WO2020232103A1 - Agents de lutte biologique séchés et leurs utilisations - Google Patents

Agents de lutte biologique séchés et leurs utilisations Download PDF

Info

Publication number
WO2020232103A1
WO2020232103A1 PCT/US2020/032650 US2020032650W WO2020232103A1 WO 2020232103 A1 WO2020232103 A1 WO 2020232103A1 US 2020032650 W US2020032650 W US 2020032650W WO 2020232103 A1 WO2020232103 A1 WO 2020232103A1
Authority
WO
WIPO (PCT)
Prior art keywords
dried composition
dried
cfu
seed
plant
Prior art date
Application number
PCT/US2020/032650
Other languages
English (en)
Inventor
Alexander P. SCHLESINGER
James D. SIEVERT
Original Assignee
AgBiome, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AgBiome, Inc. filed Critical AgBiome, Inc.
Publication of WO2020232103A1 publication Critical patent/WO2020232103A1/fr

Links

Classifications

    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas

Definitions

  • the invention relates to dried biocontrol agents and populations that have improved properties.
  • Plant diseases and pests need to be controlled to maintain the quality and quantity of food, feed, and fiber produced by growers around the world. Plant diseases are mainly caused by fungi, bacteria, viruses and nematodes. Plant pests include chewing, sucking and piercing insects from the Lepdoptera, Coleoptera, and Hemiptera, among others. Chemical pesticides are widely used in farming to protect plants and crops from such pests and diseases. These chemical products fight crop pests, diseases, and weeds, resulting in improved yield. Without crop protection and pest control, food production and the quality of food produced would decline. However, the use of chemical pesticides does impose a level of risk as many have properties that can endanger health and the environment if not used properly.
  • Pesticide resistance is the decreased susceptibility of a pest population to a control agent at doses that once killed most individuals of the species. Therefore, new products are needed with different modes of action to aid in resistance management.
  • microorganisms that lead to biocontrol can include antibiosis, competition, induction of host resistance, and predation. Screening and testing isolates have yielded a number of candidates for commercialization. Microbial biopesticides represent an important option for the management of plant diseases and pests. There is a need for biological control agents that are able to compete in field conditions particularly in the presence of herbicides and fungicides that are commonly used in commercial farming and can have antibiotic effects on microorganisms.
  • compositions and methods involving dried compositions of biological agents or biocontrol agents are provided.
  • the modified population of agents is able to grow, compete with other microbial strains and fungi, and provide protection for plants from pathogens.
  • modified biological control agents promote plant growth and yield.
  • modified biological agents and modified populations of such agents that are biocide- tolerant or -resistant; herbicide-tolerant or -resistant; fungicide-tolerant or -resistant; pesticide- tolerant or -resistant; or tolerant or resistant to crop protection chemicals are selected or
  • the modified biological agents are able to grow in the presence of at least one herbicide, fungicide, pesticide, or other crop protection chemical that is used in commercial farming. Such modified biological agents are able to grow and reproduce in soils where such herbicides, fungicides, pesticides, or other crop protection chemicals have been applied.
  • the modified biological agents render the soils suppressive or resistant to disease-causing pathogens or pests.
  • Such modified populations of biological agents can be added to soils to prevent fungal pathogens and the diseases they cause, or to inhibit feeding by insect pests or nematodes, promoting plant growth and increasing plant or crop yield.
  • compositions of the invention include selected or engineered biological agents and modified populations of biocontrol agents.
  • the dried compositions can be used as an inoculant or as a seed coating for plants and seeds. They can also be applied, after reconstitution with a liquid, as a spray application directly to the aerial parts of plants, and can be mixed with a biocide, such as the herbicide or other chemical to which they have been modified to become tolerant. As indicated, the presence of the modified biological agents under field conditions enhances resistance of the plants to pathogens and promotes plant growth.
  • the dried biological agents of the invention can be used with other agents to promote plant growth and yield.
  • a dried composition comprising at least one biological control agent comprising a Pseudomonas.
  • composition of embodiment 8, wherein said fungus comprises at least one of Pythium aphanadermatum, Phytophthora parasitica, Phytophthora nicotianae, Phytophthora inf e stans, Phytophthora capisici, Rhizoctonia solani, and/or Botrytis cinerea.
  • a coated seed comprising a seed and a coating on said seed, wherein said coating comprises a dried composition of at least one biological control agent comprising a Pseudomonas.
  • a method for growing a plant comprising planting in an area of cultivation a coated seed as set forth in any one of embodiments 15-30.
  • a method for growing a plant comprising applying to a plant, a crop, or to an area of cultivation an effective amount of a dried composition comprising at least one biological control agent comprising a Pseudomonas.
  • said at least one biological control agent comprises Pseudomonas fluorescens or Pseudomonas chloroaphis.
  • fungus comprises at least one of Pythium aphanadermatum, Phytophthora parasitica, Phytophthora nicotianae, Phytophthora inf e stans, Phytophthora capisici, Rhizoctonia solani, and/or Botrytis cinerea.
  • a biological agent or biocontrol agent for purposes of the present invention is used to describe a microorganism that is used to control disease-causing plant pathogens and plant pests.
  • the biological control agents of the invention include those that have been modified such that they are able to grow in the presence of at least one biocide.
  • a biocide is a chemical substance which can exert a controlling effect on an organism by chemical or biological means.
  • Biocides include pesticides, such as fungicides; herbicides; insecticides, other crop protection chemicals, and the like.
  • compositions of the invention include one or more isolated biocontrol agents that has been selected for resistance to biocides such as a herbicide, fungicide, pesticide, or other crop protection chemical; a recombinant biocontrol agent that has been transformed to contain a herbicide, fungicide, pesticide, or other crop protection chemical resistant gene; a modified population of biocontrol agents wherein the population is resistant to at least one herbicide, fungicide, pesticide, or other crop protection chemical; and compositions comprising these modified populations of biocontrol agents.
  • the modified population may comprise
  • the invention comprises substantially pure cultures, or biologically pure cultures, of such modified biocontrol agents or modified biological agents.
  • a "biologically pure bacterial culture” refers to a culture of bacteria containing no other bacterial species in quantities to be detected by normal bacteriological techniques. Stated another way, it is a culture wherein virtually all of the bacterial cells present are of the selected strain.
  • a modified biocontrol agent includes biocontrol agents that have acquired a trait due to selection pressure and recombinant biocontrol agents that have been transformed with a gene that confers resistance or tolerance to at least one herbicide, fungicide, pesticide, or other crop protection chemical.
  • the invention further encompasses a particular biological control agent.
  • a particular biological control agent includes AIP1620.
  • AIP1620 is a Pseudomonas strain that has been selected for glyphosate tolerance.
  • Additional agents include AIP050999.
  • AIP050999 is a Pseudomonas strain that has been selected for glufosinate tolerance.
  • AIP1620 was deposited with the Patent Depository of the National Center for Agricultural Utilization Research Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S. A. on January 31, 2014 and assigned NRRL No. B- 50897.
  • AIP050999 was deposited with the Patent Depository of the National Center for
  • NRRL No. B-50897 and NRRL No. B-50999 Some methods to identify groups of derived and functionally identical or nearly identical strains are Multi-locus sequence typing (MLST), concatenated shared genes trees, Whole Genome Alignment (WGA), and Average Nucleotide Identity (ANI). Each will be considered below. While many are also useful for higher-level groupings (species and above), the cutoffs and methodology below will be focused on those appropriate for a fine level of resolution.
  • One approach to increasing the resolution of the rRNA gene is to use multiple genes or loci, particularly those that change more rapidly but are still universally present.
  • the various references cited herein provide publically available gene sets and either set cut off values or provide methods to determine the cut off that will provide the same cut off for the same group of organisms. The larger the number of genes, the more resolution is possible, but the lack of those genes in other species make them difficult to analyze in the same framework. Cutoffs to define a group will differ based on the number and specific genes used.
  • MLST One extension of MLST is to use all universally shared genes (therefore the maximum possible loci) for a group of strains [for instance, as implemented in Benedict, M.N., et al, ((2014) BMC Genomics 15(1):8) in a concatenated shared gene alignment and inferred tree.
  • WGA is a related series of method that aligns the entire genome sequence (not just genes or defined loci) between two or many organisms (see, for example, Angiuoli, S.V., et al, (2011) Bioinformatics 27(3):334-42; Darling, A.E., et al, (2010) PLoS ONE 5(6):el l l47; and Treangen, T.J., et al, (2014) Genome Biology 15(11):524).
  • ANI see, for example, Konstantinidis, K.T., et al, (2005) PNAS USA 102(7):2567-72; and Richter, M., et al. , (2009) PNAS 106(45): 19126-31
  • derivatives see, for example, Varghese, N.J., et al. , Nucleic Acids Research (July 6, 2015): gkv657
  • Methods may differ slightly, for one widely implemented ANI method a cutoff of 99% defines a functional group (see, for example, Konstantinidis, K.T., et al , (2005) PNAS USA
  • herbicide, fungicide, pesticide, or other crop protection chemical tolerance or herbicide, fungicide, pesticide, or other crop protection chemical resistance is intended the ability of an organism (i.e, the plant, the biocontrol agent, the biocontrol bacterial agent, etc.) to survive and reproduce following exposure to a dose of the herbicide, fungicide, pesticide, or other crop protection chemical that is normally lethal to the wild type organism.
