ISOLATED BACTERIAL STRAIN OF THE GENUS BURKHOLDERIA AND

PESTICIDAL METABOLITES THEREFROM

PRIORITY CLAIM

This application is a continuation-in-part of U.S. App. No. 13/843,971, filed March 15, 2013, which is a continuation-in-part of U.S. App. No. 13/034,575, filed February 24, 201 1, which claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional App. Nos.

61/308,287, filed February 25, 2010, and 61/406,541 , filed October 25, 2010, all of which are herein incorporated by reference.

Provided herein is a species of Burkholderia sp with no known pathogenicity to vertebrates, such as mammals, fish and birds but exhibits herbicidal activity. Also provided are products derived from a culture of said species and methods of controlling germination and growth of dicotyiedenous, monocoryledonous, and sedge weeds.

BACKGROUND

The Burkholderia genus, β-subdivisioii of the proteobacteria, comprises more than 40 species that inhabit diverse ecological iches (Compant et al., FEMS Microbiol. Rev. 32:607- 626, 2008). The bacterial species in the genus Burkholderia are ubiquiious organisms in soil and rhizosphere (Coenye et al., Environ. Microbiol. 5:719-729, 2003; Parke et al., Ann. Rev. Phytopathology 39:225-258, 2001), Traditionally, they have been known as plant pathogens, B. cepacia being the first one discovered and identified as the pathogen causing disease in onions (Burkholder, Phytopathology 40: 1 15- 1 17, 1950). Several Burkholderia species have developed beneficial interactions with their plant hosts (see, for example, Cabballero- Mellado et al., Int. J. Sysi. Evol. Microbiol. 54: 1 165-1 172, 2004, Chen et al, Int. J. Sysl. Evol. Microbiol. 57: 1055-1059, 2007). Some Biirkhoideria species have also been found to be opportunistic human pathogens (see, for example, Cheng et al. Clin. Microbiol. 18:383- 416, 2005 and Nierman et al, PNAS 101 : 14246-14251, 2004). Additionally, some

Burkholderia species have been found to have potential as biocontrol products (see for example, Burkhead et al, Appl. Environ. Microbiol. 60:2031-2039, 1994; Knudsen et al, Plant Disease 71 :442-445, 1987; Jansiewicz et al., Phytopathology 78: 1697-1700, 1988; US Pub. No. 2003/0082147; US Patent No. 6,077,505; US Patent No. 6,689,357;

WO2001055398; US Patent No. 7, 141,407), Some species in this genus have been effective in bioreinediaiion to decontaminate polluted soil or groundwater (see, for example, Leahy et al, Appl, Environ. Microbiol. 62:825-833, 1996). Further, some Burkholderia species have been found to secrete a variety of extracellular enzymes with proteolytic, lipolytic and hemolytic activities, as well as toxins, antibiotics, and siderophores (see, for example, Ludovic et al., J. Microbiol. Biolechnol. 17: 1407-1429, 2007; Nagamatsu, Rec. Res. Devel Org. Bioorg. Chem. 4:97- 121, 2001 ).

BRIEF SUMMARY

Provided herein is an isolated strain of Burkholderia A396 strain (NRRL Accession No. B-50319). The A396 strain is a non-Burkholderia cepacia, non-Burkholderia plantari, xioa-Burkholderia gladioli, Burkholderia sp. that has the following characteristics:

a. Has a 16S rRNA gene sequence comprising a forward sequences having at least 99.0% identity to the sequences set forth in SEQ ID NQs:8, 1 1, and 12 and a reverse sequence having at least 99.0% identity to SEQ ID NOs:9, 10, and 13-15; b. Has pesticidal, in particular, herbicidal, insectieidal, fungicidal and nematicidal activity;

c. Produces at least one of the compounds selected from templazole A and

templazole B;

d. is non-pathogenic (non-infectious) to vertebrate animals, such as mammals, birds and fish;

e. is susceptible to kanamycin, chloramphenicol, ciprofloxacin, piperacillin,

imipenem, and a combination of sulphamethoxazole and trimethoprim; and

£ contains the fatty acids 16:0, cyclo 17:0, 16:0 3- OH, 14:0, cyclo 19:0 U)8c, 18:0. In a related aspect, disclosed is a method for modulating emergence and/or growth of a monocotyledonous, sedge, or dicotyledonous weed comprising applying to said weed or soil prior to emergence of said weed or after emergence of said weed a composition comprising an isolated strain of Burkholderia sp. A396 or a composition derived therefrom, in an amount effective to modulate emergence and/or growth of the monocotyledonous, sedge, or dicotyledonous weed. In an embodiment, the composition may be a whole cell broth comprising the isolated strain of Burkholderia sp. A396. In another embodiment, the composition may be a supernatant or an extract obtained from the composition comprising the isolated strain of Burkholderia sp. A396.

In another aspect, disclosed is a method for modulating emergence and/or growth of a monocotyledonous, sedge, or dicotyledonous weed comprising applying to the weed or soil a composition comprising an isolated templazole A or templazole B in an amount effective to modulate emergence and/or growth of the monocotyledonous, sedge, or dicotyledonous weed.

The weeds may be grass weeds (e.g., Digiiaria sanguinalis, Echinochloa crus-gali, Phalaris minor and Lolium perenne), sedge weeds (e.g., Cyperus difformis) or broadleaf weeds (e.g., Brassica juncea, TrifoUum repens, Conyza canadensis, Conyza bonariensis, Amaranthus palmeri, Amaranthus rudis, Ambrosia artemisifolia, Ambrosia trifida, Kochia scoparia, Solanum nigrum, Oxalis stricta, Chenopodium album, Medicago poiymorpha, Taraxacum oficinale, Convolvulus arvensos, Pueraria lobata, Malva parviflora, and Gallium aparine). Further provided are seeds coated with the cultures, extracts, strains, compounds, supernatant, whole cell broth, or cell fractions set forth herein. The seeds may he genetically modified seeds that may be herbicide resistant.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the comparison of the growth rate of Burkholderia A396 to

Burkholderia multivorans ATCC 17616.

Figure 2 shows the effect of Burkholderia A396 extract on bindweed.

Figure 3 shows the effect of Burkholderia A396 extract on pigweed.

Figure 4 is a schematic representation of purification scheme for obtaining the templazole and templamide compounds.

DETAILED DESCRIPTION OF EMBODIMENTS

While the compositions and methods heretofore are susceptible to various modifications and alternative forms, exemplary embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives failing within the spirit and scope of the invention as defined by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

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

As defined herein, "derived from" means directly isolated or obtained from a particular source or alternatively having identify ing characteristics of a substance or organism isolated or obtained from a particular source.

As defined herein, an "isolated compound" is essentially free of other compounds or substances, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods, electrophoretic methods.

As defined herein, "whole cell broth" refers to a liquid culture containing both cells and media. If bacteria are grown on a plate the ceils can be harvested in water or other liquid, whole culture.

The term "supernatant" refers to the liquid remaining when cells that are grown in broth or harvested in another liquid are removed by centrifugation, filtration, sedimentation, or other means well known, in the art.

As defined herein, "filtrate" refers to liquid from a whole broth culture that has passed through a. membrane.

