REFERENCE TO GOVERNMENT GRANT

This invention was supported in part by funds obtained from the U.S. Government (USDA SBIR Grant Number: 2011-33610-30455). The U.S. Government may have certain rights in the invention.

FIELD

This invention relates to compositions and methods for controlling the germination and growth of broadleaf, sedge and grass weeds using compounds comprising thaxtomin, a cyclic dipeptide produced by Streptomyces sp., as an active ingredient.

BACKGROUND

Natural products are substances produced by microbes, plants, and other organisms.

Microbial natural products offer an abundant source of chemical diversity, and there is a long history of utilizing natural products for pharmaceutical purposes. However, secondary metabolites produced by microbes can also be successfully used for weed and pest control in agricultural applications.

Thaxtomins (4-nitroindol-3-yl-containing 2,5-dioxopiperazines) are a family of dipeptide phytotoxins produced by plant-pathogenic Streptomyces sp. (5. scabies, S. acidiscabies) that cause scab diseases in potato {Soianum tuberosum) (King, Lawrence et al. 1992). Toxin production occurs in diseased tissue and can also be elicited in vitro in an optimal growth medium containing oat bran (Loria, Bukhalid et al. 1995; Beausejour, Goyer et al. 1999). King and her coworkers (King, Lawrence et al. 2001) demonstrated that all plant pathogenic species in the Streptomyces family produce one or more thaxtomins with herbicidal activity. Hiltunen et al. (Hiltunen, Laakso et al. 2006) purified four thaxtomin analogs (thaxtomin A, thaxtomin A ortho isomer, thaxtomin B and thaxtomin D) from cultures of 5. scabies and 5. turbidiscabies and showed that all four compounds induced similar symptoms of reduced shoot and root growth, root swelling, (at 10-200 ppb) and necrosis (at 200-1000 ppb) on micropropagated in vitro cultures of potato. In addition, thaxtomins applied in combinations, showed additive effects, but no synergism (Hiltunen, Laakso et al. 2006). According to Duke et al. (Duke, Baerson et al. 2003), both thaxtomin A (Figure 1) and thaxtomin D have marked activity as pre and post emergent, non-systemic herbicides, and concentrations of less than 1 uM of thaxtomin A causes cell swelling, necrosis and growth inhibition in mono and dicotyledonous seedlings (Healy, Wach et al. 2000). Thaxtomin has been evaluated as an herbicide by Dow Agro Sciences, Inc., and while active, it lacked systemic action (King, Lawrence et al.

2001). The presence of the nitro group in the indole ring required for an L,L- configuration of the diketopiperazine appears to be the minimal requirement for phytotoxicity. The position of the nitro group in the indole ring is very site specific , and the phenyl portion of the phenylalanine plays a necessary role in structural requirements of phytotoxicity (King, Lawrence et al. 1989; King, Lawrence et al. 1992; King, Lawrence et al. 2003). The herbicidal mode of action is based on disruption of cell wall synthesis (Fry and Loria 2002), with inhibition of cellulose biosynthesis being the main target (King et al., 2001 ; Duval et al., 2005; Johnson et al. 2007). Recently, Kang et al. (Kang, Semones et al. 2008) have described the use of thaxtomin and thaxtomin compositions as algaecides to control algae in water environments . SUMMARY

Provided is a method for modulating growth of weeds comprising applying to said weeds or soil an amount of thaxtomin effective to modulate growth of a weed, wherein said weed is a member of the genus set forth in Table 1.

TABLE 1. Weed Genera to be Controlled

Or alternatively members of the species set forth in Table 2.

Table 2: Weed Species to be Controlled

In a related aspect, also provided is the use of thaxtomin in modulating one or more weeds set forth above in a growth system. Also provided is a combination or compositions that may comprise in addition to thaxtomin, at least one or more herbicides. Thus the invention may comprise a thaxtomin and a chemical herbicide and/or bioherbicide. In a particular embodiment, the herbicide is a bioherbicide selected from the group consisting of bialaphos, acetic acid, sarmentine analog, manuka oil, m-tyrosine, Phoma macrostoma, chelated iron or a microbial herbicide. In an even more particular embodiment, the bioherbicide is bialophos, manuka oil, acetic acid, m-tyrosine or chelated iron.