  • Bio agents or biocontrol agents of the invention include microorganisms and fungi that control disease-causing plant pathogens and promote plant health, growth, and yield. Any of these biological or biocontrol agents can be modified by selection or transformation and produce a modified biological or biocontrol agent or recombinant biological or biocontrol agent.
  • the invention encompasses an isolated biocontrol agent.
  • the biocontrol agents can be grown to produce a population of biocontrol agents.
  • modified population of biological or biocontrol agents is intended a population of agents that substantially comprises a culture of the selected agent or the recombinant agent having the trait of interest such as resistance to a herbicide, fungicide, pesticide, or other crop protection chemical. By substantially comprises is intended that the population has been grown and produced from the modified or the recombinant biocontrol agent. That is, the modified or recombinant biocontrol agents can be grown to produce a
  • biologically pure culture It is recognized that such biologically pure cultures can be used together to enhance plant health, growth, or yield.
  • Any biological or biocontrol agent can be used in the methods of the invention.
  • Particular microorganisms of interest include strains of the bacteria Pseudomonas, Bacillus, Agrobacterium, Lysobacter, Gliocladium, Pythium, Chromobacterium, Penicillium, Pantoea, Lactobacillus, Paenibacillus, Burkholderia, Streptomyces, Variovorax, Pasteur ia, Xanthomonas, etc.
  • Fungi of interest include Aureobasidium, Ampelomyces, Beauveria, Metarhizium, Metschnikowia,
  • biocontrol agents are on the market and any of them can be modified according to the present invention.
  • Such agents include: Agrobacterium radiobacter K84; Trichoderma atroviride; Bacillus subtilis GB03; Bacillus firmus 1-1582; Trichoderma asperellum (ICC 012); T. gamsii (ICC 080); Bacillus pumilus strain QST 2808; Bacillus subtilis strain QST 713; B. subtilis strain MBI 600; Paecilomyces fumosoroseus; Gliocladium catenulatum; Trichoderma harzianum rifai strain KRL-AG2;
  • oligandrum DV 74 Bacillus subtilis GB03; Trichoderma asperellum ; T gamsii, Pseudomonas syringae ESC- 10; Metschnikowia fructicola; Trichoderma harzianum T-22; Pseudomonas chlororaphis MA 342; B. amyloliquifaciens ; Chrombacterium subtsugae strain PRAA4-1; B.
  • subtilis amyloliquefaciens FZB24 Penicillium bilaii ; Paecilomyces fumosoroseus FE 9901;
  • Biocontrol agents of the invention are those that target any of the plant pathogens.
  • Target pathogens include but are not limited to species of Alternaria, Botrytis, Fusarium, Erwinia, Pseudomonas,
  • Mycosphaerella Phomopsis, Taphrina, Elsinoe, Sclerotinia, Verticillum, Gnomonia, Fusicladium, Nectria, Phyllosticta, Diplocarpon, Albugo, Guignardia, Botrytis, Exobasidium, Entomosporium, Exobasidium, Pestalotia, Phoma, Cristulariella, Phakopsora, Thelaviopsis, Puccinia, Peronospora, Bremia, Pantoea, and Clavibacter.
  • the biological agents disclosed herein control at least one, two or all of Pythium, Phytophthora, or Rhizoctonia.
  • the biological agents disclosed herein e.g., NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof
  • the biological agents disclosed herein e.g., NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof
  • controlling and“protecting a plant from a pathogen” refers to one or more of inhibiting or reducing the growth, germination, reproduction, and/or proliferation of a pathogen of interest; and/or killing, removing, destroying, or otherwise diminishing the occurrence and/or activity of a pathogen of interest.
  • the biocontrol agent controls one or more fungi (such as for example, Pythium, Phytophthora, and/or Rhizoctonia).
  • the biocontrol agent controls Phakopsora.
  • assays to measure such activity are disclosed elsewhere herein.
  • an antipathogenic composition reduces the disease symptoms resulting from pathogen challenge by a statistically significant amount, including for example, at least about 2% to at least about 6%, at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, or at least about 50% to about 90% or greater.
  • the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens, including, for example, Fusarium spp., Phakopsora pachyrhizi , Rhizoctonia solani , Botrytis cinerea , Phytophthora spp., Pythium spp., turf pathogens, and the like.
  • plant pathogens including, for example, Fusarium spp., Phakopsora pachyrhizi , Rhizoctonia solani , Botrytis cinerea , Phytophthora spp., Pythium spp., turf pathogens, and the like.
  • Herbicide, fungicide, pesticide, or other crop protection chemical resistance is the ability of an organism to survive and reproduce following exposure to a dose of the herbicide, fungicide, pesticide, or other crop protection chemical that would normally be lethal to the wild type organism or would substantially reduce growth of the wild type organism. Resistance may be induced or identified due to selection or it may be induced through genetic engineering. To identify and produce a modified population of biocontrol agents through selection, the biocontrol agents are grown in the presence of the herbicide, fungicide, pesticide, or other crop protection chemical as the selection pressure. Susceptible agents are killed while resistant agents survive to reproduce without competition.
  • resistant biocontrol agents As the biocontrol agents are grown in the presence of the herbicide, fungicide, pesticide, or other crop protection chemical, resistant biocontrol agents successfully reproduce and become dominant in the population, becoming a modified population of biocontrol agents.
  • Methods for selecting resistant strains are known and include U.S. Patent Nos. 4,306,027 and 4,094,097, herein incorporated by reference. Therefore, the invention includes a biologically pure culture of a resistant biocontrol strain.
  • the resistant strains of the invention have the same identification characteristics as the original sensitive strain except they are significantly more tolerant to the particular herbicide, fungicide, pesticide, or other crop protection chemical. Thus, their identification is readily possible by comparison with characteristics of the known sensitive strain.
  • Herbicides include glyphosate, ACCase inhibitors (Arloxyphenoxy propionate (FOPS)); ALS inhibitors (Sulfonylurea (SU)), Imidazonlinone (IMI), Pyrimidines (PM)); microtubule protein inhibitor (Dinitroaniline (DNA)); synthetic auxins (Phenoxy (P)), Benzoic Acid (BA), Carboxylic acid (CA)); Photosystem II inhibitor (Triazine (TZ)), Triazinone (TN), Nitriles (NT),
  • BZ Benzothiadiazinones
  • US Ureas
  • US EPSP Synthase inhibitor
  • GC glycines
  • PA Glutamine Synthesis inhibitor
  • DOXP synthase inhibitor Isoxazolidinone (IA)
  • HPPD inhibitor Prazole (PA)
  • Triketone TE
  • PPO inhibitors Diphenylether (DE), N- phenylphthalimide (NP) (Ary triazinone (AT)
  • VLFA inhibitors chloroacetamide (CA)
  • Pesticides include imidacloprid clothianidin, arylpyrazole compounds (W02007103076); organophosphates, phenyl pyrazole, pyrethoids caramoyloximes, pyrazoles, amidines, halogenated hydrocarbons, carbamates and derivatives thereof, terbufos, chloropyrifos, fipronil, chlorethoxyfos, telfuthrin, carbofuran, imidacloprid, tebupirimfos (5,849,320).
  • Fungicides include aliphatic nitrogen fungicides (butylamine, cymoxanil, dodicin, dodine, guazatine, iminoctadine); amide fungicides (benzovindiflupyr, carpropamid, chloraniformethan, cyflufenamid, diclocymet, diclocymet, dimoxystrobin, fenaminstrobin, fenoxanil, flumetover, furametpyr, isofetamid, isopyrazam, mandestrobin, mandipropamid, metominostrobin,
  • acylamino acid fungicides (benalaxyl, benalaxyl-M, furalaxyl, metalaxyl, metalaxyl-M, pefurazoate, valifenalate); anilide fungicides (benalaxyl, benalaxyl-M, bixafen, boscalid, carboxin, fenhexamid, fluxapyroxad, isotianil, metalaxyl, metalaxyl-M, metsulfovax, ofurace, oxadixyl, oxycarboxin, penflufen, pyracarbolid, sedaxane, thifluzamide, tiadinil, vanguard); benzanilide fungicides (benodanil, flutolanil,
  • sulfonamide fungicides (amisulbrom, cyazofamid); valinamide fungicides (benthiavalicarb, iprovalicarb); antibiotic fungicides (aureofungin, blasticidin-S, cycloheximide, griseofulvin, kasugamycin, moroxydine, natamycin, polyoxins, polyoxorim, streptomycin, validamycin); strobilurin fungicides (fluoxastrobin, mandestrobin); methoxyacrylate strobilurin fungicides (azoxystrobin, bifujunzhi, coumoxystrobin, enoxastrobin, flufenoxystrobin, jiaxiangjunzhi, picoxystrobin, pyraoxystrobin); methoxycarbanilate strobilurin fungicides
  • pyraclostrobin pyrametostrobin, triclopyricarb
  • methoxyiminoacetamide strobilurin fungicides diimoxystrobin, fenaminstrobin, metominostrobin, orysastrobin
  • methoxyiminoacetate strobilurin fungicides kresoxim-methyl, trifloxystrobin
  • aromatic fungicides biphenyl
  • chlorodinitronaphthalenes chloroneb, chlorothalonil, cresol, dicloran, fenjuntong,
  • hexachlorobenzene pentachlorophenol, quintozene, sodium pentachlorophenoxide, tecnazene, trichlorotrinitrobenzenes
  • arsenical fungicides (asomate, urbacide); aryl phenyl ketone fungicides (metrafenone, pyriofenone); benzimidazole fungicides (albendazole, benomyl, carbendazim, chlorfenazole, cypendazole, debacarb, fuberidazole, mecarbinzid, rabenzazole, thiabendazole); benzimidazole precursor fungicides (furophanate, thiophanate, thiophanate-methyl); benzothiazole fungicides (bentaluron, benthiavalicarb, benthiazole, chlobenthiazone, probenazole); botanical fungicides (allicin,
  • conazole fungicides triazoles
  • triazoles azaconazole, bromuconazole, cyproconazole, diclobutrazol, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, etaconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, furconazole, furconazole-cis, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, penconazole, propi conazole,
  • triazoles azaconazole, bromuconazole, cyproconazole, diclobutrazol, difenoconazole, diniconazole, diniconazole-M, epoxiconazole, etaconazole, fenbuconazole, fluquinconazole, flus
  • prothioconazole quinconazole, simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triti conazole, uniconazole, uniconazole-P
  • copper fungicides acypetacs-copper, Bordeaux mixture, Burgundy mixture, Cheshunt mixture, copper acetate, copper carbonate, basic, copper hydroxide, copper naphthenate, copper oleate, copper oxychloride, copper silicate, copper sulfate, copper sulfate, basic, copper zinc chromate, cufraneb, cuprobam, cuprous oxide, mancopper, oxine-copper, saisentong, thiodiazole-copper); cyanoacrylate fungicides (benzamacril, phenamacril); dicarboximide fungicides (famoxadone, fluoroimide); dichlorophenyl dicarboximi
  • pyridine fungicides (boscalid, buthiobate, dipyrithione, fluazinam, fluopicolide, fluopyram, parinol, picarbutrazox, pyribencarb, pyridinitril, pyrifenox, pyrisoxazole, pyroxychlor, pyroxyfur, triclopyricarb); pyrimidine fungicides (bupirimate, diflumetorim, dimethirimol, ethirimol, fenarimol, ferimzone, nuarimol, triarimol); anil