As defined herein, "extract" refers to a liquid substance removed from cells by a solvent (water, detergent, buffer, chemical such as acetone) and separated from the cells by centrifugation, filtration or other method.

Tlse Bssrk siderfs Strata

The Burkhoideria strain set forth herein is a non-Burkholderia cepacia complex, non- Burkhoideria plantar i, non-Burkholderia gladioli, Burkhoideria sp and non-pathogenic to vertebrates, such as birds, mammals and fish. This strain may be isolated from a soil sample using procedures known in the art and described by Lorch et al., In Methods in Applied Soil Microbiology and Biochemistry, K. Alef and P. Nannipieri, Eds, San Diego, CA, Academic Press, p. 146-161, 1995. The Burkhoideria strain may be isolated from many different types of soil or growth medium. The sample is then plated on potato dextrose agar (PDA). The bacteria are gram negative, and it forms round, opaque cream-colored colonies that change to pink and pinkish-brown, in color and mucoid or slimy over time.

Colonies are isolated from the potato dextrose agar plates and screened for those that have biological, genetic, biochemical and/or enzymatic characteristics of the Burkhoideria strain of the present invention sei forth in the Examples below . In particular, the

Burkhoideria strain has a 16S rRNA gene comprising a forward sequence that is at least about 99.0%, preferably about 99.5%, more preferably about 99.9% and most preferably about 100% identical to the sequence set forth in SEQ ID NOs: 8, 1 1 and 12 and a forward sequence that is at least about 99.0%, preferably about 99.5%, more preferably about 99.9% and most preferably about 100% identical to the sequence set forth in SEQ ID NOs: 9, 10, 13, 14 and 15 as determined by clustal analysis. Furthermore, as set forth below, this

Burkhoideria strain has, as sei forth below, pesticidal activity, particularly, herbieidal activity. It is not pathogenic to vertebrate animals, such as mammals, birds, and fish.

Additionally, the Burkhoideria strain produces at least templazole A and templazole

B set forth in the instant disclosure.

The Burkhoideria strain is susceptible to kanamyein, chloramphenicol, ciprofloxacin, piperacillin , imipenem, and a combination of sulphamethoxazole and trimethoprim and contains the fatty acids 16:0, cyclo 17:0, 16:0 3- OH, 14:0, cyclo 19:0, 18:0.

This Burkhoideria strain may be obtained by culturing a microorganism having the identifying characteristics of Burkhoideria A396 (NRRL Accession No. B-50319) on Potato Dextrose Agar (PDA) or in a fermentation medium containing defined carbon sources such as glucose, maltose, fructose, galactose, and undefined nitrogen sources such as peptone, tryptone, soytone, and NZ amine.

Herbieidal Compounds

The herbieidal compounds disclosed herein may be produced by culturing the Burkhoideria strain described herein and producing the compounds. The compounds may be isolated from the culture or supernatant of the Burkhoideria strain. Alternatively, the compounds may be synthesized by methods known in the art.

In a particular embodiment, the herbieidal compounds obtainable from the

Burkhoideria strain is templazole A and templazole B having the following structures:

Templazole A and templazoie B are shown to have herbicidal activity in the Example section below.

A substantially pure culture, whole ceil broth, ceil fraction, supernatant, extract, and compounds of the Burkholderia strain of the present invention, may be formulated into herbicidal compositions.

The substances set forth above can be formulated in any manner. Non-limiting formulation examples include but are not limited to emulsiflable concentrates (EC), wettable powders (WP), soluble liquids (SL), aerosols, ultra- low volume concentrate solutions (ULV), soluble powders (SP), microencapsulation, water dispersed granules, flowables (FL), microemulsions (ME), nano-emulsions (NE), etc. In particular, the concentrate, powders, granules and emulsions may be ireeze-dried. In any iOrmulation described herein, percent of the active ingredient is within a range of 0.01% to 99.99%.

The compositions may be in the form of a liquid, gel or solid. Liquid compositions comprise pesticidal compounds derived from said Burkholderia strain, e.g. Burkholderia A396 (NRRL Accession No. B-50319).

A solid composition can be prepared by suspending a solid carrier in a solution of pesticidal compounds and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65°C or lower.

A composition of the invention may comprise gel-encapsulated compounds derived from the Biirkhoideria strain of the present invention. Such gel-encapsulated materials can be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.

The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA List 4B. In another particular embodiment, the nonionic surfactant is

polyoxyethyiene (20) monolaurate. The concentration of surfactants may range between 0.1 - 35% of the total formulation, preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and canonic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of these compositions.

The composition may further comprise another microorganism and/or pesticide (e.g, nematocide, fungicide, insecticide). The microorganism may include but is not limited to an agent derived from Bacillus sp., Pseudomonas sp„ Brevabacillus sp., Lecanicillium sp., non- Ampelomyces sp., Pseudozyma sp., Slreptomyces sp, Burkholderia sp, Trichoderma sp, and Gliocladium sp. Alternatively, the agent may be a natural oil or oil-product having fungicidal and/or insecticidal activity (e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil).

The composition, in particular, may further comprise an insecticide. The insecticide may include but is not limited to avermectin, Bacillus thuringiensis, neem oil and azadiractin, spinosads, Chromobacterium subtsugae, eucalyptus extract, entomopathogenic bacterium or fungi such a Beauveria hassiana, and Metarrhizium anisopliae and chemical insecticides including but not limited to organochlorine compounds, organophosphorous compounds, carbamates, pyrethroids, and neonicotinoids.

The composition my further comprise a nematicide. The nematicide may include, but is not limited to chemical nematicides such as tenamiphos, aldicarb, oxamyl, carbofuran, natural product neamtieide, avermectin, the fungi Paecilomyces lilacinas and Muscodor spp. , the bacteria Bacillus firmus and other Bacillus spp. and Pasteuria penetrans.

The composition may further comprise a biofungicide such as extract of R.

sachalinensis (Regalia) or a fungicide. Such fungicides include, but are not limited to, a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMT) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholme, hydroxypyiimidine, anilinopyrimidine, phosphorothioiate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenyiamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine). In yet a further embodiment, the antifungal agent is a demethylation inhibitor selected from the group consisting of imidazole (e.g., triflumizole), piperazine, pyrimidine and triazole (e.g., bitertanol, myclobutaniL penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole, propiconazole).

The antimicrobial agent may also be a multi-site non-inorganic, chemical fungicide selected from a nitrile (e.g., chloronitrile or fludioxonii), quinoxaline, suiphamide, phosphonate, phosphite, dithiocarbamate, chioraikythios, phenylpyridm-amine, cyano- acetamide, and oxinie.

The compositions may be applied using methods .known in the art. Specifically, these compositions may be applied to plants or plant parts. Plants are to be understood as meaning in the present context all plants and plant populations such as desired and undesired wild plants or crop plants (including naturally occurring crop plants and weeds). Crop plants can be plants which can be obtained by conventional plant breeding and optimization methods or by biotechnological and genetic engineering methods or by combinations of these methods, including the transgenic plants and including the plant cuitivars protectable or not protectable by plant breeders' rights. Plant parts are to be understood as meaning all parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. The plant parts also include harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offshoots and seeds.

