NZ620640B2 - Isolated bacterial strain of the genus burkholderia and pesticidal metabolites therefrom-formulations and uses - Google Patents

Isolated bacterial strain of the genus burkholderia and pesticidal metabolites therefrom-formulations and uses Download PDF

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NZ620640B2
NZ620640B2 NZ620640A NZ62064012A NZ620640B2 NZ 620640 B2 NZ620640 B2 NZ 620640B2 NZ 620640 A NZ620640 A NZ 620640A NZ 62064012 A NZ62064012 A NZ 62064012A NZ 620640 B2 NZ620640 B2 NZ 620640B2
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New Zealand
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spp
paraben
burkholderia
active
substituted
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NZ620640A
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NZ620640A (en
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Ratnakar Asolkar
Marja Koivunen
Pamela MARRONE
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Marrone Bio Innovations Inc
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Priority claimed from PCT/US2012/050807 external-priority patent/WO2013032693A2/en
Publication of NZ620640A publication Critical patent/NZ620640A/en
Publication of NZ620640B2 publication Critical patent/NZ620640B2/en

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    • A—HUMAN NECESSITIES
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Abstract

Discloses an insecticidal or herbicidal composition comprising: (A) an isolated strain of Burkholderia sp. A 396 (NRRL Accession No. B-50319); (B) a C1-C8 paraben, and (C) a C2-C17 alcohol, wherein said C1-C8 paraben is formed by incubating (A) and (C) at a temperature sufficient to produce said C1-C8 paraben. C1-C8 paraben.

Description

wo 2013/032693 ED BACTERIAL STRAIN OF THE GENUS BURKHOLDER/A AND PESTICIDAL METABOLITES THEREFROM-FORMULATIONS AND USES TECHNICAL FIELD Provided herein is a species of lderia sp with no known pathogenicity to vertebrates, such as mammals, fish and birds but pesticidal ty against plants, algae, insects, fungi, arachnids, such as mites and nematodes and formulations and compositions comprising said species. Also provided are natural products, formulations and compositions derived from a culture of said species and methods of controlling algae and arachnids, such as mites, using said lderia and/or said natural products.
BACKGROUND l products are substances produced by es, plants, and other organisms.
Microbial natural products offer an abundant source of chemical diversity, and there is a long history of utilizing natural products for pharmaceutical purposes. One such compound is FR901228 isolated from Chromobacterium and has been found to be useful as an antibacterial agent and antitumor agent (see, for example, Ueda et al., US Patent No.7,396,665).
However, ary metabolites produced by microbes have also been successfully found to have uses for weed and pest control in agricultural applications (see, for example, Nakajima et al. 1991; Duke et al., 2000; Lydon & Duke, 1999; Gerwick et al., US Patent No. 7,393,812). Microbial natural products have been also successfully developed into agricultural icides (see, for example, Salama et al. 1981; Thompson et al., 2000; Krieg et al. 1983).
Sometimes, such natural products have been combined with chemical pesticides (see, for example, Gottlieb, US Patent No. 4,808,207).
Acaricides Acaricides are compounds that kill mites (miticides) and ticks (ixodicides). This class of pesticides is large and includes antibiotics, carbamates, idine acaricides, roids, mite growth regulators, and organophosphate acaricides. Besides chemical ides, diatomaceous earth and fatty acids can be used to control mites. They typically work through disruption ofthe e, which dries out the mite. In addition, some essential oils such as mint oil, are used to control mites. In spite ofthe great variety ofknown acaricide compounds, mites remain a serious problem in agriculture because ofthe damage they cause to wo 2013/032693 the crops. They can produce several tions during one season, which facilitates rapid development of resistance to the acaricide ts used. Hence, new pesticide products with new target sites and novel modes of action are critically .
Algicides Algae come in many forms. These include: (1) microscopic, lled algae, filamentous algae that resemble hair, algae that grow in sheets and macroalgae that look like plants; (2) algae that live inside the outer integument ("skin") or calcium shell of some corals, anemones, and other e invertebrates called zooxanthellae; (3) very hard-to-remove little dots of green that sometimes grow on aquarium panels which also are not algae, but diatom or radiolarian colonies (microscopic, lled, animals with hard shells) with algae incorporated in their matrix.
Growth of algae in a small amount of water retained in the container over a significant period of time can be considerable, which is highly rable. As a result, algae can cause clogging of filters in water filtration devices, undesirable smells and appearance in pools, exhaustion of dissolved oxygen, and suffocation of fishes and shellfishes to death. In addition to being t in water, algae may also be present in industrial materials which are exposed to the weather and light, such as coatings containing organic film formers on mineral substrates, e es, wood paints and also materials made of cs.
Algae control can be divided into four categories: biological, ical, physical and chemical controls. A few pertinent facts hold for all methods of algae control. For example, Turbo and Astrea snails, some blennies, some tangs, among others are good grazers. Snails are the most widely used scavengers, and generally the best choice. Some parts of the country seem to favor the use of sea urchins, dwarf angels. The former die too easily and move the decor about, and the latter can be problematical with eating expensive invertebrates. Other methods include functional protein skimmers, with or without ozone and ultraviolet sterilizers. These physical filters remove and destroy algae on exposure and help oxidize nutrients as the water is circulated. Antibiotics may also be used. However, they treat the symptoms only without dealing with the cause(s) of the algae problem. The factors can contribute to water system being out of balance. Copper, usually in the form of copper sulfate solution has been employed as an algicide~ as well as a general epizootic parasite preventative. This metal is useful in treatment and quarantine tanks, dips and fish-only ements but it is persistent and toxic to all life, especially non-fish. wo 2013/032693 Burkholderia The Burkholderia genus, 13-subdivision of the proteobacteria, comprises more than 40 species that inhabit diverse ecological niches nt et al., 2008). The bacterial species in the genus Burkholderia are tous organisms in soil and rhizosphere (Coenye and Vandamme, 2003; Parke and -Sherman, 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, 1950). Several Burkholderia species have developed beneficial interactions with their plant hosts (see, for example, Cabballero-Mellado et al., 2004, Chen et al., 2007). Some Burkholderia species have also been found to be opportunistic human pathogens (see, for example, Cheng and Currie, 2005 and Nierman et al., 2004). onally, some Burkholderia species have been found to have ial as biocontrol products (see for example, Burkhead et al., 1994; Knudsen et al., 1987; Jansiewicz et al., 1988; Gouge et al., US Patent Application No. 2003/0082147; Parke et al., US Patent No. 6,077 ,505; Casida et al., US Patent No. 6,689,357; Jeddeloh et al., W02001055398; Zhang et al., US Patent No.7,141 ,407).
Some species of in this genus have been effective in bioremediation to aminate polluted soil or groundwater (see, for example, Leahy et al. 1996). Further, some Burkholderia species have been found to e a variety of extracellular enzymes with proteolytic, lipolytic and hemolytic activities, as well as toxins, antibiotics, and siderophores (see, for example, Ludovic et al., 2007; Nagamatsu, 2001).
PCT/US2011!026016 discloses a Burkholderia species, particularly Burkholderia A396 and compounds derived from said species with no known pathogenicity to vertebrates with activity against plants, insects, fungi and nematodes.
Oxazoles, Thiazoles and s Oxazoles, thiazoles and indoles are widely distributed in plants, algae, sponges, and microorganisms. A large number of natural products n one or more ofthe five-membered oxazole, thiazole and indole s/moieties. These l products exhibit a broad spectrum ofbiological activity of demonstrable therapeutic value. For example, bleomycin A (Tomohisa et al.), a widely prescribed anticancer drug, effects the oxidative degradation of DNA and uses a bithiazole moiety to bind its target DNA ces (Vanderwall et al., 1997). acin (Ming et al., 2002), a thiazoline-containing peptide antibiotic, interdicts bacterial cell wall new thesis by complexation with C55-bactoprenolpyrophosphate. Thiangazole (Kunze et al., 1993) contains a tandem array of one oxazole and three thiazolines and exhibits ral activity (Jansen et al., 1992). Yet other oxazole/thiazole-containing natural products such as thiostrepton (Anderson et al., 1970) and A (Selva et al., 1997) inhibit translation steps in bacterial protein synthesis. More than 1000 alkaloids with the indole skeleton have been reported from microorganisms. One-third of these compounds are peptides with masses beyond 500 Da where the indole is tryptophan derived. The structural variety of the remaining two thirds is higher, and their biological activity seems to cover a broader range, including antimicrobial, ral, cytotoxic, icidal, antithrombotic, or enzyme inhibitory activity.
BRIEF SUMMARY According to a first aspect of the present ion there is provided an insecticidal or herbicidal composition comprising: (A) an isolated strain of Burkholderia sp. A 396 (NRRL Accession No. B-50319); (B) a C1-C8 paraben, and (C) a C2-C17 alcohol, n said C1-C8 paraben is formed by incubating (A) and (C) at a temperature sufficient to produce said C1-C8 paraben.
According to a second aspect of the present invention there is provided a method for obtaining a C1-C8 paraben comprising (A) providing a ition comprising an isolated strain of lderia sp. A 396 (NRRL Accession No. B-50319); (B) providing a C2-C17 alcohol; (C) incubating the composition of (A) and the alcohol of (B) for a time and at a temperature sufficient to produce said C1-C8 paraben; and (D) isolating said C1-C8 paraben.
According to a third aspect of the present invention there is provided a method of modulating pest infestation, and/or monocotyledonous, sedge, or dicotyledonous weeds, comprising applying the composition according to the first aspect of the invention, to a location where modulation is desired in an amount effective to modulate said pest ation, and/or monocotyledonous, sedge, or dicotyledonous weeds.
According to a fourth aspect of the present invention there is ed a method for making an insecticidal or herbicidal ition sing (A) providing a composition comprising an ed strain of Burkholderia sp. A 396 (NRRL Accsession No. B-50319); (B) providing a C2-C17 alcohol; and (C) incubating the composition of (A) and the alcohol of (B) for a time at a temperature sufficient to produce C1-C8 paraben.
AH26(10414908_1):JIN ed herein is an isolated strain of a rkholderia cepacia, non-Burkholderia ri, non-Burkholderia li, Burkholderia sp. which has the following characteristics: (a) Has a 16rRNA gene sequence comprising a forward sequences having at least 99.5% identity to the sequences set forth in SEQ ID N0:8, 11 and 12 and a reverse sequence having at least 99.5% ty to SEQ ID N0:9, 10, 13-15; (b) Has pesticidal, in particular, idal, algicidal, acaricidal, insecticidal, fungicidal and nematicidal activity; (c) Produces at least one of the compounds ed from the group consisting of: (i) a compound having the following properties: (a) a molecular weight of about 525-555 as determined by Liquid Chromatography/Mass oscopy (LC/MS); (b) 1H NMR values of 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23. 1.74, 1.15, 1.12, 1.05, 1.02; (c) has 13C NMR values of 172.99, 172.93, 169.57, 169.23, 20 167.59, 130.74, 130.12, 129.93, 128.32, 73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90, .36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51 and (c) an High Pressure Liquid Chromatography (HPLC) retention time of about 10-15 minutes, on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) gradient; (ii) a compound having an oxazolyl-indole structure comprising at least one indole moiety, at least one oxazole moiety, at least one substituted alkyl group and at least one carboxylic ester group; at least 17 s and at least 3 oxygen and 2 nitrogens; (iii) a compound having an oxazolyl-benzyl structure comprising at least one benzyl moiety, at least one oxazole moiety, at least one substituted alkyl group and at least one amide group; at least 15 carbons and at least 2 oxygen and 2 nitrogens; (iv) a compound having at least one ester, at least one amide, at least three methylene groups, at least one tetrahydropyranose moiety and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least twenty five carbons and at least eight oxygen and one nitrogen and (d) is non-pathogenic (non-infectious) to vertebrate animals, such as mammals, birds and fish; AH26(10414908_1):JIN wo 2013/032693 (e) is susceptible to kanamycin, chloramphenicol, ciprofloxacin, piperacillin, imipenem, and a combination of sulphamethoxazole and hoprim and (f) contains the fatty acids 16:0, cyclo 17:0, 16:0 3- OH, 14:0, cyclo 19:0 w8c, 18:0.
In a particular embodiment, the strain has the fying characteristics of a Burkholderia A396 strain (NRRL Accession No. B-50319).
In a particular embodiment, the first nce is a supernatant. In yet even a more particular embodiment, the supernatant is a cell-free supernatant.
Also provided is a combination, particularly a composition or ation comprising (a) a first nce selected from the group consisting of a pure culture, cell fraction or atant derived from the Burkholderia strain set forth above or extract thereof for use optionally as a pesticide; and (b) ally at least one of a carrier, diluent, surfactant, adjuvant, or chemical or biological pesticide (e.g., algicide, acaricide, herbicide, fungicide, icide, nematocide and particularly, algicide or acaricide (e.g., miticide)). In a related aspect, provided herein is a seed coated with said combination or composition.
In a particular embodiment, the composition or formulation may comprise: (a) a first nce selected from the group ting of a pure culture, cell fraction or supernatant derived from the Burkholderia strain set forth above or extract thereof for use optionally as a pesticide; (b) fatty acids 16:0, cyclo 17:0, 16:0 3-0H, 14:0, cyclo 19:0 w8c, 18:0, C1-C7 paraben, C2-C 17 alcohol and detergent and (c) ally another substance wherein said other substance is a pesticide (e.g., fungicide, insecticide, algicide, acaricide (e.g., miticide), herbicide, nematocide).
In a particular embodiment, the C1-C7 aliphatic paraben is present in the amount of about 0.01 - 5 %, the C2-C 17 alcohol is t in the amount of about 0.00-10 % and the ent is present in the amount of about 0.001-10 %.