  • quinconazole simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triti conazole, uniconazole, uniconazole-P); triazolopyrimidine fungicides (ametoctradin); urea fungicides (bentaluron, pencycuron, quinazamid); zinc fungicides (acypetacs-zinc, copper zinc chromate, cufraneb, mancozeb, metiram, polycarbamate, polyoxorim-zinc, propineb, zinc naphthenate, zinc thiazole, zinc trichlorophenoxide, zineb, ziram); unclassified fungicides (acibenzolar, acypetacs, allyl alcohol, benzalkonium chloride, bethoxazin, bromothalonil, chitosan, chloropicrin, DBCP, dehydroacetic acid,
  • recombinant biocontrol agents having resistance to a herbicide, fungicide, pesticide, or other crop protection chemical can be made through genetic engineering techniques and such engineered or recombinant biocontrol agents grown to produce a modified population of biocontrol agents.
  • a recombinant biocontrol agent is produced by introducing polynucleotides into the biocontrol host cell by transformation. Methods for transforming microorganisms are known and available in the art. See, generally, Hanahan, D. (1983) Studies on transformation of
  • Transformation may occur by natural uptake of naked DNA by competent cells from their environment in the laboratory.
  • cells can be made competent by exposure to divalent cations under cold conditions, by electroporation, by exposure to polyethylene glycol, by treatment with fibrous nanoparticles, or other methods well known in the art.
  • Herbicide resistance genes for use in transforming a recombinant biocontrol agent include, but are not limited to, fumonisin detoxification genes (U.S. Patent No. 5,792,931); acetolactate synthase (ALS) mutants that lead to herbicide resistance, in particular the sulfonylurea-type herbicides, such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene)); and HPPD resistance (WO 96/38576, U.S. Patent Nos.
  • the bar gene encodes resistance to the herbicide basta
  • the nptll gene encodes resistance to the antibiotics kanamycin and geneticin
  • the ALS-gene mutants encode resistance to the sulfonylurea herbicides including chlorsulfuron, metsulfuron, sulfometuron, nicosulfuron, rimsulfuron, flazasulfuron, sulfosulfuron, and triasulfuron
  • the imadizolinone herbicides including imazethapyr, imazaquin, imazapyr, and imazamethabenz.
  • compositions provided herein are dried compositions.
  • a“dried composition” or“dried formulation” of a biocontrol agent refers to a population of a biocontrol agent that is substantially free of water or other liquid solvent.
  • substantially free of water or other liquid solvent it is meant that the composition comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less by weight of water or liquid solvent, such that the composition is in the form of a solid (e.g., powder, granule, pellet).
  • the amount of water in a composition can also be expressed in terms of its water activity, which is the partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water.
  • the dried compositions have a water activity of less than 1, including but not limited to less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, or less.
  • the dried compositions provided herein can be in any solid form, such as a wettable powder (WP), water-soluble powder (WSP), dust (D), granule (G), water-dispersible granule (WDG), dry flowable (DF), or pellet (P).
  • WP wettable powder
  • WSP water-soluble powder
  • D dust
  • G granule
  • WDG water-dispersible granule
  • DF dry flowable
  • P pellet
  • a“wettable powder” refers to a composition wherein the active ingredient is present in a finely ground state (generally micronized to about 5 pm or less in size) and the composition is designed to be applied as a dilute suspension following suspension in a liquid solvent (e.g., water).
  • the wettable powder further comprises a wetting agent and/or dispersing agent.
  • Dry flowable (DF) formulations are aggregated particles of a wettable powder that is water insoluble and form a suspension when mixed with water.
  • WSP Water-soluble powders
  • a dispersing agent is not generally required because of the total water solubility of the components.
  • water-soluble powders can comprise water-soluble inert diluents and/or surfactants.
  • Dust (D) formulations comprise a low percentage of active ingredient(s) (about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less by weight) and a very fine, dry inert carrier made from adsorptive or absorptive inert materials (e.g., talc, chalk, clay, nut hulls, or volcanic ash). The size of the individual dust particles varies. Dust formulations are ready-to-use, requiring no suspension or dispersion in a liquid diluent. Dust formulations are widely used as seed treatments and sometimes for field applications.
  • active ingredient(s) about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less by
  • Granules (G) are coarse dry particles. Granular formulations are similar to dust
  • the dry inert carrier of granular formulations is made from adsorptive or absorptive inert materials (e.g., clay, corn cobs, nut shells, wood fibre, recycled newspaper).
  • adsorptive or absorptive inert materials e.g., clay, corn cobs, nut shells, wood fibre, recycled newspaper.
  • the amount of active ingredient in granular formulations is relatively low, usually ranging from less than 1-15% by weight.
  • Granular formulations are generally ready-to-use, requiring no mixing with a liquid solvent, although some granules require soil moisture to release the active ingredient.
  • Water-dispersible granules are formulations consisting of granules to be applied after disintegration and dispersion in water.
  • the granular composition has distinct particles within the range of 0.2-4 mm.
  • water-dispersible granules can comprise a wetting agent (anionic or nonionic) and/or dispersing agent (anionic).
  • Pellets are uniform small- to medium-sized particles that are round in cross-section. Pellets are ready-to-use and not water-soluble.
  • Dried formulations of the biological control agents will comprise the biological control agents, and in some embodiments, carriers and other agents.
  • the formulations can be used as field inoculants for biocontrol, seed coatings, etc. That is, the dried biocontrol populations can be used in any manner known in the art, including coating seeds with an effective amount of the biocontrol agents, in furrow application of the biocontrol populations directly into the soil, in foliar application, mixing into a potting mixture, and in post-harvest disease control.
  • Such methods are known in the art and are described, for example, in U.S. Patent No. 5,348,742 and in published European Application EP0472494 A2, both of which are herein incorporated by reference.
  • Biocontrol includes management of resident populations of organisms and introductions of specific organisms to reduce disease.
  • the dried compositions of the invention can be dried using any method known in the art, including spray drying, freeze drying, drum drying, convection drying (e.g. tray drying) and fluidized bed drying.
  • Spray drying involves the use of an atomizer or spray nozzle to disperse the liquid or slurry composition into a controlled drop size spray.
  • the liquid or slurry composition is contacted with a drying fluid or gas before, during or after atomization or spraying.
  • a drying gas that is usually air, and is generally heated.
  • the drying gas can be heated to temperatures of up to about 180-200° C.
  • freeze drying also known as lyophilization or cryodesiccation
  • lyophilization is a low temperature dehydration process which involves freezing the liquid or slurry composition, lowering the pressure, and then removing ice by sublimation.
  • the material is cooled below its triple point (the lowest temperature at which the solid, liquid, and gas phases coexist) to ensure sublimation occurs in the later steps.
  • the pressure is lowered (generally within the range of a few millibars) and often sufficient heat is supplied to allow the ice to sublime.
  • Drum drying involves the use of a rotary drum in which liquid or slurry compositions are dried at relatively low temperatures over rotating drums that produce sheets of dried product, which can subsequently be milled to a flake or powder form.
  • Convection drying or direct drying, involves the application of hot air. Air heating increases the drying force for heat transfer and accelerates drying.
  • An example of this is tray drying, in which the input materials may be placed onto trays and into a convection oven.
  • Fluidized bed drying involves the application of hot air that is introduced at high pressure through a perforated bed of moist solid particulate.
  • the gas will move upwards through the spaces between the particles and as the velocity increases, upward drag forces on the particles increase and at a stage become equal to the gravitation forces beneath, at which point, the wet solids are lifted from the bottom and suspended in a stream of air (fluidized state).
  • Heat transfer is accomplished by direct contact between the wet solid and hot gases.
  • the vaporized liquid is carried away by the drying gases.
  • the population of biological control agents present in the dried compositions can comprise both viable and dead microorganisms.
  • the dried compositions comprise about 1% to about 100% viable microorganisms, including but not limited to about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or more viable microorganisms. In other embodiments, the dried compositions comprise only dead microorganisms.
  • the dried compositions disclosed herein can comprise a biological control agent, such as NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof, in an effective amount.
  • a biological control agent such as NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof, in an effective amount.
  • Such an effective amount can comprise a concentration of the biological control agent of at least about 10 5 CFU/gram to about 10 11 CFU/gram, about 10 5 CFU/gram to about 10 10 CFU/gram, about 10 5 CFU/gram to about 10 12 CFU/gram, about 10 5 CFU/gram to about 10 6 CFU/gram, about 10 6 CFU/gram to about 10 7 CFU/gram, about 10 7 CFU/gram to about 10 8 CFU/gram, about 10 8 CFU/gram to about 10 9 CFU/gram, about 10 9 CFU/gram to about 10 10 CFU/gram, about 10 10 CFU/gram to about 10 11 CFU/gram, about 10 11 CFU/gram
  • CFU/gram at least about 10 12 CFU/gram.
  • Wettable powers can comprise a biological control agent, such as NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof, in an effective amount.
  • a biological control agent such as NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof, in an effective amount.
  • Such an effective amount can comprise a concentration of the biological control agent of at least about 10 5