Treatment of the plants and plant parts with the compositions set forth above may be carried out directly or by allowing the compositions to act on their surroundings, habitat or storage space by, for example, immersion, spraying, evaporation, fogging, scattering, painting on, injecting. In the case that the composition is applied to a seed, the composition may be applied to the seed as one or more coats prior to planting the seed using one or more coats using methods known in the art.

As noted above, the compositions may be herbicidal compositions. The composition may further comprise one or more herbicides. These may include, but are not limited to, a bioherbicide and/or a chemical herbicide. The bioherbicide may be selected from the group consisting of clove, cinnamon, lemongrass, citrus oils, orange peel oil, tentoxin, comexistisi, AAL-toxin, leptospermone, thaxtomin, sarmentine, momilactone B, sorgoleone, ascaulatoxin and ascaulatoxin aglycone. The chemical herbicide may include, but is not limited to, difiufenzopyr and salts thereof, dicamba and salts thereof, topramezone, tenibotrione, S- metofach!or, atrazine, mesotrione, primisulfuron-methyl, 2,4- dichlorophenoxyacetic acid, nicosulfuron, thifensulfuron-methyl, asulam, metrihuzin, diclofop-methyl, fluazifop, fenoxaprop-p-ethyl, asulam, oxyfiuorfen, rimsuifuron, mecoprop, and quinciorac, thiobencarb, clomazone, cyhalofop, propanil, bensulfuron-methyl, penoxsulam, triclopyr, iniazethapyr, halosulfuron-methyl, pendimethalin, bispyribac- sodium, carfentrazone ethyl, sodium bentazon/sodiurn acifluorfen, glyphosate, glufosinate and orthosulfamuron.

Herbicidal compositions may be applied in liquid or solid form as pre-emergence or post-emergence formulations.

For pre-emergence dry formulations, the granule size of the carrier is typically 1 -2 mm (diameter) but the granules can be either smaller or larger depending on the required ground coverage. Granules may comprise porous or non-porous particles.

For post-emergence formulations, the formulation components used may contain smectite clays, attapulgite clays and similar swelling clays, thickeners such as xanthan gums, gum Arabic and other polysaccharide thickeners as well as dispersion stabilizers such as nonionic surfactants (for example polyoxyethylene (20) monolaurate).

Uses

The compositions and compounds derived from the Burkholderia strain set forth herein may be used as pesticides, particularly as herbicides.

The substances and compositions may be used to modulate emergence in either a pre- emergent or post-emergent formulation of monocotyledonous, sedge or dicotyledonous weeds. In a particular embodiment, the weeds may be Chenopodium sp. (e.g., Chenopodium album, Chenopodium. murale), Abutilon sp. (e.g., Abutilo theophrasU), Helianthus sp. (e.g., Helianthus artnuus), Ambrosia sp. (e.g.. Ambrosia artemesifolia. Ambrosia trifida),

Amaranthus sp. (e.g., Amaranthus retroflexus, Amaranthus palmer i, Amaranthus rudis, Amaranthus spinosus, Amaranthus tuberculatus), Convolvulus sp. (e.g., Convolvulus arvensis), Brassica sp. (e.g., Brassica kaber), Taraxacum sp. (e.g., Taraxacum officinale), Solanum sp. (e.g., Solanum nigrum, Solanum elaeagnifolium, Solanum physalifolium,

Solanum ptycanthum), Malva sp. (e.g., Malva neglecta, Malva parvijlora), Setaria sp. (e.g., Setaria lutescens), Bromus sp. (e.g., Bromus tectorum, Bromus diandrus, Bromus hordeaceus), Poa sp. (e.g., Poa annua, Poa pratensis), Lolium sp. (e.g., Lolium perenne, Lolium rigidum, Lolium multiflorum L. var. Pace), Fesiuca sp. (e.g., Festuca arundinaceae, Fesiuca rubra), Echinochloa sp. (e.g., Echinochloa crus-galli, Echinochloa colona), Oxalis sp. (e.g., Oxalis stricta, Oxalis pes-caprae, Oxalis corniculata); Cyperus sp. (e.g., Cyperus difformis, Cyperus esculentum, Cyperus rotundas, Cyperus brevifolius); Conyza sp. (e.g., Conyza canadensis, Conyza sumairensis. Conyza honarien is); Sagina sp. (e.g., Sagina procumbens); Pueraria lobata, Veronica sp. (e.g., Veronica nederafoiia), Steilaria sp. (e.g., Slellaria media), Rorippa sp. (e.g., Rorippa islandica), Senecio sp. (e.g., Senecio vulgaris), Lamium sp. (e.g., Lamium amplexicaule), Digitaria sp. (e.g., Digitaria sanguinalis, Digitaria ischaemum), Trifolium sp. (e.g., Trifolium repens, Trifolium hirtum, Trifolium incarnatum, Trifolium pratense), Alhagi maurorum, Astragalus spp., Medicago sp. (e.g. Medicago lupulina, Medicago pol morpha), Melilolus sp., Seshania sp. (e.g. Sesbania punicea, Sesbania exaltata), Vicia sp. (e.g. Vicia sativa, Vicia villosa), Gallium sp. (e.g., Gallium aparine), Galinsoga sp. (e.g., Galinsoga aristatula), Cardamine sp. (e.g., Cardamine jlexuosa, Cardamine hirsu(a), Kochia sp. (e.g., Kochia scoparia), Eleusine sp. (e.g., Eleusine indica), Portulaca sp. (e.g., Portulaca oleraceae), Plantago sp. (e.g., Plantago lanceolata), Euphorbia sp. (e.g., Euphomia supina, Euphorbia maculate, Euphorbia esula. Euphorbia prostrata), Erodium sp. (e.g., Erodium cicutarium), Sonchus sp., (e.g., Sonchus oleraceus), Lactuca sp. (e.g., Laciuca serriola), Capsella sp. (e.g., Capsella bursa-pastoris), Leptochloa sp. (e.g., Leptochloa fascicularis, Leptochloa virgata), Raphanus sp. (e..g., Raphanus raphanistrum), Calandrinia sp. (e.g., Calandrinia ciliata), Paspalum sp. (e.g., Paspalum dilatatum), Gnaphalium sp., Cynodon sp, (e.g., Cynodon dactylon, Cynodon hirsutus), Polygonum sp. (e.g.. Polygonum arenastrum, Polygonum lapalhifolium,), Avena falua, Horaeum sp. (e.g., Horaeum leporinum), Urtica sp. (e.g., Urtica wens), Tribulus terrestris, Sisymbrium sp. (e.g., Sisymbrium irio), Cenckrus sp., Salsola sp. (e.g., Salsola tragus, Salsola kali), Amsinckia sp. (e.g., Amsinckia lycopsoides), Ipomoea sp., Claytonia perfoliala, Polypogon sp. (e.g., Polypogon monspeliensis), Xanthium sp., Hypochaeris radicata,

Physalis sp., Eragrostis sp., Verbascum sp., Chamomilla suaveolens, Centaurea sp. (e.g., Centaurea solstitialis), Epilobium brachycarpum, Panicum sp. (e.g., Panicum capilare, Panicum dichotomiflorum), Rumex acetosella, Eclipta sp. (e.g., Eclipta alba, Eclipta prostrata), Ludwigia sp., Urochloa sp. (e.g. Urochloa platyphylla, Urochloa panicoides), Leersia sp., Seshania sp. {Sesbania herbacea), Rotala sp., Ammonia sp., Alternaihera philoxeroides , Commelina sp., Sorghum halepense, Parlhenium hysterophorus, Chloris truncata, and species in the Fabaceae family.

