WO1994027444A2 - Nouvelle toxine bacterienne utilisable comme regulateur de la croissance des vegetaux et comme herbicide - Google Patents

Nouvelle toxine bacterienne utilisable comme regulateur de la croissance des vegetaux et comme herbicide Download PDF

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Publication number
WO1994027444A2
WO1994027444A2 PCT/US1994/005808 US9405808W WO9427444A2 WO 1994027444 A2 WO1994027444 A2 WO 1994027444A2 US 9405808 W US9405808 W US 9405808W WO 9427444 A2 WO9427444 A2 WO 9427444A2
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tabtoxinine
lactam
toxin
weeds
atcc
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PCT/US1994/005808
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English (en)
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WO1994027444A3 (fr
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A. Lawrence Christy
Stanley J. Kostka
Kathleen A. Herbst
James P. Mullen
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Crop Genetics International Corporation
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Priority to AU70437/94A priority Critical patent/AU7043794A/en
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Publication of WO1994027444A3 publication Critical patent/WO1994027444A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/44—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom three- or four-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams

Definitions

  • the present invention relates to a herbicidal composition produced by bacteria, methods for its production, and its use for controlling the growth of plants.
  • bialaphos L-2-amino-4- [ (hydroxy) (methyl)phosphinoyl]-butyl-L-alanyl-L-alanine, which is sold under the trade name HERBIACE®.
  • Pathovars of Pseudomonas syrin ⁇ ae can infect plants, and some phytotoxins produced by these bacteria have been identified.
  • U.S. Patent No. 4,874,706 to Durbin et al. discloses the isolation of a plant toxin (tagetitoxin) produced by Pseudomonas syrin ⁇ ae pv. taqetis. Tagetitoxin has not been developed as a commercial herbicide.
  • Another toxin, tabtoxin is produced by certain pathovars of Pseudomonas syrinqae including Pseudomonas syrin ae pv.
  • a conditioned medium may enhance the effects of a chemical herbicide is that the medium may contain a phytotoxin which is present in too low a concentration to cause measurable injury when applied alone but which enhances the action of the chemical herbicide.
  • adding low levels of a known herbicide to a medium in which bacteria have grown can be used to assay for phytotoxins excreted by bacteria into culture medium, even though the toxin may be present in amounts insufficient to cause injury when the conditioned medium alone is applied to plants.
  • TBL The active herbicidal component of the toxin produced by Pseudomonas syrinqae ATCC #55090 is tabtoxinine-0-lactarn (2- amino-4-(3-hydroxy-2-oxoazacyclobutan-3-yl)-butanoic acid; "TBL” ) (1).
  • TBL tabtoxinine-0-lactarn (2- amino-4-(3-hydroxy-2-oxoazacyclobutan-3-yl)-butanoic acid; "TBL” ) (1).
  • toxin or “phytotoxin” refers to TBL, both in its purified and unpurified forms.
  • the term “toxin” or “phytotoxin” also refers to the derivatives and salts of TBL.
  • Tabtoxin (2) is an unusual dipeptide containing a tabtoxinine- ⁇ -lactam residue linked to the amino group of threonine or serine. W.W. Stewart, Isolation and proof of structure of wildfire toxin. Nature 229:174-178 (1971).
  • Tabtoxin is produced by pathovars of Pseudomonas syrinqae, including Pseudomonas syrinqae pv. tabaci, Pseudomonas syrinqae pv. coronafaciens and Pseudomonas syrinqae pv.
  • Tabtoxin was historically thought to be the active toxin, but tabtoxin was later shown to be a precursor that undergoes hydrolysis to yield the biologically active form, TBL. Uchytil, T.F. and Durbin, R.D., "Hydrolysis of Tabtoxin by Plant and Bacterial Enzymes," Experientia 36:301-302 (1980).
  • the NMR spectrum of tabtoxin is described by W.W. Stewart, "Isolation and Proof of Structure of Wildfire Toxin," Nature, 229:174-178 (1971).
  • Tabtoxinine- ⁇ -lactam has been reported to inhibit glutamine synthetase. Thomas et al. , “Inhibition of Glutamine Synthetase from Pea by Tabtoxinine- ⁇ -lactam, " Plant Phvsiol. 71:912-915 (1983). The production and regulation of tabtoxin production has been studied at the genetic level. See T.G. Kinscherf, et al., "Cloning and Expression of Tabtoxin Biosynthetic Region From Pseudomonas syrinqae," J. Bacteriol. 173:4124-4132 (1991); T.M. Barta, et al., “Regulation of Tabtoxin Produced by the lemA Gene in Pseudomonas syrinqae. " J. Bacteriol. 174:3021-3029 (1992).
  • tabtoxinine- ⁇ -lactam has been isolated previously and its mechanism of action studied, it has never been successfully developed as a commercial herbicide. It is highly surprising and unexpected that tabtoxinine- ⁇ -lactam can be used as a commercial herbicide. Indeed, there are no reports in which tabtoxinine- ⁇ -lactam was topically applied on the surface of weeds or to soil. In addition, there have been no reports that tabtoxinine- -lactam topically applied to one leaf of a plant will move to other leaves and kill the entire plant.
