Abstract:
There is a need for a selective, low-risk herbicide that can be used to control weeds in cereal cultures and turf. The present invention discloses that a bacterial secondary metabolite, thaxtomin and optionally another herbicide is an effective herbicide on broadleaved, sedge and grass weeds. Thaxtomin A and structurally similar compounds can be used as natural herbicides to control the germination and growth of weeds in cereal, turf grass, Timothy grass and pasture grass cultures with no phytotoxicity to these crops. As a natural, non-toxic compound, thaxtomin can be used as a safe alternative for weed control in both conventional and organic farming and gardening systems.

Description:
PRIORITY CLAIM 
     This application is a continuation-in-part of application Ser. No. 13/533,677 filed Jul. 19, 2012, which is a continuation-in-part of application Ser. No. 12/650,315, the contents of which are incorporated herein by reference. Application Ser. No. 12/650,315 claims priority from U.S. application Ser. Nos. 61/142179, filed Dec. 31, 2008 and 61/261504 filed Nov. 16, 2009 under 35 USC 119(e), in which the contents of both are incorporated herein by reference and also claims priority from Taiwan Patent application no. 098144895, filed Dec. 25, 2009 under 35 USC 119(a)-(d), the contents of which are also incorporated herein by reference. 
    
    
     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 OF THE INVENTION 
     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 OF THE INVENTION 
     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. ( S. scabies, S. acidiscabies ) that cause scab diseases in potato ( Solanum 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; Beauséjour, 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  S. scabies  and  S. 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 ( FIG. 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 OF THE INVENTION 
     The present invention discloses the use of thaxtomin as a pre or post-emergence herbicide against most common weeds in the cereal, pasture grass, Timothy grasses and turf grass, residential gardens, vineyards, orchards and park growth systems. A “growth system” may be any ecosystem for growing cereal, pasture grass, Timothy grass and turf grass. 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. It can serve as a safer alternative to synthetic herbicides now on the market. A primary object of the invention is to provide novel herbicidal compositions against both broadleaf, sedge and grassy weeds, that contains thaxtomin as an active ingredient. Another object is to provide a safe, non-toxic herbicidal composition that does not harm cereal crops, pasture grass, Timothy grass or turf grass and a method that will not harm the environment. 
     The above and other objects are accomplished by the present invention which is directed to herbicidal compositions containing at least one herbicidal agent, e.g., thaxtomin with optionally certain carriers to control the growth and germination of weeds in the cereal growth system and/or turf grass growth system and/or Timothy grass growth system and/or pasture grass growth system. In particular, the invention is further directed to an herbicidal composition for use in modulating the germination and growth of monocotyledonous and/or dicotyledenous and/or sedge weeds in a cereal growth system. In a particular embodiment, the cereal growth system is a non-rice cereal growth system comprising at least one herbicide in which said herbicide is thaxtomin The compositions of the present invention may further comprise a carrier and/or diluent. In a particular embodiment, the composition is an aqueous composition. In another particular embodiment, the thaxtomin in the composition is dissolved in a diluent comprising an organic solvent such as ethanol, isopropanol, or an aliphatic ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone. 
     In a related aspect, the invention is directed to the use of at least one herbicidal agent, e.g., thaxtomin, in the formulation of an herbicide for modulating monocotyledonous and/or dicotyledenous and/or sedge weeds in a cereal growth system, e.g., a non-rice cereal growth system. Similarly, the invention is directed to the use of at least one herbicidal agent in formulation of an herbicide for modulating monocotyledonous and/or dicotyledenous and/or sedge weeds in a turf grass growth system and/or Timothy grass growth system and/or pasture grass growth system, wherein at least one herbicidal agent is thaxtomin. 
     The compositions of the present invention 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. Compositions comprising thaxtomin and at least a second herbicide may be used in cereal growth systems (e.g., wheat, triticale, barley, oats, rye, corn, sorghum, sugarcane, rice or millet) and/or turf grass growth systems and/or Timothy grass growth systems and/or pasture grass growth systems and/or residential gardens, vineyards, orchards and park systems and/or aquatic systems. In a particular embodiment these compositions may be used to modulate growth of broadleaf, sedge and grassy weeds, The compositions may also be used to modulate growth of aquatic weeds which include but are not limited to  Ammania  sp., Alisma plantago-aquatica,  Cyperus  sp.,  Leptochloa  sp. 
     Given that the invention is directed to the use of thaxtomin as a pre- or post-emergence herbicide, the invention is directed to a method for selectively modulating germination and growth of monocotyledonous, dicotyledonous and sedge weeds in a cereal crop growth system. In a particular embodiment, the cereal growth system is a non-rice cereal crop growth system comprising applying to said weeds or soil in said cereal crop growing system at least one herbicidal agent, wherein said herbicidal agent is thaxtomin, in an amount of effective to modulate germination and growth of said weeds but not modulate growth of cereal crop in said cereal crop growth system. The cereal crop may include but is not limited to corn, wheat, triticale, barley, rye, oats, sorghum, sugarcane, and millet. The invention is further directed to a method for modulating germination and growth of monocotyledonous, dicotyledonous and sedge weeds in a turf, pasture and/or Timothy grass growth system comprising applying to said weeds or soil in said turf grass growing system at least one herbicidal agent, wherein said herbicidal agent is thaxtomin, in an amount of effective to modulate growth of said weeds but not modulate germination and growth of turf grass in said turf grass growth system, pasture grass in said pasture grass growth system and/or Timothy grass in said Timothy grass growth system. 
     As noted above, the method of the present invention 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. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the structure of Thaxtomin A. 
         FIG. 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. 
     Thaxtomin utilized in this invention may be derived in fermentation of the following actinomycetes cultures:  S. scabies —ATCC 49173,  S. acidiscabies —ATCC 49003 and BL37-EQ-010—or it can be purchased from commercial sources. 
     The thaxtomin utilized in the invention 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 andring-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, hydroxythaxtomin C, thaxtomin A p-isomer, hydroxythaxtomin A and des-N-methylthaxtomin C and derivatives of any of these (See  FIG. 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 of the invention 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 of the present invention 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. In particular, the bioherbicide derived from a plant may be derived from pepper (e.g., sarmentine) or may be a plant essential oil (e.g., lemongrass oil). 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/US12/50807, 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-1,3-dione, flavesone, grandiflorone, etc.), sarmentine or sarmentine analog, momilactone B, sorgoleone, ascaulatoxin, manuka oil,  Phoma macrostoma , d-limonene, m-tyrosine, tentoxin, nethylated 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 the group consisting of 24-D, Acetochlor, Acifluorfen, Amicarbazone, Anilofos, Asulam, Atrazine, Bensulfuron, Bentazone, Byspiribac-sodium, Carfentrazone, Chlorthiamid, Clomazone, Cyhalofop-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, Metribuzin, MSMA, Napropamide, Nicosulfuron, Norflurazon, Oleic acid, Oryzalin, Oxadiazon, Oxyfluorfen, Quinclorac, 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 polyoxyethylene (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— Solanum 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 ),  Stellaria  sp. (e.g.,  Stellaria 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 ),  Erodium  sp. (e.g.,  Erodium cicutarium ),  Sonchus  sp., (e.g.,  Sonchus oleraceus ),  Lactuca  sp. (e.g.,  Lactuca serriola ),  Capsella  sp. (e.g.,  Capsella 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 lapathofolium ),  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, Centaurea  sp. (e.g.,  Centaurea 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, Chloris 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 
     In a pot study test in greenhouse conditions, 6-inch corn plants ( Zea mays  var. Sunglow) were sprayed with increasing concentrations of thaxtomin A mixed in a carrier 4% ethanol, 0.02% polysorbate 60 POE (20) sorbitan monostearate solution. The spraying solutions contained 0.125, 0.25, 0.5 and 1.0 mg thaxtomin A/mL, and the plants are sprayed until total coverage. Each treatment was done in three replicates, and a control solution consists of water with 4% ethanol and 0.02% polysorbate 60 POE (20) sorbitan monostearate as a surfactant. Prior to and after treatments, plants are grown in a greenhouse under artificial lights (12-h light/dark cycle) at 25° C. 
     Plants are evaluated in one-week intervals starting at 7 days after treatment. The final evaluation is done three weeks after treatment, at which time point, no phytotoxicity is observed in any of the test plants even at the highest thaxtomin A concentration. 
     Example 2 
     A pot study is conducted to test the phytotoxicity of thaxtomin A on corn ( Zea mays  var. Early Sunglow) and wheat ( Triticum aestivum  var. PR1404). To confirm the activity on broadleaf weeds, pigweed ( Amaranthus  sp.) is planted in the same pot with either three corn or five wheat seeds, and sprayed simultaneously with the cereal test plants. The less than 3-inch tall plants grown under growth lights (12-h light/12-h dark) at 28° C. are sprayed with thaxtomin A solutions derived from a liquid culture of  S. acidiscabies  containing 0.5, and 1.0 mg thaxtomin A per mL of solvent (4% ethanol and 0.2% non-ionic surfactant). A solution of 4% ethanol +0.2% non-ionic surfactant without thaxtomin A is used as a control treatment. Each treatment is conducted in three replicates. Treated plants are kept at 28° C. under growth lights and observed at three time points—7, 14 and 21 days after treatment—for visual symptoms of phytotoxicity on corn and wheat and % control of pigweed. 
     At each time point, no symptoms of phytotoxicity are observed in the cereal plants treated with thaxtomin A. The highest concentration of thaxtomin A (1.0 mg/mL) results in a complete control of pigweed grown in the same pots with corn and wheat. 
     Example 3 
     To test the phytotoxicity of thaxtomin A on sorghum plants, five seeds of sorghum ( Sorghum bicolor ) are planted in each 4″×4″ plastic pot filled with soil. Plants were grown under optimal conditions in a greenhouse before and after treatment with solutions containing 0.5 and 1.0 mg thaxtomin A/mL. At the time of the treatment, the plants are about 3 inches tall. Each treatment is applied in three replicates, and a control treatment included plants treated with just the carrier (4% EtOH, 0.02% polysorbate 60 POE (20) sorbitan monostearate). Evaluations for phytotoxicity are performed at 7-day intervals starting one week after treatment. The last evaluation is performed three weeks after the treatment at which point, no phytotoxicity is observed in the treated plants in any treatment concentration. 
     Example 4 
     A strain of  S. acidiscabies  (ATCC-49003) is grown in oat bran broth for 5 days (25° C., 200 rpm). The whole cell broth with thaxtomin A is extracted using XAD resin. The dried crude extract was resuspended in 4% ethanol and 0.02% non-ionic surfactant at a concentration of 10 mg/mL, and the solutions with two different concentrations of thaxtomin A (0.5 and 1.0 mg/mL) are tested the following broadleaf weed species: 
     Lambsquarter— Chenopodium album    
     Velvetleaf— Abutilon theophrasti    
     Sunflower— Helianthus annuus    
     Ragweed, Common— Ambrosia artemesifolia    
     Pigweed, Redroot— Amaranthus retroflexus    
     Bindweed, Common— Convolvulus arvensis    
     Mustard, Wild—Brassica kaber 
     Dandelion— Taraxacum officinale    
     Nightshade, Black— Solanum nigrum    
     Mallow, Common—Malva neglecta 
     and on the following grass weed species: 
     Foxtail— Setaria lutescens    
     Brome, Downy— Bromus tectorum    
     Bluegrass, Annual— Poa annua    
     Bluegrass, Kentucky— Poa pratensis    
     Ryegrass, Perennial—( Lolium perenne  L. var. Pace) 
     Fescue, Tall—( Festuca arundinaceae Schreb . var. Aztec II, Anthem II, LS 1100) 
     Barnyard Grass— Echinochloa crus - galli    
     All plant species are tested in 4″×4″ plastic pots in three replicates. The untreated control plants are sprayed with the carrier solution (4% Ethanol, 0.02% glycosperse) and the positive control plants with Roundup at a rate corresponding to 1 fl. oz/acre. Treated plants are kept in a greenhouse under 12 h light/12 h dark conditions. Data for broadleaf species from weekly evaluations are presented in Table 1. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Weed control efficacy of a  S. acidiscabies  extract containing thaxtomin A on different weed species. Rating 
               