Also provided are the pesticidal substances derived from the formulation set forth above, combinations comprising said pesticidal subtances and another chemical or biological pesticide and methods for producing these pesticidal substances. In a ular embodiment, these pesticidal substances comprise at least one ofthe following characteristics: (a) has pesticidal properties and in particular, idal, insecticidal, nematicidal, and fungicidal properties; (b) has a molecular weight of about 210-240 and more particularly, 222 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (e) has 1H NMR values of b 7.90, 6.85, 4.28, 1.76, 1.46, 1.38, 1.37, 0.94; wo 32693 (d) has 13C NMR values of b 166.84, 162.12, 131.34 (2C), 121.04, 114.83 (2C), 64.32, 31.25, 28.43, 25.45, 22.18. 12.93; (e) has an High Pressure Liquid Chromatography (HPLC) retention time of about 15-20 minutes, more specifically about 17 minutes and even more specifically about 17.45 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5JL C18(2) 100 A, 100 x 4.60 mm) column using a water: acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0- 90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm; (f) The 13C NMR spectrum exhibited 13 discrete carbon signals which were attributed to one methyl, five methylene carbons, four methines, and three quaternary s; (g) has a molecular formula of C13H180 3 which was determined by interpretation of the ESIMS and NMR data analysis; (h) has UV absorption bands between about 0 nm and most particularly at about 248 nm.
Also provided are compounds having the structure shown below: R3 0 R1 R4 Wherein X, is ndently -0, -NR, or -S, wherein R isH or C1-C10 alkyl; R1, Rz, R3, ~, Rs, and R6 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, yl, amide, or sulfuryl.
In particular, the substance may have the structure x-Rs Wherein wo 2013/032693 X, is independently -0, -NR, or -S, wherein R isH or C1-C10 alkyl; R1, Rz, R3, ~, Rs, and R6 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aryl, substituted heteroaryl, heterocyclic, tuted heterocyclic, cycloalkyl, substituted lkyl, alkoxy, substituted alkoxy, thioalkyl, substituted kyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or yl.
In a more particular embodiment, the compound is butyl parben with the following structure: ~O~CH3 HOAJ In a more particular embodiment, the compound is hexyl parben with the following structure: In a more ular ment, the compound is octyl parben with the following structure: The pesticidal substance(s) derived from the formulation set forth above may ed by (a) providing the formulation set forth above; (b) incubating or storing the ation provided for a sufficient time (e.g., between about 1 day to about 6 months) and at a sufficient temperature (e.g., n about 3C to about 50 C) to produce the pesticidal substance(s) and (c) isolating the pesticidal substance.
In a related aspect, disclosed is a method for modulating proliferation and/or growth of a pest including but not limited to insect, fungi, weeds, nematode, arachnid, algae and particularly, algae, arachnid (e.g., mites. ticks) comprising applying to a location where modulation of proliferation and/or growth of a pest is desired an amount of: (I)(a) at least one or more substances selected from the group consisting of a substantially pure cell culture, cell fraction, supernatant derived from the Burkholderia strain set forth above or extract f and (b) optionally another substance, wherein said substance is a pesticide, or wo 2013/032693 (II) the combination, composition or formulation or pesticidal substances derived from said formulation set forth above, effective to modulate proliferation and/or growth of a pest at said location.
Disclosed herein are isolated compounds which are optionally able or derived from Burkholderia species, or atively, organisms capable of producing these compounds that can be used to control various pests, particularly plant phytopathogenic pests, examples of which include but are not limited to insects, nematodes, bacteria, fungi. These compounds may also be used as herbicides, acaricides or algicides.
In particular, the isolated pesticidal compounds may include but are not limited to: (A) a compound having the ing properties: (i) a molecular weight of about 525- 555 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (ii) 1H NMR values of 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23. 1.74, 1.15, 1.12, 1.05, 1.02; (iii) has 13C NMR values of 172.99, , 169.57, 169.23, 167.59, 130.74, 130.12, 129.93, 128.32, 73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51 and (iv) an High Pressure Liquid Chromatography (HPLC) retention time of about 10-15 minutes, on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) gradient; (B) a compound having an oxazolyl-indole structure sing at least one indole moiety, at least one oxazole moiety, at least one substituted alkyl group and at least one carboxylic ester group; at least 17 carbons and at least 3 oxygen and 2 nitrogens; (C) a nd having an oxazolyl-benzyl structure comprising at least one benzyl moiety, at least one oxazole moiety, at least one substituted alkyl group and at least one amide group; at least 15 carbons and at least 2 oxygen and 2 nitrogens; (D) a compound having at least one ester, at least one amide, at least three methylene groups, at least one tetrahydropyranose moiety and at least three olefinic double bonds, at least six methyl groups, at least three yl groups, at least twenty five carbons and at least eight oxygen and one nitrogen and (E) a compound having at least one ester, at least one amide, an epoxide methylene group, at least one tetrahydropyranose moiety, at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl , at least 25 carbons, at least 8 oxygens and at least 1 nitrogen.
In a particular embodiment, the isolated nds may include but are not limited to: (A) a compound having an oxazolyl-indole structure comprising at least one indole moiety, at least one oxazole moiety, at least one tuted alkyl group, at least one carboxylic ester group, at least 17 carbons, at least 3 s and at least 2 nitrogens; and which has at wo 2013/032693 least one ofthe following: (i) a lar weight of about 275-435; (ii) 1H NMR b values at 8.44, 8.74, 8.19, 7.47, 7.31, 3.98, 2.82, 2.33, 1.08; (iii) 13C NMR values of b 163.7, 161.2, 154.8, 136.1, 129.4, 125.4, 123.5, 123.3, 121.8, 121.5, 111.8, 104.7, 52.2, 37.3, 28.1, 22.7, 22.7; (iv) an High Pressure Liquid Chromatography (HPLC) retention time of about 10-20 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) with a gradient solvent system and UV detection of210 nm; (v) UV absorption bands at about 226,275, 327 nm.; (B) a compound having an oxazolyl-benzyl structure comprising at least one benzyl , at least one oxazole moiety, at least one substituted alkyl group and at least one amide group; at least 15 carbons and at least 2 oxygens, at least 2 nitrogens; and at least one ofthe following characteristics: (i) a molecular weight of about 240-290 as ined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (ii) 1H NMR b values at about 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93; (iii) 13C NMR values of b 158.2, 156.3, 155.5, 132.6, 129.5, 129.5, 127.3, 121.8, 115.2, 115.2, 41.2, 35.3, 26.7, 21.5, 21.5; (iv) a High Pressure Liquid Chromatography (HPLC) retention time of about 6-15 minutes, on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) gradient and (v) UV absorption bands at about 230, 285, 323 nm; (C) a non-epoxide compound comprising at least one ester, at least one amide, at least three methylene groups, at least one tetrahydropyranose moiety and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl , at least twenty five carbons, at least eight oxygens and one nitrogen and at least one ofthe following characteristics: (i) a lar weight of about 530-580 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (ii) 1H NMR values of b 6.40, 6.39, 6.00, 5.97, .67, 5.54, 4.33, 3.77, 3.73, 3.70, 3.59, 3.47, 3.41, 2.44, 2.35, 2.26, 1.97, 1.81, 1.76, 1.42, 1.37, 1.16, 1.12, 1.04; (iii) 13C NMR values of b 173.92, 166.06, 145.06, 138.76, 135.71, , 126.20, 123.35, 99.75, 82.20, 78.22, 76.69, 71.23, 70.79, 70.48, 69.84, 60.98, 48.84, 36.89, 33.09, 30.63, 28.55, 25.88, 20.37, 18.11, 14.90, 12.81, 9.41; (iv) a High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes, on a reversed phase C-18 HPLC column using a water: itrile (CH3CN) with a gradient solvent system and UV detection of 210 nm; (v) a molecular formula of C28H45N010 which was determined by retation of the ESIMS and NMR data analysis; (vi) UV absorption bands n about 210-450 nm; (D) a compound sing (i) at least one ester, at least one amide, an epoxide methylene group, at least one tetrahydropyranose moiety and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least 25 carbons, at least 8 wo 2013/032693 oxygens and at least 1 nitrogen, (ii) 13C NMR values of ()174.03, 166.12, 143.63, 137.50, 134.39, 128.70, 126.68, , 98.09, 80.75, 76.84, 75.23, 69.87, 69.08, 68.69, 68.60, 48.83, 41.07, 35.45, 31.67, 29.19, 27.12, 24.55, 19.20, 18.95, 13.48, 11.39, 8.04, (iii) a molecular formula of N09 and at least one of: (a) 1H NMR b values at about 6.41, 6.40, 6.01, 5.97, 5.67, 5.55, 4.33, 3.77, 3.75, 3.72, 3.64, 3.59, 3.54, 3.52, 2.44, 2.34, 2.25, 1.96, 1.81, 1.76, 1.42, 1.38, 1.17, 1.12, 1.04; (b) an High Pressure Liquid Chromatography (HPLC) retention time of about 6-15 minutes, on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) nt; (c) UV absorption band between about 0 nm and most particularly at about 234 nm.
In a more particular embodiment, provided are compounds including but not limited to: (A) a compound having the structure ##STR001## s ------------=2_ s---~ n or a pesticidally acceptable salt or steriosomers f, wherein M is 1, 2, 3 or 4; n is 0, 1, 2, or 3; p and q are independently 1 or 2; X is 0, NH or NR; R1, R2 and R3 are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative and R is a lower chain alkyl, aryl or arylalkyl moiety; (B) a compound having the structure ##STR002## ##STR002## wo 2013/032693 wherein X, Y and Z are each independently --0, --NR1, or --S, wherein R1 is--H or C1-C10 alkyl; R1, R2 and mare each independently --H, alkyl, substituted alkyl, l, substituted alkenyl, l, substituted alkynyl, aryl, tuted aryl, heteroaryl, tuted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, y, halogen, amino, amido, carboxyl, --C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl and "m" may be located anywhere on the oxazole ring; (C) a compound having the structure ##STR002a##.
##STR002a## wherein R1 is--H or C1-C10 alkyl; R2 is an alkyl ester; (D) a nd having the structure ##STR003## wherein: X and Y are each independently --OH, --NR1, or --S, wherein R1 is --H or C1-C10 alkyl; R1, R2 and m, a substituent on the oxazole ring, are each ndently --H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted , thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, --C(O)H, acyl, oxyacyl, ate, sulfonyl, sulfonamide, or sulfuryl.
(E) a compound having the structure ##STR003a## 1" ##STR003a## wo 2013/032693 wherein R1 is--H or C1-C10 alkyl; (F) a compound having the structure ##STR004a## Wherein X, Y and Z are each independently -0, -NR, or -S, wherein R isH or C1-C10 alkyl; R1, Rz, R3, ~, Rs, R6, R7, Rs, R9, Rw, Rn, R12, and R13 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cyclic, substituted heterocyclic, lkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, tuted thioalkyl, y, halogen, amino, amido, carboxyl, , acyl, oxyacyl, carbamate, yl, sulfonamide, or sulfuryl.
(G) a compound having the structure ##STR004b## n R1, Rz, R3, ~, Rs, R6, R7, Rs, R9, Rw, Rn, R12, and R13 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cyclic, substituted heterocyclic, lkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, , acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; (H) a compound having the structure ##STR004c## ~'& R~- . ~>1• . .} . .. CH-;:; R·t:t:x::c,,,,,"':;c:t:,~, R_~. ' HO'""'Oit ##S'fR004~## wherein R1, Rz, R3, ~, Rs, R6, R7, Rs, Rn, are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, , acyl, l, carbamate, sulfonyl, sulfonamide, or sulfuryl; (I) a compound having the structure ##STR005## wo 2013/032693 R,~~R, ##STR005## wherein X andY are each ndently --OH, --NR1, or --S, wherein R1, R2 are each independently --H, alkyl (e.g., C1-C10 alkyl), substituted alkyl, alkenyl, substituted alkenyl, alkynyl, tuted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, tuted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, --C(O)H, acyl, oxyacyl, carbamate, yl, sulfonamide, or sulfuryl; (J) a nd having the structure ##STR006a## Wherein X, Y and Z are each independently -0, -NR, or -S, wherein R isH or C1-C 10 alkyl; R1, R2 , R3, R4 , R5 , R6, R7, R8, R11 , R12, and R13 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted cyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, kyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or yl.
In a most particular embodiment, the compounds may include but are not limited to (i) templazole A; (ii) templazole B; (iii) templamide A; (iv) templamide B; (v) FR901228; (vi) H (vii) wo 32693 (viii) of)l))'N (ix) (xi) (xii) (xiii) wo 32693 (xiv) (xv) (xvi) (xvii) (xviii) wo 32693 Br OH OH (xix) (xx) (xxi) (xxii) wo 2013/032693 2012/050807 (xxiii) wo 2013/032693 2012/050807 (xl) FR901465; (xli) F8H17, an active compound from fraction F8, which has been assigned a molecular weight wo 2013/032693 of 1080 based on the molecular ion peak at 1081.75 (M + H) in positive ESI mode and further confirmed by the negative ESIMS with base peak at 1079.92. This compound showed UV absorption at 234 nm.
In a related aspect, disclosed is a method for modulating proliferation and/or growth of a pest (e.g., algae, arachnid, nematode, insect, fungus) comprising ng to a location where modulation of proliferation and/or growth of a pest (e.g., algae, id, nematode, insect, fungus) is desired an amount of (I) (a) the isolated compounds set forth above and (b) optionally another substance, wherein said substance is an algicide or (II) the composition or ation set forth above in an amount effective to modulate proliferation and/or growth of pest at said on.
In another related aspect, disclosed is a method for modulating proliferation and/or growth of algae and/or modulating pest infestation in a plant and/or a method for modulating emergence and/or growth of monocotyledonous, sedge or dicotyledonous weeds comprising applying to a location where modulation of proliferation and/or growth of algae and/or modulation of infestation of an arachnid and/or modulation of emergence and/or growth of said weed is desired an amount of (A) the formulation set forth above or pesticidally effective substance derived therefrom; (B) the combination set forth above; (C) mide A; (D) templamide B; (E) 65; (F) 28 rffective to modulate said proliferation and/or growth of algae and/or pest infestation and/or emergence or growth of monocotyledonous, sedge or dicotyledonous weeds at said location.
The nematode and/or insect infestation is modulated with templamide A, mide B, FR901465 and/or FR901228. In a more particular embodiment, ation of insects, specifically Oncopeltus sp. (e.g., O.fasciatus) and/or Lygus sp. and/or free living nematodes and/or parasitic nematodes (e.g., M. ita) are modulated.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the comparison of the growth rate of Burkholderia A396 to Burkholderia orans ATCC 17616.