In yet another particular embodiment, the herbicide is a chemical herbicide selected from the group consisting of Amicarbazone, Glufosinate, Chlorthiamid, 2,4-D,Triclopyr, Linuron, Flumioxazin, Fluridone, MCPP, Asulam, , Sulcotrione, Simazine, Thiazopyr, Isoxaben,

Norflurazon, Napropamide, Indazifam, EPTC, Fomesafen, Thiencarbazone, Methiozolin,

Metolachlor, Acetochlor, Pyrithrobac-sodium, Picloram, Oleic acid, Trioxysulfuron-sodium MCPA, Fluroxypyr . In specific embodiment, the herbicide is glufosinate. In a related aspect provided is a method for modulating the growth and/or emergence of a weed by applying said combination to soil or the weed.

The thaxtomin in the methods set forth above may be applied prior to and/or after emergence of said weeds . In a particular embodiment said weed is selectively modulated in a growth system without causing significant injury to non-weed plants in said growth system. In a particular embodiment, the practice is to control one or more weeds without causing significant injury to other non-weed plants in the growth system means the application of thaxtomin in a manner that results in less than 50% phytotoxicity to the non-weed plants relative to an untreated control, more particularly, less than 33% phytotoxicity to the non-weed plants relative to an untreated control, more particularly, less than 25% phytotoxicity to the non-weed plants relative to an untreated control, even more particularly, less than 10% phytotoxicity to non-weed plants s relative to an untreated control, and even more particularly, less than 5% phytotoxicity to the non-weed plants relative to an untreated control. The cereal crop may include but is not limited to corn, wheat, triticale, barley, rye, oats , sorghum, sugarcane, and millet. Alternatively, weed emergence and/or growth are modulated in a rice growing system, a turf or Timothy grass growth system.

In a related aspect, provided is the use of thaxtomin and a second herbicidal agent in modulating the growth and/or emergence of a weed in a growth system. In particular, the second herbicidal agent is a bioherbicide. In a particular embodiment, the herbicide is a bioherbicide selected from the group consisting of bialaphos , sarmentine analog, manuka oil, m-tyrosine, Phoma macrostoma, chelated iron or a microbial herbicide. In an even more particular embodiment, the bioherbicide is bialophos , manuka oil, m-tyrosine or chelated iron.

In yet another particular embodiment, the herbicide is a chemical herbicide selected from the group consisting of A rmcarbazone, Glufosinate, Paclobutrazol, Methiozolin, Cblorthiamid , 2,4-D,Triclopyr, Linuron, , Flumioxazin, Fluridone, MCPP, Asularn , , Sulcotrione, Simazine,

Thiazopyr, Isoxaben, Norflurazon, Napropamide, Indazifam, EPTC , Fomesafen, Thiencarbazone, Metolachlor, Acetochlor, Pyrithrobac-sodium, Picloram, Oleic acid, Trioxysulfuron, -sodium MCPA, Fluroxypyr .

As noted above, the method set forth above may also involve the use of at least a second herbicidal agent. The two herbicidal agents may be applied together in one formulation or separately in two formulations . Thus , provided is a combination comprising thaxtomin and a second herbicidal agent. The combination may be a synergistic combination. In a particular embodiment, the E/Ee ratio is at least about 1.2, 1.5 , 1.7 , 1.9, 2.0, 2.5 , or higher. Control of weeds can be achieved by using thaxtomin A in a tank mix or rotation with other herbicidally active compounds known to have good activity against grass weeds but no or low phytotoxicity against cereal crops and/or turf grass and/or, pasture grass and/or Timothy grasses . In particular, the invention relates to a method for modulating growth of monocotyledonous , dicotyledonous and sedge weeds comprising applying to said weeds an amount of thaxtomin and amount of at least a second herbicidal agent to modulate growth of said weeds. The two herbicidal agents may be applied together in one formulation or separately in two formulations . The thaxtomin and second herbicidal agent may be applied in a cereal growth system (e.g., wheat, triticale, barley, oats, rye, corn, sorghum, sugarcane, rice or millet) and/or turf grass growth system and/or pasture grass growth system and/or Timothy grass growth system. In a particular synergistic combination, the combination may be a thaxtomin and acetic acid, thaxtomin and manuka oil, thaxtomin and m-tyrosin and thaxtomin and bialophos.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the structure of Thaxtomin A.

Figure 2 shows the pre-emergent effect of MBI-005 + Manuka oil on crabgrass.