  • CFU/gram to about 10 11 CFU/gram about 10 7 CFU/gram to about 10 10 CFU/gram, about 10 7 CFU/gram to about 10 11 CFU/gram, about 10 6 CFU/gram to about 10 10 CFU/gram, about 10 6 CFU/gram to about 10 11 CFU/gram, about 10 11 CFU/gram to about 10 12 CFU/gram, about 10 5 CFU/gram to about 10 10 CFU/gram, about 10 5 CFU/gram to about 10 12 CFU/gram, about 10 5 CFU/gram to about 10 6 CFU/gram, about 10 6 CFU/gram to about 10 7 CFU/gram, about 10 7 CFU/gram to about 10 8 CFU/gram, about 10 8 CFU/gram to about 10 9 CFU/gram, about 10 9 CFU/gram to about 10 10 CFU/gram, about 10 10 CFU/gram to about 10 11 CFU/gram, about 10 11 CFU/gram to about 10 12 CFU/gram.
  • the concentration of the biological control agent comprises at least about 10 5 CFU/gram, at least about 10 6 CFU/gram, at least about 10 7 CFU/gram, at least about 10 8 CFU/gram, at least about 10 9 CFU/gram, at least about 10 10 CFU/gram, at least about 10 11 CFU/gram, at least about 10 12 CFU/gram, at least about 10 13 CFU/gram.
  • a coated seed which comprises a seed and a coating on the seed, wherein the coating comprises at least one biological control agent, such as NRRL No. B-50999 or NRRL No.
  • the seed coating can be applied to any seed of interest (i.e., for a monocot or a dicot). Various plants of interest are disclosed elsewhere herein.
  • a seed coating can further comprise at least one nutrient, at least one herbicide or at least one pesticide, or at least one biocide. See, for example, US App Pub. 20040336049, 20140173979, and 20150033811.
  • the biocontrol agent provided herein can be mixed with a fungicide, insecticide, or herbicide to enhance its activity or the activity of the chemical to which it has been added.
  • the modified biological control agents of the invention can be used to significantly reduce disease, to promote plant growth and yield, and to reduce reliance on traditional pesticides.
  • the modified agents of the invention can be used with other pesticides for an effective integrated pest management program.
  • the modified biocontrol populations can be mixed in formulation with known pesticides in a manner described in WO 94/10845, herein incorporated by reference.
  • the biocontrol populations are applied in an effective amount.
  • An effective amount of a biocontrol population is an amount sufficient to control or inhibit the pathogen.
  • the effective amount of the biocontrol agent is an amount sufficient to promote or increase plant health, growth or yield in the presence of an agricultural field application rate of a biocide.
  • the rate of application of the biocontrol agent and/or the biocide may vary according to the pathogen being targeted, the plant or crop to be protected, the efficacy of the biocontrol
  • the percent viability of the biocontrol populations the percent viability of the biocontrol populations, the severity of the disease, the climate conditions, and the like.
  • the rate of biocontrol agent application is about 1 g to about 100 kg of active ingredient per hectare, including but not limited to about 1 g, 10 g, 20 g, 30 g, 40 g, 50 g, 100 g, 200 g, 300 g, 400 g, 500 g, 1 kg, 2 kg, 5 kg, 10 kg, 20 kg, 30 kg, 40 kg, 50 kg, 60 kg, 70 kg, 80 kg, 90 kg, and 100 kg.
  • the rate of biocontrol agent application is 10 12 to 10 16 colony forming units (CFU) per hectare. (This corresponds to about 10 g to 10 kg of active ingredient per hectare if the a.i. is 100 billion CFU per g.). In other embodiments, for a field inoculation, the rate of biocontrol agent application is 3 x 10 15 to 1 x 10 17 colony forming units (CFU) per hectare. (This corresponds to about 30 kg to 1000 kg of active ingredient per hectare if the a.i. is 100 billion CFU per g.).
  • the rate of biocontrol agent application is 3 x 10 15 to 1 x 10 17 colony forming units (CFU) per hectare; about lxlO 12 to about lxlO 13 colony forming units (CFU) per hectare, about lxlO 13 to about lxlO 14 colony forming units (CFU) per hectare, about lxlO 14 to about lxlO 15 colony forming units (CFU) per hectare, about lxlO 15 to about lxlO 16 colony forming units (CFU) per hectare or about lxlO 16 to about lxlO 17 colony forming units (CFU) per hectare.
  • the rate of biocontrol agent application is at least about lxlO 13 , about lxlO 14 , lxlO 15 , about lxlO 16 or about lxlO 17 colony forming units (CFU) per hectare.
  • the rate of biocontrol agent application is lOg to 50kg, 50kg to 100kg, 100kg to 200kg, 200kg to 300kg, 300kg to 400kg, 400kg, to 500kg, 500kg to 600kg, 600kg to 700kg, 700kg to 800kg, 800kg to 900kg, 900kg to 1000kg of active ingredient per hectare if the a.i. is 100 billion CFU per g.
  • the rate of biocontrol agent application is at least lOg, 50kg, 100kg, 200kg, 300kg, 400kg, 500kg, 600kg, 700kg, 800kg, 900kg, 1000kg of active ingredient per hectare if the a.i. is 100 billion CFU per g.
  • biocontrol agent applied comprises the strain deposited as NRRL No. B-50897 and/or the strain AIP050999 deposited as NRRL No. B- 50999.
  • Any appropriate agricultural application rate for a biocide can be applied to the plant or crop, for example, an effective amount of the biocide that controls a given organism (i.e., a pest of interest, such as fungus, insects, weeds, disease, etc) may be applied.
  • Methods to assay for the effective amount of the modified biocontrol agent include, for example, any statistically significant increase in plant health, yield and/or growth that occurs upon application of an effective amount of the biocontrol agent and a field application rate of a biocide when compared to the plant health, yield and/or growth that occurs when the same concentration of a non-modified biocontrol agent is applied in combination with the effective amount of the biocide.
  • a further embodiment of the invention provides a method for controlling or inhibiting the growth of a plant pathogen by applying the population of biological control agents of the invention to an environment in which the plant pathogen may grow.
  • the application may be to the plant, to parts of the plant, to the seeds of the plants to be protected, or to the soil in which the plant to be protected are growing or will grow.
  • Application to the plant or plant parts may be before or after harvest.
  • Application to the seeds will be prior to planting of the seeds.
  • a method for growing a plant comprises planting in an area of cultivation a coated seed as described elsewhere herein.
  • the seed is coated with NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof.
  • concentrations of CFUs per seed are disclosed elsewhere herein.
  • a method for growing a plant comprising applying to a plant, a crop, or an area of cultivation an effective amount of a composition comprising at least one biological control agent, such as NRRL No. B-50999 or NRRL No. B-50897 or an active derivative thereof.
  • Various effective amounts of biological control agent are disclosed elsewhere herein and in one, non-limiting example, the effective amount of the biological control agent comprises at least about 10 12 to 10 16 colony forming units (CFU) per hectare.
  • an“area of cultivation” comprises any region in which one desires to grow a plant.
  • Such areas of cultivations include, but are not limited to, a field in which a plant is cultivated (such as a crop field, a sod field, an ornamental plant field, a tree field, a managed forest, a field for culturing fruits and vegetables, any other plant field, etc.), a greenhouse, a growth chamber, etc.
  • a plant, a crop, area of cultivation, seed and/or weed can be treated with a combination an effective amount of the biological control agent and an effective amount of a biocide.
  • a biocide By“treated with a combination of’ or“applying a combination of’ biocontrol agent and a biocide to a plant, a crop, area of cultivation or field it is intended that one or more of a particular field, plant crop, seed and/or weed is treated with one or more of the biocontrol agent and one or more biocide so that a desired effect is achieved.
  • the application of one or both of the biocontrol agent and the biocide can occur prior to the planting of the plant or crop (for example, to the soil, the seed, or the plant).
  • the application of the biocontrol agent and the biocide may be simultaneous or the applications may be at different times (sequential), so long as the desired effect is achieved.
  • the modified biocontrol agent is resistant to glyphosate and further increases plant health, yield or growth when applied in an effective amount
  • the biocide comprises glyphosate or an active derivative thereof.
  • a seed, plant or area of cultivation is treated with a combination of an effective amount of the modified biocontrol agent that is resistant to glyphosate and an effective amount of glyphosate, wherein the effective amount of glyphosate is such as to selectively control weeds while the crop is not significantly damaged.
  • the effective amount of the modified biocontrol agent is sufficient to result in a statistically significant increase in plant health, yield and/or growth when compared to the plant health, yield and/or growth that occurs when the same concentration of a non-modified biocontrol agent is applied in combination with the effective amount of the glyphosate or active derivative thereof.
  • the biocontrol agent comprises an effective amount of AIP1620.
  • the modified biocontrol agent is resistant to glufosinate and further increases plant health, yield or growth when applied in an effective amount, and the biocide comprises glufosinate or an active derivative thereof.
  • a seed, plant or area of cultivation is treated with a combination of an effective amount of the modified biocontrol agent that is resistant to glufosinate and an effective amount of glufosinate, wherein the effective amount of glufosinate is such as to selectively control weeds while the plant or crop is not significantly damaged.
  • the effective amount of the modified biocontrol agent is sufficient to result in a statistically significant increase in plant health, yield and/or growth when compared to the plant health, yield and/or growth that occurs when the same concentration of a non-modified biocontrol agent is applied in combination with the effective amount of the glufosinate or active derivative thereof.
  • the biocontrol agent comprises an effective amount of AIP050999.
  • the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.
  • Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species.
  • Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced
  • the biocontrol agent can be employed with any plant species, including, but not limited to, monocots and dicots.
  • plant species of interest include, but are not limited to, corn ( Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea ), particularly those Brassica species useful as sources of seed oil, alfalfa (.
  • Medicago sativa rice ( Oryza sativa ), rye ( Secale cereale), sorghum ⁇ Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet ⁇ Pennisetum glaucum), proso millet ⁇ Panicum miliaceum), foxtail millet ( Setaria italica), finger millet ⁇ Eleusine coracana )), sunflower ⁇ Helianthus annuus), safflower ⁇ Carthamus tinctorius), wheat ⁇ Triticum aestivum), soybean ( Glycine max), tobacco ⁇ Nicotiana tabacum), potato ( Solanum tuberosum), peanuts ( Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato ( Ipomoea batatus), cassava ⁇ Manihot esculenta), coffee ⁇ Coffea s