Figure 2 shows the general scheme used to obtain fractions from formulated MBI-206. wo 32693 Figure 3 shows the l scheme used to obtain fractions and compounds from an MBI-206 e.
Figure 4 shows insecticidal (sucking) activities oftested compounds against milkweed bugs (Oncopeltus fasciatus).
Figure 5 shows insecticidal (feeding) activities ofpure compounds t Lygus Hesperus.
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 tood, r, that there is no intent to limit the invention to the particular forms sed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling 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 ce or testing of the present invention, the preferred methods and materials are now bed.
It must be noted that as used herein and in the ed claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.
As defined herein, ed from" means directly isolated or obtained from a particular source or alternatively having identifying 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 ably about 90% pure, and even most preferably about 95% pure, as ined by analytical methods, ing but not limited to chromatographic methods, electrophoretic methods. wo 2013/032693 As used herein, the term "alkyl" refers to a monovalent ht or branched chain hydrocarbon group having from one to about 12 carbon atoms, including methyl, ethyl, npropyl , isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like.
As used herein, "substituted alkyl" refers to alkyl groups further bearing one or more substituents selected from hydroxy, alkoxy, mercapto, cycloalkyl, substituted cycloalkyl, cyclic, substituted cyclic, aryl, substituted aryl, heteroaryl, substituted aryl, aryloxy, substituted aryloxy, halogen, cyano, nitro, amino, amido, --C(O)H, acyl, oxyacyl, carboxyl, yl, sulfonamide, yl, and the like.
As used herein, "alkenyl" refers to straight or ed chain hydrocarbyl groups having one or more carbon-carbon double bonds, and having in the range of about 2 up to 12 carbon atoms, and "substituted alkenyl" refers to alkenyl groups further bearing one or more substituents as set forth above.
As used herein, "alkynyl" refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon triple bond, and having in the range of about 2 up to 12 carbon atoms, and "substituted alkynyl" refers to alkynyl groups further bearing one or more substituents as set forth above.
As used herein, "aryl" refers to aromatic groups having in the range of 6 up to 14 carbon atoms and "substituted aryl" refers to aryl groups further bearing one or more substituents as set forth above.
As used herein, oaryl" refers to aromatic nngs containing one or more heteroatoms (e.g., N, 0, S, or the like) as part of the ring structure, and having in the range of 3 up to 14 carbon atoms and "substituted aryl" refers toheteroaryl groups r bearing one or more substituents as set forth above.
As used herein, "alkoxy" refers to the moiety -alkyl-, wherein alkyl is as defined above, and "substituted alkoxy" refers to alkoxyl groups further bearing one or more substituents as set forth above.
As used herein, "thioalkyl" refers to the moiety --S-alkyl-, wherein alkyl is as defined above, and "substituted thioalkyl" refers to thioalkyl groups further bearing one or more substituents as set forth above.
As used herein, "cycloalkyl" refers to ring-containing alkyl groups ning in the range of about 3 up to 8 carbon atoms, and "substituted cycloalkyl" refers to cycloalkyl groups further bearing one or more substituents as set forth above.
As used , "heterocyclic", refers to cyclic (i.e., ring-containing) groups containing one or more heteroatoms (e.g., N, 0, S, or the like) as part of the ring structure, and having in wo 2013/032693 the range of 3 up to 14 carbon atoms and "substituted heterocyclic" refers to heterocyclic groups further bearing one or more substituent's as set forth above.
As used herein "algae" refers to any ofvarious y aquatic, eukaryotic, photosynthetic organisms, ranging in size from single-celled forms to the giant kelp. The term may further refer to photosynthetic protists responsible for much ofthe photosynthesis on Earth.
As a group, the algae are polyphyletic. Accordingly, the term may refer to any ts considered to be algae from the ing groups, alveolates, raachniophytes, cryptomonads, ids, glaucophytes, hytes, red algae such as Rhodophyta, stramenopiles, and viridaeplantae. The term refers to the green, yellow-green, brown, and red algae in the eukaryotes. The term may also refer to the cyanobacteria in the prokaryotes. The term also refers to green algae, blue algae, and red algae.
As used herein "algicide" refers to one or more agents, compounds and/or compositions having tatic and/or algaecidal ty.
As used herein "algicidal" as used herein means the killing of algae.
As used herein "algistatic" as used herein means inhibiting the growth of algae, which can be reversible under certain conditions.
The Burkholderia Strain The Burkholderia strain set forth herein is a non-Burkholderia cepacia complex, non- Burkholderia ri, non-Burkholderia gladioli, Burkholderia 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 bed by Lorch et al., 1995. The Burkholderia 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 h-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 Burkholderia strain of the present invention set forth in the Examples below. In particular, the Burkholderia strain has a 16S rRNA gene comprising a forward sequence that is at least about 99.5%, more preferably about 99.9% and most preferably about 100% identical to the sequence set forth in SEQ ID NO: 8, 11 and 12 and a forward sequence that is at least about 99.5%, more preferably about 99.9% and most preferably about 100% identical to the sequence set forth in SEQ ID NO: 9, 10, 13, 14 and 15 as determined by clustal analysis. Furthermore, as set forth below, this Burkholderia strain may, as set forth below, have pesticidal activity, particularly, virucidal, wo 2013/032693 herbicidal, germicidal, fungicidal, nematicidal, bactericidal and insecticidal and more particularly, herbicidal, algicidal, acaricidal, insecticidal, idal and nematicidal activity. It is not pathogenic to vertebrate animals, such as mammals, birds, and fish.
Additionally, the Burkholderia strain produces at least the pesticidal compounds set forth in the instant disclosure.
The Burkholderia strain 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, 18:0.
This Burkholderia strain may be obtained by culturing a microorganism having the identifying characteristics of Burkholderia A396 (NRRL Accession No. B-50319) on Potato Dextrose Agar (PDA) or in a fermentation medium containing d carbon s such as glucose, maltose, fructose, galactose, and undefined nitrogen s such as peptone, tryptone, soytone, and NZ amine.
Algicidal and Acaricidal Compounds The algicidal and acaricidal compounds disclosed herein may have the following properties: (a) is obtainable from a novel Burkholderia species, e.g., A396; (b) is, in particular, toxic to most common agricultural insect pests; (c) has a molecular weight of about 525-555 and more particularly, 540 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (d) has 1H NMR values of 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23. 1.74, 1.15, 1.12, 1.05, 1.02; (d) has 13C NMR values of , , 169.57, 169.23, 167.59, , 130.12, 129.93, 128.32, 73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51 (e) has an High Pressure Liquid Chromatography (HPLC) retention time of about 10-15 minutes, more specifically about 12 minutes and even more specifically about 12.14 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5JL C18 (2) 100A, 100 x 4.60 mm) column using a water: acetonitrile (CH3CN) with a gradient solvent system (0-20 min 90-0 % aqueous CH3CN, -24 min 100% CH3CN, 24-27 min, 0-90% aqueous CH3CN, 27-30 min 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm (f) has a lar a, C24H36N40 determined by interpretation of 1H, 13C NMR and LC/MS data (g) a 13C 6S2 , which is NMR spectrum with signals for all24 carbons, including 5 methyl, 4 methylene, 9 methine, and 6 quaternary carbons and (g) 1H NMR spectrum displaying characteristics of a typical depsipeptide, illustrating three -amino protons [4.63, 4.31, 3.93], and one ester carbinol proton . In a particular embodiment, the nd has the structure ##STROOl##: wo 2013/032693 Or a pesticidally able salt or stereoisomers thereof, wherein M is 1, 2, 3 or 4; n is 0, 1, 2, or 3; p and q are independently 1 or 2; X is 0, NH or NR; R1, R2 and R3 are the same or different and independently an amino acid hain moiety or an amino acid side-chain derivative and R is a lower chain alkyl, aryl or arylalkyl moiety.
In an even more particular embodiment, the compound has the structure of FR901228: Provided herewith are compounds set forth in 02##: ##STR002## wherein: X, Y and Z are each independently --0, --NR1, or --S, wherein R1 is--H or C1-C10 alkyl; R1, R2 and mare each independently --H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, lkyl, substituted cycloalkyl, alkoxy, substituted wo 2013/032693 , thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, --C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or yl.
In an even another particular embodiment, Family ##STR002## compounds may be the compounds set forth in (vi)-(xix). (vii) (viii) v:lf) (ix) (xi) wo 32693 (xii) (xiii) (xiv) (xv) (xvi) (xvii) wo 2013/032693 (xviii) (xix) These are from either l materials or compounds obtained from commercial sources or by chemical synthesis. Natural sources of Family ##STR002## compounds include, but are not limited to, microorganisms, alga, and sponges. In a more particular embodiment, microorganisms which include the Family ##STR002## compounds include but are not limited to, or alternatively, Family ##STR002## compounds may be derived from species such as overticillium waksmanii (compound vi) (Umehara, et al., 1984), Streptomyces pimprina (compound vii) (Naiket al., 2001), Streptoverticillium olivoreticuli (compounds viii, ix, x) (Koyama Y., et al., 1981), Streptomyces sp (compounds xi, xii) (Watabe et al., 1988), monas syringae unds xiii, xiv) (Pettit et al., 2002). Family ##STR002## compounds may also be derived from algae including but not limited to red alga (compound xv) (N'Diaye,et al., 1996), red alga siafragilis (compound xvi) (Takahashi S. et al., 1998), wo 2013/032693 Diazona chinensis (compounds xvii & xviii) (Lindquist N. et al., 1991), Rhodophycota haraldiophyllum sp (compound xix) a et al., 1994).
Also provided is ##STR003##: wherein: X and Y are each independently --OH, --NR1, or --S, wherein R1 is --H or C1-C10 alkyl; R1, R2 and m, a tuent on the oxazole ring, are each independently --H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, tuted heteroaryl, cyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, --C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.
Further provided is ##STR005##: wherein X andY are each independently --OH, --NR1, or --S, wherein R1, R2 are each independently --H, alkyl (e.g., C1-C10 alkyl), substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted aryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, kyl, tuted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, --C(O)H, acyl, l, carbamate, sulfonyl, sulfonamide, or sulfuryl.
In a particular embodiment, Family 05## compounds such as compounds from xx-xxiii set forth below may be derived from natural or commercial sources or by chemical synthesis. (xx) (xxi) wo 2013/032693 (xxii) (xxiii) Natural sources of Family ##STR005## compounds include, but are not limited to plants, , microorganisms, and sponges. The microorganisms include, but are not limited to Streptomyces griseus (compound xx) (Hirota et al., 1978), Streptomyces albus (compound xxi) (Werner et al., 1980). Family STR004 compounds may also be derived from algae ing but not limited to Haraldiophyllum sp (compound xxii a et al., 2006), and red algae (compound xxiii) (N'Diayeet al., 1994).
In one embodiment, the compound may be derived from or is obtainable from a microorganism, and in particular from Burkholderia species and characterized as having a structure comprising at least one ester, at least one amide, at least three methylene groups, at least one tetrahydropyranose moiety and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least twenty five carbons and at least eight oxygen and one en. The compound further comprises at least one ofthe following characteristics: (a) pesticidal properties and in particular, nematicidal, fungicidal, icidal, acaricidal, algicidal and herbicidal properties; (b) a molecular weight of about 0 and more particularly, 555 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (c) 1H NMR values of b 6.40, 6.39, 6.00, 5.97, 5.67, 5.54, 4.33, 3.77, 3.73, 3.70, 3.59, 3.47, 3.41, 2.44, 2.35, 2.26, 1.97, 1.81, 1.76, 1.42, 1.37, 1.16, 1.12, 1.04; wo 2013/032693 (d) 13C NMR values of b 173.92, 166.06, 145.06, 138.76, 135.71, , 126.20, 123.35, 99.75, 82.20, 78.22, 76.69, 71.23, 70.79, 70.48, 69.84, 60.98, 48.84, 36.89, 33.09, .63, 28.55, 25.88, 20.37, 18.11, 14.90, 12.81, 9.41; (e) an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes, more specifically about 10 minutes and even more specifically about 10.98 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5JL C18(2) 100 A, 100 x 4.60 mm) column using a water: acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm; (f) 13C NMR spectrum which exhibits 28 discrete carbon signals which may be attributed to six methyls, four ene carbons, and en methines including five sp2 , four quaternary carbons; (g) a molecular formula of C28H45N010 which was determined by interpretation of the ESIMS and NMR data analysis; (h) UV absorption bands n about 210-450 nm and most particularly at about 234 Also provided are compounds having the structure ##STR004a##: Wherein X, Y and Z are each independently -0, -NR, or -S, wherein R isH or C1-C10 alkyl; R1, Rz, R3, ~, Rs, R6, R7, Rs, R9, R10, Rn, R12, and R13 are each independently H, alkyl, tuted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted lkyl, alkoxy, tuted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.
In a ular embodiment, the compound has the structure set forth in ##STR004b##: wherein R1, Rz, R3, ~, Rs, R6, R7, Rs, R9, R10, Rn, R12, and R13 are as previously defined for ##STR004a##. wo 2013/032693 In a more particular embodiment, the compound is Templamide A with the ing structure: In another embodiment, provided is a compound having formula ##STR004c##: ~.,.. R;:: ct.,. ~~ .-.,. .·~ .,, .· $'"' CH-~ "t ··+--·~:"leo/"-~., ,..~:., #',. .<;~~·''•,. .-~{'·' ....·v, l ·· I H ' ~ - f l~~ ·:::.:::,,.••..·•'''-...t ~ '······· .R~T ~"'·. ..•., . . ·.,.. .·•''' R{ Hd~....~H #*'$1'Rot1:~)-4(:,~ Wherein R1, Rz, R3, ~, Rs, R6, R7, Rs, and Rn are as usly defined for ##STR004a##.