DETAILED DESCRIPTION OF THE INVENTION

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits , ranges excluding either both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

As defined herein, "modulate" means to modify and/or control the extent and/or rate of emergence and/or growth of one or more weeds.

As defined herein, a "growth system" may be any ecosystem for growing plants, including but not limited to cereal, pasture grass, Timothy grass, turf grass and aquatic plants that are not weeds. For example, a "cereal growth system" may be a cereal growth culture or may be a field containing planted cereal crops or cereal seeds . Similarly, a "turf grass growth system" may be a turf grass growth culture or may be a field, lawn or golf course containing planted turf grass or turf grass seeds. Thaxtomin utilized may be derived in fermentation of the following actinomycetes cultures: 5. scabies - ATCC 49173 , 5. acidiscabies - ATCC 49003 and BL37-EQ-010 - or it can be purchased from commercial sources .

The thaxtomin utilized include but are not limited to agents described as cyclic dipeptides having the basic structure cyclo-(L-4-nitrotryptophyl-L-phenylalanyl). In embodiments, suitable diketopiperazne moieties may be N-methylated, and include congeners carrying phenylalanyl alpha and ring-carbon hydroxyl groups. The chemical in a particular embodiment comprises:

wherein R ₁ is methyl or H, R ₂ is hydroxy or H, R ₃ is methyl or H, R ₄ is hydroxy or H, R ₅ is hydroxy or H, R ₆ is hydroxy or H, and combinations thereof.

Non limiting examples of suitable thaxtomin is for use in accordance with the present invention include but are not limited to thaxtomin A, thaxtomin A ortho isomer, thaxtomin B, thaxtomin C , hydroxy thaxtomin C, thaxtomin A p-isomer, hydroxythaxtomin A and

des-N-methylthaxtomin C and derivatives of any of these (See Figure 1).

Compositions

The compositions of the present invention may be sprayed on the plant or applied to soil. Particular embodiments are described in the Examples, infra. These compositions may be in the form of dust, coarse dust, micro granules, granules, wettable powder, emulsifiable concentrate, liquid preparation, suspension concentrate, water degradable granules or oil suspension.

The compositions set forth herein do comprise a carrier and/or diluent. The term, 'carrier' as used herein means an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to plant or other object to be treated, or its storage, transport and/or handling. Examples of diluents or carriers for the pre- and post-emergence herbicides include, but are not limited to, water, milk, ethanol, mineral oil, glycerol.

The compositions set forth herein may comprise at least two herbicidal agents. One herbicidal agent is thaxtomin set forth above. It may be present in one embodiment thaxtomin is present in an amount ranging from about 0.01 to about 5.0 mg/mL. The other herbicidal agent may be a bioherbicide and/or a chemical herbicide. The bioherbicide may be derived from a plant, or may be a microbial bioherbicide. The microbial bioherbicide may be derived from bacteria (e.g. Streptomyces sp., Burkholderia sp., particularly Burkholderia sp. or fractions, supernatant, filtrate, or compounds derived therefrom which disclosed in WO/2011/106491 and PCT/US 12/50807, the contents of which are incorporated herein by reference or fungus. The bioherbicide may be selected from the group consisting of clove, cinnamon, lemongrass, citrus oils, orange peel oil, bialaphos, cornexistin, AAL-toxin, leptospermone and other triketones (such as

2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-l ,3-dione, flavesone, grandiflorone, etc.), sarmentine analog, which in particular are disclosed in US patent publication 20110021358 (the disclosure of which is herein incorporated by reference) but there 25 may be mentioned

N-(Decanoyl)pyrrolidine, N-(Decenoyl)pyrrolidine, N-(Decanoyl)piperidine,

N-(trans-Cinnamoyl)pyrrolidine, (2E,4Z-Decadienoyl)pyrrolidine, N-(Decenoyl)piperidine, (2E ,4Z-Dec adienoy l)piperidine , (2E ,4Z-Decadienoy l)hexamethyleneimine ,