               
                 scale: 0 - no control, 1 - 10% control, 2 - 25% control, 3 - 50% control, 4 - 75% control, 5 - 100% control. 
               
             
          
           
               
                   
                   
                 THAXTOMIN SOLUTION 
                 THAXTOMIN SOLUTION 
               
               
                   
                 UTC 
                 0.5 mg/mL 
                 1.0 mg/mL 
               
             
          
           
               
                 Weed species 
                 7 DAYS 
                 14 DAYS 
                 21 DAYS 
                 7 DAYS 
                 14 DAYS 
                 21 DAYS 
                 7 DAYS 
                 14 DAYS 
                 21 DAYS 
               
               
                   
               
               
                 Dandelion 
                 0.0 
                 0.0 
                 0.0 
                 2.0 
                 2.3 
                 4.0 
                 2.0 
                 2.0 
                 3.7 
               
               
                 Nightshade 
                 0.0 
                 0.0 
                 0.0 
                 2.7 
                 2.2 
                 2.3 
                 2.7 
                 2.0 
                 2.3 
               
               
                 Lambsquarter 
                 0.0 
                 0.0 
                 0.0 
                 2.0 
                 2.0 
                 2.0 
                 2.0 
                 2.0 
                 2.0 
               
               
                 Ragweed 
                 0.0 
                 0.0 
                 0.0 
                 1.0 
                 0.5 
                 0.0 
                 1.0 
                 0.5 
                 0.0 
               
               
                 Velvetleaf 
                 0.0 
                 0.0 
                 0.0 
                 1.7 
                 1.0 
                 1.0 
                 2.0 
                 1.0 
                 0.3 
               
               
                 Bindweed 
                 0.0 
                 0.0 
                 0.0 
                 1.0 
                 1.0 
                 0.0 
                 1.2 
                 1.0 
                 0.0 
               
               
                 Mustard 
                 0.0 
                 0.0 
                 0.0 
                 3.3 
                 4.0 
                 4.5 
                 3.5 
                 2.8 
                 3.5 
               
               
                 Sunflower 
                 0.0 
                 0.0 
                 0.0 
                 1.0 
                 2.0 
                 0.5 
                 1.0 
                 1.7 
                 0.5 
               
               
                 Mallow 
                 0.0 
                 0.0 
                 0.0 
                 1.0 
                 1.0 
                 1.0 
                 1.2 
                 1.0 
                 1.0 
               
               
                 Pigweed 
                 0.0 
                 0.0 
                 0.0 
                 3.5 
                 4.0 
                 4.0 
                 4.2 
                 3.0 
                 3.7 
               
               
                   
               
             
          
         
       
     
     The extract from a bacterial culture of  S. acidiscabies  with a thaxtomin A concentration of 0.5 mg/mL or higher showed good efficacy (&gt;50%) against at least three of the most common broadleaf weed species (dandelion, mustard and pigweed) in both cereal and turf growing systems. Control of some weeds such as Black nightshade and Common lambsquarter was not complete but thaxtomin A even at the lower concentration (0.5 mg/mL) results in severe stunting of these weeds. In this same study, no adverse effects are observed in grass species treated with either 0.5 or 1.0 mg/mL thaxtomin A. In all tested grass species, no phytotoxic effects were visible at even the higher thaxtomin A concentration. 
     Example 5 
     The combined effect of thaxtomin A and two commercial herbicides (Bipyribac-sodium formulated as Regiment and Lemongrass oil formulated as GreenMatch EX) on small-flower umbrella sedge and watergrass is tested in a field study using small (1-sq foot) plots. All single product treatments and tank mix combinations were sprayed at 57 gal per acre. Evaluation of % control was done 14 days after treatment and the results are presented in Table 2 below. Means in each column marked with the same letter in Table 2 are not statistically different from each other at p&lt;0.05 
     According to the results, lemongrass oil at 1.25% weight does not improve the efficacy of thaxtomin A (at 0.25 mg/mL) on sedge but it significantly increases the efficacy on grass weeds such as watergrass (field test) and sprangletop (greenhouse test). 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Effect of thaxtomin A alone and in combination with 
               
               
                 bispyribac-sodium and lemongrass oil on two rice weeds, 
               
               
                 small-flower umbrella sedge and watergrass. 
               
