treatment: the ants in each of the three dishes were in contacted with 1 mL of 2.5% formic acid for 10 sec; mixture of ethanol extract (2.5%) and formic acid (2.5%) treatment: the ants in each of the three dishes were in contacted with 1 mL of a mixture of ethanol extract and formic acid (each had 2.5% in concentration in the mixture) for 10 sec; 5% ethanol extract treatment: the ants in each of the three dishes were in contacted with 1 mL of 5% ethanol extract for 10 sec; 5% formic acid treatment: the ants in each of the three dishes were in contacted with 1 mL of 5% formic acid for 10 sec; and mixture of ethanol extract (5%) and formic acid (5%) treatment: the ants in each of the three dishes were in contacted with 1 mL of a mixture of ethanol extract and formic acid (each had 5% in concentration in the mixture) for 10 sec. The surviving number of the fire ants in each treatment was counted by 15 min interval for 90 min. (4) The impacts of combined application of ethanol extract and formic acid on the injured fire ants: The 450 workers of the red imported fire ants were placed in 15 Petri Dishes (85 mm in diameter) with 30 in each dish. The ants were anesthetized by CO.sub.2 and then each ant was injured in its gaster by pin. Control: the ants in three dishes had no treatment; Water treatment: the ants in each of the three dishes were in contact with 1 mL water for 10 sec; 1.25% ethanol extract treatment: the ants in each of the three dishes were in contacted with 1 mL of 1.25% ethanol extract for 10 sec; 1.25% formic acid treatment: the ants in each of the three dishes were in contact with 1 mL of 1.25% formic acid for 10 sec; and mixture of ethanol extract (1.25%) and formic acid (1.25%) treatment: the ants in each of the three dishes were in contacted with 1 mL of a mixture of ethanol extract and formic acid (each had 1.25% in concentration in the mixture) for 10 sec. The surviving number of the fire ants in each treatment was counted by 15 min interval for 90 min. (5) The impacts of ethanol extract of the red imported fire ants on subterranean termite (Reticulitermes flavipes (Kollar)) (family Rhinotermitidae): the experiment included five treatments and each treatment had 150 subterranean termites with 20 workers, 10 soldiers and 5 winged reproductive ants in each of the three 100 mL beakers (as three replications). The termites in each beaker were topically sprayed with a total 1 mL of 0.1%, 1%, 5%, or 10% solutions of ethanol extract, respectively. The control group was sprayed with 1 mL pure water. The beakers in all treatments were covered by cloth to prevent the escape of fire ants. The surviving number of the fire ants in each treatment was counted by 1 h interval for 7 h. Fumigation Toxicity Assays of Formic Acid: The experiment included approximately 300 workers of the fire ants in each container with the filter paper treated with 10 mL of 0.1%, 1%, or 5% formic acid, respectively. The surviving number of the fire ants in each treatment was counted by 1 h interval for 2 h. LD.sub.50 (the dose required to kill half of the exposed fire ants) and LD.sub.90 (the dose required to kill 90% of the exposed fire ants) were calculated by the PROBIT procedure of SPSS 13.0 for Windows.

(160) Results:

(161) The topical applications of both the extracts of the red imported fire ants and the organic acids inhibited the fire ants. Both formic acid and acetic acid were not detected in chloroform, acetone, or ethanol extract according to the NMR analysis. The ethanol extract showed more significant toxicity against the fire ants than the acetone extract. During the 7 h contact toxicity bioassays, an average of approximately 60%, 70%, or 80% of the fire ants in contact with 1%, 5%, or 10% ethanol extract were dead, respectively (FIG. 36). In contact toxicity bioassays, 7 h LD.sub.50 and LD.sub.90 of formic acid, acetic acid, ethanol extract, and acetone extract for the fire ants was 1.9% and 5.09%, 6.42% and 9.96%, 1.81% and 15.58%, and 18.67% and 34.18%, respectively. Formic acid had a more significant impact on survival of fire ants than acetic acid. During the 90 min of contact toxicity bioassay, the average mortality with 2.5% and 5% formic acid was more than 70% or 100%, respectively (FIG. 37). In fumigation bioassays, 2 h LD.sub.50 and LD.sub.90 of formic acid for the fire ants was 0.5% and 0.9%, respectively. The combined application of ethanol extract of the fire ants and formic acid had more significant effects on fire ant survival than use of either ethanol extract or formic acid alone (FIGS. 37 and 38). However, the ethanol extract of the fire ants had no impacts on subterranean termite.