In another embodiment, provided is a compound which may be derived from Burkholderia s and characterized as having a ure comprising at least one ester, at least one amide, an epoxide methylene group, at least one tetrahydropyranose moiety and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl , at least 25 carbons and at least 8 oxygen and 1 en, and pesticide activity. The compound further comprises at least one ofthe following characteristics: (a) pesticidal properties and in particular, insecticidal, fungicidal, nematocidal, acaricidal, algicidal and herbicidal properties; (b) a molecular weight of about 520-560 and particularly 53 7 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (c) 1H NMR b values at about 6.41, 6.40, 6.01, 5.97, 5.67, 5.55, 4.33, 3.77, 3.75, 3.72, 3.64, 3.59, 3.54, 3.52, 2.44, 2.34, 2.25, 1.96, 1.81, 1.76, 1.42, 1.38, 1.17, 1.12, 1.04; (d) 13C NMR values of b 174.03, , 143.63, 137.50, 134.39, 128.70, 126.68, 124.41, 98.09, 80.75, 76.84, 75.23, 69.87, 69.08, 68.69, 68.60, 48.83, 41.07, 35.45, 31.67, 29.19, 27.12, 24.55, 19.20, 18.95, 13.48, 11.39, 8.04; (e) High re Liquid Chromatography (HPLC) retention time of about 6-15 minutes, more specifically about 8 minutes on a reversed phase C-18 HPLC column using a water: acetonitrile (CH3CN) nt, ularly, an High Pressure Liquid Chromatography (HPLC) retention time of about 8-15 minutes, more specifically about 11 minutes and even more specifically about 11.73 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5~-t C18(2) 100 A, 100 x 4.60 mm) column using a water:acetonitrile (CH3CN) with a gradient wo 2013/032693 solvent system (0-20 min; 90-0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0- 90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm; (f) a lar formula of C28H43N09 which was determined by interpretation of the ESIMS and NMR data analysis; (g) UV absorption bands at about 210-450 nm and most particularly at about 234 nm.
In a particular embodiment, the compound has the structure ##STR006a##: n X, Y and Z are each independently -0, -NR, or -S, wherein R isH or C1-C10 alkyl; R1, Rz, R3, ~, Rs, R6, R7, Rs, Rn, R12, and R13 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, l, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, tuted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or yl.
In a particular embodiment, the compound has the structure: T~mptamid~ A In another ment, provided is a compound having formula ##STR006b##: Wherein R1, Rz, R3, ~, Rs, R6, R7, Rs, and Rn are as previously d for ##STR006a##.
In a more particular embodiment, the compound is Templamide B with the following structure: wo 2013/032693 Q"''t''") " yH:t "' K,c1~-~:(r~:")"':)~f= H 0 T~'tllp~amkh~ a In yet another particular ment, the compound may be derived from Burkholderia species and characterized as having a structure comprising at least one ester, at least one amide, an epoxide methylene group, at least one tetrahydropyranose moiety and at least three ic double bonds, at least six methyl groups, at least three yl groups, at least 25 carbons and at least 8 oxygen and at least 1 nitrogen. The compound r comprises at least one of the following characteristics: (a) pesticidal properties and in particular, insecticidal, fungicidal, acaricidal, nematicidal, dal and herbicidal properties; (b) a molecular weight of about 510-550 and particularly about 523 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (c) 1H NMR b values at about 6.41, 6.40, 6.01, 5.98, 5.68, 5.56, 4.33, 3.77, 3.75, 3.72, 3.65, 3.59, 3.55, 3.50, 2.44, 2.26, 2.04, 1.96, 1.81, 1.75, 1.37, 1.17, 1.04; (d) 13C NMR values of b 172.22, 167.55, 144.98, 138.94, 135.84, 130.14, 125.85, 123.37, 99.54, 82.19, 78.28, 76.69, 71.31, 70.13, 69.68, 48.83, 42.52, 36.89, 33.11, 30.63, .99, 21.20, 20.38, 18.14, 14.93, 12.84; (e) an High Pressure Liquid Chromatography (HPLC) retention time of about 6-15 s, more specifically about 8 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) gradient, particularly, an High Pressure Liquid tography (HPLC) retention time of about 8-15 s, more specifically about 10 minutes and even more specifically about 10.98 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5~-t C18(2) 100 A, 100 x 4.60 mm) column using a acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90- 0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0- 90 % aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm; (f) a molecular formula of C27H41N09 which was determined by interpretation of the ESIMS and NMR data analysis; (g) UV absorption bands at about 210-450 nm and most particularly at about 234 nm.
In a more particular embodiment, the compound is a known compound FR901465 which was isolated r from culture broth of a bacterium of Pseudomonas sp. No. 2663 wo 2013/032693 (Nakajima et al. 1996) and had been reported to have anticancer ty with the ing structure: ,,,., ..CH~ H,c l~o f'clol~"4 J"""'"(o{:~1 yH~~ '·<.--··' 'N··' H 'v-··' 'CHi He,.. .,)'< .,OH , {y""' FR%)'14$$ In an even another ular ment, Family ##STR006a## compounds may be the compounds set forth in xxiv to xxxix. These are from either natural materials or compounds obtained from commercial sources or by chemical synthesis. Natural sources of Family ##STR006a## compounds include, but are not d to, microorganisms, alga, and sponges. In a more ular embodiment, microorganisms which include the Family ##STR006a## compounds which may be derived from species such as Pseudomonas sp. No. 2663 (compounds xxiv-xxvi) (Nakajima et al., 1996), The synthetic analogues of the FR901464 (xxvii-xxxix) which have been synthesized and patented as anticancer compounds (see Koide et al., US Patent ation No. 2008/0096879 AI).
Also provided are the pesticidal compounds produced by the formulation set forth above which comprises at least one ofthe following characteristics: (a) has pesticidal properties and in particular, herbicidal, icidal, nematicidal, and fungicidal properties; (b) has a molecular weight of about 210-240 and more particularly, 222 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (e) has 1H NMR values of b 7.90, 6.85, 4.28, 1.76, 1.46, 1.38, 1.37, 0.94; (d) has 13C NMR values of b 166.84, 162.12, 131.34 (2C), 121.04, 114.83 (2C), 64.32, 31.25, 28.43, 25.45, 22.18. 12.93; (e) has an High Pressure Liquid Chromatography (HPLC) retention time of about 15-20 minutes, more specifically about 17 minutes and even more specifically about 17.45 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5JL C18(2) 100 A, 100 x 4.60 mm) column using a water:acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90- 0 % aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0-90 % aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm; (f) The 13C NMR spectrum exhibited 13 discrete carbon s which were attributed to one methyl, five methylene carbons, four methines, and three quaternary s; (g) has a molecular formula of C13H180 3 which was determined by interpretation of the ESIMS and NMR data analysis; wo 32693 (h) has UV absorption bands between about 210-450 nm and most particularly at about 248 nm.
Also provided are compounds having the structure shown below R3 0 R2«x-Rs R1 R4 Wherein X, is independently -0, -NR, or -S, wherein R isH or C1-C10 alkyl; R1, Rz, R3, ~, Rs, and R6 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, tuted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, , substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.
In a more ular embodiment, the nd is butyl parben with the following structure: ~O~CH3 HOAJ In a more particular embodiment, the compound is hexyl parben with the following ure: In a more particular embodiment, the compound is octyl parben with the following structure: In yet another embodiment, the compound is F7H18, which has a molecular weight of about 1080. wo 2013/032693 Compositions A substantially pure culture, cell fraction or supernatant and compounds produced by the Burkholderia strain disclosed herein, all of which are alternatively referred to as "active ingredient(s)",may be formulated into pesticidal compositions. In a particular embodiment, the supernatant may be a ree supernatant.
The active ingredient(s) set forth above can be formulated in any . Non-limiting formulation examples include but are not limited to emulsifiable concentrates (EC), wettable powders (WP), soluble liquids (SL), aerosols, low volume concentrate solutions (ULV), soluble powders (SP), microencapsulation, water dispersed granules, flowables (FL), microemulsions (ME), nano-emulsions (NE), dusts, emulsions, liquids, flakes etc. In any formulation described herein, percent of the active ingredient is within a range of 0.01% to 99.99%.
A solid composition can be prepared by suspending a solid carrier in a solution of idal compounds and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65°C or lower. atively, a solid composition may be derived via spray-drying or freeze-drying.
When ing to solid compositions, it should be understood by the artisan of ordinary skill that physical forms such as dusts, beads, powders, particulates, pellets, s, agglomerates, granules, floating solids and other known solid formulations are included. The artisan of ordinary skill will be able to y optimize a particular solid formulation for a given application using methods well known to those of ordinary skill in the art.
The composition may comprise gel-encapsulated nds derived from the Burkholderia strain set forth above. Such gel-encapsulated materials can be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a on of algicidal compounds and inducing gel formation of the agent.
The composition may additionally comprise a tant 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 yethylene (20) monolaurate. The concentration of surfactants may range between 0.1-35% of the total ation, preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, eric and cationic sing and emulsifying agents, and the amount ed is determined by the nature of the composition and the ability of the agent to tate the dispersion of these compositions. wo 2013/032693 In order to provide compositions containing the active ingredient(s) set forth above in the form of dusts, granules, water dispersible powders, aqueous dispersions, or emulsions and dispersions in organic s, the carrier or t agent in such compositions may be a finely divided solid, an organic liquid, water, a wetting agent, a dispersing agent, humidifying agent, or emulsifying agent, or any suitable combination of these. Generally, when liquids and wettable powders are prepared, a conditioning agent comprising one or more surface-active agents or surfactants is t in amounts sufficient to render a given composition containing the active material, the microorganism, dispersible in water or oil.
Since these compositions can be applied as a spray utilizing a liquid r, it is contemplated that a wide variety of liquid rs such as, for example, water, organic solvents, decane, dodecane, oils, vegetable oil, mineral oil, alcohol, glycol, polyethylene glycol, agents that result in a differential distribution of pathogenic bacterium in water being treated. combinations thereof and other known to artisan of ry skill can be used.
The present compositions can also include other substances which are not detrimental to the active ient(s) such as adjuvants, tants, binders, stabilizers and the like, which are commonly used in algicides, either singly or in combination as needed.
It will be understood by the artisan of ordinary skill that s additives or agents that predispose pests susceptible to the active ingredient set forth above are added to e its pesticidal action. By the phrase "additive that enhances the pesticidal action of the active ingredient" is meant any compound, solvent, reagent, substance, or agent that increases the effect of the active ingredient toward pests and more particularly, mites as compared to the pesticidal effect of the active ingredient in the absence of said additive. In some embodiments, these additives will increase the susceptibility of a particular pest to the active ingredient.
Additional additives e but are not limited to agents which weaken the biological defenses of tible pests. Such agents can include salts, such as NaCl and CaC12.
The composition may further comprise another microorganism and/or pesticide (e.g, nematocide, fungicide, insecticide, ide, algicide, aracicide). 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., Streptomyces sp, lderia sp, Trichoderma sp, Gliocladium sp. Alternatively, the agent may be a natural oil or oduct having fungicidal, herbicidal, aracidal, algicidal, nematocidal and/or insecticidal activity (e.g., paraffinic oil, tea tree oil, rass 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 gae, eucalyptus extract, entomopathogenic bacterium or wo 2013/032693 fungi such a Beauveria bassiana, 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 nematocide. The nematocide may include, but is not limited to al nematocides such as fenamiphos, aldicarb, , carbofuran, natural product neamticide, avermectin, the fungi omyces nas and Muscodor spp., the ia Bacillus firmus and other us spp. and Pasteuria penetrans.
The composition may further comprise a gicide such as extract of R. sachalinensis (Regalia) or a fungicide. Such fungicides include, but are not limited to, a single site ungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, pyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyamine, calamine, phthalimide, benzenoid (xylylalanine). In yet a further embodiment, the antifungal agent is a demethylation inhibitor selected from the group consisting of imidazole (e.g., mizole), piperazine, pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, nazole, tetraconazole, onazole).
The antimicrobial agent may also be a multi-site non-inorganic, chemical ide selected from the group consisting of a nitrile (e.g., chloronitrile or fludioxonil), quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine, cyano-acetamide oxime.
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 ing crop ). 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 cultivars 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 al, for example cuttings, tubers, rhizomes, offshoots and seeds. wo 2013/032693 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 e space by, for e, 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 ition 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 oil, cinnamon oil, lemongrass oil, citrus oil, orange peel oil, tentoxin, cornexistin, xin, manuka oil, leptospermone, min, sarmentine, momilactone B, sorgoleone, ascaulatoxin and ascaulatoxin aglycone. The chemical herbicide may include, but is not limited to, diflufenzopyr and salts thereof, dicamba and salts thereof, topramezone, tembotrione, S-metolachlor, atrazine, mesotrione, primisulfuron-methyl, 2,4- dichlorophenoxyacetic acid, nicosulfuron, thifensulfuron-methyl, , metribuzin, diclofopmethyl , fluazifop, fenoxaprop-p-ethyl, asulam, oxyfluorfen, rimsulfuron, mecoprop, and quinclorac, thiobencarb, clomazone, cyhalofop, propanil, bensulfuron-methyl, penoxsulam, triclopyr, imazethapyr, halosulfuron-methyl, pendimethalin, ibac-sodium, carfentrazone ethyl, sodium bentazon/sodium orfen, 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 ing on the required ground coverage. Granules may comprise porous or non-porous les.
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 ners as well as dispersion stabilizers such as nonionic surfactants (for example polyoxyethylene (20) monolaurate).
In a particular embodiment, the composition may comprise in addition to the active ient another microorganism and/or de and/or ide. The microorganism may include but is not limited to an agent derived from us sp., Brevibacillus sp., and Streptomyces sp.
The compositions may also as set forth above, be algicidal compositions which can further se other algicides such as copper sulphate, diquat or thaxtomin A. wo 2013/032693 The compositions may be acaricidal compositions which can r comprise other acaricides such as antibiotics, carbamates, formamidine acaricides, pyrethroids, mite growth regulators, organophosphate acaricides and diatomaceous earth.
Uses The compositions and pesticidal compounds derived from the Burkholderia strain set forth herein may be used as pesticides, particularly as insecticides, nematocides, fungicides, algicides, acaricides and herbicides.