             
          
           
               
                   
                   
                 Sedge 
                 Watergrass 
               
               
                   
                 Treatment 
                 control (%) 
                 control (%) 
               
               
                   
               
               
                   
                 Thaxtomin 0.25 mg/mL 
                  95a 
                  5d 
               
               
                   
                 Thaxtomin 0.5 mg/mL 
                 100a 
                  5d 
               
               
                   
                 Bispyribac-sodium (12 g/acre) 
                  87.5a 
                 32.5a 
               
               
                   
                 (12 g/acre) 
                   
                   
               
               
                   
                 Bispyribac-sodium ½ (6 g/acre) 
                  47.5c 
                  15c 
               
               
                   
                 Bispyribac-sodium ½ + 
                  67.5b 
                 25ab 
               
               
                   
                 Thaxtomin 0.5 mg/mL 
                   
                   
               
               
                   
                 Bispyribac-sodium ½ + 
                  55bc 
                  7.5c 
               
               
                   
                 Thaxtomin 0.25 mg/mL 
                   
                   
               
               
                   
                 Lemongrass oil 5% 
                  15d 
                 10c 
               
               
                   
                 Lemongrass oil 2.5% 
                  12.5d 
                 10c 
               
               
                   
                 Lemongrass oil 1.25% 
                  20d 
                  5d 
               
               
                   
                 Lemongrass oil 1.25% + 
                 100a 
                 10c 
               
               
                   
                 Thax 0.25 mg/mL 
                   
                   
               
               
                   
                 Lemongrass oil 1.25% + 
                 100a 
                 20b 
               
               
                   
                 Thaxtomin 0.5 mg/mL 
               
               
                   
               
             
          
         
       
     
     According to the results, lemongrass oil at 1.25% does not improve the efficacy of thaxtomin A (at 0.25 mg/mL) on sedge but it significantly increases the efficacy on grass weeds such as watergrass (field test) and sprangletop. Thaxtomin A (at 0.5 mg/mL) improves the efficacy of an ALS inhibitor, bipyribac sodium; used at half label rate on both sedge and grasses. 
     Example 6 
     The efficacy of thaxtomin A derived from a liquid culture of  S. acidiscabies  is tested in a field study on rice using 4.9 sq-ft plots surrounded by a metal ring. Treatments with either thaxtomin A or thaxtomin A in combination with lemongrass oil (formulated as GreenMatch EX) or cyhalofop (formulated as Clincher CA) were done using a hand-held sprayer with a water volume corresponding to 57 gallons per acre. Rice (variety M209) was grown until maturity and harvested by hand for yield and weed count assessment. Results of yield (kg/ha), and numbers of redstem, small-flower umbrella sedge, and sprangletop in each plot are presented in Table 3 below. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effect of thaxtomin A alone and in combination with lemongrass 
               
               
                 oil and cyhalofop on rice yield and weed control. 
               
             
          
           
               
                 Treatment 
                 Yield (kg/ha) 
                 # of redstem 
                 # of sedge 
                 # of sprangletop 
               
               
                   
               
             
          
           
               
                 1 
                  7516b 
                 10.3 
                 0.8a 
                 86.0a 
               
               
                 2 
                  7876b 
                 0.5b 
                 1.0a 
                 76.0a 
               
               
                 3 
                  9054ab 
                 0.3b 
                 0.5a 
                 69.3a 
               
               
                 4 
                 11296a 
                 12.8a 
                 0.5a 
                 4.0b 
               
               
                   
               
               
                 1. UTC; 
               
               
                 2. Thaxtomin A (180 g/acre); 
               
               
                 3. Lemongrass oil 1.25% + thaxtomin A (90 g/acre); 
               
               
                 4. Cyhalofop (half label rate; 52 g/acre) + thaxtomin A (90 g/acre) + veg oil 2.5% 
               
               
                 Means in each column marked with the same letter are not statistically different from each other at p &lt; 0.05. 
               
             
          
         
       
     
     Results indicate that thaxtomin at 180 g/acre significantly reduced the number of sedges but had no effect on sprangletop or yield. When used at half rate (thaxtomin A 90 g/acre), a combination with lemongrass oil had better effect on sedges than a combination with cyhalofop (used at half label rate 52 g/acre). Good grass weed (sprangletop) control is achieved when thaxtomin (90 g/acre) is combined with cyhalofop at half the label rate—this combination also improves the yield significantly. 
     Example 7 
     Cyhalofop (2-[4-(4-cyano-2-fluorophenoxy)phenoxy]propanoic acid, butyl ester) is also mixed together with adjuvant containing ethyl oleate, polyethylene dialky ester and ethoxylated nonylphenol (2.5% v/v) and increasing concentrations of thaxtomin A (purified from the ATCC strain 49003) at concentrations 0.1, 0.2 and 0.4 mg/ml. The concentrations of the 2-[4-(4-cyano-fluorophenoxy)phenoxy]propanoic acid, butyl ester before dilution are 29.6% (2.38 lb/gal) and 21.7% (2 lb/gal), respectively. The effect of these mixtures on the growth of common water plantain, red stem, smallflower sedge and sprangletop is determined in the greenhouse. Similarly, rice plants of variety M104 are grown and tested for phytotoxic effects, and all plants are evaluated 7, 14, and 21 days after treatment. Results of from the study with cyhalofop formulated as Clincher CA at the 21-day evaluation point are presented in Table 4 below. 
                                                                 TABLE 4                   Effect of thaxtomin A alone and with cyhalofop       on rice yield and weed control            Cyhalofop                       (6.5 oz/acre) +                       Thaxtomin A   Redstem   Waterplantain   Sedge   Sprangletop       (mg/mL)   % control   % control   % control   % control                    UTC   0   0   0   0       0 - no thx A   75   8   0   90       0.1   100   85   87   100       0.2   97   87   88   100       0.4   100   85   100   100                    
As a conclusion, Clincher CA (29.6% cyhalofop by weight) applied at half label rate (6.5 oz/acre) has good efficacy against grass weeds—not so good on broadleaves and poor on sedges. A combination of Clincher CA (cyhalofop) and thaxtomin A provides good control of all rice weeds tested in this study. Efficacy of thaxtomin A against grass weeds is substantially improved if combined with Clincher. Combination of thaxtomin A with Clincher CA did not cause phytotoxicity on rice at any tested concentration.
 
     Example 8 
     Penoxsulam(2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5c]pyrimidin-2-yl)-6-trifluoromethyl)benzenesulfonamide) is mixed together with adjuvant containing ethyl oleate, polyethylene dialky ester and ethoxylated nonylphenol (2.5% v/v) and increasing concentrations of thaxtomin A (purified from the ATCC strain 49003) at concentrations 0.1, 0.2 and 0.4 mg/ml. The concentrations of the 2-[4-(4-cyano-fluorophenoxy)phenoxy]propanoic acid, butyl ester or 2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5c]pyrimidin-2-yl)-6-trifluoromethyl)benzenesulfonamide before dilution are 29.6% (2.38 lb/gal) and 21.7% (2 lb/gal), respectively. The effect of these mixtures on the growth of common water plantain, red stem, smallflower sedge and sprangletop is determined in the greenhouse. Similarly, rice plants of variety M104 are grown and tested for phytotoxic effects, and all plants are evaluated 7, 14, and 21 days after treatment. 
     Example 9 
     A strain of  S. acidiscabies  was grown in oat bran broth for 5 days (25° C., 200 rpm). The whole cell broth was extracted using XAD resin, and the dried crude extract was resuspended in 4% ethanol and 0.2% non-ionic surfactant at a concentration of 10 mg/mL. The diluted extracts containing 0.2 and 0.4 mg thaxtomin A per mL were tested on three weed species (redstem;  Ammania  spp., smallflower umbrella sedge;  Cyperus difformis  and sprangletop:  Leptochloa uninervia ). Other treatments included sarmentine at 2.5 and 5.0 mg/mL, and a combination treatment containing 0.2 mg thaxtomin A and 2.5 mg sarmentine per mL. Each treatment was applied in three replicates. Treated plants were kept in a greenhouse under 12 h light/12 h dark conditions. Results from an evaluation performed 25 days after treatment are presented in Table 5. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Efficacy of herbicidal treatments using thaxtomin A (0.2 and 0.4 mg/mL) 
               
               
                 or sarmentine (2.5 or 5.0 mg/mL) alone or in combination 
               
               
                 (0.2 + 2.5 mg/mL) to control broadleaf, 
               
               
                 sedge and grass weeds in a greenhouse study. 
               