  • Vegetables include tomatoes (. Lycopersicon esculentum ), lettuce (e.g., Lactuca sativa ), green beans ⁇ Phaseolus vulgaris ), lima beans ⁇ Phaseolus limensis ), butter beans, kidney beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), pigeon peas (Cajanus cajan), yam beans, jicama, a legumes, peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus ), cantaloupe (C. cantalupensis ), and musk melon (C. meld).
  • Ornamentals include azalea ⁇ Rhododendron spp.), hydrangea ⁇ Macrophylla hydrangea ), hibiscus ⁇ Hibiscus rosasanensis ), roses ⁇ Rosa spp.), tulips ⁇ Tulipa spp.), daffodils ⁇ Narcissus spp.), petunias ⁇ Petunia hybrida ), carnation ⁇ Dianthus
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine ⁇ Pinus taeda ), slash pine ⁇ Pinus elliotii ), ponderosa pine ⁇ Pinus ponder osa ), lodgepole pine ⁇ Pinus contorta ), and Monterey pine ⁇ Pinus radiata ); Douglas-fir ⁇ Pseudotsuga menziesii) ; Western hemlock ⁇ Tsuga canadensis ); Sitka spruce ⁇ Picea glauca) redwood ⁇ Sequoia sempervirens) true firs such as silver fir ⁇ Abies amabilis ) and balsam fir ⁇ Abies balsamea ); and cedars such as Western red cedar ⁇ Thuja plicata ) and Alaska yellow-cedar (C hamaecyparis nootkatensis).
  • plants of the present invention can be crop plants (for example, com, alfalfa, sunflower, Brassica , soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.) or any other plants that benefit from the present invention.
  • a com or soybean plants is employed.
  • Example 1 Production of glyphosate resistant mutants of AIP0069.
  • Glyphosate is a chemical herbicide that accounts for about 25% of the global herbicide market and is applied at a rate of around 200 million pounds per year. This herbicide inhibits the enzyme, 5 -enolpymvylshikimate-3 -phosphate (EPSP) synthase (EPSPS) which catalyzes one step in aromatic amino acid biosynthesis in plants and many bacteria.
  • EPP 5 -enolpymvylshikimate-3 -phosphate
  • EPSPS 5 -enolpymvylshikimate-3 -phosphate
  • glyphosate decreases viability of organisms including any biocontrol agents that rely on EPSPS, and it has been reported to alter the plant microbial community.
  • glyphosate may inhibit the growth of desirable biological control or plant growth promoting bacteria.
  • examples include the herbicides glufosinate (glutamine synthase inhibitor), sulfonylurea and imidazolinone herbicides (branched chain aminio acid synthesis inhibitors) and the antibiotics streptomycin, oxytetracycline and kasugamycin.
  • the biological control strain Pseudomonas fluorescens AIP0069 was streaked onto agar plates containing 0 or 5 mM glyphosate.
  • the basal medium consisted of 11.3 g Na2HPCri 7H2O, 3 g KH2PO4, 1 g NH4CI, 10 g monosodium glutamate, 31 g molasses, 493 mg MgSCri TEhO, 50 mg ZnSCri 7H2O, 5 mg FeSCri 7H2O, and 0.3 g thiamine per liter of deionized water.
  • numerous bacterial colonies were visible after incubating overnight at 25C.
  • glyphosate In the presence of 5 mM glyphosate no colonies were visible after a similar incubation, however, after extended incubation of several days a few colonies were seen. These colonies were isolated and grown in liquid medium at multiple glyphosate concentrations.
  • glyphosate can be used as a selective agent during the production, formulation and/or storage of this strain to prevent contamination by other bacteria.
  • Example 2 Biological control activity of glyphosate resistant mutants.
  • Bacteria were inoculated into 50 ml of broth medium consisting of 11.3 g Na2HPCri 7H2O, 3 g KH2PO4, 1 g NH4CI, 10 g Monosodium glutamate, 30 g molasses, 493 mg MgSCri 7H2O, 50 mg ZnSCri 7H2O, and 5 mg FeSCri 7H2O per liter of deionized water. Cultures were grown in 250 ml baffled flasks in a shaking incubator at 28C, 250 rpm for 2 days. Cells were collected by centrifugation at 3500 xg for 10 minutes.
  • AIP0323 a mutant of AIP0069 which does not have antifungal activity, was included as a negative control.
  • Rhizoctonia solani infested rice grain was produced as described previously (K.A. Holmes and D.M. Benson, 1994. Evaluation of Phytophthora parasitica var. nicotianae as a biocontrol for Phytophthora parasitica on Catharanthus roseus. Plant Disease 78: 193-199.) Infested rice was pulverized in a blender and screened through a #10 sieve (2 mm opening). The pulverized grain was mixed with germination medium at the rate of 2 g per liter.
  • Impatiens seeds were planted into the infested germination medium in size 402 plug trays, treated with 0.3 ml of resuspended bacteria per plug and grown under standard greenhouse production conditions. There were 2 replicates for each experimental treatment with 20 plugs per replicate. The number of healthy seedlings was assessed after two weeks.
  • Table 1 demonstrate that the glyphosate resistant variants AIP0404 and AIP1620 retain full antifungal activity, compared to the progenitor strain, AIP0069.
  • the biocontrol strain can be used to control Fusarium head blight, Asian Soybean Rust,
  • Rhizoctonia Botrytis, Pythium, turf diseases, and the like.
  • Genes encoding glyphosate tolerant EPSPS enzymes may be obtained from various bacteria (A. Schulz et al, 1985. Differential sensitivity of bacterial 5 -enolpyruvylshikimate-3 -phosphate synthases to the herbicide glyphosate. FEMS Microbiology Letters, 28:297-301).
  • the EPSPS genes from Agrobacterium tumefaciens CP4 G.F. Barry et al, 1992. Inhibitors of amino acid biosynthesis: Strategies for imparting glyphosate tolerance to crop plants p. 139-145.
  • B.K. Singh et al. Biosynthesis and molecular regulation of amino acids in plants. Am. Soc. Plant Physiologists, Rockville, MD), and Arthrobacter globiformis (C.L. Peters et al, 2010, GRG23 and GRG51 genes conferring herbicide resistance. US patent 7,674,958), are highly resistant.
  • a suitable gene is amplified by PCR or made synthetically using techniques well known in the art.
  • the open reading frame is cloned into the plasmid vector pKK223-3 (Pharmacia) between the tad promoter and the rrnB transcriptional terminator.
  • the tad promoter provides strong constitutive expression of genes in Pseudomonas.
  • Genomic DNA sequences from strain AIP0069 are incorporated on each side of the promoter - gene - terminator cassette to direct homologous recombination into the AIP0069 chromosome.
  • the resulting plasmid is mobilized from E. coli to Pseudomonas fluorescens AIP0069 by conjugation, a technique well known in the art, and selection on defined medium containing 100 mM glyphosate.
  • the plasmid contains the narrow host range colEl origin of replication and thus cannot replicate in Pseudomonas. Glyphosate resistant colonies will be obtained when the promoter - gene - terminator cassette integrates into the Pseudomonas chromosome by homologous recombination. Single crossover events (where the entire plasmid is integrated into the
  • chromosome are distinguished from double crossover events (where only the desired promoter - gene - terminator cassette is integrated) by PCR, Southern blotting, or other techniques well known in the art. A double crossover event is selected for use.
  • AIP1620 starter cultures were inoculated using colonies from Luria agar plates and grown in 0. IX NBY broth (0.8 g of Difco Nutrient Broth powder and 0.5 g of yeast extract powder per liter of deionized water) and grown at 28C, 250 rpm. Production cultures were grown in a broth containing, per liter of deionized water: 11.3 g Na2HP047H2O, 3.0 g KH2PO4, 1.0 g NEECl, 10 g monosodium glutamate, 3.0 g molasses, 0.49 g MgSCri YEbO, 50 mg ZnSCri TEbO, 5 mg
  • FeS047H2O and sufficient hydrochloric acid to adjust the pH to approximately 6.2 Fifty ml of production broth was placed in a 250 ml baffled culture flask, inoculated with 0.5 ml of starter culture and incubated at 28C, 250 rpm. The production cultures were inoculated at various times and then harvested simultaneously to yield cultures with incubation times of 15, 24, 33, and 43 hours. Forty ml of each culture was harvested by centrifugation. The spent culture broth was discarded and the cells were re-suspended in autoclaved deionized water to 40 ml final volume.
  • Fungal inoculum was prepared using the rice grain method described by Holmes and Benson (K.A. Holmes and D.M. Benson, 1994. Evaluation of Phytophthora parasitica var.
  • the inoculated germination mix was placed in 392 greenhouse plug trays (Landmark Plastic Corporation, Akron, OH) and one impatiens seed was planted into each cell.
  • AIP1620 cell suspensions were applied at the rate of 0.3 ml per cell.
  • the seeds were germinated under standard greenhouse conditions. There were 3 replicates of each treatment with 20 cells per replicate. After 10 to 14 days the assays were scored by counting the number of healthy seedlings in each treatment. Results are summarized in Table 2 below.
  • AIP1620 cells were performed over a 10 month period. For each trial AIP1620 cultures were grown, harvested and re-suspended in autoclaved deionized water essentially as described in Example 4 using a culture time of approximately 24 hours. The greenhouse germination trials also were performed as described in Example 4, but the R. solani inoculum rate varied from 0.25 to 1.0 g of pulverized rice grain per liter of germination mix, depending on the trial. The results compiled from 17 trials are shown in Table 3 below and demonstrate consistent performance of AIP1620 in controlling damping off disease. Table 3. Performance AIP1620 cells in multiple greenhouse seed germination assays
  • AIP1620 cell paste Fifty grams of AIP1620 cell paste was mixed with 50 g of Min-U-Gel 400 or Min-U-Gel 200 attapulgite clay (Active Minerals International, LLC, Sparks, MD) dried to a water activity of less than 0.3. One portion of each formulation was stored at 4°C and another was stored at
  • AIP1620 cell paste was mixed with 20 g of synthetic calcium silicate (MicroCel E, Imerys Filtration Minerals, Lompoc, CA) using a food processor.
  • the resulting material contained 2.7 x 10 10 colony forming units per gram (CFU/g) of AIP1620, as determined by dilution plating.
  • This material was dried at 40C to a water activity of less than 0.30 at which time it contained 1.4 x 10 9 CFU/g of AIP1620.
  • the dried powder formulation was stored in vacuum sealed mylar pouches at 22C. After 85 days the powder contained 1.1 x 10 6 CFU/g of AIP1620 and retained antifungal activity against Rhizoctonia solani as determined by a greenhouse seed germination assay.
  • AIP1620 cell paste was mixed with 5 g of glycerol and 20 g of synthetic calcium silicate using a food processor.
  • the resulting material contained 5.7 x 10 11 CFU/g of AIP1620, as determined by dilution plating.
  • This material was dried at 40C to a water activity of less than 0.30 at which time it contained 3.1 x 10 9 CFU/g of AIP1620.
  • the dried powder formulation was stored in vacuum sealed mylar pouches at 22C. After 61 days the powder contained 6.2 x 10 8 CFU/g of AIP1620 and retained antifungal activity against Rhizoctonia solani as determined by a greenhouse seed germination assay.