The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLES

The compositions and methods set forth above will be further illustrated in the following, non- limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein.

Example 1, isolation and identification of the microbe

/. / isolation of the microorganism

The microbe is isolated using established techniques know to the art from a soil sample collected under an e vergreen tree at (he Rinnoji Temple, Nikko, Japan. The isolation is done using potato dextrose agar (PDA) using a procedure described in detail by Lorch et al., In Methods in Applied Soil Microbiology and Biochemistry, K. Aief and P. Nannipieri, Eds, San Diego, CA, Academic Press, p. 146- 161 , 1995. In this procedure, the soil sample is first diluted in sterile water, after which it is plated in a solid agar mediu such as potato dextrose agar (PDA). The plates are grown at 25°C for five days, after which individual microbial colonies are isolated into separate PDA plates. The isolated bacterium is gram negative, and it forms round, opaque cream-colored colonies that change to pink and pinkish- brown in color and mucoid or slimy over time.

1.2. Identification on the microorganism

The microbe is identified based on gene sequencing using universal bacterial primers to amplify the 1 6S rRNA region. The following protocol is used: Burkholderia sp A396 is cultured on potato-dextrose agar plates. Growth from a 24 hour-old plate is scraped with a sterile loop and re-suspended in DNA extraction buffer, DNA is extracied using the MoBio Liitra Clean Microbial DNA extraction kit, D A extract is checked for quality/quantity by miming 5μ1 on a 1% agarose gel.

PC reactions are set up as follows: 2 μΐ DNA extract, 5 μΐ PGR buffer, 1 μΐ dNTPs (10 mM each), 1.25 μΐ forward primer (27F; 5 ' - AGAGTTTG A.TCCTG G CTCAG- 3 ' (SEQ ID NO: 1 ), 1.25 μΐ reverse primer (907R; 5 '-CCGTCAATTCCTTTG AGTTT-3 ' (SEQ ID NO:2)) and 0.25 μΐ Taq enzyme. The reaction volume is made up to 50 μΐ using sterile nuclease- free water. The PCR reaction includes an initial denaturation step at 95°C for 10 minutes, followed by 30 cycles of 94°C/30 sec, 57°C/20 sec, 72°C/30 sec, and a final extension step at 72°C for 10 minutes.

The product's approximate concentration and size is calculated by running a 5 μΐ volume on a 1% agarose gel and comparing the product band to a mass ladder.

Excess primers, dNTPs and enzyme are removed from the PCR product with the MoBio PCR clean up kit. The cleaned PCR product as directly sequenced using primers 27F (same as above), 530F (5'-GTGCCAGCCGCCGCGG-3' (SEQ ID NO:3)), 1 1 14F (5'- GCAACGAGCGCAACCC (SEQ ID NO:4)) and 1525R (5 ' - AAGG AGGTGWTCC ARCC- 3' (SEQ ID NO:5)), 1100R (5'-GGGTTGCGCTCGTTG-3 ⁵ (SEQ ID N0:6)), 519R (5'- GWATTACCGCGGCKGCTG-3' (SEQ ID N0:7).

The 16S rRNA gene sequence of strain A396 is compared with the available 16s rRNA gene sequences of representatives of the β-proteobacteria using BLAST. Strain A395 A396 is closely related to members of the Burkholderia cepacia complex, with 99% or higher similarity to several isolates of Burkholderia multivorans, Burkholderia Vietnam ens is, and Burkholderia cepacia. A BLAST search excluding the B. cepacia complex, showed 98% similarity to B. plantarii, B. gladioli and Burkholderia sp. isolates.

A distance tree of results using the neighbor joining method, showed that A396 is related to Burkholderia multivorans and other Burkholderi cepacia complex isolates, Burkholderia plantarii and Burkholderia glumae grouped in a separate branch of the tree.

The isolated Burkholderia strain was found to contain the following sequences:

forward sequence, D A sequence with 27F primer, 815 nucleotides (SEQ ID NO:8);

reverse sequence, 1453 bp, using primers 1525R, 1 100R, 519R (SEQ ID NO:9);

reverse sequence 824 bp using primer 907R (SEQ NO: 10); forward sequence 1 152 bp using primer 530F (SEQ ID NO: l 1); forward sequence 1067 bp using 11 14F primer (SEQ ID NO: 12); reverse sequence 1223 bp using 1525R primer (SEQ NO: 13); reverse sequence 1216 bp using 1 100R primer (SEQ ID NO: 14); reverse sequence i 194 bp using 519R primer (SEQ ID NO: 15).

1.3. Proof that Burkholderia A396 does not belong to Burkholderia cepacia complex 1.3.1 Molecular Biology work using specific PGR primers

In order to confirm the identification of Burkholderia A396 as Burkholderia multivorans, additional sequencing of housekeeping genes is performed. Burkholderia multivorans is a known member of the Burkholderia cepacia complex. Efforts are focused on PGR of recA genes, as described by Mahenthiralingam et aL, 2000. The following primers are used: (a) BCR1 and BCR2 set forth in Mahenthiralingam et a!,, 2000 to confirm B. cepacia complex match and (b) BCRBM1 and BCRBM2 set forth Mahenthiralingam et af, 2000 to confirm B. multivorans match. A product-yielding PGR reaction for the first primer set would confirm ihai ihe microbe belongs to the B. cepacia complex. A product-yielding PGR reaction for the second primer set would confirm that the microbe is indeed B.

multivorans.

No PGR product is obtained for either pair of primers. The performance of the PGR reaction and primers is tested using Burkholderia multivorans ATCC 17616 (positive control) and Pse domonas jluorescens (negative control). Strong bands are observed both for B. multivorans using both sets of primers. No bands are observed for Pseudomonas fluorescens. The results indicate that A396 is a Burkholderia, but not a member of the B. cepacia complex, and not Burkholderia multivorans. This is also demonstrated in a comparative culture experiment in which both A396 and a type culture of B. multivorans are grown side- by-side in a shake culture, and the growth is monitored daily using optical density measurements at 600 nm. Under the set conditions, the novel species A396 grew much faster than the B. multivorans type strain (Figure 1).

1.3.2 DNA-DNA Hybridization

In order to confirm that isolate A396 is a new species of Burkholderia, a DNA-DNA hybridization experiment with Burkholderia multivorans (the closest 16S rRNA sequence match) is conducted. Biomass for both A396 and B. multivorans is produced in 1SP2 broth, grown over 48 hours at 200 rpm/25 ^c C in Fernbach flasks. The biomass is aseptically harvested by centrifugation. The broth is decanted and the cell pellet is resuspended in a 1 : 1 solution of water: isopropanoJ. DNA-DNA hybridization experiments are performed by the DSMZ, the German Collection of Microorganisms and Cell Cultures in Germany. DNA is isolated using a French pressure cell (Thermo Spectronic) and is purified by chromatography on hydroxyapatite as described by Cashion et al,, Anal Biochem. 81 :461-466 (1977), DNA- DNA. hybridization is carried out as described by De Ley et al, Eur, J. Biochem. 12: 133-142 (1970) under consideration of the modifications described by Huss et al, System. Appl.