             
          
           
               
                   
                 Control of 
                 Control of 
                   
               
               
                   
                 redstem 
                 sedge 
                 Control of 
               
               
                 Treatment 
                 (%) 
                 (%) 
                 sprangletop (%) 
               
               
                   
               
               
                 UTC 
                  0a 
                  0a 
                  0a 
               
               
                 Thaxtomin A 0.2 mg/mL 
                  5.0b 
                 48.3b 
                  8.3ab 
               
               
                 Thaxtomin A 0.4 mg/mL 
                 11.7c* 
                 91.7d 
                 10.0b 
               
               
                 Thaxtomin 0.2 + 
                 11.7c* 
                 61.7c 
                 73.3c 
               
               
                 Sarmentine 2.5 mg/mL 
                   
                   
                   
               
               
                 Sarmentine 2.5 mg/mL 
                  0a 
                  8.3a 
                 80.0c 
               
               
                 Sarmentine 5.0 mg/mL 
                  2.5ab 
                  6.7a 
                 92.3d 
               
               
                   
               
               
                 *stunted. In a column, Means followed by the same letter are not statistically different from each other at p &lt; 0.05. 
               
             
          
         
       
     
     Thaxtomin A at the highest concentration of 0.4 mg/mL provides excellent control of sedge but poor control of the grass weed (sprangletop). When combined with sarmentine, the efficacy against grass weeds improves significantly. Also, efficacy against sedge is improved with the combination treatment compared with the single application of thaxtomin A alone at the corresponding concentration. In this study, the control of the broadleaf weed (redstem) is poor with all treatments. 
     Example 10 
     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 ⅔ 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 6, 7, and 8). At higher rates of the mixtures, 100% control was achieved (Table 8). Synergy was observed when bialaphos at 0.178 mg/mL was mixed with MBI-005 at 0.25 mg/mL, and about 42% efficacy was achieved when the rate of bialaphos was increased close to 1.0 mg/mL from 10% control with bialaphos alone (Table 7). 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Effects of bialaphos, MBI-005 (thaxtomin A), and the combinations 
               
               
                 of bialaphos with MBI-005 in controlling barnyard grass. 
               
             
          
           
               
                   
                 Bialaphos 
                 MBI-005 
                 % Control 
                   
               
               
                 Treatment 
                 (mg/mL) 
                 (mg/L) 
                 (14 days) 
                 E/Ee #   
               
               
                   
               
             
          
           
               
                 Untreated Control 
                   
                   
                  0.0a* 
                   
               
               
                 (deionized water) 
                   
                   
                   
                   
               
               
                 Bialaphos 
                 0.089 
                   
                  0.0 a 
                   
               
               
                 Bialaphos 
                 0.178 
                   
                  0.0 a 
                   
               
               
                 Bialaphos 
                 0.356 
                   
                  0.0 a 
                   
               
               
                 Bialaphos 
                 0.534 
                   
                  0.0 a 
                   
               
               
                 Bialaphos 
                 0.712 
                   
                  0.0 a 
                   
               
               
                 Bialaphos 
                 0.890 
                   
                  1.3 ab 
                   
               
               
                 Bialaphos 
                 1.068 
                   
                  5.0 abc 
                   
               
               
                 MBI-005 (thaxtomin A) 
                   
                 0.25 
                  1.3 ab 
                   
               
               
                 Bialaphos + MBI-005 
                 0.089 
                 0.25 
                  1.3 ab 
                 1.0 
               
               
                 Bialaphos + MBI-005 
                 0.178 
                 0.25 
                  3.8 abc 
                 3.0 
               
               
                 Bialaphos + MBI-005 
                 0.356 
                 0.25 
                 11.9 c 
                 9.5 
               
               
                 Bialaphos + MBI-005 
                 0.534 
                 0.25 
                 29.4 d 
                 23.5 
               
               
                 Bialaphos + MBI-005 
                 0.712 
                 0.25 
                 34.4 d 
                 27.5 
               
               
                 Bialaphos + MBI-005 
                 0.890 
                 0.25 
                 59.4 e 
                 23.9 
               
               
                 Bialaphos + MBI-005 
                 1.068 
                 0.25 
                 62.5 e 
                 10.1 
               
               
                   
               
               
                 *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&#39;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 &lt;1 the combination is antagonistic; if E/Ee = 1 the combination is additive; if E/Ee &gt;1 the combination is synergistic). 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Effects of bialaphos, MBI-005 (thaxtomin A), 
               
               
                 and the combinations of bialaphos with 
               
               
                 MBI-005 in controlling barnyard grass. 
               
             
          
           
               
                   
                 Bialaphos 
                 MBI-005/011 
                 % Control 
                   
               
               
                 Treatment 
                 (mg/mL) 
                 (mg/mL) 
                 (14 days) 
                 E/Ee 
               
               
                   
               
             
          
           
               
                 Untreated Control 
                   
                   
                  0.0 a* 
                   
               
               
                 (deionized water) 
                   
                   
                   
                   
               
               
                 Bialaphos 
                 0.18 
                   
                  6.7 bc 
                   
               
               
                 Bialaphos 
                 0.53 
                   
                  3.3 ab 
                   
               
               
                 Bialaphos 
                 1.07 
                   
                 10.0 c 
                   
               
               
                 MBI-005 (thaxtomin A) 
                   
                 0.25 
                  6.7 bc 
                   
               
               
                 Bialaphos + MBI-005 
                 0.18 
                 0.25 
                  5.0 abc 
                 0.4 
               
               
                 Bialaphos + MBI-005 
                 0.53 
                 0.25 
                 25.0 d 
                 2.6 
               
               
                 Bialaphos + MBI-005 
                 1.07 
                 0.25 
                 41.7 e 
                 2.6 
               
               
                   
               
               
                 *Treatment means in each column marked with the same letter are not statistically different with LSD test at p = 0.05 level 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Effects of bialaphos, MBI-005 (thaxtomin A), and the combinations of 
               
               
                 bialaphos with MBI-005 or MBI-011 in controlling barnyard grass. 
               
             
          
           
               
                   
                   
                   
                 % 
                   
                   
               
               
                   
                 Bialaphos 
                 MBI-005 
                 Control 
                 % Control 
                 E/Ee 
               
               
                 Treatment 
                 (mg/mL) 
                 (mg/mL) 
                 (7 days) 
                 (14 days) 
                 (7 days) 
               
               
                   
               
               
                 Untreated Control 
                   
                   
                   
                  0.0 a 
                   
               
               
                 (deionized water) 
                   
                   
                  0.0 a* 
                   
                   
               
               
                 Bialaphos 
                 1.1 
                   
                  5.0 ab 
                  3.7 a 
                   
               
               
                 Bialaphos 
                 1.4 
                   
                 10.8 b 
                  23.3 b 
                   
               
               
                 Bialaphos 
                 1.8 
                   
                 62.5 c 
                  66.7 c 
                   
               
               
                 MBI-005 
                   
                 0.38 
                  6.7 ab 
                  21.7 b 
                   
               
               
                 (thaxtomin A) 
                   
                   
                   
                   
                   
               
               
                 Bialaphos + 
                 1.1 
                 0.38 
                 87.5 d 
                 100.0 d 
                 7.7 
               
               
                 MBI-005 
                   
                   
                   
                   
                   
               
               
                 Bialaphos + 
                 1.4 
                 0.38 
                 87.5 d 
                 100.0 d 
                 5.2 
               
               
                 MBI-005 
                   
                   
                   
                   
                   
               
               
                 Bialaphos + 
                 1.8 
                 0.38 
                 87.5 d 
                 100.0 d 
                 1.3 
               
               
                 MBI-005 
               
               
                   
               
               
                 *Treatment means in each column marked with the same letter are not statistically different with LSD at p = 0.05 level. 
               