N-(Decenoyl)hexamethyleneimine, N-(Decanoyl)hexamethyleneimine, decanoic acid, 2E-Decenoic acid. , momilactone B, sorgoleone, ascaulatoxin, manuka oil, Phoma macrostoma, d-limonene, m-tyrosine, tentoxin, methylated seed oil, chelated iron, acetic acid and ascaulatoxin aglycone. In a particular embodiment, the composition may comprise thaxtomin, lemongrass oil and optionally a surfactant and/or vegetable oil. In another embodiment, the composition may comprise thaxtomin, sarmentine and optionally a nonionic surfactant and/or vegetable oil. In another particular embodiment, the composition may comprise thaxtomin, bialaphos (also known as bialafos) and optionally a nonionic surfactant and/or vegetable oil. The bioherbicide such as lemongrass oil, bialaphos (bialfos), manuka oil, sarmentine, sarmentine analog, Burkholderia species may be present in an amount ranging from about 0.01 mg/mL to about 100 mg/mL and more preferably between about 0.025 mg/mL to about 50 mg/mL

The chemical herbicide may be selected from, or related to a member of, the group consisting of 2,4-D, Acetochlor, Acifluorfen, Ami.carbaz.one, Amiofos, Asutam, Atrazine,

Bensulfuron, Bentazone, Byspiribac-sodiurrs, Carfentrazone, Chiorthiamid, Clomazone,

Cy alof op-butyl , Dicamba, Diclofop, Dithiopyr, Diuron, EPTC, Fenoxaprop, Flumioxazin,

Fluridone, Fluroxypyr, Fomesafen, Glufosinate, Glyphosate, Halosulfuron, Imazethapyr, Indazifam, Isoxaben, Linuron, MCPA (4-chloro-2-methylphenoxy acetic acid), MCPP, Mesotrione,

Metolachlor, Methiozolin, Metribuzin, MSMA, Napropamide, Nicosulfuron, Norflurazon, Oleic acid, Oryzalin, Oxadiazon, Oxyfluorfen, Quinclorac, Paclobutrazol, Pelargonic acid, Pendimethalin, Penoxsulam, Picloram, Propanil, Pyrithrobac-sodium, Rimsulfuron, Saflufenacil, Sethoxydim, Simazine, Sulcotrione, Thiazopyr, Thiencarbazone, Thiobencarb, Triclopyr, Trifluralin,

Trioxysulfuron-sodium. The chemical herbicide such as pendimethalin or clomazone, atrazine, oryzalin, trifluralin and metolachlor may be present in a pre-emergent weed control application in an amount ranging from about 0.01 mg/mL to about 50 mg/mL and a chemical herbicide such as clomazone, cyhalofop, oryzalin, S -metolachlor, bispyribac-sodium, glyophosate, glufosinate, mesotione, penoxsulam, carfentrazone, quinclorac, triclopyr-ester, trioxysulfuron-sodium, thiobencarb, propanil, 2,4-D, dicamba in a pre or post-emergent application from about 0.1 mg/mL to about 60 g a.i./mL or 0.03 kg a.i./ha to 5.6 kg a.i./ha. The composition may further comprise an adjuvant which may be vegetable oil comprising ethyl oleate, polyethylene dialkyl ester and ethoxylated nonylphenol. The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, improvement of fluidity or rust inhibition. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the herbicidal compositions of the present invention.

For post-emergent formulations , the formulation components used may contain smectite clays , attapulgite clays and similar swelling clays , thickeners such as xanthan gums , gum Arabic and other polysaccharide thickeners as well as dispersion stabilizers such as nonionic surfactants (for example polyoxy ethylene (20) monolaurate or polysorbate 60 POE (20) sorbitan monostearate, ethylene glycol monostearate). The concentration of the clays may vary between about 0-2.5% w/w of the total formulation, the polysaccharide thickeners may range between about 0-0.5% w/w of the total formulation and the surfactants may range from about 0-5% w/w of the total formulation.

Uses

The compounds , combinations and compositions set forth herein may be used to modulate the emergene and/or growth of various weeds and can thus be used in pre-emergent and

post-emergent applications. Said weeds may include but are not limited to Echinochloa