Microbiol. 4: 184- 192 (1983) using a model Gary 100 Bio UV/VIS-speetrophotometer equipped with a Peltier thermostatted 6x6 muiticell changer and a temperature controller with in-situ temperature probe (Varian). DSMZ reported % DNA-DNA similarly between A396 and Burkholderia multivorans of 37.4%. The results indicate that Burkholderia sp strain A396 does not belong to the species Burkholderia multivorans when the recommendations of a threshold value of 70% DN A-DNA similarity for the definition of bacterial species by the ad hoc committee (Wayne et al, Int. J. Syst. Evol. Microbiol. 37:463-464, 1987) are considered.

1.4. Biochemical profile using Biolog GN2 plates

For the carbon source utilization profile, A396 is grown overnight on Potato Dextrose Agar (PDA). The culture is transferred to BUG agar to produce an adequate culture for Biolog experiments as recommended by the manufacturer (Biolog, Hayward, CA).

The biochemical profile of the microorganism is determined by inoculating onto a Biolog GN2 plate and reading the plate after a 24-hour incubation using the MicroLog 4- automated microstation system. Identification of the unknown bacteria is attempted by comparing its carbon utilization pattern with the Microlog 4 Gram negative database.

No clear definitive matches are found to the Biolog profile. The closest matches all had less than 35% similarity with A396: Pseudomonas spinosa (Biirkhoideria). Biirkhoideria cepacia, and Biirkhoideria pseudomaUei. The results are shown in Table L

Table Ϊ. Biochemical Profile of A396

Substrate Result Substrate 1 Result j i Cyclodextrin L-arabinose

i Dextrin - D-arabitol - ; Glycogen D-cellobiose

: Tween 40 ÷ Erythritol -

: Tween 80 + D-Fruclose -

N-aeetyl-D-Gal actoseamine - L-Fucose -

N-acetyl-D-giucosamine D-Galactose ! + ^■ /- ;

; Adonitoi - Gentibiose - i Succinic Acid Mon-methyl ester - D-Glucose 1 +■ ;

Acetic acid rn -Inositol

i Cis-aconitic acid - D-Lactose - : Citric acid Lactulose

i Formic acid Maltose - D-Galactonie Acid Lactone D-Mannitol

; D-Galacturonie Acid - D-Mannose - 1 D-Giuconic acid D-Melibiose

D-Glucosamiiiic acid - β-methyl-D-glucoside - ; D-Glucuronic Acid D-Psicose

^: a-hydroxvburytic acid - D-Raffinose -

: β-hydroxvbirtyric acid L-Rharnonose - i y-hydroxvbutyric acid - D-Sorbitol - i p-hydroxyphenyiacetic acid - Sucrose ! - i

; Itaconic acid - D-Trehalose ! + ; i a-keto butyric acid - Turanose ! - :

; a-keto glutaric acid - Xylitol

i a-ket valeric acid - Pyruvic Ac id Meshy; es;her - i D,L-Lactic acid Urid ine

: Malonie acid Thymidine - ; Propionic a id + Pheny e thy 1 - amin e

Quinic acid - Putrescine - 1 D- Saccharic acid 2-aminoethanol

Sebacic acid 2,3-ButanedioI

Succinic Acid Glycerol ! +/- \

; Bromosuccinic acid - D,L-a-glycerol phosphate : + - i i Succinamic acid a-D-Glucose- 1 -phosphate

; Glueuron amide D-gUicose-6-phosphate + ; L-alaninamide + γ-ainino butyric acid + \ ; D- Alanine Urocanic acid

i L-alanine ; + Inosine

; L-alanvl-aiycine L-phenylalanme +

1 L-asparagine + L-proline

i L-aspartic acid : H-/„ L-pyroglutamic acid

i L-glutamic acid ; + D-serine

i Glyc l-L-Aspartic acid ! · s rine

; Glycyl-L-glutamic acid - L-threonine

i L-histidine - D,L-carnitine

i Hydroxy-L-proline L-ornithine

L-leucine

1.5. Fatty acid composition

After incubation for 24 hours at 28°C, a ioopful of well-grown ceils are harv ested and fatty acid methyl esters are prepared, separated and identified using the Sherlock Microbial identification System (MIDI) as described (see Vandamme et al., Int. J. Syst. BActeriol. 42:344-356, 1992). The predominant fatty acids present in the Burkholderia A396 are as follows: 16:0 (24.4%), cycle 1 7:0 (7.1 %), 16:0 3- OH (4.4%), 14:0 (3.6%), 19:0 <o8c (2.6%) cycle, 18:0 (1.0%). Summed feature 8 (comprising 18: 1 (o7c) and summed feature 3 (comprising of 16: 1 0)7e and 16: 1 co6c) corresponded to 26.2% and 20.2 % of the total peak area, respectively. Summed feature 2 comprising 12:0 ALDE, 16: 1 iso I, and 14:0 3-OH) corresponded to 5.8% of the total peak area while summed feature 5 comprising 1 8:0 ANTE and 18:2 a)6,9c corresponded to 0.4%. Other fatty acids detected in A396 in minor quantities included: 13: 1 at 12- 13 (0.2%), 14: 1 to5c (0.2%), 15:0 3-OH (0.13%), 17: 1 to 7c (0.14%), 17:0 (0.15%), 16:0 iso 3-OH (0.2%), 16:0 2-OH (0.8%), 18: 1 co7c 1 1 -methyl (0.15%), and 1 8: 1 2-OH (0.4%).

A comparison of the fatty acid composition of A396 with those of known microbial strains in the MIDI database suggested that the fatty acids in the novel strain A396 were most similar with those of Burkholderia. cenocepacia. 1.6 Resistance to Antibiotics

Antibiotic susceptibility of Burkholderia A396 is tested using antibiotic disks on

Muller-Hinton medium as described in PML Microbiological' s technical data sheet #535.

Results obtained after 72-hour incubation at 25°C are presented in Table 2 below.