  • TBL tabtoxinine-0- lactam
  • TBL can be topically delivered to weeds or soil via standard commercial application methods to reproducibly achieve commercial levels of weed control in the field. Topically applied TBL moves rapidly across the plant epidermis and inside plants it moves from the site of application throughout the plant, resulting in highly effective control of unwanted target vegetation.
  • the present invention comprises methods for the control of weeds by topically applying a herbicidal composition comprising tabtoxinine- ⁇ -lactam to the weeds as a plant growth regulator and herbicide.
  • a herbicidal composition comprising tabtoxinine- ⁇ -lactam
  • tabtoxinine-/9- lactam and "toxin” as used herein encompasses not only tabtoxinine- ⁇ -lactam but also its herbicidal salts, analogues and derivatives, including precursor compounds such as tabtoxin.
  • the present invention further comprises methods for the production of tabtoxinine- -lactarn produced by fermentation by a bacterium, Pseudomonas syrinqae ATCC #55090.
  • Another embodiment of the invention comprises employing tabtoxinine- ⁇ -lactam produced by Pseudomonas syrinqae ATCC #55090 for the control of weeds.
  • the present invention also comprises a naturally occurring herbicidal composition produced by Pseudomonas syrinqae ATCC #55090 that is useful as a broad spectrum herbicide.
  • the present invention comprises methods for the production of this herbicidal composition by fermentation of the bacterium Pseudomonas syrinqae ATCC #55090.
  • Another embodiment of the present invention comprises a strain of Pseudomonas syrinqae ATCC #55090.
  • Another embodiment of the invention comprises methods for the control of weeds by the topical application of tabtoxinine- ⁇ -lactam in combination with chemical herbicides.
  • Another embodiment of the invention comprises herbicidal compositions comprising mixtures of tabtoxinine- ⁇ -lactam and chemical herbicides.
  • compositions comprising tabtoxinine- ⁇ -lactam for use as harvest aids, and the use of such compositions as a harvest aid to defoliate crops such as potatoes, peanuts and cotton.
  • Another embodiment of the invention comprises a method of controlling the growth of weeds by topically applying tabtoxinine- ⁇ -lactam to the foliage or roots of emergent weeds.
  • Another embodiment of the invention comprises a method for controlling weeds to prevent or inhibit weed growth which comprises applying tabtoxinine- ⁇ -lactam to soil.
  • Another embodiment of the invention comprises a method for controlling aquatic weeds by applying tabtoxinine- ⁇ - lacta to water, including ponds, lakes, streams, rivers and estuaries.
  • a weed is "any plant that is objectionable or interferes with the activities or welfare of man” at the location where it is growing.
  • a herbicide is a “chemical used to control, suppress, or kill plants, or to severely interrupt their normal growth processes. " Herbicide Handbook of the Weed Society of America, Fifth Edition f1983. , xxi-xxiv.
  • Tabtoxin and tabtoxinine- ⁇ -lactam can be produced by chemical synthesis according to methods known to those skilled in the ar:. See, e.g. , J.E. Baldwin, et al., "Stereospecific Synthesis of Tabtoxin,” Tetrahedron, 40(19):3695-3700 (1984); J. Baldwin et al., “Synthetic Studies on Tabtoxin. Synthesis of a Naturally Occurring Inhibitor of Glutamine Synthetase, Tabtoxinine-0-lactam, and Analogues," Tetrahedron 42(12) :3097-3110 (1986); R.E.
  • Tabtoxin may be isolated from culture media in which certain pathovars of Pseudomonas syrinqae have grown. Methods for purification of tabtoxin produced by fermentation are known in the art and have been described in the literature. E.g. , W.W. Stewart, Isolation and proof of structure of wildfire toxin. Nature 229:174-178 (1971).
  • tabtoxinine-j ⁇ -lactam produced by Pseudomonas syringae ATCC #55090 is described below in the Examples.
  • the purified tabtoxinine- ⁇ -lactam may be stored as a dry powder.
  • This strain was originally selected for its ability to infect various target weeds. It is particularly virulent on tobacco. Tobacco plants (Nicotiana tabacum L. ) were used periodically to confirm the virulence of this strain of Pseudomonas syringae.
  • the fatty acid profiles of this bacterium resemble the fatty acids of Pseudomonas syringae pv. papulans.
  • this pathovar differs from Pseudomonas syringae pv. papulans in its virulence toward tobacco and in its production of the toxin described here, which differs from any toxin reported to be produced by Pseudomonas syringae pv. papulans.
  • liquid media can be used to cultivate Pseudomonas syringae ATCC #55090.
  • Compositions of media appropriate for growing microorganisms are well-known in the art, as are methods for growing microorganisms and liquid media on a commercial scale. See, e.g., E.L. Demain and N.A. Solomon (Eds. ), Manual of Industrial Microbiology and Biotechnology, Washington, D.C., American Society for Microbiology, 1986.
  • the media for cultivating Pseudomonas syringae ATCC #55090 and other TBL-producing strains should contain soyflour.