phyllopogon, Echinochloa colona, Echinochloa oryzoides, Echinochloa crus-galli, Schoenoplectus spp., Heteranthera spp., Monochoria spp., Bacopa spp., Sagittaria spp., Oryza punctata and Oryza sativa (weedy). The application we filed also includes the following targets: Chenopodium sp. (e.g., Chenopodium album, Chenopodium murale), Abutilon sp. (e.g., Abutilon theophrasti), Helianthus sp. (e.g., Helianthus annuus), Ambrosia sp. (e.g., Ambrosia artemesifolia, Ambrosia trifida), Amaranthus sp. (e.g., Amaranthus retroflexus, Amaranthus palmeri, Amaranthus rudis, Amaranthus spinosus, Amaranthus tuberculatus), Convolvulus sp. (e.g., Convolvulus arvensis), Brassica sp. (e.g., Brassica kaber), Taraxacum sp. (e.g., Taraxacum officinale), Solanum sp. (e.g., Solanum nigrum, Solanum elaeagnifolium, Solanum physalifolium, Solanum ptycanthum), Malva sp. (e.g., Malva neglecta), Setaria sp. (e.g., Setaria lutescens), Bromus sp. (e.g., Bromus tectorum, Bromus diandrus, Bromus hordeaceus), Poa sp. (e.g., Poa annua, Poa pratensis), Lolium sp. (e.g., Lolium perenne, Lolium rigidum, Lolium multiflorum L. var. Pace), Festuca sp. (e.g., Festuca arundinaceae, Festuca rubra), Echinochloa sp. (e.g., Echinochloa crus-galli, Echinochloa colona), and particularly, Lambsquarter - Chenopodium album, Redroot Pigweed - Amaranthus retroflexus, Wild Mustard - Brassica kaber, Dandelion - Taraxacum officinale, and Black Nightshade -

Solatium nigrum, Oxalis sp. (e.g., Oxalis stricta, Oxalis pes-caprae, Oxalis corniculata); Cyperus sp. (e.g., Cyperus difformis); Conyza sp. (e.g., Conyza canadensis, Conyza sumatrensis, Conyza bonariensis); Sagina sp. (e.g., Sagina procumbens); Veronica sp. (e.g., Veronica hederafolia) , Stellar ia sp. (e.g., Stellar ia media), Rorippa sp. (e.g., Rorippa islandica), Senecio sp. (e.g., Senecio vulgaris), Lamium sp. (e.g., Lamium amplexicaule) , Digitaria sp. (e.g., Digitaria sanguinalis,

Digitaria ischaemum), Galium sp. (e.g., Galium aparine), Galinsoga sp. (e.g., Galinsoga aristatula), Cardamine sp. (e.g., Cardamine flexuosa, Cardamine hirsuta), Kochia sp. (e.g., Kochia scoparia), Eleusine sp. (e.g., Eleusine indica), Portulaca sp. (e.g., Portulaca oleraceae), Plantago sp. (e.g., Plantago lanceolata), Euphorbia sp. (e.g., Euphornia supina, Euphorbia maculate, Euphorbia esula, Euphorbia prostrata), Er odium sp. (e.g., Er odium cicutarium), Sonchus sp., (e.g., Sonchus oleraceus), Lactuca sp. (e.g., Lactuca serriola), Cap sella sp. (e.g., Cap sella bursa-pastoris) , Leptochloa sp. (e.g., Leptochloa fascicularis , Leptochloa virgata) , Raphanus sp. (e..g., Raphanus raphanistrum) , Calandrinia sp. (e.g., Calandrinia ciliata), Paspalum sp. (e.g., Paspalum dilatatum), Gnaphalium sp., Cynodon sp. (e.g., Cynodon dactylon, Cynodon hirsutus), Polygonum sp. (e.g., Polygonum arenastrum, Polygonum lap atho folium,) , Avena fatua, Hordeum sp. (e.g., Hordeum leporinum), Urtica sp. (e.g., Urtica urens), Tribulus terrestris, Sisymbrium sp. (e.g., Sisymbrium irio), Cenchrus sp., Salsola sp. (e.g., Salsola tragus, Salsola kali), Amsinckia sp. (e.g., Amsinckia lycopsoides), Ipomoea sp., Claytonia perfoliata, Polypogon sp. (e.g., Polypogon monspeliensis), Xanthium sp., Hypochaeris radicata, Physalis sp., Eragrostis sp., Verbascum sp., Chamomilla suaveolens, Centaur ea sp. (e.g., Centaur ea solstitialis) , Epilobium brachycarpum, Panicum sp. (e.g., Panicum capilare, Panicum dichotomiflorum) , Rumex acetosella, Eclipta sp. (e.g., Eclipta alba, Eclipta prostrata) , Ludwigia sp., Urochloa sp. (e.g. Urochloa platyphylla, Urochloa panicoides), Leersia sp., Sesbania sp. (Sesbania herbacea) , Rotala sp. , Ammania sp., Alternathera philoxeroides, Commelina sp., Sorghum halepense, Parthenium hysterophorus, C Moris truncata

EXAMPLES

The composition and method of the present invention will be further illustrated in the following, non-limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein. Example 1

Bialaphos is produced by bacteria Streptomecyes spp. and its synthesized active ingredient glufosinate is marketed as Rely ^® 200 (Bayer CropScience, Research Triangle Park, NC). Bialaphos has a non-selective property and inhibits the activity of glutamine synthetase, an enzyme involved in the synthesis of the amino acid glutamine.