Table 2: Susceptibility of ΜΒΙ-2Θ6 to various antibiotics. +++ very susceptible, ++ susceptible, - resistant

Concentration (ug) Susceptible

Tetracycline 30 Kananiycin 30 +-·--·

Erythromycin 15 -

Streptomycin 10

Penicillin 10

Ampicillin 10 -

Oxytetracycline 30 -

Chloramphenicol 30 +4-

Ciprofloxacin 5 +4-

Gentamicin 10 -

Piperacillin 100 444

Ο

Imipenem 10 444 ulphametboxazole ^■

Trimethoprim 23,75/ 25 +-;-

The results indicate that the antibiotic susceptibility spectrum of Burkholderia A396 is quite different from pathogenic B. cepacia complex strains. Burkholderia A396 is susceptible to kanamycin, chloramphenicol, ciprofloxacin, piperacillin, imipenem, and a combination of sulphamethoxazole and trimethoprim. As a comparison, Zhou et al,, Antimicrobial Agents and Chemotherapy 51 : 1085-1088, 2007 tested the susceptibility of 2,621 different strains in B, cepacia complex isolated from cystic fibrosis patients, and found that only 7% and 5% of all strains were susceptible to imipenem or ciprofloxacin, respectively. They also found 85% of all strains to be resistant to chloramphenicol (15% susceptible), and 95% to be resistant (5% susceptible) to the combination of sulphamethoxazole and trimethoprim. Results of

Zhou et al., 2007 are similar to those of Pitt et ah, J. Med. Microbiol. 44:203-210, 1996, who determined antibiotic resistance among 366 B, cepacia isoiaies and reported ihai most of them are resistant to ciprofloxacin, cefuroxime, imipenem, chloramphenicol, tetracycline, and sulphametoxacole.

Example 2, Burkholderia sp. as an Herbicide

2.1 Study #1

To confirm the activity found in the initial herbicide screen, an in vivo study is conducted using the Amberlite 7 XAD resin extract derived from a 5 -day old whole cell broth of the novel Burkholderia species. The dried crude extract is resuspended in 4% ethanol and 0.2 % non-ionic surfactant (glycosperse) at a concentration of 10 mg/mL, and further diluted to a concentration of 5.0 mg mL. The two samples are sprayed on 4-week old plants of bindweed (Convolvulus: arvensis), and the plants are kept under growth lights at 25°C for 2 weeks, at which point, the phytotoxicity evaluations are performed. In the same study, 2- week old redroot pigweed plants are sprayed with increasing concentrations of the crude extract derived from the bacterial culture. The test concentrations are 1.25, 2.5, 5.0 and 10.0 mg/mL, and the plants are incubated as described above before phytotoxicity evaluations.

Results presented in Figures 2 (bindweed) and 3 (pigweed) show the phytotoxic effect of Burkholderia crude extract at different concentrations, and they show good herbicidai effect on pigweed even at low treatment concentrations. Both extract treaiments (5 and 10 mg/mL) result in stunting on bindweed.

2.2 Study #2

A novel strain of Burkholderia sp. A396 is gro wn in an undefined mineral medium for 5 days (25°C, 200 rpm). The whole cell broth is extracted using XAD7 resin. The dried crude extract is resuspended in 4% ethanol and 0.2 % non-ionic surfactant at a concentration of 10 mg/mL, and further diluted to concentrations of 5.0, 2.5, and 1.25 mg/mL. All four test solutions are then tested on the following broadieaf and grass weed species listed in Table 3:

Table 3, Broadieaf and Grass Weed Species Tested

Common Name Sciesiiiiic Name

Larabsquarter Chenopodium album

Horseweed Conyza canadensis

Curlydock Rumex crispus

Crabgrass Digitaria sanguina s

Bluegrass Poa annua

Dandelion Taraxacum officinale

Nightshade Solanum nigrum

Mustard Brassica kaher

Mallow Malva neglecta

Cocklebur Xanthium pensyivanicum

Bermuda Grass Cynodon dactylon

Foxiail Setaria luiescens

Sowthistle Sonchus oleraceus

A solution of 0.2 % glycosperse and Roundup at 6 fl oz per gallon rate is used as negative and positive controls, respectively.

Ail plant species are tested in 4"x4" plastic pots in three replicates. The untreated control plants are sprayed with the carrier solution (4% Ethanol, 0.2% glycosperse) and the positive control plants with Roundup at a rate corresponding to 6 fl. oz/acre. Treated plants are kept in a greenhouse under 12h light/12h dark conditions. Phytotoxicity data taken 22 days after treatment for species #1 -8 and 12 days for species #9- 12 are presented in Tables 5 and 6, respectively. The rating scale for both tables is shown in Table 4:

Table 4. Rating Scale Rating Scale % Control

0 0

1 <10

2 25

3 50

4 75

5 100

Table 5. Phytotoxicity Data for Species #1-8

* stunting that resulted in plants approximately half the size of untreated plants

Table 6. Phytotoxicity Data for Species #9-12

Based on the results obtained in these studies, the compounds extracted from fermentation broths of the isolated Burkho!deria species had herbicidal activity against several weed species are tested. Of ihe twelve species tested, Lambsquarters and mustard are most susceptible, followed by mallow and horseweed. Extract concentration as low as 1.25 mg/mL is able to provide almost complete control of Lambsquarters and mustard, whereas higher concentration is required for the mallow and horseweed.

In a separate experiment, using the same design as described above, systemic activity is tested. A 10 mg/mf crude extract supernatant of Burkholderia sp. A396 is painted onto first trite leaves of Ragweed, Mustard, Nightshade, Crabgrass, Wheat and Barnyard Grass.

Seedlings are evaluated 7 days after treatment. Observed symptoms include: burning, warping, bleaching Herbicidal activity is observed in the next leaf above the treated leaf in Ragweed, Mustard and Nightshade. No systemic activity is observed in the tested grasses. In a second experiment, five fractions of the same crude extract (10 mg/ml) are evaluated using the same experimenta l design as described abo v e. Seedlings of Mustard, Wheat and Crabgrass are treated. Seven and 20 days after treatment, symptoms of herbicidai activity are observed in Mustard from four out of the five fractions (091 1 13B4F6, 091 1 13B4F7, 091 1 13B4F8 and 091 1 13B4F9) using a C-18 column (Phenomenex Sepra C18-E, 50 μτη, 65 A). Symptoms are observed in the next leaf above the treated leaf. No systemic activity is observed in the tested grasses.

Example 3, Isolation of Templazole A and B

Methods and Materials

The following procedure is used for the purification of Tem lazole A and B extracted from cell culture of Burkholderia sp (see Figure 4):

The culture broth derived from the 10-L fermentation Burkholderia (A396) in Hy soy growth medium is extracted with Amberlite XAD-7 resin (Asolkar et a]., J. Nat. Prod, 69: 1756-1759, 2006) by shaking the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell mass are collected by filtration through cheesecloth and washed with DI water to remove salts. The resin, cell mass, and cheesecloth are then soaked for 2 h in acetone after which the acetone is filtered and dried under vacuum using rotary evaporator to give the crude extract. The crude extract is then fractionated by using reversed- phase CI 8 vacuum liquid chromatography (H ₂ O/CH ₃ OH; gradient 90:20 to 0: 100%) to give 10 fractions. These fractions are then concentrated to dryness using rotary evaporator and the resulting dry residues are screened for biological activity using 96 well plate lettuce seeding assay. The active fractions are then subjected to reversed phase FIPLC (Spectra System P4000 (Thermo Scientific) to give pure compounds, which are then screened in above mentioned bioassays to locate/identify the active compounds. To confirm the identity of the compound, additional spectroscopic data such as LC/MS and NMR is recorded.