  • optimization of other parameters can also be determined by a person of ordinary skill by means of simple comparative testing.
  • Well-known techniques for the isolation or selection of mutants, either naturally occurring or induced, can be used to obtain mutants and variants of Pseudomonas syringae ATCC #55090 that produce greater quantities of tabtoxinine- ⁇ - lactam. This can also be done by modifying the microorganism using well-known techniques of classical strain improvement, genetic engineering, recombinant DNA and fermentation research.
  • Classical strain improvement involves the use of mutagens such as nitrosoguanidine (NTG), hydroxylamine, ethyl methanesulfonate (EMS), diethylsulfate (DES), and ultraviolet light (UV light).
  • NTG nitrosoguanidine
  • EMS ethyl methanesulfonate
  • DES diethylsulfate
  • UV light ultraviolet light
  • the bacterial cells to be mutagenized are exposed to the mutagen for the desired period of time and grown overnight on Petri plates containing KB media. Individual colonies are then selected from the plates and tested for increased tabtoxinine- ⁇ -lactam production. Once the new strain has demonstrated increased tabtoxinine- ⁇ - lactam production, it becomes the new parent strain and is exposed to the mutagen. This cycle is repeated until the desired level of production is achieved.
  • Strains for tabtoxinine- / 9-lactam production can also be improved by genetic engineering, as is known to those skilled in the art. Basically, genes affecting tabtoxinine-9-lactam production can be isolated, allowing for manipulation of expression to optimize toxin production. More specifically, genes affecting tabtoxinine- -lactam production may be isolated from the current production strain, other tabtoxin or tabtoxinine- -lactam producers, other irelated Pseudomonas species, or unrelated organisms.
  • genes for tabtoxinine-0-lactam biosynthetic enzymes include, for example, genes for tabtoxinine-0-lactam biosynthetic enzymes, genes for regulatory factors affecting biosynthetic gene expression, genes for resistance to tabtoxinine- ⁇ - lactam, genes affecting the stability of tabtoxinine- ⁇ - lactam, or genes for enzymes involved in primary metabolism involved in availability of toxin precursors.
  • genes may be isolated by means known to those skilled in the art, such as transposon mutagenesis and tagging, by genetic selections, by DNA or protein homology with known sequences, or other such means.
  • tabtoxinine- ⁇ -lactam production-related genes may be manipulated, for example, by controlling the level of transcriptional regulatory factors, by changing gene copy number using either plasmids or chromosomally-integrated copies, by using other promoter sequences, by deleting genes, or by similar means.
  • the genes of interest can be transferred to a suitable production host, such as the current production strain, a different toxin producer, a related Pseudomonas species or an unrelated bacterium or fungus.
  • a suitable production host such as the current production strain, a different toxin producer, a related Pseudomonas species or an unrelated bacterium or fungus.
  • the engineered genes may be moved into the production host by those methods known in the art, such as transformation or electrotransformation using phage or plasmid vectors, or by conjugation or transfection.
  • the production strain may be improved by isolating the genes for biosynthesis of tabtoxinine- - lactam, replacing the promoter(s) with strong inducible heterogeneous promoters, such as lac or tac and then moving the new biosynthetic operon into Escherichia coli on a high copy number plasmid, thus allowing for high levels of production in a fermenter upon induction.
  • the bacteria should be removed from the medium.
  • the microorganisms can be removed by well-known methods such as sedimentation and microfiltration or ultrafiltration or reverse osmosis. If desired, the organisms may be killed by well-known methods such as heating, irradiation, or the use of bacteriocidal chemicals.
  • the dead bacteria can be removed from the conditioned medium by sedimentation, filtration or other means.
  • the cell free fermentation medium can then be passed through a column containing a cation exchange resin which binds the toxin and allows most of the spent media components to pass through and be discarded.
  • a cation exchange resin which binds the toxin and allows most of the spent media components to pass through and be discarded.
  • cation exchange resins that may be used are Dowex 50W or Amberlite 200 in the H form.
  • Tabtoxinine-0-lactam can be eluted from the cation exchange resin with an appropriate buffer, concentrated by various drying methods such as lyophilization, rotary evaporation or spray drying, and then formulated for application.
  • the present invention is also directed to salts and derivatives of tabtoxinine-0-lactam.
  • Derivatives of the amino group may be prepared by protonation, alkylation, and/or acylation.
  • Protonated derivatives are prepared by treatment with acids such as sulfuric acid, phosphoric acid and carboxylic acids such as acetic acid, propionic acid and benzoic acid.
  • Alkylated derivatives are prepared by well- known alkylation processes such as treatment of the amine with methyl iodide, bromide, or chloride.
  • Other alkyl and aryl halides can also be employed, such as isopropyl chloride, n-butyl chloride and cyclohexyl chloride.
  • Acylation is accomplished by well-known acylating methods, which include acylation with sulfuric acid derivatives, phosphoric acid derivatives and carboxylic acid derivatives such as acetic anhydride, acetyl chloride, benzoyl chloride and other carboxylic acid chloride or halide derivatives.