The MBI-005 and bialaphos were prepared at various concentrations either as single ingredients or in combination with bialaphos . The treatments were applied at approximately 2/3 ml per replicate with a hand-help spray nozzle to barnyard grass or sedge. There were 3 replicates per treatment which were randomized after spray and were kept in a greenhouse at 25°C for evaluation of phytotoxicity (% control) .

When MBI-005 was mixed with bialaphos, the efficacy was increased several times more than when they were used alone (Table 3 , 4, and 5). At higher rates of the mixtures, 100% control was achieved (Table 6). Synergy was observed when bialaphos at 0.178 mg/niL was mixed with

MBI-005 at 0.25 mg/niL, and about 42% efficacy was achieved when the rate of bialaphos was increased close to 1.0 mg/niL from 10% control with bialaphos alone (Table 4).

Table 3. Effects of bialaphos, MBI-005 (thaxtomin A), and the combinations of bialaphos with MBI-005 in controlling barnyard grass.

* Treatment means in each column marked with the same letter are not statistically different at LSD at p=0.05 level.

* Synergy is calculated from Colby's formula ( Colby, 1967. Weeds 15:20-22): Ee = X + Y - (XY/100) (Where E is the observed efficacy of product A + B, Ee is expected efficacy of A + B , and X and Y are the efficacy of product A or B when used alone. If E/Ee < 1 the combination is antagonistic; if E/Ee = 1 the combination is additive; if E/Ee > 1 the combination is synergistic). Table 4. Effects of bialaphos , MBI-005 (thaxtomin A), and the combinations of bialaphos with

MBI-005 in controlling barnyard grass

Table 5. Effects of bialaphos, MBI-005 (thaxtomin A), and the combinations of bialaph* MBI-005 in controlling barnyard grass.

Treatment means in each column in Tables 4 and 5marked with the same letter are not statistically different with LSD at p=0.05 level. Example 2

To further describe the spectrum of pre-emergence activity, tests were conducted in laboratory and field conditions. In laboratory testing, 35 seeds were placed on a ring of blotter paper inside a 3 cm petri dish and supplied with 4 ml of MBI-005 (≤0.1 mg MBI-005/ml) . Water was used as a negative control and oryzalin applied as a positive control. Petri dishes were randomly placed in a growth room at 25 °C and 50% RH. Treatments were replicated three times and germinated seeds were counted 7 and 14 days after application; water was added as necessary to maintain moisture levels inside each petri dish.

In field testing, field soil containing weed seeds were treated with 38.2 L MBI-005/ha at a spray volume of 280 L/ha using a C02 sprayer with flat fan nozzles . Treatments , including water as the negative control and Gallery as the positive control, were replicated four times and applied twice at a four week interval. Treatments were evaluated visually as % cover and above-ground fresh weights per container were taken.

Results in Table 6 indicate broad spectrum activity on both annual grasses and broadleaves, as well as on some perennials . Field results showing control over an eight-day period also suggest soil residual activity.

Table 6: Pre-Emergent Effect of MBI-005. An (R) indicates an herbicide resistant population.

Key-Table 6A

Example 3. Further Studies on the Post-Emergent Effect of MBI-005

To further describe the spectrum of post-emergence activity, tests were conducted in laboratory and field conditions. For laboratory testing, 3-10 plants (depending on the species) at the 1-2 leaf stage plants in 2.5 cm square pots containing Sunshine Mix No. 2 planting media were sprayed at lmg MBI-005/mL at a rate of 40 gal/A using a cabinet track sprayer. Negative controls were sprayed with water and positive controls with glufosinate. Pots were randomly placed in a growth room at 25 °C and 50% RH, and watered as necessary. Treatments were replicated five times and evaluated at 7 and 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death.