Specifically, nematodes that may be controlled using the method set forth above include but are not limited to parasitic nematodes such as not, ring, sting, lance, cyst, and lesion nematodes, including but not limited to free living nematodes, Meloidogyne, Heterodera and Globodera spp; particularly Meloidogyne incognita (root knot nematodes), as well as Globodera rostochiensis and globodera pailida (potato cyst nematodes); Heterodera glycines (soybean cyst nematode); Heterodera tii (beet cyst nematode); Oligonychus pratensis (Banks grass mite); Eriophyes cynodoniensis da grass mite); a praetiosa (Clover mite) -and Heterodera avenae l cyst nematode).
Phytopathogenic insects controlled by the method of the present invention include, but are not limited to, insects from the order (a) Lepidoptera, for e, Acleris spp., Adoxophyes spp., a spp., s spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp ., la fusca, Cadra cautella, Carposina ensis, Chilo spp ., toneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., hora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis ea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, ria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia lis, Pammene spp., Pandemis spp., Panolisflammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., nothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example, Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., wo 2013/032693 Rhizopertha spp., eidae, Sitophilus spp., Sitotroga spp., io spp., Tribolium spp. and Trogoderma spp.; (c) Orthoptera,for e, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp ., Periplaneta spp. and Schistocerca spp .; (d) Isoptera,for example, litermes spp.; (e) Psocoptera,for e, Liposcelis spp.; (f) Anoplura,for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.; (g) Mallophaga,for example, Damalinea spp. and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniella spp., otnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii; (i) Heteroptera,for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp., Oncopeltus spp. Lygys spp. and Tniatoma spp.; (j) Homoptera,for example, Aleurothrixus floccosus, Aleyrodes brassicae, ella spp., dae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., ria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., phis spp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae and Unaspis citri; (k) Hymenoptera,for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp., Monomorium nis, Neodiprion spp., Solenopsis spp. and Vespa spp.; (l) Diptera,for example, Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp., na spp., Hypoderma spp., Hyppobosca spp., yza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinellafrit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis lla, Sciara spp., Stomoxys spp., s spp., Tannia spp. and Tipula spp.; (m) Siphonaptera,for example, Ceratophyllus spp. und Xenopsylla cheopis and (n) from the order Thysanura, for example, a saccharina. The active ingredients according to the invention may further be used for controlling crucifer flea beetles (Phyllotreta spp.), root maggots (Delia spp.), cabbage seedpod weevil (Ceutorhynchus spp.) and aphids in oil seed crops such as canola (rape), mustard seed, and hybrids thereof, and also rice and maize. In a particular embodiment, the insect may be a member of the Spodoptera, more particularly, Spodoptera exigua, Myzus persicae, Plutella xylostella or Euschistus sp.
The substances and compositions may also be used to modulate ernergence in either a pre--emergent or post--emergent formulation of monocotyledonous, induding sedges and wo 2013/032693 2012/050807 grasses, or dicotyledonous •Needs. In a particular e1nbodiment, the weeds 1nay include, but not be limjted to, odium ,;p. (e.g. C. a/burn), on sp. (e.g. A. theophrasti), Helianthus sp. (e.g. H. annuus), Lwb'V·igiasp. (e.g. L. hexapetala), llmbrosia sp. (e.g. A. artemesifolia), Amaranthus sp. (e.g., A. retn:~fle.xus, A.pa!meri), Convolvulus sp. (e.g.(-;, arvensis), lpomoeae sp., Brassica sp. (e.g. B. kaber), Raplwnus sp., Taraxacum sp. (e.g. T. (~fficinale), Centaureu sp. (e.g. C. solstitalis), Conyza sp. (e.g. C. bonariensis), Cirsium sp. (e.g. C. arvense), Lepidiwn sp., Ga!iium sp., Solanum :-rp. (e.g. S. nigrwn), lvfalva sp. (e.g.
M. neglecta), Cyperus sp. (e.g. C. rotundus), Oxalis sp., Euphorbia sp., Tr{folium sp., lvledicugo sp., H:,;drilla sp., Azollu sp., Digitaria sp. (e.g. D. sanguinalis), Setaria sp. (e.g.
S. !utescens), Cynodon dact_vlon, Brmnus sp. (e.g .. B. tectonm?), Poo sp. (e.g. P. onnua. P. pratensis), Lolliunt sp. (e.g, L perenne), Sorghum sp. (e.g. S. ha!epense), iirundo donax, Festuc·a sp. (e.g. F. naceue), Ecllinoc·hloa sp. (e.g., E. cms~galli, E. phyllopogon).
The Burkholderia strain, nds and compositions set forth above may also be used as a fungicide. The targeted fungus may be a Fusarium sp., is sp., Monilinia sp., Colletotrichum sp, Verticillium sp.; Microphomina sp., Phytophtora sp, Mucor sp., Podosphaera sp., Rhizoctonia sp., Peronospora sp., Geotrichum sp., Phoma, and Penicillium.
In another most particular embodiment, the bacteria are Xanthomonas.
The substance or compositions can be used to control, reduce and or ate the growth and eration of marine and non-marine micro and macro algae including but not limited to lular, multicellular and diatom, red-, green- and bluegreen- algae such as Pseudokirchneriella subcapitata, Rhizoclonium sp., Cladophoera sp., Anabaena sp., Nostoc sp., Hydrodictyon sp., Chara sp, Microcystis and Didymo sp., Chlamydomonas sp., Scenedesmus sp., Oscillatoria sp., Volvox sp., Navicula sp, nium sp., Spirogyra sp., Batrichospermum sp., Rhodymenia sp., Callithamnion sp.,Undaria sp., through algaecide and algaestatic ty.
The active ingredient(s) and itions set forth above may be applied to locations containing algae. These include but are not d to a body of water such as a pond, lake, stream, river, aquarium, water treatment facility, power plant or a solid surface, such as plastic, concrete, wood, fiberglass, pipes made of iron and polyvinyl choride, surfaces covered wih coating als and/or paints.
As noted above, the active ingredient(s) and compositions set forth above may be applied to locations ning arachnids, such as mites, including but not limited to, Panonychus sp. such as Panonychus citri (citrus red mite), and Panonychus ulmi (red spider mite), Tetranychus sp. such as Tetranychus kanzawi (Kanzawa spider mite), Tetranychus urticae (2 spotted spider mite), Tetranychus pacificus (Pacific spider mite), Tetranychus wo 2013/032693 turkestanii (Strawberry mite) and Tetranychus cinnabarinus (Carmine spider mite), Oligonychus sp. such as Oligonychus panicae (avacado brown mite), ychus perseae (persea mite), Oligonychus pratensis (Banks grass mite) and Oligonychus coffeae, Aculus sp. such as Aculus cornatus (Peach silver mite), Aculus fockeni (plum rust mite) and Aculus lycopersici (tomato russet mite), Eotetranychus sp. such as Eotetranychus wilametti, Eotetranychus yumensis (yuma spider mite) and Eotetranychus sexmaculatis (6-spotted mite), Bryobia rubrioculus (brown mite), Epitrimerus pyri (pear rust mite), Phytoptus pyri (Pear leaf blister mite), is essigi (red berry mite), Polyphagotarsonemus latus (Broad mite), Eriophyes ni (citrus bud mite), Brevipalpus lewisi (citrus flat mite), Phylocoptruta oleivora s rust mite), ia s (Brown wheat mite), Oxyenus li (olive mite), Rhizoglyphus spp., Tyrophagus spp., Diptacus gigantorhyncus (bigheaded plum mite) and leaa major (winter grain mite), Avocado red mite, Flat mite, black and red Mango spider mite, Papaya leaf edgeroller mite, Texas citrus mite, European red mite, Grape m mite (blister mite), Pacific spider mite, Willamette spider mitePink citrus rust mite.
Such locations may include but are not limited to crops that are infested with such mites or other arachnids (e.g., aphenids).
The ion will now be described in greater detail by reference to the following nonlimiting examples.
EXAMPLES The compositions and methods set forth above will be further illustrated in the following, non-limiting Examples. The examples are illustrative of s embodiments only and do not limit the d invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein. 1. Example 1. Isolation and identification of the microbe 1.1 Isolation ofthe microorganism The microbe is ed using established techniques know to the art from a soil sample collected under an evergreen tree at the Rinnoji Temple, Nikko, Japan. The isolation is done using potato dextrose agar (PDA) using a procedure described in detail by Lorch et al. , 1995.
In this procedure, the soil sample is first diluted in sterile water, after which it is plated in a solid agar medium 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. wo 2013/032693 2012/050807 1.2. Identification on the rganism The microbe is identified based on gene sequencing using universal bacterial primers to amplify the 16S rRNA region. The following ol 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 . DNA is extracted using the MoBio Ultra Clean Microbial DNA extraction kit. DNA extract is checked for quality/quantity by running S]tl on a 1% agarose gel.
PCR reactions are set up as follows: 2 Jtl DNA extract, 5 Jtl PCR buffer, 1 Jtl dNTPs (10 mM each), 1.25 Jtl forward primer (27F; (SEQ ID N0:1), 1.25 Jtl reverse primer (907R; (SEQ ID N0:2)) and 0.25 Jtl Taq enzyme. The reaction volume is made up to 50 Jtl 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, 0 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 Jtl 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 d PCR product as directly sequenced using s 27F (same as above), 530F (SEQ ID , 1114F (SEQ ID N0:4)) and 1525R (SEQ ID N0:5)), 1100R (SEQ ID N0:6)), 519R (SEQ ID N0:7).
The 16S rRNA gene sequence of strain A396 is compared with the ble 16S rRNA gene sequences of representatives of the ~-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 vietnamensis, and Burkholderia cepacia. A BLAST search excluding the B. cepacia complex, showed 98% similarity to B. rii, B. gladioli and lderia sp. isolates.
A distance tree of results using the neighbor joining method, showed that A396 is related to Burkholderia multivorans and other Burkholderia cepacia complex isolates.
Burkholderia plantarii and Burkholderia glumae grouped in a separate branch of the tree.
The isolated Burkholderia strain was found to n the following sequences: Forward sequence, DNA sequence with 27F primer, 815 nucleotides (SEQ ID N0:8); Reverse sequence, 1453 bp, using primers 1525R, 1100R, 519R (SEQ ID N0:9); Reverse sequence 824 bp using primer 907R (SEQ ID NO: 10); Forward sequence 1152 bp using primer 530F (SEQ ID NO: 11); Forward sequence 1067 bp using 1114F primer (SEQ ID NO: 12); Reverse sequence wo 2013/032693 1223 bp using 1525R primer (SEQ NO: 13); Reverse sequence 1216 bp using 1100R primer (SEQ ID ; Reverse sequence 1194 bp using 519R primer (SEQ ID N0:15) 1.3. Proofthat Burkholderia A396 does not belong to Burkholderia cepacia complex 1.3 .1 Molecular Biology work using specific PCR primers In order to confirm the identification of Burkholderia A396 as Burkholderia multivorans, additional sequencing of housekeeping genes is performed. Burkholderia orans is a known member of the Burkholderia cepacia complex. Efforts are focused on PCR of recA genes, as described by Mahenthiralingam et al., 2000. The following primers are used: (a) BCR1 and BCR2 set forth in Mahenthiralingam et al., 2000 to confirm B. cepacia complex match and (b) BCRBM1 and BCRBM2 set forth Mahenthiralingam et al, 2000 to confirm B. multivorans match. A product-yielding PCR reaction for the first primer set would confirm that the microbe belongs to the B. cepacia complex. A product-yielding PCR reaction for the second primer set would m that the microbe is indeed B. multivorans.
No PCR product is obtained for either pair of primers. The performance of the PCR reaction and primers is tested using Burkholderia multivorans ATCC 17616 (positive control) and Pseudomonas scens (negative control). Strong bands are observed both forB. 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. a 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 y-side in a shake culture, and the growth is monitored daily using optical density measurements at 600 nm.
Under the set conditions, 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 ization ment with Burkholderia multivorans (the t 16SrRNA sequence match) is conducted. Biomass for both A396 and B. multivorans is produced in ISP2 broth, grown over 48 hours at 200 °C in Fernbach flasks. The s is cally harvested by fugation. The broth is decanted and the cell pellet is resuspended in a 1: 1 solution of water: isopropanol. 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., 1977. DNA-DNA hybridization is carried out as described by wo 2013/032693 De Ley et al., 1970 under eration of the modifications described by Huss et al., 1983 using a model Cary 100 Bio UV/VIS-spectrophotometer ed with a Peltier thermostatted 6x6 multicell changer and a temperature controller with in-situ ature 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 s lderia multivorans when the recommendations of a threshold value of 70% DNA-DNA similarity for the tion of bacterial species by the ad hoc committee (Wayne et al., 1987) are considered. 1.4. Biochemicalprofile 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 te 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% rity with A396: Pseudomonas spinosa (Burkholderia), Burkholderia cepacia, and Burkholderia pseudomallei. The results are shown in Table 1. wo 2013/032693 Table 1. Biochemical Profile of A396 Substrate Result Substrate Result <:::y~l9.~t?~~r.iJ:l L-arabinose Dextrin D-arabitol (]lyc9.gt?11 D-cellobiose Tween 40 + f.:r.Y.~Ilr.it()~ Tween 80 + D-Fructose ..~>acetykJ?~Qa1ac;tosea1Iline... L-Fucose .. N~a.~~~YkP~gll1C:9.~<t1Ilill~ .. D-Galactose +1- Adonitol Gentibiose .. Sl1cci11ic. J\ci~l\19.J:l~1Ilet]1yl.t?ster ... ose + Acetic acid m-Inositol Cis-aconitic acid D-Lactose Citric acid Lactulose Formic acid + Maltose D-Galactonic Acid Lactone D-Mannitol D-Galacturonic Acid D-Mannose D-Gluconic acid D-Melibiose osaminic acid yl-D-glucoside D-Glucuronic Acid D-Psicose .. <l~hydroxyburytic.a.ci~··· D-Raffinose ...~.~]1y~r9.~Y.bl}tyfic.a.ci~··· + L-Rhamonose .. Y~?Y~!()X..Y?..tJtyr.~C:.<tC:~~··· D-Sorbitol p-hydroxyphenylacetic acid Sucrose Itaconic acid D-Trehalose + .. (l~l\:~~9.. ?..tJtyr.~c; .etC:~~ ... Turanose a-keto glutaric acid l a-ket valerie acid ............ lJ\'ic.J\ci~ J\1t?t?yl. ?r ...