             
          
         
       
     
     Example 11 
     The test species barnyard grass, ragweed, sedge, and broad-leaf mustard were used for the valuation of synergy between MBI-005 and the rice herbicides clomazone, penoxsulam, cyhalofop, fenoxaprop-p-ethyl, bispyribac-sodium, thiobencarb, and propanil. 
     The common turf weeds dandelion and plantain were used in testing for synergy between MBI-005 and 2, 4- or dicamba, two common turf herbicides. 
     Three other herbicides commonly used for field crops, glyphosate, glufosinate, synthetic version of bialaphos, and mesotrione were also tested for synergy with MBI-005 on crabgrass and ragweed. 
     There were 3 replicates per treatment which were sprayed with approximately ⅔ ml per replicate. The treatments were completely randomized and kept in a greenhouse at 25° C. The efficacy was rated at 7 and 14 days post treatment. The results are shown in Table 9 to 11. For barnyard grass control, MBI-005 had synergistic effects when combined with clomazone, (penoxsulam, bispyribac-sodium, thiobencarb, and propanil (Table 9). MBI-005 had additive effects when combined with cyhalofop, and fenoxaprop-p-ethyl (Table 10). 
     MBI-005 showed great synergy with glyphosate for controlling ragweed and also showed synergy with both turf herbicides. The synergistic effect of MBI-005 with glufosinate (synthetic bialaphos) (Table 11) on crabgrass was likely less since the rate of MBI-005 was too low. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Summary of synergistic or additive effects between MBI-005 (thaxtomin 
               
               
                 A) and commercial products for rice weed control. The data are means 
               
               
                 of percentage control of three replicates 14 days post treatment. 
               
             
          
           
               
                   
                   
                 Product 
                 MBI-005 
                 % 
                 % 
                 % Control 
                   
               
               
                   
                 Test 
                 Rate 
                 Rate 
                 Control 
                 Control 
                 Product + 
               
               
                 Active Ingredient 
                 Species 
                 (mg/mL) 
                 (mg/mL) 
                 Product 
                 MBI-005 
                 MBI-005 
                 E/Ee 
               
               
                   
               
             
          
           
               
                 clomazone 
                 Barnyard 
                 0.513 
                 0.25 
                 37.5 
                 28.3 
                 91.7 
                 1.7 
               
               
                   
                 grass 
               
               
                   
                 Mustard 
                 0.513 
                 0.25 
                 25.0 
                 58.3 
                 70.8 
                 1.0 
               
               
                   
                 Sedge 
                 0.501 
                 0.02 
                 0.0 
                 37.5 
                 50.0 
                 1.3 
               
               
                 penoxsulam 
                 Barnyard 
                 0.051 
                 0.125 
                 25.0 
                 17.5 
                 75.0 
                 2.0 
               
               
                   
                 grass 
               
               
                   
                 Sedge 
                 0.047 
                 0.01 
                 66.7 
                 20.0 
                 87.5 
                 1.2 
               
               
                 cyhalofop 
                 Mustard 
                 0.051 
                 0.125 
                 11.7 
                 13.3 
                 15.0 
                 0.6 
               
               
                   
                 Sedge 
                 1.176 
                 0.01 
                 3.3 
                 20.0 
                 8.3 
                 0.4 
               
               
                 fenoxaprop-p-ethyl 
                 Barnyard 
                 0.006 
                 0.25 
                 91.7 
                 66.7 
                 87.5 
                 0.9 
               
               
                   
                 grass 
               
               
                   
                 Mustard 
                 0.006 
                 0.125 
                 0.0 
                 45.8 
                 33.3 
                 0.7 
               
               
                   
                 Sedge 
                 0.116 
                 0.02 
                 0.0 
                 70.8 
                 66.7 
                 0.9 
               
               
                 bispyribac-sodium 
                 Barnyard 
                 0.032 
                 0.125 
                 0.0 
                 3.3 
                 62.5 
                 18.8 
               
               
                   
                 grass 
               
               
                   
                 Mustard 
                 0.0216 
                 0.25 
                 53.3 
                 37.5 
                 95.8 
                 1.4 
               
               
                   
                 Ragweed 
                 0.0216 
                 0.125 
                 5.0 
                 23.3 
                 50.0 
                 1.8 
               
               
                 thiobencarb 
                 Barnyard 
                 1.743 
                 0.25 
                 41.7 
                 41.7 
                 79.2 
                 1.2 
               
               
                   
                 grass 
               
               
                   
                 Mustard 
                 1.743 
                 0.125 
                 10.0 
                 54.2 
                 54.2 
                 0.9 
               
               
                   
                 Sedge 
                 3.15 
                 0.02 
                 58.3 
                 37.5 
                 70.8 
                 1.0 
               
               
                 propanil 
                 Barnyard 
                 0.365 
                 0.125 
                 32.5 
                 10.0 
                 79.2 
                 2.0 
               
               
                   
                 grass 
               
               
                   
                 Mustard 
                 0.036 
                 0.25 
                 1.67 
                 58.3 
                 50.0 
                 0.9 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Summary of synergistic or additive effects between MBI-005 (thaxtomin 
               
               
                 A) and commercial products for turf weed control. The data are means 
               
               
                 of percentage control of three replicates 14 days post treatment. 
               
             
          
           
               
                   
                   
                   
                 MBI-005 
                   
                   
                 % Control 
                   
               
               
                 Active 
                 Weed 
                 Product Rate 
                 Rate 
                 % Control 
                 % Control 
                 Product + 
               
               
                 Ingredient 
                 Species 
                 (mg/mL) 
                 (mg/mL) 
                 Product 
                 MBI-005 
                 MBI-005 
                 E/Ee 
               
               
                   
               
             
          
           
               
                 2, 4-D 
                 Dandelion 
                 0.176 
                 0.1 
                 25.0 
                 20.0 
                 91.7 
                 2.3 
               
               
                   
                 Plantain 
                 2.340 
                 0.24 
                 50.0 
                 25.0 
                 83.3 
                 1.3 
               
               
                 dicamba 
                 Dandelion 
                 0.121 
                 0.1 
                 45.8 
                 20.0 
                 75.0 
                 1.3 
               
               
                   
                 Plantain 
                 6.025 
                 0.12 
                 50.0 
                 10.0 
                 79.2 
                 1.4 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Summary of synergistic or additive effects between MBI-005 (thaxtomin 
               
               
                 A) and commercial products with broad spectrum. The data are means 
               
               
                 of percentage control of three replicates 14 days post treatment. 
               
             
          
           
               
                   
                   
                   
                 MBI-005 
                   
                   
                 % Control 
                   
               
               
                 Active 
                 Weed 
                 Product Rate 
                 Rate 
                 % Control 
                 % Control 
                 Product + 
               
               
                 Ingredient 
                 Species 
                 (mg/mL) 
                 (mg/mL) 
                 Product 
                 MBI-005 
                 MBI-005 
                 E/Ee 
               
               
                   
               
             
          
           
               
                 glyphosate 
                 Crabgrass 
                 0.754 
                 0.125 
                 45.8 
                 28.3 
                 83.3 
                 1.4 
               
               
                   
                 Ragweed 
                 2.198 
                 0.125 
                 15.0 
                 6.7 
                 29.2 
                 1.4 
               
               
                 glufosinate 
                 Crabgrass 
                 0.151 
                 0.125 
                 83.3 
                 20.0 
                 75.0 
                 0.9 
               
               
                 (bialaphos) 
                 Ragweed 
                 0.194 
                 0.125 
                 22.5 
                 37.5 
                 75.0 
                 1.5 
               
               
                 mesotrione 
                 Crabgrass 
                 0.24 
                 0.125 
                 62.5 
                 24.2 
                 75.0 
                 1.1 
               
               
                   
                 Ragweed 
                 0.96 
                 0.125 
                 25.0 
                 15.0 
                 45.8 
                 1.3 
               
               
                   
               
             
          
         
       
     
     Example 12 
     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 CO2 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 12 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 12 
               
               
                   
               
             
             
               
                 Pre-Emergent Effect of MBI-005. An (R) indicates an herbicide resistant population. 
               