In field testing, field soil containing weeds at the 1-5 leaf stage were treated with 38.2 L

MBI-005/ha at a spray volume of 374 L/ha. Treatments , including water as the negative control and WeedBGon or Preen Broadleaf as the positive control, were replicated four times and applied twice at a four week interval. The positive control on Late watergrass, Ricefield Bulrush, Smallflower sedge and Arrowhead was an herbicide containing as active ingredient 2,4-D and an organosilicone surfactant was added to MBI-005. Treatments were assessed for % control and % ground cover.

Results, shown in Table 7, indicate good (between 80 and 100% control) post-emergence activity on annual many broadleaves and sedges, and limited post-emergent activity on annual grasses, unless surfactants are added. Post-emergent control of perennials was generally limited except for Oxalis pes-caprae.

Table 7: Post-Emergent Effect of MBI-005

Key-Table 7A

Example 4. Synergistic Effect of MBI-005 +Manuka Oil on the Emergence of Crabgrass

Synergistic effects of MBI-005 and Manuka oil (containing leptospermone and other triketones) were evaluated on crabgrass seed germination in a laboratory experiment. Thirty-five seeds were placed on a ring of blotter paper inside a 3 cm petri dish, covered with 10 cm ³ of inert baked clay particle media and supplied with 7 ml of 0.035 mg/ml MBI-005, a Manuka oil concentration, or a combination of

MBI-005 and Manuka oil (Figure 2). Water was used as a negative control. Petri dishes were randomly placed in a growth room at 25 °C and 50% RH. Treatments were replicated three times and germinated seeds were counted 7 days after application; water was added as necessary to maintain moisture levels inside each petri dish.

Synergy occurred between MBI-005 and Manuka oil at 0.003%v/v. Mild synergy was seen between MBI-005 and Manuka oil at 0.01%v/v. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) reduction in germination using Colby's formula. Results are shown in Figure 2.

Example 5. 005 + m-Tyrosine Pre-Emergent Synergies in Crabgrass

Synergistic effects of MBI-005 and m-Tyrosine were evaluated on crabgrass seed germination in a laboratory experiment. Forty-five seeds were placed on a ring of blotter paper inside a 3 cm petri dish, covered with 10 cm ³ of inert baked clay particle media and supplied with 7 ml of 0.035 mg/ml MBI-005, a m-Tyrosine concentration, or a combination of MBI-005 and m-Tyrosine (Table 8). Water was used as a negative control. Petri dishes were randomly placed in a growth room at 25 °C and 50% RH.

Treatments were replicated three times and germinated seeds were counted 7 days after application; water was added as necessary to maintain moisture levels inside each petri dish.

Synergy occurred between MBI-005 and m-Tyrosine at 0.44 mg/mL but not for MBI-005 and m-Tyrosine at 22 mg/mL. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) reduction in germination using Colby's formula.

Table 8; 005 + m-Tyrosine Pre-Emergent Synergies in Crabgrass (7 DAT)

Example 6: MBI-005 + Chelated Fe Post-Emergent Synergistic Effect in Crabgrass

Post-emergent synergistic effects of MBI-005 and Chelated iron were evaluated on crabgrass at the 1-2 leaf stage in a laboratory experiment. Ten seedlings in 2.5 cm square pots containing plant growth mix were sprayed with two suboptimal rates of MBI-005, 14.49 mg/mL of Chelated Fe, or a combination thereof, at a volume of 40 gal/A using a cabinet track sprayer. Negative controls were sprayed with water. Pots were randomly placed in a growth room at 25 °C and 50% RH, and watered as necessary. Treatments were replicated five times and evaluated at 7 and 15 days for visual % damage, with 0% indicating no damage and 100% indicating plant death.

A substantial synergistic effect occurred between MBI-005 and chelated Fe, at both

concentrations of MBI-005. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby's formula.

Table 9: MBI-005 + Chelated Fe Post-Emergent Synergies in Crabgrass (15 DAT)

Example 7. Synergy between MBI-005 and acetic acid

Post-emergent synergistic effects of MBI-005 and acetic acid were evaluated on mustard at the 1-2 leaf stage in a laboratory experiment. Two seedlings in 2.5 cm square pots containing plant growth mix were sprayed with two suboptimal rates of MBI-005, 14.49 mg/mL of Acetic Acid, or a combination thereof, at a volume of 40 gal/A using a cabinet track sprayer. Negative controls were sprayed with water. Pots were randomly placed in a growth room at 25 °C and 50% RH, and watered as necessary. Treatments were arranged in a central composite design (CCD) with three repeat measures and evaluated at 21 days after application for fresh weight. Table 10: Treatments for MBI-005 + acetic acid interaction test

A substantial synergistic effect occurred between MBI-005 and acetic acid on mustard. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby's formula. An E/Ee score above 1.0 indicates synergy. Percent control was calculated in relation to the untreated control (0% control) with 100% control denoting no living green tissue to weigh.