D ,L-Lactic acid Uridine ...........................................
Malonic acid ine .. J>r9.pi9.11ic ac;ici ... + J>?~llyet]1yk<t1Ilille lic acici Putrescine D-Saccharic acid 2-aminoethanol Sebacic acid 2,3-Butanediol ...................................................
Succinic Acid + (Jlyc~rol +1- Bromosuccinic acid .............P ,~~a~glycer()l.Pl19.sphate ... +1- Succinamic acid ............. <l~P~Q~l1~().S.~~~ ~Pll()S.Pil<ttt? ...
Glucuronamide .............P.~gll}cose~6~phosp]1at~··· + L-alaninamide + ............ Y~a1Ilino ~l}tyric;.aci~ .. + D-Alanine Urocanic acid L-alanine + e .. ~~ala.nyl~glyciJ:lt? ... ............. ~~pheJ:lyl[tl[tlline .. + ..~~ (tSP(t!(lgiJ:l~ .. + ~~pr.()l~Il~ L-aspartic acid +1- L-pyroglutamic acid .. r<gll}ta1Ilic. ·· + D-serine .. Qlycyl~~~Aspa.rtic.a.ci~... L-serine Glycyl-L-glutamic acid L-threonine idine D ,L-carnitine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . .
.. IIY~r9.:KY~.l.~pr()liJ:l~ ... + L-ornithine L-leucine wo 2013/032693 2012/050807 1.5. Fatty acid composition After incubation for 24 hours at 28°C, a loopful ofwell-grown cells are harvested and fatty acid methyl esters are ed, separated and identified using the Sherlock Microbial Identification System (MIDI) as described (see Vandamme et al., 1992). The predominant fatty acids present in the Burkholderia A396 are as follows: 16:0 (24.4%), cyclo 17:0 (7.1 %), 16:0 3- 0H (4.4%), 14:0 (3.6%), 19:0 w8c (2.6%) cyclo, 18:0 (1.0%). Summed feature 8 ising 18:1 m7c) and summed feature 3 ising of 16:1 m7c and 16:1 m6c) corresponded to 26.2% and 20.2% ofthe total peak area, respectively. Summed feature 2 comprising 12:0 ALDE, 16:1 iso I, and 14:0 3-0H) corresponded to 5.8% ofthe total peak area while summed feature 5 comprising 18:0 ANTE and 18:2 ru6,9c corresponded to 0.4%. Other fatty acids detected in A396 in minor ties included: 13:1 at 12-13 (0.2%), 14:1 ru5c (0.2%), 15:0 3-0H (0.13%), 17:1 m7c (0.14%), 17:0 (0.15%), 16:0 iso 3-0H (0.2%), 16:0 2-0H (0.8%), 18:1 m7c 11-methyl (0.15%), and 18:1 2-0H (0.4%).
A comparison ofthe fatty acid composition ofA396 with those ofknown microbial strains in the MIDI database suggested that the fatty acids in the novel strain A396 were most similar with those ofBurkholderia cenocepacia. 1.6 Resistance to Antibiotics Antibiotic susceptibility ofBurkholderia A396 is tested using antibiotic disks on Muller- Hinton medium as bed in PML Microbiological's technical data sheet #535. Results obtained after 72-hour incubation at 25°C are presented in Table 2 below. wo 2013/032693 Table 2: Susceptibility of MBI-206 to various antibiotics.+++ very susceptible,++ susceptible, - resistant Concentration (tJ.g) Susceptible Tetracycline 30 cin 30 +++ Erythromycin 15 Streptomycin 10 llin 10 Ampicillin 10 Oxytetracycline 30 Chloramphenicol 30 ++ Ciprofloxacin 5 ++ Gentamicin 10 Piperacillin 100 +++ Cefuroxime 30 Imipenem 10 +++ Sulphamethoxazole- Trimethoprim 25 ++ The results indicate that the antibiotic susceptibility spectrum ofBurkholderia A396 is quite different from pathogenic B. cepacia complex strains. Burkholderia A396 is susceptible to kanamycin, chloramphenicol, ciprofloxacin, cillin, imipenem, and a combination of sulphamethoxazole and trimethoprim. As a comparison, Zhou et al., 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 s to be resistant to chloramphenicol (15% tible), and 95% to be ant (5% susceptible) to the ation of sulphamethoxazole and trimethoprim. Results of Zhou et al., 2007 are similar to those of Pitt et al., 1996 who determined antibiotic resistance among 366 B. cepacia isolates and reported that most ofthem are resistant to ciprofloxacin, cefuroxime, imipenem, chloramphenicol, tetracycline, and sulphametoxacole. wo 2013/032693 2. e 2: Burkholderia formulation and isolation of Fractions from Formulated Product The following procedure is used for the purification of compounds extracted from a ated product of MBI-206 containing a whole cell broth of a culture of Burkholderia sp.: The culture broth derived from the 10-L fermentation Burkholderia (A396) in Hy soy growth medium and formulated using methyl 0.1 %and propylparaben, 0.1 % l 0.67% and Glycosperse .67% is extracted with Amberlite XAD-7 resin (Asolkar et al., "Weakly cytotoxic polyketides from a marine-derived mycete of the genus Streptomyces strain CNQ-085." J. Nat. Prod. 69:1756-1759. 2006) by shaking the cell sion 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 ed and dried under vacuum using rotary evaporator to give the crude extract (MBIFP-CE). The crude extract is then fractionated by using reversed-phase C18 vacuum liquid chromatography (H20/CH30H; gradient 80:20 to 0:100%) to give 10 fractions (see Figure 1 for schematic). These fractions are then trated to dryness using rotary ator and the resulting dry residues are ed for biological activity using a whole plant herbicidal assay. The active fractions, fractions 3, 4, 5 and 6 and indicated as MBIFP-3, MBIFP-4, MBIFP-5, and MBIFP-6 tively are then subjected to repeatedly to ed phase HPLC separation (Spectra System P4000 (Thermo ific) to give pure compounds, which are then screened in abovementioned bioassays to locate/identify the active compounds (see Figure 2). 2.1 Analysis ofFormulation fractions These fractions are analyzed on a Thermo high performance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm x 100 mm Luna C18 5 f..Lm column (Phenomenex). The solvent system consisted 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 is 10 JLL and the samples are kept at room temperature in an auto sampler.
To discover the identity of the compound, additional spectroscopic data such as LC/MS and UV are recorded. Compound corresponding to fraction 5, with a retention time of 17.45 s is not found in any of the starting materials, which indicates that the compound is a product of a chemical reaction between natural products in the ial fermentation broth and one or more of the compounds found in the formulation agents. Specifically, this fraction was wo 32693 2012/050807 analyzed using ESI-LCMS on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100- 1500 Da) on a LCQ DECA XPplus Mass Spectrometer (Thermo Electron Corp., San Jose, CA).
Mass spectroscopy is 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 med with a spray e 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 additional new compounds found in fraction 5 were found to have a molecular weight (MW) of 194 (RT = 14.74 min) and 222 (RT = 17.43 min). 2.2 Bioassay Healthy radish plants with two to three true leaves were selected for testing. The radish plants are 13 days old at treatment. The plants are sorted so that all treatments are equivalent in e surface area and plant height. The pots are labeled with treatment number and repetition number. Three repetitions per treatment are tested.
Ten fractions of MBI-206 formulated product are . The fractions are at a concentration of 10 mg/ml. The crude extracts of the formulated product and broth are also tested. An untreated l (treated with deionized water) and a positive control (RoundUp Super Concentrate at a rate of 2.5 fluid ounces per gallon) are included in the test.
The following treatments were tested as shown in Table 3: Table 3: Test escription Treatment SampleiD Description 1 MBIFP-Fl 4% ethanol!water (0.2% perse) 2 MBIFP-F2 4% ethanol/water (0.2% Glycosperse) .:) MBIFP-F3 4% ethanol/water (0.2% Glycosperse) 4 MBIFP-F4 4% ethanol!water (0.2% G1ycosperse) MBlFP-FS 4% ethanol/water (0.2% G1ycosperse) 6 6-FP-F6 4% ethanol/water (0.2% Glycosperse) 7 MBIFP-F7 4% ethanol!water (0.2% Glycosperse) 8 6-FP-F8 4% ethanol/water (0.2% G1ycosperse) 9 MBIFP-F9 4% l/water (0.2% Glycosperse) MBIFP-FlO 4% ethanol!water (0.2% Glycosperse) 11 MBIFP-CE 4% ethanol!water (0.2% G1ycosperse) 12 MBICE (broth) 4% ethanol/water (0.2% Glycosperse) 13 UTC UTC (DI water) Pos. Control (RoumtUp cg 2.) 11 oz/gal (AI: 14 Positive Control glyphosate @ 50.2%)) wo 2013/032693 All products and treatments are well shaken prior to application. Treatments are d using a nozzle from a 2-ounce spray . Separate spray nozzles were used for each treatment. The plant foliage is sprayed evenly and with a moderate volume (i.e. r a light misting nor a heavy ation that resulted in runoff). Two milliliters of each treatment are d simultaneously over the three repetitions of each treatment so that each plant is treated with approximately 0.67 milliliters of treatment solution.
The plants are allowed to air dry and are then randomized in holding trays. Each tray is labeled with the experiment name and treatment date and placed on the laboratory greenhouse shelves. The tory greenhouse maintains a temperature of 70-80°F and a relative humidity of 30-40%. Throughout the ay, plants are watered from below by filling the holding trays with an appropriate amount of water so that plant foliage remained dry.
Results are taken at 3, 8, and 14 days after treatment. Symptoms included foliage burning and plant stunting. The following rating scale, shown in Table 4 is used to quantify cy. Ratings are determined by observing the following factors relative to the plants of the untreated control: overall plant health, average plant height, and foliage health. Symptoms of affected plants may include ored/spotted/burnt/bleached foliage, warped/twisted/curled leaves, side branching (due to damaged apical meristem), plant k, or death.
Table 4: Rating Scale 0 - 0% control symptoms 0.5 - ::; 5% control symptoms 1 - 10% control symptoms 2 - 25% control symptoms 3 - 50% control symptoms 4 -75% control symptoms 5 - 100% control symptoms The mean of three gs is shown in Figure 2. In a whole plant herbicide test, fractions 4 and 5 show good herbicidal activity (see Figure 2). 2.3 Isolation ofPesticidal Compounds from Formulation This fraction was further purified using a HPLC C-18 column (Phenomenex, Luna 1Ou C18(2) 100 A, 250 x 30), water:acetonitrile gradient solvent system (0-10 min; 80% aqueous CH3CN, 10-25 min; 80- 65% s CH3CN, 25-50 min; 65- 50% aqueous CH3CN, 50-60 min; 50 - 70 % aqueous CH3CN, 60-80 min; 70 - 0 % aqueous CH3CN, 80-85 min; 0 - 20 % aqueous CH3CN) at 8 mL/min flow rate and UV detection of 210 nm, to give butyl paraben, retention time 59.15 min (MBI206-FP-F5H32) and hexyl paraben, retention time 74.59 min (MBI206-FP-F5H40) respectively. wo 2013/032693 2.3.1 NMR Spectroscopy Analysis of Compounds NMR spectra were measured on a Bruker 600 MHz gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm). 2.3.1.1 Structure elucidation ofhexyl paraben (MBI206-FP-F5H40) The active compound was isolated as a colorless solid, with UV tion at 248 nm.
The(-) ESIMS showed lar ion at 221 (M-H) corresponding to the molecular weight of 222. The compound exhibited 1H NMR () singals at 7 .90, 6.85, 4.28, 1.76, 1.46, 1.38, 1.37, 0.94 and has 13C NMR values of 166.84, 162.12, 131.34 (2C), 121.04, 114.83 (2C), 64.32, 31.25, 28.43, 25.45, 22.18. 12.93. The molecular formula ofC 13H180 3 (5 degrees of unsaturation), was assigned by combination ofNMR and ESI mass ometry data. The 1H NMR spectrum exhibited signals for an A2B2-type aromatic signals at() 7.90, 2H d, J = 8.5 Hz, and 6.85, 2H d, J = 8.5 Hz. Furthermore, the 1H NMR spectrum ed the presence of -CH CHz-CHz-CHz-CHz-CH3 group, at b 4,28, 2H, t, J= 7.3 Hz; 1,76, 2H, m; 1.46, 2H, m; 1.38, 2H. m; 1.37, 2H, m, and 0.94, 3H, t, J = 7.3 Hz. From an analysis ofthe foregoing spectral data, the structure ofthe aromatic polyketide was established as hexyl paraben, which was confirmed by detail analysis ofthe COSY, HMQC and HMBC ments. A literature search revealed that this compound has been reported as synthetic compound. 2.3.1.2 ure elucidation ofbutylparaben (MBI206-FP-F5H32) This compound was obtained as a colorless solid with UV max at 248 nm. The LCMS analysis in the negative mode showed molecular ion at mlz 193 corresponding to the molecular formula 194. By comparison ofthe UV, MS and NMR data with that ofhexyl paraben with MW 222, this compound was found to be the analogue ofhexyl paraben. The only ence between them was only in the side chain. Thus, the structure ofbutyl paraben was assigned to this compound with MW 194. A search in the literature suggested that this nd is also known as a synthetic nd. 2.3.2 Herbicidal Activity The pure compounds (butyl paraben [MBI206-FP-F5H32] and hexyl paraben 6- FP-F5H40]) obtained from fraction 5 were tested at a concentration of 10 mg/ml. An untreated control (treated with deionized water), the formulation blank (at 3% v/v & 10% v/v), and a positive control Up Super Concentrate at a rate of 2.5 fluid ounces per gallon) are included in the test.
The ing treatments were tested as shown in Table 5: wo 2013/032693 Table 5: Treatment Regimen Test descnp"IOnf Vol (mL) Treatment Sample ID Description of Sample 1 UTC UTC (DI water) 2 Butyl paraben 2 (MBI206-FP-F5H32) 4% ethanol/water (0.2% Glycosperse) 2 Hexyl paraben 3 (MBI206-FP-F5H40) 4% ethanol/water (0.2% Glycosperse) 2 4 Blank Formulation Blank (jiJ 3% v/v 2 Blank Formulation Blank (jiJ 10% v/v 2 Positive Control (RoundUp@ 2.5 fl 6 Positive Control oz/gal (AI: sate@ 50.2%)) 2 Results obtained are set forth in Table 6.