  • acylating methods include acylation with sulfuric acid derivatives, phosphoric acid derivatives and carboxylic acid derivatives such as acetic anhydride, acetyl chloride, benzoyl chloride and other carboxylic acid chloride or halide derivatives.
  • derivatives of the amino group which provide increased stability to the toxin and/or increased lipophilicity to the molecule to aid in leaf and membrane permeability.
  • the herbicidal composition of the invention comprises not only the free acid of tabtoxinine- ⁇ -lactam, but also its salts.
  • salts of tabtoxinine-0-lactam are used as herbicides.
  • the cation should preferably be one that is biodegradable and does not pose environmental hazards.
  • Such salts are known to the art and include salts of alkali metals, alkaline earth metals, or sulfur, phosphorus or nitrogen based salts such as sulfonium, phosphonium, and ammonium as well as organic ammonium salts such as alkylammonium and arylammonium derivatives.
  • alkali metal encompasses lithium, sodium, potassium, cesium and rubidium; and the term “alkaline earth metal” includes beryllium, magnesium, calcium, strontium and barium.
  • Organic ammonium salts are those prepared from low molecular weight organic amines, i.e. having a molecular weight below about 300, and such organic amines include the alkyl amines, alkylene amines and alkanol amines, such as methylamine, ethylamine, n-propylamine, isopropylamine, n- butylamine, isobutylamine, sec-butylamine, n-amylamine, iso- amylamine, hexylamine, heptylamine, octylamine, nonylamine, decylamine, undecylamine, dodecylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, heptadecylamine, octadecylamine, methylethylamine, methylisopropylamine, methylhe
  • tabtoxinine-0-lactam as a herbicide, it may be applied topically to the foliage of weeds or to soil, by methods of application well-known in the art, such as spraying.
  • the toxin may be isolated and used in its purified form, or it may be applied in unpurified form, for example, as a constituent of culture medium in which Pseudomonas syrinqae ATCC #55090 and other TBL-producing strains have grown.
  • the amount of toxin applied in is the range of from 0.3 kg/ha to 12 kg/ha, more preferably 1.3 kg/ha to 2.0 kg/ha.
  • the toxin may be applied to the foliage of weeds or soil as a buffered solution.
  • Buffers may be any of the buffers known to those skilled in the art.
  • the buffer is a 0.1 M salt solution. More preferably, the buffer is a 0.1 M solution of potassium phosphate (K 2 P0 4 ).
  • tabtoxinine- ⁇ -lactam may be post- emergent, as a nonselective contact herbicide.
  • Application may also be pre-emergent, by applying the toxin directly to soil in which weed growth is to be controlled.
  • the invention can be used to control weeds in an agricultural setting and can also be used for nonagricultural applications.
  • the invention can be used to control weeds in residential applications and as a contact herbicide for the management of roadside vegetation.
  • Tabtoxinine-0-lactam begins to act soon after application. In some field experiments, signs of injury to the weeds such as water soaking of the leaves was observed within a few hours after the toxin was applied. In most experiments the degree of injury was rated quantitatively at 13-16 days after application of tabtoxinine- -lactam, since long lasting control of weeds is of commercial interest.
  • tabtoxinine- -lactam The herbicidal effects of tabtoxinine- -lactam involve processes that require light. When tabtoxinine-0-lactam was sprayed on plants and the plants are kept in the dark overnight or for one or two days, no injury was observed. Signs of injury begin to appear within a few hours after the plants were returned to the light without application of additional tabtoxinine- ⁇ -lactam.
  • the invention can also be used as a harvest aid to defoliate crops such as potatoes, peanuts and cotton.
  • crops such as potatoes, peanuts and cotton.
  • Several major crops often require defoliation prior to harvesting. This reduces the amount of dirt, leaves and other contaminants in the final product. For example, as much as 75% of the cotton harvested in the U.S. is treated with defoliants.
  • the current defoliants are chemically based and pose potential health and environmental risks. In addition, undesirable odors are often noticeable for several days after some of the commonly used defoliants are applied.
  • Desiccants are used as a harvest aid to eliminate unwanted green plant material and dry out or harden off seed crops such as alfalfa and clover prior to harvesting.
  • the herbicidal composition of the invention can be used as a defoliant or desiccant on some of the major crops such as potatoes, peanuts and cotton, thus eliminating some of the undesirable side effects associated with the current chemical based products.
  • compositions for practice of the invention can be formulated in numerous ways, including flowables, dry flowables, water dispersible granules, emulsified or liquid concentrates, or encapsulated using various techniques and materials.
  • the toxin can also be used in combination with other chemical herbicides to control weeds.
  • Many chemical herbicides such as sulfosate (TOUCHDOWN®), glyphosate (ROUNDUP®), alachlor (LASSO® or MICRO-TECH®), 2,4-D, and dicamba (BANVEL®) have shortcomings in either the speed of activity or the weed control spectrum.