Table 11. Control and synergistic effects on mustard of MBI-005 and Acetic acid combinations.

Example 8: Effects of MBI-005 on Egeria densa

A tube bioassay was set up on 5 cm sprigs of the submerged aquatic plant, Egeria densa. In this assay, sprigs from different locations along the plant were placed in 40 mL of test solution in 50 mL centrifuge tubes. The test solution was comprised of water mixed with the tested treatment. Treatments tested were 300 of 0.01% Diquat, 300 of 0.01% 2,4-D, 2 mL, 5 mL, and 10 mL of fermentation medium, and 2 mL, 5 mL and 10 mL of MBI-005 (90 mg/L Thaxtomin A). The initial fresh weight of each sprig was recorded. The tubes were incubated under fluorescent lights for 3 weeks before being scored visually and evaluating the fresh weight and length. Since the sprigs were not taken from the same location on the plant, there was a good deal of variation within the five replicates of each treatment for these endpoints, so visual scoring gave the best assessment of the health of the sprigs.

Only the apical tips grew lengthwise or significantly increased in fresh weight. All other sprigs only grew by either sprouting a new apical tip or by sending out roots. The negative control sprigs remained generally healthy; one sprig was rooting and one sprouted an apical tip. The sprigs treated with Diquat were dead after 3 weeks. The sprigs treated with 2,4-D - while less healthy than the control, were generally unaffected. The sprigs dosed with fermentation medium were very healthy and grew significantly. In all three doses, most sprigs were rooting and had new apical tips growing. Only one of the total 15 sprigs treated with fermentation medium died.

After 2 weeks the sprigs treated with MBI-005 supernatant began to fall apart when the tubes were gently swirled. The stems became brittle and broke into pieces and the leaves began to turn yellow and fall off the stems. In addition, the MBI-005 -treated sprigs tended to turn brown along the stem. This activity is particularly evident in the apical tips, which disintegrated to a larger extent than sprigs taken from further down the length of the plant.

This test demonstrates that MBI-005 supernatant has herbicide activity against Egeria densa. Example 9: Dose-response on Egeria densa to MBI-005 supernatant

Egeria densa apical tips were collected and measure for length and weight. The plants were then and placed in 50 ml centrifuge tubes filled with water to cover the plants. The water in the tubes was spiked with MBI-005 supernatant. The supernatant had a concentration of 90 mg/L thaxtomin (MBI-005). The treatments were: 0 (water only), 1.25%, 2.5%, 4.5% and 10% supernatant. The positive control was Diquat. The test tubes were covered with plastic caps that were loosened to allow air exchange, while minizing evaporation of the liquid. Stems began turning brown after 5 days. Sprigs began to fall apart starting from the tips after 7 days. At 7 days after treatment, the plants were evaluated for change in weight (W), and change in length (L): - 1.25% MBI-005 (1.125mg/L Tax): Brown stems. 34% reduction in weight, 17% reduction in length.

- 2.5 % MBI-005 (2.25mg/L Tax): Brown stems. 48 % Reduction in weight, 16 % reduction in length.

- 5 % MBI-005 (4.5 mg/L Tax):Brown stems. 58 % reduction in weight, 25 % reduction in length.

10 % MBI-005 (9 mg/L Tax): Brown stems. 86 % reduction in weight, 72% reduction in length. Diquat: No effect. It is possible that the dose was too low

MBI-005 at 3.5%v/v (0.35mg/mL) and Acetic acid at 7.0% v/v had an average synergy value of 2.03. A response surface regression of the data shows that 95% control can be achieved with between 9-10% Acetic acids combined with between 3.75-4.75% v/v MBI-005(0.375-0.475 mg/mL).

Although this invention has been described with reference to specific embodiments , the details thereof are not to be construed as limiting, as it is obvious that one can use various equivalents, changes and modifications and still be within the scope of the present invention.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

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