  • AIP1620 cell paste was mixed with 5 g of trehalose and 20 g of synthetic calcium silicate using a food processor.
  • the resulting material contained 5.7 x 10 11 CFU/g of AIP1620, as determined by dilution plating.
  • This material was dried at 40C to a water activity of less than 0.30 at which time it contained 4.0 x 10 8 CFU/g of AIP1620.
  • the dried powder formulation was stored in vacuum sealed mylar pouches at 22C. After 54 days the powder contained 2.7 x 10 7 CFU/g of AIP1620.
  • xanthan gum Four grams of xanthan gum and was dispersed into 4 g of soybean oil. The resulting mixture was combined with 100 g of AIP1620 cell paste and allowed to thicken for about 5 minutes at room temperature. The thickened mixture was blended into 20 g of synthetic calcium silicate using a food processor. The resulting material contained 9.4 x 10 11 CFU/g of AIP1620 and was divided into two 50 g portions. One portion was dried at 40C to a water activity of ⁇ 0.30 at which time it contained 7.0 x 10 8 CFU/g of AIP1620.
  • the other portion was dried over silica gel at room temperature to a water activity of ⁇ 0.10 at which time it contained 1.18 x 10 10 CFU/g of AIP1620.
  • Fungal inoculum was prepared using the rice grain method described by Holmes and Benson (K.A. Holmes and D.M. Benson, 1994. Evaluation of Phytophthora parasitica var.
  • Infested rice grains were pulverized in a blender and screened through a #10 sieve. This inoculum was mixed into Fafard superfine germinating mix at the rate of 0.25 g per liter.
  • the inoculated mix was divided and formulated AIP1620 was added at the rate of 5 g per liter. Impatiens seed were planted into the inoculated and treated mixes. The seeds were germinated under standard greenhouse conditions. After 10 days the assays were scored by counting the number of healthy seedlings in each treatment. Results are summarized in Table 6 below.
  • Fungal inoculum was prepared using the rice grain method described by Holmes and Benson (K.A. Holmes and D.M. Benson, 1994. Evaluation of Phytophthora parasitica var.
  • Infested rice grains were pulverized in a blender and screened through a #10 sieve. This inoculum was mixed into Fafard superfine germinating mix at the rate of 0.25 g per liter.
  • the inoculated mix was divided and formulated AIP1620 was added at the rate of 5 g per liter. On the same day, a subsample of each formulation was dilution plated to determine the CFU/g of AIP1620. Impatiens seed were planted into the inoculated and treated mixes. The seeds were germinated under standard greenhouse conditions. After 10 to 14 days the assays were scored by counting the number of healthy seedlings in each treatment. Results are summarized in Table 8 below.
  • AIP1620 cell paste Fifty grams of AIP1620 cell paste was mixed with 50 g of Min-U-Gel 400 or Min-U-Gel 200 attapulgite clay (Active Minerals International, LLC, Sparks, MD) dried to a water activity of less than 0.2, and stored at 22C. The viability of these formulations was tested at various times by dilution plating and the results are shown in Table 9 below. After 21 days both formulations were tested in a greenhouse seed germination assay and found to have retained antifungal activity against Rhizoctonia solani.
  • AIP1620 starter cultures were inoculated using colonies from Luria agar plates and grown in 0. IX NBY broth (0.8 g of Difco Nutrient Broth powder and 0.5 g of yeast extract powder per liter of deionized water) and grown at 28C, 250 rpm. Production cultures were grown in a broth containing, per liter of deionized water: 11.3 g NaiHPCE 7EhO, 3.0 g KH2PO4, 1.0 g NEECl, 10 g monosodium glutamate, 3.0 g molasses, 0.49 g MgSCN 7EhO, 50 mg ZnSCE 7EhO, 5 mg
  • FeSCE 7EhO and sufficient hydrochloric acid to adjust the pH to approximately 6.2 Fifty ml of production broth was placed in a 250 ml baffled culture flask, inoculated with 0.5 ml of starter culture and incubated at 28C, 250 rpm. The production cultures were inoculated at various times and then harvested simultaneously to yield cultures with incubation times of 15, 24, 33, and 43 hours. Forty ml of each culture was harvested by centrifugation. The spent culture broth was discarded and the cells were re-suspended in autoclaved deionized water to 40 ml final volume.
  • Botrytis cinerea was grown on potato dextrose broth for 1 to 2 weeks without shaking. The resulting mycelial mat was removed from the broth and homogenized in autoclaved deionized water to produce liquid inoculum.
  • AIP1620 starter cultures were inoculated using colonies from Luria agar plates and grown in 0. IX NBY broth (0.8 g of Difco Nutrient Broth powder and 0.5 g of yeast extract powder per liter of deionized water) and grown at 28C, 250 rpm. Production cultures were grown in a broth containing, per liter of deionized water: 11.3 g Na2HP047H2O, 3.0 g KH2PO4, 1.0 g NH l, 10 g monosodium glutamate, 3.0 g molasses, 0.49 g MgSCL VEhO, 50 mg ZnS047H2O, 5 mg
  • FeS047H2O and sufficient hydrochloric acid to adjust the pH to approximately 6.2 Fifty ml of production broth was placed in a 250 ml baffled culture flask, inoculated with 0.5 ml of starter culture and incubated at 28C, 250 rpm. The production cultures were inoculated at various times and then harvested simultaneously to yield cultures with incubation times of 15, 24, 33, and 43 hours. Forty ml of each culture was harvested by centrifugation. The spent culture broth was discarded and the cells were re-suspended in autoclaved deionized water to 40 ml final volume.
  • Inoculum of P. aphanadermatum was prepared using the rice grain method described by Holmes and Benson (K.A. Holmes and D.M. Benson, 1994. Evaluation of Phytophthora parasitica var. nicotianae for biocontrol of Phytophthora parasitica on Catharanthus roseus. Plant Disease, 78: 193-199.). Infested rice grains were pulverized in a blender and screened through a #10 sieve. This inoculum was mixed into Fafard superfine germinating mix at the rate of 6.0 g per liter (trials 1 - 4) or 7.0 g per liter (trial 5).
  • the inoculated germination mix was placed in 392 greenhouse plug trays (Landmark Plastic Corporation, Akron, OH) and one impatiens seed was planted into each cell.
  • AIP1620 cell suspensions were applied at the rate of 0.3 ml per cell.
  • the seeds were germinated under standard greenhouse conditions. There were 2 or 3 replicates of each treatment with 20 cells per replicate. After 7 to 17 days the assays were scored by counting the number of healthy seedlings in each treatment. Results are summarized in Table 11 below.
  • Example 16 Control of Asian Soybean rust with AIP1620
  • AIP1620 cells were produced as described in the previous examples. Phakopsora pachyrhizi was grown on susceptible soybean plants and ureidinospores were harvested by vacuum suction from infected leaves which manifested erupted pustules (Twizeyimana, M., and Hartman,
  • Williams 82 soybean plants were grown in plant growth chambers using techniques well known in the art. When plants were at V3 -stage, the first fully expanded trifoliate leaf was sprayed with re-suspended AIP1620 cells, a chemical fungicide standard, or deionized water (inoculated control). One day later the leaves were inoculated with a suspension of P. pachyrhizi ureidinospores (1 x 10 5 /ml). Both inoculation and strain/fungicide were applied using an atomizer attached to an air compressor. The plants were maintained in a growth chamber at 95% RH with a daily cycle of 12 h of light and 12 h of darkness at 21 and 23°C, respectively.
  • Example 17 AIP1620 compatibility with commercial fungicides
  • AIP1620 The viability of AIP1620 was measured after mixing the strain 3 commercial fungicides, each containing a different active ingredient (Table 13). Fungicide concentrations were selected to simulate those in a typical tank mix for field application.
  • AIP1620 was grown in 3 mL of LB medium in a 10 mL tube for 24 hours at 28°C, 250 rpm. Cell pellets were harvested by centrifugation and suspended in 3 mL of dFbO. Nine hundred microliters of cell suspension was mixed with 100 microliters of 10X fungicide stock and incubated at 28°C for 5 minutes or 120 minutes.
  • Example 18 Evaluation of the protectant activity of mixtures of AIP1620 and fungicides against Asian soybean rust caused by Phakoysora yachyrhizi.
  • Bacteria are inoculated into 50 ml of broth medium consisting of 11.3 g NaiHPCE 7EhO, 3 g KH2PO4, 1 g NH4CI, 10 g Monosodium glutamate, 30 g molasses, 493 mg MgSCE 7EhO, 50 mg ZnSCE 7EEO, and 5 mg FeSCE 7EEO per liter of deionized water. Cultures are grown in 250 ml baffled flasks in a shaking incubator at 28C, 250 rpm for 2 days. Cells are collected by
  • AIP0323 a mutant of AIP0069 which does not have antifungal activity, is included as a negative control.
  • Urei dinospores of Phakopsora pachyrhizi are harvested by vacuum suction from leaves infected with the fungus that manifest erupted pustules.
  • the spores are re-suspended in water at 10 A 5/mL and inoculated onto detached soybean leaves as an aerosol using an airbrush using techniques known in the art (Twizeyimana, M., and Hartman, G. L. 2010. Culturing Phakopsora pachyrhizi on detached leaves and urediniospore survival at different temperatures and relative humidities. Plant Disease 94: 1453-1460).
  • AIP1620 cells re-suspended in water and fungicidal active ingredients are prepared in various ratios comprising 10 L 6, 10 L 7, 10 L 8, 10 L 9 or 10 L 10 AIP1620 cells/mL, mixed with fungicidal active ingredient at 1/lOX, 1/3X, 1/2X, or IX normal field use rate, calculated by converting the field rate from the published label to g/mL, based on an assumption about spray volume per hectare or acre.
  • the inoculated soybean leaves are treated the biocontrol agent at the titers above, and with the fungicides at the rates above, as well as with mixtures of biocontrol agent and fungicide in various combinations at the titers and rates specified above.
  • some inoculated detached leaves are left untreated, or treated with AIP0323 as controls.
  • At least 3 leaves (or leaf segments) are used for each treatment of biocontrol agent, chemical, or mixture.
  • the detached leaves are incubated in high humidity in a growth chamber on a 12 light, 21°C / 12 hour dark, 23°C. After 10- 14 days, the leaves are observed and scored according to the number of visible uredinia/cm A 2.
  • Colby’s equation is used to determine the fungicidal effects expected from the mixtures.
  • Example 19 Selection of a population of the biological control strain Pseudomonas fluorescens AIP000069 that has acquired resistance to the herbicide Glufosinate
  • M63 Plus medium 50 microliters of AIP000069 culture, grown in 0.5X LB for 24 hours at 28°C, was spread onto plates containing M63 Plus medium with 0 or 100 mM Glufosinate.
  • the M63 Plus medium consisted of 13.6 g KH2PO4, 9.92g CeHiiOe, 2g (NH ) 2 S0 , 5.5 mg CaCh, 0.278mg FeSOr 7H 2 0, and 10.16 mg MgCh 6H 2 0 per liter of deionized water. In the absence of Glufosinate numerous bacterial colonies (a lawn) were visible after incubating plates for 2 days at 28°C.