Table 6: Bioassay Results D ay-1Read"mg D ay-7Read"mg Replicates Control Replicate Control STD STD 1 2 3 AVG 1 2 3 AVG Treatment DEV DEV 1 0 0 0 0.0 0.0 0 0 0 0.0 0.0 2 25 5 25 18.3 11.5 25 10 37.5 24.2 13.8 3 75 75 75 75.0 0.0 87.5 87.5 87.5 87.5 0.0 4 0 0 0 0.0 0.0 0 0 0 0.0 0.0 0 0 0 0.0 0.0 0 0 0 0.0 0.0 6 0 0 0 0.0 0.0 87.5 87.5 75 83.3 7.2 Based on the data presented in the table above, hexyl paraben was found to be the most potent herbicidal compound. 2.3 .3 Insecticidal Acitivity The insecticidal activity of butyl paraben (MBI206-FP-F5H32) and hexyl n 6-FP-F5H40) were tested in a tory assay using a l diet overlay assay with 1st instar Beet Armyworm ptera exigua) larvae using microtiter plates with 200 ul of solid, artificial Beet Armyworm diet in each well. One hundred (100) microliters of each test sample (containing 40 ug of sample) is pipetted on the top of the diet (one sample in each well), and the sample is let dry under flowing air until the e is dry. Each sample was tested in six replicates, and water and a commercial Dipel product are used as negative and positive controls, respectively. One first instar larvae of the test insect (Beet armyworm- Spodoptera exiqua) was placed in each well, and the plate was covered with plastic cover with airholes. The plates wo 32693 with insects were incubated at 26 oc for 6 days with daily mortality evaluations. Based on the results presented in Table 7, hexyl paraben and butyl paraben resulted in 71% and 9% mortality, respectively.
Table 7. Insecticidal Bioassay data for butyl paraben (MBI206-FP-F5H32) and hexyl paraben (MBI206-FP-F5H40) against 1st instar Beet Army Worm (Spodoptera exigua).
Sample information Day3 Day4 Butyl paraben MBI206-FP-F5H32) @ 40 !-!g/well 8.93 8.9286 Hexyl parben (MBI206-FP-F5H40)@ 40 ell 50.00 70.833 2% Dipel 0.00 0 4% Dipel 25.00 25 8% Dipel 0.00 25 16% Dipel 0.00 0 32% Dipel 0.00 0 64% Dipel 25.00 100 40% EtOH 14.29 14.286 Dipel 33.33 100 H20 0.00 0 2.3.4 Nematicidal Acitivity; In vitro g ofbutyl paraben (MBI206-FP-F5H32) and hexyl paraben 6-FP-F5H40): The pure sample ofbutyl paraben and hexyl n was used in an in vitro l plastic cell-culture plate bioassay. 15-20 nematodes in a 50 !-!1 water solution were exposed to 3 !-!1 of a 20 mg/ml peak concentrate for a 24 hour period at 25°C. Once the incubation period was ted, results were recorded based on a visual grading of immobility ofthe juvenile nematodes (J2's) in each well treated with compounds; each treatment was tested in replicate of 4 wells. Results are shown in Table 8, which shows the results oftwo different 96-well plate extract bioassays ofcompounds. Three controls are included in each trial; 1 positive (1% Avid) & 2 negative (DMSO & water). Trials (T1) was carried out using M. incognita nematodes and and trail (T2) was carried out using M. hapla nematodes, the samples were dissolved in 100% DMSO. The hexyl paraben 6-FP-F5H40) showed the excellent control with the immobility of93.75% against M. incognita as compared to butyl n with 81.25% immobility.
Table 8: Effect of hexyl paraben and butyl paraben on M. incognita and M. hap/a.
Sample information % immobility % immobility Mean% (trial# T1) (trial# T2) immobility MBI206-FP-F5H32 (butyl paraben) 75 87.5 81.25 MBI206-FP-F5H40 (hexyl paraben) 87.5 100 93.75 Avid (1%) 75 75 75 DMSO 6.25 0 3.12 Water 0 0 0 wo 2013/032693 2.3 .5 Study of formation of Parabens during formulation ofthe product In order to tand the formation ofthese parabens, the effect ofchange in alcohol in the formulation was taken into eration. The different carbon chain alcohols were used in the formulation and the formation ofthe new parabens were monitored using LCMS.
Four separate formulation experiments were performed using butanol, hexanol, octanol and cetyl alcohol and all other ients were kept same. The formulation products were extracted over the period of 2 days and 3 weeks. The crude extract obtained from these formulations were analysed by LCMS. The corresponding parabens formed for all alcohols except for cetyl l. The yield ofthe parabens was found to be the highest for butyl n, followed by hexyl paraben and then octyl n for the one day old formulation product. The is result even after 3 weeks remain the same order ie, butyl paraben > hexyl paraben > octyl paraben. Thus, the rate of ion ofthese parabens such as butyl paraben, hexyl paraben & octyl paraben was found to depend on the carbon chain (number of carbon) of the t (alcohol) ofthe corresponding alcohol used in the formulation (butanol (C4) > hexanol (C6) > octanol (C8) etc). The formation of cetyl paraben was not detected till 3 weeks.
The yields ofthese parabens were found to increase over the time.
Another set of experiments were carried out to understand the role ofwhole cell broth (WCB) in the ion ofthe new paraben analogues. In 4 different expt. with were carried out with following changes in the formulation- Expt-1 : Propy1paraben (No methy1paraben) + WCB + other ingredients Expt-2: Methylparaben (No Propylparaben)+ WCB +other ingredients Expt-3: No parabens (both)+ WCB +other ients.
Expt-4: MethylParaben+ Propylparaben+ other ingredients+ No WCB.
The above formulations were extracted separately and the crude extract obtained were then analysed using LCMS. The formation ofthe hexyl paraben was observed only in the first two experiments. Thus, these experiments suggested that WCB plays a very important role in the formation ofthese parabens. 3. Example 3. Isolation of Templazole A and B s and Materials The following procedure is used for the purification of Templazole A and B extracted from cell culture ofBurkholderia sp (see Figure 3): 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 al., 2006) by shaking the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell wo 2013/032693 mass are ted 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 t is then fractionated by using reversed-phase Cl8 vacuum liquid chromatography (H20/CH30H; gradient 90:10 to 0:100%) to give 11 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 HPLC (Spectra System P4000 (Thermo Scientific) to give pure compounds, which are then screened in above mentioned bioassays to /identify the active compounds. To confirm the ty ofthe compound, onal spectroscopic data such as LC/MS and NMR is recorded.
The active fraction 5 is purified further by using HPLC C-18 column (Phenomenex, Luna lOu Cl8(2) 100 A, 250 x 30), water:acetonitrile gradient solvent system (0-10 min; 80% aqueous CH3CN, 10-25 min; 80- 65% aqueous CH3CN, 25-50 min; 65- 50% aqueous CH3CN, 50-60 min; 50-70% CH3CN, 60-80 min; 70-0% aqueous CH3CN, 80-85 min; 0-20% aqueous CH3CN) at 8 mL/min flow rate and UV detection of 210 nm, to give templazole B, ion time 46.65 min. The other active fraction 7 is also purified using HPLC C-18 column (Phenomenex, Luna lOu Cl8(2) 100 A, 250 x 30), water:acetonitrile gradient solvent system (0- min; 80% aqueous CH3CN, 10-25 min; 80- 60% aqueous CH3CN, 25-50 min; 60- 40% aqueous CH3CN, 50-60 min; 40% CH3CN, 60-80 min; 40-0% aqueous CH3CN, 80-85 min; 0- % s CH3CN) at 8 mL/min flow rate and UV detection of 210 nm, to give zole A, retention time 70.82 min.
Mass spectroscopy analysis of pure compounds is performed on a Thermo an LCQ Deca XP Plus ospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XPplus Mass Spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high mance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm x 100 mm Luna Cl8 5 11m 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 ed 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 11L and the samples are kept at room temperature in an auto r. 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 wo 2013/032693 2012/050807 and aux/sweep gas flow rate, respectively. ospray 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 mlz ion at 297.34 in negative ionization mode. The LC-MS chromatogram for templazole B suggests a molecular mass of 258 and exhibited mlz ion at 257.74 in negative tion mode. 1H, 13C and 2D NMR spectra were measured on 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 oftemplazole A, the purified nd with a molecular weight 298 is further analyzed using a 500 MHz NMR instrument, and has 1H NMR b values at 8.44, 8.74, 8.19, 7.47, 7.31, 3.98, 2.82, 2.33, 1.08 and has 13C NMR values of b 163.7, 161.2, 154.8, 136.1, 129.4, 125.4, 123.5, 123.3, 121.8, 121.5, 111.8, 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, C17H18N20 3, was determined by interpretation of 1H, 13C NMR and HRESI MS data mlz 96 (M+Ht (Calcd for C17H19N20 3, 299.1397), which entails a high degree ofunsaturation shown by 10 double bond equivalents. The 13C 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 ed from 1H -1H COSY and HMBC spectral data. The 1H -1H COSY and HMBC also indicated the presence of a ylic acid methyl ester group and a -CH2-CH-(CH3) 2 side chain. From the detailed analysis of 1H-1H COSY, 13C, 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.
The second herbicidally active compound, templazole B, with a lar weight 258 is further analyzed using a 500 MHz NMR instrument, and has 1H NMR b values at 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93 and 13C NMR values of b 158.2, 156.3, 155.5, 132.6, 129.5, 129.5, 127.3, 121.8, 115.2, 115.2, 41.2, 35.3, 26.7, 21.5, 21.5. The lar formula, is assigned as C15H18N20 2, which is ined by interpretation of 1H, 13C NMR and mass data.
The 13C NMR um revealed signals for all 15 carbons, including two methyls, two methylene carbons, one aliphatic e, one amide carbonyl, and nine aromatic carbons. The general nature of the structure was deduced from 1H and 13C NMR spectra that showed a para- substituted aromatic ring [b 7.08 (2H, d, J = 8.8 Hz), 6.75 (2H, d, J = 8.8 Hz), and 132.7, 129.5, wo 2013/032693 115.2, 127.3, 115.2, 129.5]. The 1H NMR spectrum of this structure together with the 1H- 1H COSY and HSQC spectra, displayed characteristic signals for an isobutyl moiety [b 0.93 (6H, d, J = 6.9 Hz), 2.15 (IH, sept., J = 6.9 Hz), 2.57 (2H, d, J = 6.9 Hz). In addition, an olefinic/aromatic proton at (b 7.06, s), and a carbonyl carbon group (b 158.9) were also found in the 1H and 13C NMR spectra. On inspection of the HMBC um, the H-1' signal in the isobutyl moiety correlated with the olefinic carbon (C-2, b 156.3), and the olefinic proton H-4 correlated with (C-5, b 155.5; C-2, 156.3 & C-1", 41.2). The methylene signal at b 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 tivity among the isobutyl, and the para-substituted benzyl es for the skeleton ofthe structure as shown. In addition, the carboxamide group is assigned at the para position of the benzyl moiety based on the HMBC correlation from the ic proton at H-4"& H-6" position. Thus, based on the above data, the structure was designated as templazole B. 4. Example 4. ion of FR901228 The whole cell broth from the fermentation of Burkholderia sp. in an undefined growth medium is extracted with Amberlite XAD-7 resin (Asolkar et al., 2006) by shaking the cell suspension with resin at 225 rpm for two hours at room ature. The resin and cell mass are ted 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 e 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 C18 vacuum liquid chromatography (H20/CH30H; gradient 90: 10 to 0:100%) to give 11 ons. These fractions are then trated to dryness using rotary evaporator and the resulting dry residues are screened for ical activity using both insect bioassay as well as herbicidal bioassay. The active ons are then subjected to reversed/normal phase HPLC (Spectra System P4000; Thermo Scientific) to give pure compounds, which are then screened in herbicidal, insecticidal and nematicidal bioassays described below to locate/identify the active compounds. To confirm the identity of the compound, additional spectroscopic data such as LC/MS and NMR is recorded.
Mass spectroscopy analysis of active peaks is performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XPplus Mass Spectrometer o Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm x 100 mm Luna C18 5 f..Lm column (Phenomenex). The solvent system consists wo 2013/032693 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 . The ion volume is 10 JLL and the samples are kept at room temperature in an auto sampler. The compounds are analyzed by LC-MS utilizing the LC and ed phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the ing conditions: The flow rate of the nitrogen gas is fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, respectively. Electrospray ionization is performed with a spray voltage set at 5000 V and a capillary voltage at 35.0 V. The capillary temperature is set at 400°C. The data is analyzed on Xcalibur re. Based on the LC-MS analysis, the active icidal compound from fraction 6 has a molecular mass of 540 in negative ionization mode.
For ure elucidation, the purified insecticidal compound from fraction 6 with molecular weight 540 is further ed using a 500 MHz NMR instrument, and has 1H NMR values at 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23. 1.74, 1.15, 1.12, 1.05, 1.02; and has 13C NMR values of 172.99, 172.93, 169.57, 169.23, 167.59, 130.74, 130.12, 129.93, 128.32, 73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90, .36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51. The NMR data tes that the compound contains amino, ester, carboxylic acid, aliphatic methyl, ethyl, methylene, oxymethylene, methine, oxymethine and sulfur . The detailed 1D and 2D NMR analysis confirms the structure for the compound as FR901228 as a known compound. . e 5. Isolation of Templamide A, B, 65 and FR901228 Methods and Materials The culture broth d from the 10-L fermentation Burkholderia (A396) in Hy soy growth medium is extracted with Amberlite XAD-7 resin (Asolkar et al., 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 C18 vacuum liquid chromatography (H20/CH30H; gradient 90: 10 to 0:100%) to give 11 fractions. These fractions are then concentrated to dryness using rotary evaporator and the ing dry residues are screened for biological activity using 96 well plate lettuce seeding (herbicidal) and early 3rd instar Beet Armyworm (insecticidal) assay. The active fractions are then subjected to repeatedly to reversed phase HPLC separation (Spectra System P4000 (Thermo Scientific) to give pure wo 2013/032693 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, HRMS and NMR are recorded.