             
          
           
               
                   
                   
                   
                   
                 Test Scale 
               
               
                   
                   
                   
                 Rating at 
                 (lab/ 
               
               
                 Plant Category 
                 Species (common name) 
                 Species (scientific name) 
                 0.1 mg/ml 
                 field) 
               
               
                   
               
               
                 Grass, annual 
                 Crabgrass 
                 
                   Digitaria sanguinalis 
                 
                 ++++ 
                 lab &amp; field 
               
               
                   
                 Bluegrass 
                 
                   Poa annua 
                 
                 ++++ 
                 lab &amp; field 
               
               
                   
                 Barnyardgrass 
                 
                   Echinochloa crus-gali 
                 
                 ++++ 
                 lab 
               
               
                   
                 Late watergrass (R) 
                 
                   Echinochloa phyllopogon 
                 
                 ++++ 
                 lab 
               
               
                   
                 Canarygrass 
                 
                   Phalaris minior 
                 
                 ++++ 
                 lab 
               
               
                   
                 Bearded Sprangletop 
                 
                   Leptochloa fascicularis 
                 
                 ++++ 
                 lab 
               
               
                   
                 Bristlegrass/Foxtail 
                 
                   Setaria viridis 
                 
                 ++++ 
                 lab 
               
               
                   
                 Fall Panicum 
                 
                   Panicum dichotomiflorum 
                 
                 ++++ 
                 lab 
               
               
                 Grass, perennial 
                 Ryegrass 
                 
                   Lolium perenne 
                 
                 ++++ 
                 lab 
               
               
                 Broadleaf, annual 
                 Ragweed 
                 
                   Ambrosia artemisifolia 
                 
                 ++++ 
                 lab 
               
               
                   
                 Buckhorn plantain 
                 
                   Plantago lanceolata 
                 
                 ++++ 
                 lab 
               
               
                   
                 Mustard 
                 
                   Brassica juncea 
                 
                 ++++ 
                 lab 
               
               
                   
                 Lambsquarters 
                 
                   Chenopodium album 
                 
                 +++ 
                 lab 
               
               
                   
                 Black nightshade 
                 
                   Solanum nigrum 
                 
                 ++++ 
                 lab 
               
               
                   
                 Clover 
                 
                   Trifolium repens 
                 
                 ++++ 
                 lab 
               
               
                   
                 Horseweed (R) 
                 
                   Conyza canadensis 
                 
                 ++++ 
                 lab 
               
               
                   
                 Palmer pigweed (R) 
                 
                   Amaranthus palmerii 
                 
                 +++ 
                 lab 
               
               
                   
                 Chickweed 
                 
                   Stellaria media 
                 
                 ++++ 
                 Field 
               
               
                   
                 Eclipta 
                 
                   Eclipta alba 
                 
                 ++ 
                 Field 
               
               
                   
                 Bittercress 
                 
                   Cardamine hirsuta 
                 
                 +++ 
                 Field 
               
               
                   
                 Henbit 
                 
                   Lamimum amplexicaule 
                 
                 +++ 
                 Field 
               
               
                   
                 Common groundsel 
                 
                   Senecio vulgaris 
                 
                 ++++ 
                 Field 
               
               
                   
                 Velvetleaf 
                 
                   Abutilon theophrasti 
                 
                 ++ 
                 Field 
               
               
                   
                 Shepherd&#39;s purse 
                 
                   Capsella bursa-pastora 
                 
                 ++++ 
                 Field 
               
               
                   
                 Hairy galinsoga 
                 
                   Galinsoga ciliata 
                 
                 +++ 
                 Field 
               
               
                   
                 New Zealand moss 
                 
                   Sagina procumbens 
                 
                 ++++ 
                 Field 
               
               
                   
                 Marsh watercress 
                 
                   Rorippa islandica 
                 
                 ++++ 
                 Field 
               
               
                   
                 Flexuous bittercress 
                 
                   Cardamine flexuosa 
                 
                 ++ 
                 Field 
               
               
                   
                 Common yellow oxalis 
                 
                   Oxalis stricta 
                 
                 ++++ 
                 Field 
               
               
                 Broadleaf, 
                 Dandelion 
                 
                   Taraxacum oficinale 
                 
                 ++++ 
                 Field 
               
               
                 perennial 
               
               
                   
               
             
          
           
               
                 Key-Table 12A 
                   
               
               
                 Reduction of 
                   
               
               
                 Germination (%) 
                 Rating 
               
               
                  0-59 
                 + 
               
               
                 60-84 
                 ++ 
               
               
                 85-94 
                 +++ 
               
               
                 95-100 
                 ++++ 
               
               
                   
               
             
          
         
       
     
     Example 13 
     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 1 mg 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 13, 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 13 
               
               
                   
               
             
             
               
                 Post-Emergent Effect of MBI-005 
               
             
          
           
               
                   
                   
                   
                   
                 Test 
               
               
                 Plant 
                 Species (common 
                 Species (scientific 
                 Rating at 
                 Scale 
               
               
                 Category 
                 name) 
                 name) 
                 1 mg/ml 
                 (Lab/Field) 
               
               
                   
               
               
                 Grass, annual 
                 Late watergrass 
                 
                   Echinochloa phyllopogon 
                 
                 +++ 
                 Field 
               
               
                   
                 Bluegrass 
                 
                   Poa annua 
                 
                 ++ 
                 Field 
               
               
                 Sedge, annual 
                 Smallflower 
                 
                   Cyperus difformis 
                 
                 ++++ 
                 Field 
               
               
                   
                 Ricefield bullrush 
                 
                   Scirpus mucronatus 
                 
                 ++++ 
                 Field 
               
               
                 Sedge, 
                 Yellow nutsedge 
                 
                   Cyperus esculentus 
                 
                 + 
                 Lab 
               
               
                 perennial 
                   
                   
                   
                   
               
               
                 Broadleaf, 
                 Redstem filaree 
                 
                   Erodium cicutarium 
                 
                 ++ 
                 Lab 
               
               
                 annual 
                 Mustard 
                 
                   Brassica juncea 
                 
                 ++++ 
                 Lab 
               
               
                   
                 Lambsquarters 
                 
                   Chenopodium album 
                 
                 +++ 
                 Lab 
               
               
                   
                 Arrowhead 
                 
                   Sagittaria montevidensis 
                 
                 ++++ 
                 Field 
               
               
                   
                 Velvetleaf 
                 
                   Abutilon theophrasti 
                 
                 ++ 
                 Field 
               
               
                   
                 Shepherd&#39;s purse 
                   Capsella bursa - pastoris   
                 ++++ 
                 Field 
               
               
                   
                 Hairy galinsoga 
                 
                   Galinsoga ciliata 
                 
                 +++ 
                 Field 
               
               
                   
                 Nightshade, Eastern 
                 
                   Solanum ptychanthum 
                 
                 ++ 
                 Field 
               
               
                   
                 Black 
                   
                   
                   
               
               
                   
                 Common yellow 
                   Oxalis pes - caprae   
                 ++++ 
                 Field 
               
               
                   
                 oxalis 
                   
                   
                   
               
               
                   
                 Clover 
                 
                   Trifolium hirtum 
                 
                 ++ 
                 Field 
               
               
                   
                 Sunflower 
                 
                   Helianthus anuus 
                 
                 ++ 
                 Lab 
               
               
                   
                 Mallow 
                 
                   Malva parviflora 
                 
                 + 
                 Lab 
               
               
                   
                 Common chickweed 
                 
                   Stellaria media 
                 
                 ++ 
                 Field 
               
               
                   
                 Eclipta 
                 
                   Eclipta alba 
                 
                 ++ 
                 Field 
               
               
                   
                 Hairy bittercress 
                 
                   Cardamine hirsuta 
                 
                 ++ 
                 Field 
               
               
                   
                 Henbit 
                 
                   Lamimum amplexicaule 
                 
                 ++++ 
                 Field 
               
               
                   
                 Common groundsel 
                 
                   Senecio vulgaris 
                 
                 ++++ 
                 Field 
               
               
                   
                 Marsh watercress 
                 
                   Rorippa islandica 
                 
                 ++ 
                 Field 
               
               
                   
                 Flexuous bittercress 
                 
                   Cardamine flexuosa 
                 
                 ++ 
                 Field 
               
               
                 Broadleaf, 
                 Dandelion 
                 
                   Taraxacum oficinale 
                 
                 ++ 
                 Lab 
               
               
                 perennial 
                   
                   
                   
                   
               
               
                   
                 Bindweed 
                 
                   Convolvulus arvensis 
                 
                 + 
                 Lab 
               
               
                   