  • TOUCHDOWN® sulfosate
  • ROUNDUP® glyphosate
  • alachlor LASSO® or MICRO-TECH®
  • 2,4-D 2,4-D
  • dicamba dicamba
  • Herbicides that can be combined with tabtoxinine- ⁇ - lactam in improved herbicidal compositions are listed in Table 1. Examples of additional chemical herbicides that can be used are described in Herbicide Handbook of the Weed Society of America, Sixth Ed. (1989), Farm Chemicals Handbook, Willoughby, Ohio: Meister Publishing Co., (1990), and in H. J. Lorenzini and L.S. Jeffrey, Weeds of the United States and Their Control, N.Y.: Van Nostrand 1987, Table 1.9.
  • Herbicides comprising tabtoxinine- -lactam may also contain adjuvants such as surfactants and suspension agents.
  • adjuvants such as surfactants and suspension agents.
  • adjuvants include spreaders such as ORTHO-77® (Chevron Chemical Co.), ATTAGEL-40® and X-77®, and organosilicones such as SILWET L-77®, SILWET L-7607®, X2- 5309 (SYLGARD 309®) (Dow Corning), Genapol LRO (Hoechst- Roussel Agri Vet Company), X2-5395 (Dow Corning), Kinetic, and Dyne-amic (Helena).
  • spreaders such as ORTHO-77® (Chevron Chemical Co.), ATTAGEL-40® and X-77®
  • organosilicones such as SILWET L-77®, SILWET L-7607®, X2- 5309 (SYLGARD 309
  • active agents in X-77® are alkylarylpolyoxyethylene glycols, free fatty acids and isopropanol.
  • Polyoxyethylene surfactants are preferably employed as adjuvants, more preferably alcohol polyoxyethylene surfactants such as MARLIPAL-34®.
  • Crop oils that can be used as adjuvants include Sure Crop Agridex, Crop Surf, CIDE-KICK®, and Crop Oil Concentrate (Crop Surf Spray Oil, Universal Cooperatives, Inc., 7801 Metro Parkway, Minneapolis, MN 55440).
  • Other adjuvants include SOY-DEX®, AGRI-DEX®, TRITON AG-98®, STEROX®, ammonium sulfate. Atlas G-3780A, urea, vegetable oil, Tween A and invert emulsions.
  • Certain adjuvants have proven to be particularly advantageous for use with tabtoxinine-0-lactam. When isolated toxin is applied to weeds with these preferred adjuvants, the degree of weed control is unexpectedly greater than is seen with other adjuvants.
  • the preferred adjuvants are neutral surfactants and neutral organosilicone surfactants. Examples of currently preferred adjuvants are ORTHO-77®, SYLGARD-309®, SILWET-7604® and SILWET-7614®.
  • Phloem or xylem translocation is a desirable property for a herbicide. See J.R. Hay, "Herbicide Transport in Plants," L.J. Audus (ed.). Herbicides, Physiology, Biochemistry, Ecology, 2nd Ed., Vol. I., N.Y. : Academic Press, 1976, pp. 365-396. Total weed control can be achieved much more readily with a phloem- or xylem-mobile herbicide as compared to non-mobile contact herbicides. As illustrated below, we have demonstrated that when tabtoxinine- ⁇ -lactam is painted on a single leaf of a plant it moves throughout the plant by vascular transport and produces a high degree of injury to the entire plant.
  • Monobactams such as tabtoxinine- ⁇ -lactam that are known to inhibit glutamine synthetase may have utility as antibiotics. Preliminary tests have indicated biocidal activity of both Gram-positive and Gram-negative bacteria. Because of the high activity of the antibiotic against selected strains, the compound may find utility in controlling pathogens of medical, veterinary and agricultural importance. In addition, based on the mode of action the compound may find utility for controlling fungi and arthropods.
  • Seed stocks of stationary phase Pseudomonas syringae ATCC #55090 were maintained in liquid culture in a modified King's B medium (Table 3).
  • a two liter flask containing 500 ml of King's B medium was inoculated with 0.5 ml of Pseudomonas syringae ATCC #55090 and were grown overnight at 28°C on an orbital shaker at 180 rpm.
  • Seed cultures were prepared by inoculating 0.5 ml of a stationary phase culture of Pseudomonas syringae ATCC #55090 into two liter flasks containing 500 ml of King's B Medium. The flasks were shaken overnight at 180 rpm and 28°C. Two S. seed cultures were used to inoculate two Microferm fermentors each containing 10 liters of King's B Medium (S 2 ). The S 2 seed cultures were grown for 7 h at 25°C, 400 rpm, with an air sparge of 1 wm (volume of air spray/volume of fermentor vessel/min).
  • the pH was not controlled. Twenty liters of S ⁇ was transferred to a B. Braun 500 liter free-standing fermentor with a 350 liter working volume. The fermentation was carried out in 350 liters of UWF medium for 67-72 h at 25°C with an agitation rate of 150 rpm and an air sparge of 1 wm, with pH maintained between 6.0 and 6.8. A one time nutrient spike was added after 24 h consisting of 5 kg of glucose.
  • medium Fl-12 or medium Fl-16 (Table 4a) is employed as the fermentation medium.