Abstract

La présente invention concerne des compositions et des procédés impliquant des compositions séchées d'agents biologiques ou d'agents de lutte biologique, en particulier ceux qui sont modifiés pour pouvoir rivaliser et survivre en plein champ. Par l'amélioration de la population d'agents biologiques, la population d'agents modifiée peut se développer, rivaliser avec d'autres souches microbiennes et champignons et fournir une protection destinée aux plantes contre les pathogènes. De plus, des agents de lutte biologique modifiés favorisent la croissance et le rendement des plantes. En particulier, des agents biologiques modifiés et des populations modifiées de tels agents qui sont tolérants ou résistants aux biocides ; tolérants ou résistants aux herbicides ; tolérants ou résistants aux fongicides ; tolérants ou résistants aux pesticides ; ou tolérants ou résistants aux produits chimiques de protection des cultures sont sélectionnés ou modifiés. De cette manière, la protection des plantes et des cultures contre des agents pathogènes ou des organismes nuisibles est améliorée. Ces agents biologiques peuvent être utilisés comme un inoculant ou comme un enrobage de semences destiné aux plantes et aux semences.
PCT/US2020/032650 2019-05-13 2020-05-13 Agents de lutte biologique séchés et leurs utilisations WO2020232103A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962846845P 2019-05-13 2019-05-13
US62/846,845 2019-05-13

Publications (1)

Publication Number Publication Date
WO2020232103A1 true WO2020232103A1 (fr) 2020-11-19

Family

ID=70919216

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/032650 WO2020232103A1 (fr) 2019-05-13 2020-05-13 Agents de lutte biologique séchés et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2020232103A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024026305A1 (fr) * 2022-07-26 2024-02-01 AgBiome, Inc. Compositions et procédés basés sur pseudomonas chlororaphis pour améliorer la santé des plantes et lutter contre les maladies des plantes

Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4094097A (en) 1976-09-10 1978-06-13 Cornell Research Foundation, Inc. Method for preparing a pesticidally resistant rhizobium and agronomic composition thereof
US4306027A (en) 1976-09-10 1981-12-15 Cornell Research Foundation, Inc. Pesticidally resistant rhizobium and agronomic use thereof
EP0472494A2 (fr) 1990-08-20 1992-02-26 Ciba-Geigy Ag Agents anti-pathogéniques de biocontrôle, gènes codant pour la synthèse d'antibiotiques et utilisation de ces antibiotiques
WO1994010845A2 (fr) 1992-11-17 1994-05-26 Ciba-Geigy Ag Compositions microbicides synergiques
US5348742A (en) 1991-05-24 1994-09-20 Ciba-Geigy Corporation Anti-pathogenic bacterial strains of Pseudomonas fluorescens
US5496547A (en) 1994-01-24 1996-03-05 Ciba-Geigy Corporation Pseudomonas biocontrol strains
WO1996038576A1 (fr) 1995-06-01 1996-12-05 Signal Pharmaceuticals, Inc. Lignee de cellules parentes oligodendrogliales humaines
US5756087A (en) 1996-12-06 1998-05-26 Novartis Finance Corporation Genetically modified Pseudomonas strains with enhanced biocontrol activity
US5792931A (en) 1994-08-12 1998-08-11 Pioneer Hi-Bred International, Inc. Fumonisin detoxification compositions and methods
US5849320A (en) 1996-06-13 1998-12-15 Novartis Corporation Insecticidal seed coating
US5955348A (en) 1997-11-25 1999-09-21 Novartis Ag Genetically modified pseudomonas strains with enhanced biocontrol activity
US6060051A (en) 1997-05-09 2000-05-09 Agraquest, Inc. Strain of bacillus for controlling plant diseases and corn rootworm
US6635425B2 (en) 2001-01-05 2003-10-21 Molecular Staging, Inc. 5′-thio phosphate directed ligation of oligonucleotides and use in detection of single nucleotide polymorphisms
US6758044B2 (en) 2001-10-10 2004-07-06 Goodrich Pump & Engine Control Systems, Inc. Control system for positioning compressor inlet guide vanes
US7250561B1 (en) 1996-07-16 2007-07-31 Bayer Cropscience S.A. Chimera gene with several herbicide resistant genes, plant cell and plant resistant to several herbicides
WO2007103076A1 (fr) 2006-03-01 2007-09-13 Bayer Cropscience, Lp Enrobage de semence a base de polymeres
US7674958B2 (en) 2005-12-01 2010-03-09 Athenix Corporation GRG23 and GRG51 genes conferring herbicide resistance
US8124846B2 (en) 2000-10-30 2012-02-28 Bayer CropScience S.A.S. Herbicide-tolerant plants through bypassing metabolic pathway
US20130142759A1 (en) 2011-11-04 2013-06-06 Agraquest, Inc. Biocontrol of nematodes
US20140173979A1 (en) 2012-12-21 2014-06-26 Dow Agrosciences Llc Method of coating seed
US20150033811A1 (en) 2013-08-05 2015-02-05 Specialty Fertilizer Products, Llc Micronutrient-enhanced polymeric seed coatings
WO2015116838A1 (fr) * 2014-01-31 2015-08-06 AgBiome, Inc. Agents de lutte biologique modifiés et leurs utilisations
WO2017019448A1 (fr) * 2015-07-24 2017-02-02 AgBiome, Inc. Agents de lutte biologique modifiés et leurs utilisations
US9675081B2 (en) * 2014-01-31 2017-06-13 AgBiome, Inc. Modified biological control agents and their uses