                 Bermuda buttercup 
                   Oxalis pes - caprae   
                 ++++ 
                 Field 
               
               
                   
               
             
          
           
               
                 Key-Table 13A 
               
             
          
           
               
                   
                 Post-Emergence 
               
               
                 Rating 
                 % control 
               
               
                 + 
                  0-50 
               
               
                 ++ 
                 51-80 
               
               
                 +++ 
                 81-90 
               
               
                 ++++ 
                 91-100 
               
               
                   
               
             
          
         
       
     
     Example 14 
     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 ( FIG. 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&#39;s formula. Results are shown in  FIG. 2   
     Example 15 
     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 14). 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&#39;s formula. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 005 + m-Tyrosine Pre-Emergent Synergies in Crabgrass (7 DAT) 
               
             
          
           
               
                   
                   
                   
                   
                 E (% 
                   
                   
               
               
                   
                   
                 AVG No. 
                   
                 controlled 
                 Ee 
               
               
                 Treat- 
                   
                 germi- 
                   
                 compared 
                 (expected 
                 E/ 
               
               
                 ment 
                 Description 
                 nated 
                 SE 
                 to UTC) 
                 control) 
                 Ee 
               
               
                   
               
             
          
           
               
                 1 
                 Untreated 
                 44.3 
                 .9 
                 0.0 
                 — 
                 — 
               
               
                   
                 control 
               
               
                 2 
                 .22 mg/mL 
                 40.7 
                 2.6 
                 8.3 
                 — 
                 — 
               
               
                   
                 M-tyrosine 
               
               
                 3 
                 .44 mg/mL 
                 44.0 
                 3.8 
                 0.8 
                 — 
                 — 
               
               
                   
                 M-tyrosine 
               
               
                 4 
                 MBI-005 at 
                 38.0 
                 4.0 
                 14.3 
                 — 
                 — 
               
               
                   
                 0.0035 mg/mL 
               
               
                 5 
                 MBI-005 at 
                 35.0 
                 2.1 
                 21.1 
                 21.4 
                 1.0 
               
               
                   
                 0.0035 mg/ 
               
               
                   
                 mL + .22 
               
               
                   
                 mg/mL M- 
               
               
                   
                 tyrosine 
               
               
                 6 
                 MBI-005 at 
                 26.0 
                 2.1 
                 41.4 
                 14.9 
                 2.8 
               
               
                   
                 0.0035 mg/ 
               
               
                   
                 mL + .44 
               
               
                   
                 ml/mL M- 
               
               
                   
                 tyrosine 
               
               
                   
               
             
          
         
       
     
     Example 16 
     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&#39;s formula. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 MBI-005 + Chelated Fe Post-Emergent Synergies in Crabgrass (15 DAT) 
               
             
          
           
               
                   
                 Crabgrass (15 DAT) 
                   
               
             
          
           
               
                 Description 
                 AVG E 
                 STD DEV 
                 Ee 
                 E/Ee 
               
               
                   
               
             
          
           
               
                 Untreated Control 
                 0.0 
                 0.0 
                 — 
                 — 
               
               
                 12.5% MBI-005 
                 1.3 
                 2.5 
                 — 
                 — 
               
               
                 (0.125 mg/mL) 
                   
                   
                   
                   
               
               
                 25% MBI-005 (0.25 mg/mL) 
                 1.3 
                 2.5 
                 — 
                 — 
               
               
                 95% Chelated Fe Field Rate 
                 6.3 
                 2.5 
                 — 
                 — 
               
               
                 (14.49 mg/ml) 
                   
                   
                   
                   
               
               
                 12.5% MBI-005 
                 25.0 
                 0.0 
                 7.4 
                 3.4 
               
               
                 (0.125 mg/mL)/95% Chelated 
                   
                   
                   
                   
               
               
                 Fe 
                   
                   
                   
                   
               
               
                 25% MBI-005 (.25 mg/mL)/ 
                 25.0 
                 0.0 
                 7.4 
                 3.4 
               
               
                 95% Chelated Fe 
               
               
                   
               
             
          
         
       
     
     Example 17 
     MBI-005+Atrazine Post-Emergent Synergies in Crabgrass (7 and 14 DAT) 
     Post-emergent synergistic effects of MBI-005 and Atrazine 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, and 35% of the atrazine label rate (0.3 gallons/A), 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 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death. 
     A substantial synergistic effect occurred between MBI-005 and atrazine, at either concentration of MBI-005 at both 7 and 14 days after treatment. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby&#39;s formula. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 MBI-005 + Atrazine Post-Emergent 
               
               
                 Synergies in Crabgrass (7 and 14 DAT) 
               
             
          
           
               
                   
                 7DAT 
                   
                 14DAT 
                   
               
               
                   
                 Crabgrass 
                   
                 Crabgrass 
               
             
          
           
               
                   
                   
                 STD 
                   
                 AVG/ 
                 STD 
                 E/ 
               
               
                 Description 
                 AVG E 
                 DEV 
                 Ee 
                 E 
                 DEV 
                 Ee 
               
               
                   
               
             
          
           
               
                 Untreated Control 
                 0.0 
                 0.0 
                 — 
                 0.0 
                 0.0 
                 — 
               
               
                 12.5% MBI-005 Label 
                 10.0 
                 0.0 
                 — 
                 10.0 
                 0.0 
                 — 
               
               
                 Rate (0.125 mg/mL) 
               
               
                 25% MBI-005 Label 
                 11.9 
                 3.8 
                 — 
                 11.9 
                 3.8 
                 — 
               
               
                 Rate(0.25 mg/mL) 
               
               
                 35% Atrazine Label 
                 11.9 
                 3.8 
                 — 
                 8.8 
                 2.5 
                 — 
               
               
                 Rate 
               
               
                 12.5% MBI-005/35% 
                 65.6 
                 12.0 
                 3.2 
                 78.1 
                 12.0 
                 4.4 
               
               
                 Atrazine 
               
               
                 25% MBI-005/35% 
                 75.0 
                 0.0 
                 3.4 
                 90.6 
                 6.3 
                 4.6 
               
               
                 Atrazine 
               
               
                   
               
             
          
         
       
     
     Example 18 
     Post-Emergent Effect of MBI-005+Rice Herbicides in Barnyard Grass 
     Post-emergent synergistic effects of MBI-005 and three common rice herbicides were evaluated on barnyard grass at the 1-2 leaf stage in a laboratory experiment. Ten seedlings in 2.5 cm square pots containing plant growth mix were sprayed to full coverage using a hand 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 three times and evaluated at 7 and 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death. 
     Synergistic effect were noted between MBI-005 and all three rice herbicides, 14 days after treatment. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby&#39;s formula. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                 Post-Emergent Effect of MBI-005 + Rice Herbicides in Barnyard Grass 
               
             
          
           
               
                   
                   
                   
                   
                   
                 % control 
                   
               
               
                   
                 % v/v of 
                 % of the 
                 % control 
                   
                 MBI-005 + 
               
               
                 Chemical 
                 label 
                 label rate 
                 chemical 
                 % control 
                 chemical 
                 Synergy 
               
               
                 Active Ingredient (AI) 
                 rate (AI) 
                 MBI 005 
                 AI 
                 MBI-005 
                 AI 
                 coefficient 
               
               
                   
               
             
          
           
               
                 Clomazone 
                 13.3 
                 25% 
                 37.50 
                 28.30 
                 91.66 
                 1.66 
               
               
                 Penoxsulam 
                 13.0 
                 12.5%     
                 25.0 
                 17.5 
                 75.0 
                 1.97 
               
               
                 Bispyribac-sodium 
                 13.3 
                 25% 
                 6.7 
                 41.7 
                 83.3 
                 1.83 
               
               
                   
               
             
          
         
       
     
     Example 19 
     Post-Emergent Effect of MBI-005+Rice Herbicides in Smallflower Umbrella Sedge 
     Post-emergent synergistic effects of MBI-005 and five common rice herbicides were evaluated on small flower umbrella sedge at the 0.5-1 leaf stage in a laboratory experiment. Twenty seedlings in 2.5 cm square pots containing plant growth mix were sprayed to full coverage using a hand 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 three times and evaluated at 7 and 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death. 
     Synergistic effects were noted 14 days after treatment between MBI-005 and all three rice herbicides, with clomazone showing the greatest synergism with MBI-005. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby&#39;s formula. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 18 
               