  • the cell free fermentation beer was adjusted with HC1 to a pH of 4.0 and passed through a cation exchange resin.
  • Any strong cation exchange resin such as Dowex 50W or Amberlite 200 in the H form can be used. Amberlite 200 in the H form is preferred.
  • the column size was adjusted according to the volume of fermentation beer to maintain the ratio of fermentation beer to resin bed volume at 10:1.
  • the buffers listed below were used to elute the phytotoxin from the resin bed. A ratio of 5 volumes of buffer to 1 bed volume as used. Buffers 2 and 3 are preferred.
  • the eluate was adjusted to pH 4.0 with HC1 and stored at 5° C. Concentration steps included rotary evaporation at 40° C under vacuum followed by lyophilization.
  • the dry product was desalted with three rinses of dry methanol, filtered, and the residue discarded. The remaining filtrate was reduced by rotary evaporation and the final extract was stored at -20° C and pH 4.0. When stored dry. the powder is redissolved in water or methanol immediately before use as a herbicide.
  • TBL can be assayed with a Beckman model 126AA amino acid analyzer or similar instrument. Using a sodium ion exchange column and sodium citrate buffers, TBL has a retention time of approximately ten minutes using a standard method for amino acids. Glycine may be used as a standard for quantitative analysis of TBL.
  • the culture medium was adjusted to pH 4.0 with HCl, and passed through a column containing activated carbon.
  • the herbicidally active material was eluted from the activated carbon with methanol acidified to a pH of 4.2.
  • the resulting eluate was reduced in volume, lyophilized, dissolved in a minimal amount of water and stored at -20° C at pH 4.0.
  • An alternative method of isolation involves direct lyophilization of the fermentation beer. After lyophilization, the dry product is extracted three times with dry methanol and the extract volume reduced under vacuum to a workable volume. The concentration of tabtoxinine- -lactam is determined in the final product.
  • the toxin was identified by H nuclear magnetic resonance (NMR) spectroscopy of a purified sample.
  • the purified sample was prepared for analysis by thin layer chromatography (TLC) on Whatman silica gel 60A (250 ⁇ m) TLC plates. Small quantities (approximately l ⁇ g) of crude toxin were spotted on the TLC plates in discrete spots. Approximately 25 plates with 30 spots per plate were required to obtain sufficient toxin for analysis.
  • the TLC plates were developed in butanol:acetic acid:water (80:20:20). After drying, the silica on the area of the plate containing the toxin was removed and extracted with methanol at pH 4.5.
  • the methanol was removed under vacuum and the dry product suspended in 1 ml of D 2 0 and allowed to sit for 30 min at 22°C. The sample was then frozen, lyophilized, resuspended in D 2 0, and analyzed by H NMR spectroscopy (500MHz, Bruker AMX-500). An aliquot of the purified toxin was tested in a bioassay to ensure that it retained its biological activity.
  • Table 5 is a comparison of the H NMR spectrum of the purified toxin and a chemically synthesized sample of tabtoxinine- -lactam (Dolle et al., J. Org. Chem. , 57:128- 132 (1992)) confirming that the toxin is TBL.
  • the effects of the purified toxin were evaluated in the greenhouse on weed species selected as being weeds of economic importance in row crops, residential and industrial markets and as representing a spectrum of plant families. The effects were also tested on tomato, another representative broadleaf plant.
  • Extracts were suspended in 8 ml of water and
  • Rates were set based on the initial amount of fermentation beer that was used to prepare the extract. Rates varied from 0.5 to 1.0
  • the toxin can be combined with chemical herbicides to enhance weed control. Effects of combining the toxin with sulfosate are shown in Table 11.
  • Table 12 provides examples of the spectrum of weeds that can be controlled with the phytotoxin.
  • Duckweed (Lemna minor) is a small aquatic weed that can be used as an indicator of herbicide activity on aquatic weeds or as a bioassay for herbicides and plant growth regulators.
  • Duckweed is grown in water containing minimal salts such as Hoagland's growth solution at pH 4.0. The plants are maintained in a stock culture in a growth chamber uunnddeerr continuous light (approximately 250 ⁇ E m -2 sec-1) and 20°C.
  • the bioassay is conducted in 24 well culture plates as follows: Under sterile conditions, 1.0 ml of medium was added to each well along with two duckweed frond doublets of equivalent size from the stock culture. Treatments were added to each well as indicated in Table 13. The plates were returned to the growth chamber for 5 days.
  • Ester derivatives of tabtoxin are prepared in the following manner, illustrated with the methyl ester of tabtoxinine-0-lactam: 5 g of tabtoxinine-0-lactam is dissolved in 25 ml of anhydrous methanol and 0.2 g of concentrated sulfuric acid is added in one portion. The homogenous solution is allowed to stir at room temperature for 12 hours. Dilute sodium hydroxide solution is added to bring the pH of the mixture to 6.5. The solution is evaporated to dryness and the methyl ester is isolated by standard techniques.
  • This example illustrates the use of ethyl chloroformate as the acid chloride to derivatize the amine group at C-2' .