Example 21

Elimination and Prohibition of the Subterranean Termite (Reticulitermes flavipes (Kollar)) by Formic Acid

(162) General Experimental Procedures:

(163) Formic acid (90.4%, certified ACS reagent grade, Fisher Scientific Company, Fair Lawn, N.J., USA) was prepared as solutions at the concentration of 0.1%, 1%, and 5% immediately before the bioassay experiments. Detection of formic acid by NMR analysis: Approximately 100 termites were anesthetized by CO.sub.2 and dissolved in 1 mL deuterated CHCl.sub.3 (chroroform). The mixture was put under ultrasound for 30 min extraction. The termites were then removed and the extract was dried with anhydrous magnesium sulfate. 500 L of the extract was transferred to NMR tube for detection. .sup.1H-NMR experiments were performed on a JEOL ECS 400 spectrometer, with spectroscopic data referenced to the solvent used. According to standard formic acid .sup.1H-NMR spectrum, the unique singlet of the aldehyde proton should appear at .sub.H 8.02. Contact Toxicity Assays: The experiment included five treatments and each treatment had total 105 subterranean termite (Reticulitermes flavipes (Kollar)) (family Rhinotermitidae) including 20 workers, 10 soldiers and 5 winged reproductives (alates) in each of the three 100 mL beakers (as three replications). The termites in each beaker were topically sprayed with a total 1 mL of 0.1%, 1%, 5%, or 10% solutions of formic acid, respectively. The control group was sprayed with 1 mL pure water. The beakers in all treatments were covered by cloth to prevent the escape of termites. The surviving number of the termites in each treatment was counted by 1 h interval for 7 h.

(164) Results:

(165) The NMR analysis indicated that the subterranean termites contained significant amount of formic acid (FIG. 39). Formic acid at higher concentrations (5 or 10%) killed all termites (including workers, soldiers, and reproductive) during the first hour of contact experiment. During the 7 h of contact toxicity bioassays, formic acid at 1% concentration killed more than 40% of the termites (FIG. 40). However, formic acid at 0.1% concentration had no impact on the termites. In the contact toxicity bioassays, 7 h LD.sub.50 and LD.sub.90 of formic acid for the termite was 4.71% and 9.08%, respectively.

(166) All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

(167) The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Abbasi & Nipaney, Environ. Conserv. 13: 235-241, 1986. Abdul-Rahman and Habib, J. Chem. Ecol. 15: 2289-2300, 1989. Ascune, et al., Science 331: 1066-1068, 2011. Baldwin & Callahan, Oecologia 94: 534-541, 1993. Barrett, Sci. Amer. 260: 90-97, 1989. Barthlott, et al., Botany. 87: 830-836, 2009. Batish, et al., Environ. Exp. Bot. 47: 149-155, 2002. Beck, et al., Invasive Pl. Sci. Manag. 1: 414-421, 2008. Chen, et al., J. Agric. Food Chem. 57: 3128-3133, 2009. Chon, et al., Crop Protect. 21(10): 1077-1082, 2002. Chou & Kuo, J. Chem. Ecol. 12(6): 1431-1448, 1986. Chou & Lin, J. Chem. Ecol. 2: 353-367, 1976. Choudhary, et al., Phytochemistry 69: 1018-1023, 2008. Ding, et al., Chem. Pharm. Bull. 47: 652-655, 1999. Finlayson, Aquat. Bot. 18: 257-262, 1984. Fowden & Lea, In: Herbivores: their interaction with secondary plant metabolites. Academic Press, New York, pp 135-160, 1979. Gog, et al., Chemoecology 15: 115-119, 2005. Hedge and Miller, Crop Sci. 30(6): 1255-1259, 1990. Heisey, In: Biological active natural products: agrochemicals. CRC Press, Florida, pp. 57-68, 1999. Hoffman, Curr. Opin. Allergy Clin. Immunol. 10: 342-346, 2010. Javaid, et al., Pak. J. Bot. 39(7): 2611-2618, 2007. Jubinsky & Anderson, Catanea 61(3): 226-231, 1996. Julien, et al., In Biological control of tropical weeds using arthropods. Cambridge University Press, 2009. Kato-Noguchi & Ota, J. Rice Res. 1: 108, 2013 Li, A system for increasing the production of indole and quinoline alkaloids, particularly camptothecins and related compounds, from plants, 2002. Li, et al., Front. Biosci. E2: 1196-1210, 2010. Maurya, et al., Phytochemistry 65: 915-920, 2004. McKey, Am. Nat. 108: 305-320, 1974. Morgan, Myrmecol. News 11: 79-90, 2008. Morrison, et al., Biol. Invasions 6: 193-191, 2004. Narasimhulua, et al., Nat. Prod. Res. 24: 1390-1394, 2010. Paudel, J. Plant Sci. 6: 85-92, 2009. Picman & Picman, Biochem. Syst. Ecol. 12(3): 287-292, 1984. Pimentel, et al. Environmental and economic costs associated with nonindigenous species in the United States, 1999. Quintana, et al., Phytochemistry 69: 2572-2578, 2008. Room, et al., Trends Ecol. Evol. 5: 74-79, 1990. Room & Thomas, J. Appl. Ecol. 23: 1013-1028, 1986. Schooler, et al., Nature 470: 86-89, 2011. Singh, et al., Crit. Rev. Plant Sci. 18: 757-772, 1999. Sirikantaramas, et al., Phytochem. Rev. 7: 467-477, 2008a. Sirikantaramas, et al., PNAS. 105: 6782-6786, 2008b. USDA Animal and Plant Health Inspection Service. Giant SalviniaPest Alert, 2000. Wang, Ann. Rev. Biochem. 65: 635-692, 1996. Wu, et al., J. Nat. Prod. 74: 989-996, 2011. Wyman-Simpson, et al., Plant Soil 135: 83-94, 1991. Xuan, et al. Agron. Sustain. Dev. 26: 89-97, 2006.