The active fraction 4 is purified further by using HPLC C- 18 column (Phenomenex, Luna l Ou CI 8(2) 100 A, 250 x 30), watenacetonitrile gradient solvent system (0- 10 min; 80% aqueous CHjCN, 10-25 min; 80 - 65% aqueous CH ₃ CN, 25-50 min; 65 - 50 % aqueous CH ₃ CN, 50-60 min; 50-70% CH ₃ CN, 60-80 min; 70-0% aqueous CH ₃ C , 80-85 min; 0 - 20% aqueous CH ₃ CN) at 8 mL/min flow rate and UV detection of 210 nm, to give templazole B, retention time 46.65 min. The other active fraction 6 is also purified using HPLC C- 18 column (Phenomenex, Luna lOu CI 8(2) 100 A, 250 x 30), water:acetonitrile gradient solvent sysiem (0- 10 min; 80 % aqueous C¾CN, 10-25 min; 80 - 60 % aqueous C¾CN, 25-50 min; 60 - 40% aqueous CH ₃ CN, 50-60 min; 40% CH ₃ CN, 60-80 min; 40-0% aqueous CFI ₃ C , 80-85 min; 0-20 % aqueous CH ₃ CN) at 8 mL/min flow rate and UV detection of 210 mn. to give templazole A, retention time 70.82 min.

Mass spectroscopy analysis of pure compounds is performed on a Thermo Finnigan

LCQ Deca XP Plus electrospray (EST) instrument using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XP ^plu£i Mass Spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (FTPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm x 100 mm Luna CT 8 5 um column (Phenomenex). The solvent system consists of water (solvent A) and acetonitrile (solvent B). The mobile phase begins at 10% solvent B and is linearly increased to 100% solvent B over 20 min and then kept for 4 min, and finally returned to 10% solvent B over 3 min and kept for 3 min. The flow rate is 0.5 mL/min. The injection volume was 10 and the samples are kept at room temperature in an auto sampler. The compounds are analyzed by LC-MS utilizing the LC and reversed phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the following conditions: The flow rate of the nitrogen gas was fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, respectively, Electrospray ionization was performed with a spray voltage set at 5000 V and a capillary voltage at 35.0 V. The capillary temperature was set at 400°C. The data was analyzed on Xcalibur software. The active compound templazole A has a molecular mass of 298 and showed m/z ion at 297.34 in negative ionization mode. The LC-MS chromatogram for templazole B suggests a molecular mass of 258 and exhibited m/z ion at 257.74 in negative ionization mode.

^J H, ¹ C and 2D NMR spectra were measured 011 a Bruker 500 MHz & 600 MHz gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm).

For structure elucidation of templazole A, the purified compound with a molecular weight 298 is further analyzed using a 500 MHz NMR instrument, and has Ή NMR δ values at 8.44, 8.74, 8.19, 7.47, 7.31, 3.98, 2.82, 2.33, 1.08 and has ¹³ C NMR δ values of 163.7, 161.2, 154.8, 136.1 , 129.4, 125.4, 123.5, 123.3, 121.8, 121.5, 1 1 1.8, 104.7, 52.2, 37.3, 28.1, 22.7, 22.7. Templazole A has UV absorption bands at 226, 275, 327 nm, which suggested the presence of indole and oxazole rings. The molecular formula, C17H18N2Q3, was determined by interpretation of ^: H, ⁱ³ C NMR and HRESI MS data m/z 299.1396 (M+H) ⁺ (Calcd for Ci7Hi N2C>3, 299.1397), which entails a high degree of unsaturation shown by 10 double bond equivalents. The ¹³ C NMR spectrum revealed signals for all 17 carbons, including two methyls, a methoxy, a methylene carbon, an aliphatic methine, an ester carbonyl, and eleven aromatic carbons. The presence of 3 '-substituted indole was revealed from Ή-Ή COSY and HMBC spectral data. The Ή-Ή COSY and HMBC also indicated the presence of a carboxylic acid methyl ester group and a -CH ₂ -CH-(C¾)2 side chain. From the detailed analysis of Ή-Ή COSY, "C, and HMBC data it was derived that the compound contained an oxazole nucleus. From the 2D analysis it was found that the iso-butyl side chain was attached at C-2 position, a carboxylic acid methyl ester at C-4 position and the indole unit at C-5 position to give templazole A, shown below.

The second herbicidally active compound, templazole B, with a molecular weight 258 is further analyzed using a 500 MHz NMR instrument, and has ^! H NMR δ values at 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93 and ⁱ³ C NMR values of δ 158.2, 156.3, 155.5, 132.6, 129.5, 129.5, 127.3, 121.8, 1 15.2, 1 15.2, 41.2, 35.3, 26.7, 21.5, 21.5. The molecular formula, is assigned as C15H1 SN2Q2, which is determined by interpretation of Ή, ^{J i} C NMR and mass data. The ^{l 3} C NMR. spectrum revealed signals for all 5 carbons, including two methyls, two methylene carbons, one aliphatic methine, one amide carbonyl, and nine aromatic carbons. The general nature of the structure was deduced from ^! H and ^l3 C NMR spectra that showed a /wra-substituted aromatic ring [δ 7.08 (2H, d, J = 8.8 Hz), 6.75 (2H, d, J = 8.8 Hz), and 132.7, 129.5, 115.2, 127.3, 115.2, 129.5]. The Ή NMR spectrum of this structure together with the Ή-Ή COSY and HSQC spectra, displayed characteristic signals for an isobutyl moiety [δ 0.93 (6H, d, J = 6.9 Hz), 2.15 (1H, sept., J = 6.9 Hz), 2.57 (2H, d, J = 6.9 Hz). In addition, an olefinic/aromatic proton at (δ 7.06, s), and a carbonyl carbon group (δ 158.9) were also found in the ¾ and ¹³ C NMR spectra. On inspection of the HMBC spectrum, the H-l ' signal in the isobutyl moiety correlated with the olefmic carbon (C-2, δ 156.3), and the oiefmic proton H-4 correlated with (C-5, δ 155,5; C-2, 156.3 & C- l ", 41.2). The methylene signal at δ 3.75 correlated with C-5, C-4 as well as the C-2" of the para-substituted aromatic moiety. All these observed correlations suggested the connectivity among the isobutyl, and the para-substituted benzyl moieties for the skeleton of the structure as shown. In addition, the carboxamide group is assigned at the para position of the benzyl moiety based 011 the HMBC correlation from the aromatic proton at H-4"& H-6" position. Thus, based on the above data, the structure was designated as templazole B, shown below.

Example 4. Herbicidal Effect of Burkholderi sp. A396 Formulations (Pre-Emergent) To determine the spectrum of pre-emergence activity, tests were conducted in a petri dish or small pot conditions. Tn laboratory testing, 35 seeds were placed on a ring of blotter paper inside a 3 cm petri dish and supplied with 4 ml of MBI-010 (<().1 mg MBI-()()5/ml). Water was used as a negative control and oryzalin applied as a positive control. Petri dishes were randomly placed in a growth room at 25 °C and 50% RH. Treatments were replicated three times and germinated seeds were counted 7 and 14 days after application; water was added as necessary to maintain moisture levels inside each petri dish.

In pot testing, potting soil was placed into 4 inch square pot, into which were then inserted five weed tubers, rhizomes or other underground perennation structure, according to species. Pots were drenched with 20 mL MBI-010 (Burkholderia sp. A396) at a range of dilutions with water. Treatments, including water as the negative control and glyphosate as the positive control, were replicated five times. Treatments were evaluated visually as number of germinating plants per pot and above-ground fresh weights per container were taken.