  • Tabtoxinine-0-lactam is dissolved in water and the pH of the solution is adjusted to 7.5.
  • the pH of the solution should be maintained at 7.5 during the course of the reaction.
  • the pH must be maintained below 8.0.
  • the reaction could also be run by maintaining a lower pH such as pH 6.5, but the reaction proceeds f ster at pH 7.5.
  • Antibiotic activity can be demonstrated by growing ATCC strain #55090 on petri plates with various indicator strains or by exposing the target strains to various concentrations of toxin.
  • indicator strains were suspended in a minimal media such as PMS and allowed to solidify in plates.
  • the producer strain .Pseudomonas syringae ATCC strain #55090) was inoculated into the minimal medium containing the indicator strain. Cultures were incubated overnight at 28°C and colony diameter was measured. The resulting inhibition of bacteria is summarized in Table 14.
  • plates containing bacterial indicator strains were prepared as described above. Sterilized filter paper discs (5mm) were treated with 20 ⁇ L of the appropriate dilution of toxin, allowed to dry and applied to the plates. The plates were incubated overnight at 28°C and the halo diameter measured to indicate antibiotic activity. (Table 15).
  • EXAMPLE 14 Genetic Engineering for Strain Improvement To improve the production strain, a broad host range multi-copy plasmid containing the toxin biosynthetic genes is obtained and moved into a toxin producing isolate, thereby increasing the copy number of the biosynthetic genes and potentially the level of production. This is accomplished by:
  • Step 1 Selection of a drug resistant toxin producing recipient.
  • Step 2 In order to select for transconjugants from the bacterial mating described in Step 2, it is necessary to have a selectable marker present in the recipient. To do this, the current production strain is plated on KB (King et al., J. Lab. Clin. Med. 44:301-307 (1954)) plus rifampicin (100 ⁇ g/ml), incubated at 28°C and resistant colonies isolated and purified as described in Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1972. Rifampicin resistant isolates are checked for maintenance of toxin production by bioassay using toxin sensitive Escherichia coli as described in Gasson, Appl. Environ. Microbiol. 39:25-29 (1980).
  • Step 2 Move plasmid containing the toxin biosynthetic genes into the recipient.
  • the entire tabtoxin biosynthetic region from Pseudomonas syringae BR2 is contained on the cosmid pRTBL823, which also contains a tetracycline resistance marker (Kinscherf et al., J. Bacteriol. 173:4124-4132 (1991)).
  • a triparental mating between the donor strain E . coli DH5 ⁇ (pRTBL823), the helper strain JE. coli pRK2013 (ATCC #37159) and the recipient strain is carried out as described in Ditta et al. , Proc. Natl. Acad. Sci. USA 77:7347-7351 (1980).
  • Transconjugants colonnies arising from the movement of pRTBL823 into recipient cells are selected on KB plates containing tetracycline (10 ⁇ g/ml) and rifampicin (100 ⁇ g/ml) at 28°C. After restreaking transconjugants on KB + tet + rif, isolates are checked for kanamycin sensitivity (the helper plasmid pRK2013 (ATCC #37159), which should not be present in the transconjugants, contains a kanamycin resistance marker) and toxin production by bioassay.
  • kanamycin sensitivity the helper plasmid pRK2013 (ATCC #37159
  • the resulting isolates have an increased copy number of the toxin biosynthetic genes, including the native genes present in the current toxin producer and multiple copies of the BR2 genes present on multiple copies of the plasmid.
  • the native genes present in the current toxin producer and multiple copies of the BR2 genes present on multiple copies of the plasmid.
  • the recipient Pseudomonas strain is prototrophic, and the donor and helper E. coli strains are both auxotrophic, so that growth on minimal media is used as the selectable phenotype of the recipient (instead of rifampicin resistance).
  • the donor strain E ⁇ _ coli HB101 pRTBL823
  • the helper strain E. coli pRK2013 [ATCC #37159]
  • the recipient strain Pseudomonas syringae pathovar tabaci 11527 [ATCC #11527] is carried out.
  • Cultures of the three strains are grown in liquid media to mid-log phase, the cells harvested by centrifugation, and the cells re-suspended at about 10-fold higher density in M9 liquid media (Miller) .
  • M9 liquid media M9 liquid media
  • Approximately equal numbers of cells of all three strains are mixed together in a total volume of 50 to 300 microliters of M9, then collected by filtration on a 0.2 micron sterile filter.
  • the filters are placed on LB plates (Miller) at 28°C overnight. Then, the cells are collected by washing the filters with 300 to 700 microliters of PBS. Serial dilutions of this cell suspension are plated on M9 plates containing 6 to 8 micrograms/milliliter tetracycline.
  • Transconjugants are restreaked on KB containing tetracycline (10 ⁇ g/ml) to check for retention of the fluorescent phenotype and then for toxin production by plate bioassay on an J ⁇ . coli lawn. Individual colonies are miniprepped and the existence of pRTBL823 in the tranconjugants is verified.
  • TBL pre-emergence herbicides are very useful to farmers by eliminating weed competition during the early phases of crop development and represent a major portion of the herbicides market.