Patent Citations (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4306027A (en) 1976-09-10 1981-12-15 Cornell Research Foundation, Inc. Pesticidally resistant rhizobium and agronomic use thereof
US4094097A (en) 1976-09-10 1978-06-13 Cornell Research Foundation, Inc. Method for preparing a pesticidally resistant rhizobium and agronomic composition thereof
EP0472494A2 (fr) 1990-08-20 1992-02-26 Ciba-Geigy Ag Agents anti-pathogéniques de biocontrôle, gènes codant pour la synthèse d'antibiotiques et utilisation de ces antibiotiques
US5348742A (en) 1991-05-24 1994-09-20 Ciba-Geigy Corporation Anti-pathogenic bacterial strains of Pseudomonas fluorescens
WO1994010845A2 (fr) 1992-11-17 1994-05-26 Ciba-Geigy Ag Compositions microbicides synergiques
US5496547A (en) 1994-01-24 1996-03-05 Ciba-Geigy Corporation Pseudomonas biocontrol strains
US5792931A (en) 1994-08-12 1998-08-11 Pioneer Hi-Bred International, Inc. Fumonisin detoxification compositions and methods
WO1996038576A1 (fr) 1995-06-01 1996-12-05 Signal Pharmaceuticals, Inc. Lignee de cellules parentes oligodendrogliales humaines
US5849320A (en) 1996-06-13 1998-12-15 Novartis Corporation Insecticidal seed coating
US7250561B1 (en) 1996-07-16 2007-07-31 Bayer Cropscience S.A. Chimera gene with several herbicide resistant genes, plant cell and plant resistant to several herbicides
US7935869B2 (en) 1996-07-16 2011-05-03 Bayer S.A.S. Chimeric gene with several herbicide tolerance genes, plant cell and plant resistant to several herbicides
US5756087A (en) 1996-12-06 1998-05-26 Novartis Finance Corporation Genetically modified Pseudomonas strains with enhanced biocontrol activity
US6060051A (en) 1997-05-09 2000-05-09 Agraquest, Inc. Strain of bacillus for controlling plant diseases and corn rootworm
US5955348A (en) 1997-11-25 1999-09-21 Novartis Ag Genetically modified pseudomonas strains with enhanced biocontrol activity
US8124846B2 (en) 2000-10-30 2012-02-28 Bayer CropScience S.A.S. Herbicide-tolerant plants through bypassing metabolic pathway
US6635425B2 (en) 2001-01-05 2003-10-21 Molecular Staging, Inc. 5′-thio phosphate directed ligation of oligonucleotides and use in detection of single nucleotide polymorphisms
US6758044B2 (en) 2001-10-10 2004-07-06 Goodrich Pump & Engine Control Systems, Inc. Control system for positioning compressor inlet guide vanes
US7674958B2 (en) 2005-12-01 2010-03-09 Athenix Corporation GRG23 and GRG51 genes conferring herbicide resistance
WO2007103076A1 (fr) 2006-03-01 2007-09-13 Bayer Cropscience, Lp Enrobage de semence a base de polymeres
US20130142759A1 (en) 2011-11-04 2013-06-06 Agraquest, Inc. Biocontrol of nematodes
US20140173979A1 (en) 2012-12-21 2014-06-26 Dow Agrosciences Llc Method of coating seed
US20150033811A1 (en) 2013-08-05 2015-02-05 Specialty Fertilizer Products, Llc Micronutrient-enhanced polymeric seed coatings
WO2015116838A1 (fr) * 2014-01-31 2015-08-06 AgBiome, Inc. Agents de lutte biologique modifiés et leurs utilisations
US9675081B2 (en) * 2014-01-31 2017-06-13 AgBiome, Inc. Modified biological control agents and their uses
WO2017019448A1 (fr) * 2015-07-24 2017-02-02 AgBiome, Inc. Agents de lutte biologique modifiés et leurs utilisations

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
A. SCHULZ ET AL.: "Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate", FEMS MICROBIOLOGY LETTERS, vol. 28, 1985, pages 297 - 301, XP023918645, DOI: 10.1111/j.1574-6968.1985.tb00809.x
ANGIUOLI, S.V. ET AL., BIOINFORMATICS, vol. 27, no. 3, 2011, pages 334 - 42
BENEDICT, M.N. ET AL., BMC GENOMICS, vol. 15, no. 1, 2014, pages 8
BISHOP, C. ET AL., BMC BIOLOGY, vol. 7, no. 1, 2009, pages 1741 - 7007,7-3
CHOI ET AL., J. MICROBIOL. METHODS, vol. 64, 2006, pages 391 - 397
COHAN, F. ET AL., CURRENT BIOLOGY, vol. 17, no. 10, 2007, pages 373 - 86
COLBY, S. R.: "Calculation of the synergistic and antagonistic response of herbicide combinations", WEEDS, vol. 15, 1967, pages 20 - 22
DARLING, A.E. ET AL., PEERJ, vol. 2, 2014, pages e243
DARLING, A.E. ET AL., PLOS ONE, vol. 5, no. 6, 2010, pages e11147
DIDELOT, X. ET AL., GENETICS, vol. 175, no. 3, 2007, pages 1251 - 66
G.F. BARRY ET AL.: "Biosynthesis and molecular regulation of amino acids in plants", 1992, AM. SOC. PLANT PHYSIOLOGISTS, article "Inhibitors of amino acid biosynthesis: Strategies for imparting glyphosate tolerance to crop plants", pages: 139 - 145
HANAHAN, D.: "Studies on transformation of Escherichia coli with plasmids", J. MOL. BIOL., vol. 166, 1983, pages 557 - 77
JOLLEY, K.A. ET AL., BMC BIOINFORMATICS, vol. 11, no. 1, 2010, pages 595
K.A. HOLMESD.M. BENSON: "Evaluation of Phytophthora parasitica var. nicotianae as α biocontrol for Phytophthoraparasitica on Catharanthus roseus", PLANT DISEASE, vol. 78, 1994, pages 193 - 199
K.A. HOLMESD.M. BENSON: "Evaluation of Phytophthoraparasitica var. nicotianae for biocontrol of Phytophthora parasitica on Catharanthus roseus", PLANT DISEASE, vol. 78, 1994, pages 193 - 199
KOEPPEL, A. ET AL., PNAS, vol. 105, no. 7, 2008, pages 2504 - 9
KONSTANTINIDIS, K.T. ET AL., PNAS USA, vol. 102, no. 7, 2005, pages 2567 - 72
LANG, P. ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 76, no. 6, 2010, pages 1913 - 25
LARSEN, M.V. ET AL., JOURNAL OF CLINICAL MICROBIOLOGY, vol. 50, no. 4, 2012, pages 1355 - 61
MENDE, D.R. ET AL., NATURE METHODS, vol. 10, no. 9, 2013, pages 881 - 84
RICHTER, M. ET AL., PNAS, vol. 106, no. 45, 2009, pages 19126 - 31
TREANGEN, T.J. ET AL., GENOME BIOLOGY, vol. 15, no. 11, 2014, pages 524
TWIZEYIMANA, M.HARTMAN, G. L.: "Culturing Phakopsora pachyrhizi on detached leaves and urediniospore survival at different temperatures and relative humidities", PLANT DISEASE, vol. 94, 2010, pages 1453 - 1460
VARGHESE, N.J. ET AL., NUCLEIC ACIDS RESEARCH, 6 July 2015 (2015-07-06), pages gkv657
WANG ET AL., J. CHEM. TECHNOL. BIOTECHNOL., vol. 85, 2010, pages 775 - 778
WU, M. ET AL., BIOINFORMATICS, vol. 28, no. 7, 2012, pages 1033 - 34
WU, M. ET AL., GENOME BIOLOGY, vol. 9, no. 10, 2008, pages R151

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024026305A1 (fr) * 2022-07-26 2024-02-01 AgBiome, Inc. Compositions et procédés basés sur pseudomonas chlororaphis pour améliorer la santé des plantes et lutter contre les maladies des plantes

Similar Documents

Publication Publication Date Title
US11760971B2 (en) Modified biological control agents and their uses
WO2017019448A1 (fr) Agents de lutte biologique modifiés et leurs utilisations
US11518977B2 (en) Modified biological control agents and their uses
WO2020232103A1 (fr) Agents de lutte biologique séchés et leurs utilisations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20729527

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20729527

Country of ref document: EP

Kind code of ref document: A1