Results in Table 7 indicate broad spectrum activity on both annual grasses and hroadieaves, as well as on some perennials.

Table 7: Pre-Emergent Effect of Burkholderia sp. A396 Formulations

Conyza

Horseweed (R) canadensis ++++ petri dish Supernatant

Arnaranihus

Palmer pigweed (R) pa.lmerii _++÷+ peiri dish Supernatant

Cyperus

Sciges, annual Smallfiower dij brmis +÷++ petri dish Sucematant

Broad!eai ^" , Field Bindweed (root Convolvulus

perennial segments) arvensis +÷÷+ pots Supernatant

Sedge, Puple Nut sedge Cyperus

perennial (tubers) rotutidus 4- ..Mi> Supernatant

Example S, Herbkidal Effect of B rkholderia sp, A396 Formulations (Post-Emergent) To determine the spectrum of post-emergence activity, tests were conducted in laboratory and field conditions. For laboratory foliar applications, 3-10 plants (depending on the species) at the 1-2 leaf stage in 2.5 cm square pots containing potting soil were sprayed with MBI-010 (Burkholderia sp, A396) at a rate of 40 gal/A using a cabinet track sprayer. Negative controls were sprayed with water and positive controls with glufosinate. Pots were randomly placed in a growth room at 25 °C and 50% RH, and watered as necessary.

Treatments were replicated five times and evaluated at 7 and 14 days for visual % damage, with 0% indicating no damage and 100% indicating plant death.

In drench testing, potting soil was placed into 4 inch square pots containing plants at the 2 -3 leaf stage. Pots were drenched with 20 mL MBI-010 (Burkholderia sp. A396) at a range of dilutions with water. Treatments, including water as the negative control and oryzalin as the positive control, were replicated five times and kept in a growth room as described above. Treatments were evaluated visually on a percent control basis and above- ground fresh weights per container were taken.

In field testing, field soil containing weeds at the 1 -5 leaf stage were treated with 50% 010 + water solutions delivered via a hand sprayer to full coverage. Treatments, including water as the negative control and glufosinate as the positive control, were replicated 3 times and applied twice at a four week interval. Treatments were assessed for % control. Results in Table 8 indicate broad spectrum post-emergence activity on broadleaves, with little to no activity on grasses, either applied as a soil drench or as a foliar application.

Table 8: Post-Emergent Effect of B rkholderia sp, A396 Formulations. An S indicates an assay that successfully showed systemic activity, a 0 indicates no activity.

Species Species

Plant (c Entn ni {scientific Test Scale Product Category name) name) Mode Rating (lab/GH/field) Embodiment

Grass, Digifaria Prototype annual Crabgrass sanguinalis Foliar Greenhouse formulation

Digifaria Prototype

Crabgrass sanguinalis Drench 0 Greenhouse formulation

Echinochioa

Bamyardgrass crus-galli Foliar + Greenhouse Supernatant

Echinochioa

Barnyardgrass crus-galli Drench 0 Greenhouse Supernatant

B!uegrass Poa annua Foliar 0 Greenhouse CE

Broadleaf, Br ^ic

annual Mustard kaber Foliar ÷+÷+ Greenhouse Supernatant

Br ^ic

Mustard kaber Drench ÷++ Greenhouse Supernatant

Trifolium

Clover repens Drench ÷+÷+ Greenhouse Supernatant

Chenopodiu

Lambsquarters m album Drench ÷+÷+ Greenhouse Supernatant

Amaranthus

Pigweed reiroflexus Spot ÷++ Greenhouse CE

Amaranthus

Pigweed reiroflexus Foliar ÷+÷+ Greenhouse Supernatant

Ambrosia.

Ragweed ariernisifoUa Foliar s Greenhouse WCB

Black Solatium

nightshade nigrum Spot s Greenhouse WCB

onyza

Horseweed canadensis Foliar Greenhouse CE

Yellow eniaurea

Starthistle solstitialis Field 0 Field Supernatant

Mallow Malva spp. Field -i Field Supernatant

Capsella

Shepherd's bursa- Purse pasiora Field Field Supernatant

Lamium

Henbit a j lexicuale. Field 0 Field Supernatant

California Medicago Field -r-H- Field Supernatant burclover polymorpka Cut! eat ^' Geranium

geranium dissection Field Fieid Supernatant

Broadieaf, Taraxacum

peretmia] Dandelion oficinaie Foliar Greenhouse Supernatant

Taraxacum

Dandelion oficinaie Drench 0 Greenhouse Supernatant

Taraxacum Drench

Dandelion oficinaie & Foliar Greenhouse Supernatant

Convoivulus

Bindweed arvensis Foliar s Greenhouse WCB

Bum ex

Curly Dock crispus Foliar Greenhouse CE

Crops Fava Beans Foliar - -H-+ Greenhouse WCB

Snap Peas Foliar -H- Greenhouse WCB

Cucumber Foliar - -H-+ Greenhouse WCB

Radish Foliar - -H-+ Greenhouse WCB

Tomato Foliar - -H-- Greenhouse WCB

Bean Foliar ++ Greenhouse WCB

Rice Foliar 0 Greenhouse CE

Wheat Foliar 0 Greenhouse CE

Sorghum Foliar 0 Greenhouse CE

Broccoli Foliar 0 Greenhouse CE

Capsicum

Peppers annum Drench 0 Greenhouse Supernatant

Com

(conventional) Zea mays Foliar 0 Greenhouse CE

Libert Link

Corn Zea mays Foliar 0 Greenhouse CE

Arackis

Peanuts hypo aea Foliar Greenhouse Supernatant

CE is concentrated extract; WCB is whole ceil broth and Prototype Formulation is whole cell broth with added surfactants (e.g. hostaphat or genapol).

Example 6: Herbicidal activity of Templazole A and Templazole B

The herbicidal activity of the active compounds templazole A and B were tested in a laboratory assay using one-week old lettuce seedlings in a 96 -well plate platform. One lettuce seedling was placed in each of the wells containing 99 microliters of deionized water, into each well, a one microliter aliquot of the pure compound in ethanol (10 mg/mL) was added, and the plate was sealed with a lid. One microliter of ethanol in 99 microliters of water was used as a negative control. The treatments were done in eight replicates, and the sealed plate was incubated in a greenhouse under artificial lights (12 hr light/dark cycle). After five days, the results were read by visual inspection.

Table 9: Herbicidal bioassay data for templazole A and templazole B. Samples were tested at 100 ug/ ^' mL concentration per well.

The following biological material has been deposited under the terms of the Budapest Treaty with the Agricultural Research Culture Collection (NRRL), 1815 N. University Street, Peoria, Illinois 61604 USA, and given the following number:

Deposit Accession Number Date of Deposit

Burkholderia sp. A396 NRRL, B-50319 September 15, 2009

The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. § 122, The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subjeci application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of se veral aspects of the invention. Any equivalent aspects are intended to he within the scope of this invention. Indeed, various modifications of the invention in addition io those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.