  • TBL was tested as a pre-emergent herbicide in the greenhouse.
  • the concentrated TBL prepared as described in
  • Example 3 was suspended in 8 ml of water and applied to pots
  • the pots (4" or 10 cm square) contained soil (Metro mix) and the seeds of the appropriate weed species. Two replications were used and arranged in a randomized complete block design. Injury was rated as outlined in Example 5. Results from the pre-emergence trials are shown in Table 16. Ratings were taken 19 days after treatment in experiment 1 and 16 days after treatment in experiment 2.
  • TWEEN 20 sorbitan monolaurate (polysarbate 20)
  • TBL 300 MHZ/D2O* Pseudomonas toxin (500 MHz/DoO)
  • phytotoxin 1.0 63 75 53 100 100 95
  • phytotoxin 3 .0 100 89 85 100 100 98
  • Phytotoxin was applied at a dose of 4.25 kg/ha.
  • Phytotoxin was applied at a dose of 4.25 kg/ha.
  • Salmonella cholorasuis (ATCC 29213)
  • Bacillus subtilis (ATCC 633)
  • Bacillus pumilus (ATCC 14884)
  • Bacillus subtilis (ATCC 9466)
  • Indicator strains were suspended in a minimal media (PMS) which was allowed to solidify in plates. Subsequently, the producer strain was inoculated into the minimal medium continuing the indicator strain. Culture were incubated and colony diameter measured.
  • PMS minimal media
  • Acetobacter diazotrophians 36.4 25.7 ND ND 1 (ATCC 49037)

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Abstract

Le nouveau composé herbicide, la tabtoxine-β-lactam, a été isolé à partir d'une bactérie phytopathogène, la Pseudomonas syringae (Réf. ATCC: 55090). L'activité herbicide peut être isolée à partir d'un milieu de culture dans lequel cette bactérie s'est développée. Une application locale sur le feuillage des végétaux provoque une rapide imprégnation d'eau et la chlorose. La toxine est véhiculée dans la mauvaise herbe par voie vasculaire depuis le lieu de l'application locale et permet une exploitation commerciale pour l'élimination des mauvaises herbes. L'association de la toxine avec les herbicides chimiques accélère et augmente les lésions des mauvaises herbes.
PCT/US1994/005808 1993-05-28 1994-05-27 Nouvelle toxine bacterienne utilisable comme regulateur de la croissance des vegetaux et comme herbicide WO1994027444A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010713A1 (fr) * 1995-09-21 1997-03-27 Novartis Ag Derives 7-azabicyclo(4,2,0)oct-4-en-8-one en tant que microbicides pour plantes
US6172004B1 (en) 1997-01-31 2001-01-09 Monsanto Company Composition and method for treating plants with exogenous chemicals
US8536095B2 (en) 2008-07-03 2013-09-17 Monsanto Technology Llc Combinations of derivatized saccharide surfactants and etheramine oxide surfactants as herbicide adjuvants

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4874706A (en) * 1986-09-10 1989-10-17 Wisconsin Alumni Research Foundation Manufacture and use of tagetitoxin
WO1992008357A1 (fr) * 1990-11-16 1992-05-29 Crop Genetics International Corporation Combinaisons herbicides de produits de fermentation microbiens et d'agents chimiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4874706A (en) * 1986-09-10 1989-10-17 Wisconsin Alumni Research Foundation Manufacture and use of tagetitoxin
WO1992008357A1 (fr) * 1990-11-16 1992-05-29 Crop Genetics International Corporation Combinaisons herbicides de produits de fermentation microbiens et d'agents chimiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NATURE vol. 229 , 15 January 1971 pages 174 - 178 WALTER W. STEWART 'Isolation and proof of structure of wildfire toxin.' cited in the application *
PLANT PHYSIOLOGY vol. 71 , 1983 pages 912 - 915 M. D. THOMAS,P J. LANGSTON-UNKEFER, TH. F. UCHYTIL & R. D. DURBIN. 'Inhibition of Glutamine Synthetase from pea by Tabtoxinine-beta-lactam.' *
PLANT PHYSIOLOGY vol. 82 , 1986 pages 1045 - 1050 TH. J. KNIGHT, R. D. DURBIN & P. J. LANGSTON-UNKEFER 'Effects of Tabtoxinin-beta-lactam on nitrogen metabolism in Avena sativa L. roots.' *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010713A1 (fr) * 1995-09-21 1997-03-27 Novartis Ag Derives 7-azabicyclo(4,2,0)oct-4-en-8-one en tant que microbicides pour plantes
US6172004B1 (en) 1997-01-31 2001-01-09 Monsanto Company Composition and method for treating plants with exogenous chemicals
US8536095B2 (en) 2008-07-03 2013-09-17 Monsanto Technology Llc Combinations of derivatized saccharide surfactants and etheramine oxide surfactants as herbicide adjuvants
US9351486B2 (en) 2008-07-03 2016-05-31 Monsanto Technology Llc Combinations of derivatized saccharide surfactants and etheramine oxide surfactants as herbicide adjuvants

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