             
             
               
                   
               
               
                 Post-Emergent Effect of MBI-005 + Rice Herbicides in Smallflower Umbrella Sedge 
               
             
          
           
               
                   
                   
                   
                   
                   
                 % control 
                   
               
               
                   
                 % v/v of 
                 % of the 
                 % 
                   
                 MBI-005 + 
               
               
                 Chemical Active 
                 label rate 
                 label rate 
                 control 
                 % control 
                 chemical 
                 Synergy 
               
               
                 Ingredient (AI) 
                 AI 
                 MBI 005 
                 AI 
                 MBI-005 
                 AI 
                 coefficient 
               
               
                   
               
             
          
           
               
                 Clomazone 
                 13% 
                 2.0% 
                 0.0 
                 20.0 
                 70.8 
                 3.54 
               
               
                 Penoxsulam 
                 13% 
                 2.0% 
                 33.3 
                 20.0 
                 66.7 
                 1.43 
               
               
                 Bispyribac-sodium 
                 13.3%     
                 0.5% 
                 37.5 
                 3.3 
                 62.5 
                 1.57 
               
               
                 Thiobencarb 
                 25% 
                 2.0% 
                 41.7 
                 20.0 
                 83.3 
                 1.56 
               
               
                 Propanil 
                  .5% 
                 2.0% 
                 62.5 
                 20.0 
                 83.3 
                 1.19 
               
               
                   
               
             
          
         
       
     
     Example 20 
     Post-Emergent Effect of MBI-005+Propanil on Barnyard Grass 
     Post-emergent synergistic effects of MBI-005 and the common rice herbicide Propanil were evaluated on barnyard grass at the 1-2 leaf stage in a laboratory experiment. Ten seedlings in 2.5 cm square pots containing plant growth mix planting media were sprayed with 12.5% or 25% of the label rate of MBI-005, 25% of the propanil label rate, or a combination thereof, to full coverage using a hand 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 three times and evaluated at 7 and 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death. 
     Synergistic effects were noted between both rates of MBI-005 and propnail, 14 days after treatment. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby&#39;s formula. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Post-Emergent Effect of MBI-005 + Propanil on Barnyard Grass 
               
             
          
           
               
                   
                   
                 Observed Average 
                   
                   
               
               
                   
                 Treatment Description 
                 % Efficacy (E) 
                 Ee 
                 E/Ee 
               
               
                   
               
             
          
           
               
                   
                 UTC (No spray) 
                 0.0 
                   
                   
               
               
                   
                 12.5% MBI-005 
                 10.0 
                   
                   
               
               
                   
                 25% MBI-005 
                 20.0 
                   
                   
               
               
                   
                 25% Propanil 
                 32.5 
                   
                   
               
               
                   
                 250% Propanil + 
                 79.2 
                 39.25 
                 2.02 
               
               
                   
                 12.5% MBI-005 
                   
                   
                   
               
               
                   
                 25% Propanil + 25% 
                 75.0 
                 46.00 
                 1.63 
               
               
                   
                 MBI-005 
               
               
                   
               
             
          
         
       
     
     Example 21 
     Post-Emergent Effect of MBI-005+Thiobencarb on Smallflower Umbrella Sedge 
     Post-emergent synergistic effects of MBI-005 and the common rice herbicide thiobencarb are evaluated on Smallflower umbrella sedge at the 0.5-1 leaf stage in a laboratory experiment. Twenty seedlings in 2.5 cm square pots containing plant growth mix were sprayed with 0.5%, 1% or 2% of the label rate of MBI-005, 25% of the thiobencarb label rate, or a combination thereof, to full coverage using a hand 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 three times and evaluated at 7 and 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death. 
     Synergistic effects were observed for three of the four thiobencarb+MBI-005 combinations, 14 days after treatment. Synergy was calculated as the ratio of observed (E) to expected (E/Ee) percent control using Colby&#39;s formula. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 005 + Thiobencarb Post-Emergent Synergies on Smallflower sedge 
               
             
          
           
               
                   
                   
                 Observed Average 
                   
                   
               
               
                   
                 Treatment Description 
                 % Efficacy (E) 
                 Ee 
                 E/Ee 
               
               
                   
               
             
          
           
               
                   
                 UTC (No spray) 
                 0.0 
                   
                   
               
               
                   
                 .5% MBI-005 
                 3.3 
                   
                   
               
               
                   
                 2% MBI-005 
                 20. 
                   
                   
               
               
                   
                 25% Thiobencarb 
                 41.7 
                   
                   
               
               
                   
                 25% Thiobencarb + 
                 62.5 
                 43.61 
                 1.43 
               
               
                   
                 .5% MBI-005 
                   
                   
                   
               
               
                   
                 25% Thiobencarb + 
                 83.3 
                 53.33 
                 1.56 
               
               
                   
                 2% MBI-005 
                   
                   
                   
               
               
                   
                 UTC (No Spray) 
                 0.0 
                   
                   
               
               
                   
                 1% MBI-005 
                 10.0 
                   
                   
               
               
                   
                 2% MBI-005 
                 79.2 
                   
                   
               
               
                   
                 15% Thiobencarb 
                 25.0 
                   
                   
               
               
                   
                 15% Thiobencarb + 
                 66.7 
                 32.50 
                 2.05 
               
               
                   
                 1% MBI-005 
                   
                   
                   
               
               
                   
                 15% Thiobencarb + 
                 79.2 
                 84.38 
                 0.94 
               
               
                   
                 2% MBI-005 
               
               
                   
               
             
          
         
       
     
     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. 
     Cited References: 
     
         
         Beauséjour, J., C. Goyer, et al. (1999). “Production of thaxtomin A by  Streptomyces scabies  strains in plant extract containing media.”  Can J Microbiol  45: 764-768. 
         Duke, S. O., S. R. Baerson, et al. (2003). “United States Department of Agriculture-Agricultural Research Service research on natural products for pest management.”  Pest Manag Sci  59: 708-717. 
         Duke, S. O., F. E. Dayan, et al. (2000). “Natural products as sources of herbicides: current status and future trends.”  Weed Research  40: 99-111. 
         Fry, B. A. and R. Loxia (2002). “Thaxtomin A: Evidence for a plant cell wall target.”  Physiological and Molecular Plant Pathology  60: 1-8. 
         Gerwick, B. C., P. R. Graupner, et al. (2005). Methylidene mevalonates and their use as herbicides. U. p. 7393812: 16. 
         Healy, F. G., M. J. Wach, et al. (2000). “The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptidesynthetase required for phytotoxin thaxtomin A prodcution and pathogenicity.”  Molecular Microbiology  38: 794-804. 
         Hiltunen, L. H., I. Laakso, et al. (2006). “Influence of thaxtomins in different combinations and concentrations on growth of micropropagated potato shoot cultures.”  J Agric Food Chem  54: 3372-3379. 
         Hoagland, R. E. (2001). “Microbial allelochemicals and pathogens as bioherbicidal agents.”  Weed Technology  15: 835-857. 
         Kang, Y., S. Semones, et al. (2008). Methods of controlling algae with thaxtomin and thaxtomin compositions. USA, Novozymes Biologicals, Inc. 
         King, R. R., C. H. Lawrence, et al. (1992). “Chemistry of phytotoxins associated with  Streptomyces scabies , the causal organism of potato common scab.”  J. Agric. Food Chem  40: 834-837. 
         King, R. R., C. H. Lawrence, et al. (1989). “Isolation and characterization of phytotoxin associated with  Streptomyces scabies.” Journal of the Chemical Society, Chemical Communications  13: 849-850. 
         King, R. R., C. H. Lawrence, et al. (2003). “More chemistry of the thaxtomin phytotoxins.”  Phytochemistry  64: 1091-1096. 
         King, R. R., C. H. Lawrence, et al. (2001). “Herbicidal properties of the thaxtomin group of phytotoxins.”  J Agric Food Chem  49: 2298-2301. 
         Loxia, R., R. A. Bukhalid, et al. (1995). “Differential production of thaxtomins by pathogenic  Streptomyces  species in vitro”  Phytopathology  85: 537-541.