The active fraction 6 is purified further by using HPLC C-18 column (Phenomenex, Luna lOu Cl8(2) 100 A, 250 x 30), water:acetonitrile gradient solvent system (0-10 min; 80% aqueous CH3CN, 10-25 min; 80- 65% s CH3CN, 25-50 min; 65- 50% aqueous CH3CN, 50-60 min; 50-70% aqueous CH3CN, 60-80 min; 70- 0% aqueous CH3CN, 80-85 min; 0 - 20 % aqueous CH3CN) at 8 mL/min flow rate and UV detection of 210 nm, to give templamide A, retention time 55.64 min and FR901465, retention time 63.59 min and FR90128, ion time 66.65 min respectively. The other active fraction 6 is also purified using HPLC C-18 column (Phenomenex, Luna lOu Cl8(2) 100 A, 250 x 30), water:acetonitrile gradient solvent system (0-1 0 min; 70-60 % aqueous CH3CN, 10-20 min; 60-40 % aqueous CH3CN, 20-50 min; 40- 15% aqueous CH3CN, 50-75 min; 15-0% CH3CN, 75-85 min; 0- 70 % aqueous CH3CN) at 8 mL/min flow rate and UV ion of 210 nm, to give templamide B, retention time 38.55 min.
Mass oscopy analysis ofpure compounds is performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) ment using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XPplus Mass Spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) ment equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm x 100 mm Luna Cl8 5 11m column (Phenomenex) is used. The solvent system consists ofwater (solvent A) and acetonitrile (solvent B). The mobile phase begins at % t B and is linearly increased to 100% solvent B over 20 min and then kept for 4 min, and finally returns to 10% t B over 3 min and kept for 3 min. The flow rate is 0.5 mL/min. The injection volume is 10 11L 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 oscopy analysis ofthe present compounds is performed under the following conditions: The flow rate ofthe nitrogen gas is fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, respectively. Electrospray ionization is performed with a spray voltage set at 5000 V and a capillary voltage at 45.0 V. The capillary temperature is set at 300°C. The data is analyzed on Xcalibur software. The active compound templamide A has a molecular mass of 555 based on the mlz peak at 556.41 [M + Ht and 578.34 [M +Nat in ve ionization mode. The LC-MS analysis in positive mode ionization for mide B suggests a molecular mass of 537 based mlz ions at 538.47 [M + Ht and 560.65 [M +Nat. The wo 2013/032693 2012/050807 molecular weight for the compounds FR901465 and FR901228 are assigned as 523 and 540 tively on the basis of LCMS analysis. 1H, 13C and 2D NMR a are measured on a Bruker 600 MHz gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm).
For structure elucidation oftemplamide A, the purified compound with molecular weight 555 is further analyzed using a 600 MHz NMR instrument, and has 1H NMR b values at 6.40, 6.39, 6.00, 5.97, 5.67, 5.54, 4.33, 3.77, 3.73, 3.70, 3.59, 3.47, 3.41, 2.44, 2.35, 2.26, 1.97, 1.81, 1.76, 1.42, 1.37, 1.16, 1.12, 1.04 and has 13C NMR values of b 173.92, 166.06, 145.06, 138.76, 135.71, 129.99, 126.20, , 99.75, 82.20, 78.22, 76.69, 71.23, 70.79, 70.48, 69.84, 60.98, 48.84, 36.89, 33.09, 30.63, 28.55, 25.88, 20.37, 18.11, 14.90, 12.81, 9.41. The 13C NMR um exhibits 28 discrete carbon signals which are attributed to six methyls, four ene s, and thirteen methines including five sp2 , four quaternary carbons. The molecular formula, C28H45N0 10, is determined by interpretation of 1H, 13C NMR and HRESI MS data.
The detailed is of 1H-1H COSY, HMBC and HMQC spectral data reveals the following substructures (I- IV) and two isolated methylene & singlet methyl groups. These substructures are ted later using the key HMBC correlations to give the planer structure for the compound, which has been not yet reported in the literature and designated as templamide A.
This polyketide molecule contains two tetrahydropyranose rings, and one conjugated amide. o~"' otvVvvv H 0~ OANH CH 3 "·~~ H,C~ ~YT¢/ H ~\ '{.0 H H y 0 I II Ill IV Substructures I-IV assigned by analysis of 1D & 2D NMR spectroscopic data.
The(+) ESIMS analysis for the second herbicidal compound, shows mlz ions at 538.47 [M + Ht and 560.65 [M +Nat corresponding to the molecular weight of 537. The molecular formula of N09 is determined by interpretation of the ESIMS and NMR data analysis.
The 1H and 13C NMR of this compound is similar to that of templamide A except that a new isolated -CH2- appear instead of the non-coupled ene group in templamide A. The small germinal coupling constant of 4.3 Hz is characteristic of the presence of an epoxide methylene group. The presence of this epoxide is further confirmed from the 13C NMR shift from 60.98 in templamide A to 41.07 in compound with MW 537. The molecular formulae difference between these two compounds is reasonably explained by elimination of the water molecule wo 2013/032693 2012/050807 followed by formation of epoxide. Thus, on the basis ofbased NMR and MS analysis the ure for the new compound was assigned and was designated as templamide B.
For structure elucidation, the purified nd from fraction 6 with molecular weight 523 is further analyzed using a 600 MHz NMR instrument, and has 1H NMR values at 6.41, 6.40, 6.01, 5.98, 5.68, 5.56, 4.33, 3.77, 3.75, 3.72, 3.65, 3.59, 3.55, 3.50, 2.44, 2.26, 2.04, 1.96, 1.81, 1.75, 1.37, 1.17, 1.04; and has 13C NMR values of 172.22, 167.55, 144.98, 138.94, 135.84, 130.14, 125.85, 123.37, 99.54, 82.19, 78.28, 76.69, 71.31, 70.13, 69.68, 48.83, 42.52, 36.89, 33.11, 30.63, 25.99, 21.20, 20.38, 18.14, 14.93, 12.84. The detailed 1H and 13C NMR analysis of compound suggested that this compound was quite similar to compound templamide B; the only difference was in the ester side chain; an acetate moiety was present instead of a propionate moiety in the side chain. The detailed 1D and 2D NMR analysis confirm the structure for the compound as FR901465 as a known compound.
Based on the LC-MS is, the other compound from fraction 6 has a molecular mass of 540 in negative ionization mode. For structure elucidation, the purified compound from fraction 5 with molecular weight 540 is further analyzed using a 500 MHz NMR instrument, and has 1H NMR values at 6.22, 5.81, 5.69, 5.66, 5.65, 4.64, 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 2.26, 2.23. 1.74, 1.15, 1.12, 1.05, 1.02; and has 13C NMR values of 172.99, 172.93, 169.57' 169.23, 167.59, 130.74, 130.12, 129.93, , 73.49, 62.95, 59.42, 57.73, 38.39, 38.00, 35.49, 30.90, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12.51. The NMR data indicates that the compound contains amino, ester, carboxylic acid, aliphatic methyl, ethyl, methylene, oxymethylene, e, oxymethine and sulfur groups. The detailed 1D and 2D NMR analysis confirm the structure for the compound as FR901228 as a known compound.
The molecular weight for the other active compound (F8H17) from Fraction F8 was assigned as 1080 based on the molecular ion peak at 5 (M +H) in positive ESI mode and further confirmed by the negative ESIMS with base peak at 1079.92. This compound showed UV absorption at 234 nm.
Example 6. Burkholderia sp. as an Algicide Burkholderia sp. A396 is grown in an ned mineral medium for 5 days (25°C, 200 rpm). Cells are separated from the atant by centrifugation at 8,000 g, and the cell-free supernatant is used to test the algaicidal activity against a unicellular algal species (P. itata) and a blue-green alga species (Anabaena sp.). A specified increasing amount of supernatant is added into wells of a l polystyrene plate that has the specified algae g in 750 micro liters of Gorham's medium to determine the dose-response curve for the test supernatant on each algae type. Each treatment is done in two replicates, and the blank wo 2013/032693 growth medium is used as a negative control. The plate is closed with a lid and incubated for 48 hours under constant growth light at room temperature. After 48 hours, the fluorescence (at 700 nm) of the suspension in each well is measured using a SpectraMax Gemini XS plate reader, and the reduction in fluorescence compared with the un-treated control is ted into percent control of algal growth. Results presented in Table 9 below show excellent control of unicellular algae and good control or algistatic effect on blue-green algae.
Table 9. Control of two algal species by Burkholderia A396 cell-free broth measured as a reduction of fluorescence at 700 nm.
Amount of broth per well %control P. subcapitata Anabaena sp. 0 1-1L 0 0 1-1L 74.2 0.0 1-1L 84.1 0.0 1-1L 85.5 0.0 40 1-1L 88.3 0.0 50 1-1L 90.6 0.0 100 1-1L 94.6 36.4 Example 7: Control of Chlamydomonas rdtii by crude extract and fractions of Burkholderia sp.
Fractions obtained from the fractionation ofcrude extract holderia sp. were tested for algaecide activity against domonas reinhardtii. An increasing volume of fraction (with concentration of 20 mg/mL in ethanol) was added to a clear 48 well polystyrene plate with 750 micro liters ofthe specified algae growing. Each treatment was done in two replicates and the t (ethanol) used as a negative control. The plate was closed with a lid and incubated for 72 hours under constant light at room temperature. After 72 hours, the fluorescence (at 680 nm) ofthe suspension in each well was measured using a aMax M2 plate , and the reduction in scence compared with the negative control was converted into percent control of algal growth. Each sample was visually compared to the negative control; a well that was visually clearer than the negative control was scored as active.
Results presented in Table 10 below shows control ofthe specified algae in fractions 5, 6, 7, 8, and 9. Tests were run in two replicates and % Control was calculated as a reduction of fluorescence at 680 nm compared with the negative control. Each sample was visually compared to the ve control; a well that was visually clearer than the ve control was scored as active. wo 2013/032693 2012/050807 Table 10: Control of Chlamydomonas reinhardtii by crude extract & fractions of Burkholderia sp. (MBI 206).
Sample 1-1L of Sample per % Inhibition Visual 750 1-1L of Algae Solvent Blank 22.5 0.00 Not Active 11 0.00 Not Active 0.00 Not Active Crude Extract 22.5 97.10 Active 11 89.54 Active 90.82 Active MBI 206F1 22.5 -74.47 Not Active 11 46.47 Not Active 46.21 Not Active MBI 206F2 22.5 12.64 Not Active 11 -214.35 Not Active -297.56 Not Active MBI 206F3 22.5 -143.92 Not Active 11 -740.16 Not Active 32.68 Not Active MBI 206F4 22.5 -98.80 Not Active 11 -155.41 Not Active 58.51 Not Active MBI 206F5 22.5 92.89 Active 11 79.45 Active 71.60 Weak MBI 206F6 22.5 94.88 Active 11 96.33 Active 86.45 Active MBI 206F7 22.5 97.32 Active 11 98.96 Active 97.89 Active MBI 206F8 22.5 94.35 Active 11 32.17 Weak -13.51 Not Active MBI 206F9 22.5 85.35 Active 11 96.49 Active 97.73 Active MBI 206F10 22.5 50.30 Not Active 11 48.54 Not Active 4 Not Active MBI 206F11 22.5 -121.50 Not Active 11 -16.21 Not Active 36.46 Not Active Example 8: Algicidal effect of crude extract and various fractions obtained from Burkholderia sp. against P. subcapitata. wo 2013/032693 2012/050807 The crude extract as well as the fractions obtained from Burkholderia sp. was tested for algicidal activity against a unicellular algal species (P. subcapitata). An increasing volume of pure ethanol on derived by re-dissolving a known amount of material (10 mg/mL concentration) corresponding to each sample was added into wells of a 24-well polystyrene plate that has the specified algae growing in 750 micro liters of Gorham's medium to determine the algicidal effect of sample (extract/fractions) on unicellular algae. Each treatment was done in three replicates, and pure l was used as a negative control. After mixing, the plate was closed with a lid and incubated for 48 hours under nt growth lights at room temperature.
After 48 hours, the fluorescence (at 700 nm) ofthe suspension in each well was measured using a SpectraMax Gemini XS plate reader, and the ion in fluorescence compared with the untreated control was converted into percent control of algal growth. s presented in Table 11 below show excellent control ofunicellular algae with fractions F5, F6 and F7 whereas no substantial algicidal effect was obtained with other samples. wo 32693 Table 11: Algicidal effect of various samples obtained from Burkholderia sp. The bioassay was run in three replicates using P. subcapitata as the test organism.
Sample sample 1-1L %control Results F1 5 0.0 Not Active 33.9 Not Active 58.2 Not Active MBI206F2 5 35.7 Not Active 6.0 Not Active 31.8 Not Active MBI206F3 5 40.9 Not Active 66.4 Not Active 68.5 Not Active MBI206F4 5 46.8 Not Active 69.8 Weak 84.7 Active MBI206F5 5 49.9 Not Active 71.5 weak 95.4 Active MBI206F6 5 62.7 Not Active 74.7 weak 90.7 Active MBI206F7 5 40.1 Not Active 88.6 Active 93.0 Active MBI206F8 5 36.8 Not Active 50.0 Not Active 65.9 Not Active MBI206F9 5 66.3 Not Active 40.7 Not Active 51.8 Not Active MBI206F10 5 26.8 Not Active 27.5 Not Active 32.9 Not Active MBI206F11 5 25.9 Not Active 32.8 Not Active 39.2 Not Active Crude extract 5 45.6 Not Active 69.6 weak 70.0 weak Solvent Blank 5 0.0 Not Active 0.0 Not Active 0.0 Not Active wo 32693 2012/050807 Example 9: Control of Chlamydomonas reinhardtii by purified compounds from Burkholderia sp. fermentation broth Purified compounds from Burkholderia sp. fermentation broth was tested for algaicidal activity against Chlamydomonas reinhardtii. An increasing volume ofthe purified compounds (20 mg/mL in ethanol) was added to a clear 48 well polystyrene plate with 750 micro liters of the specified algae growing. Each treatment was done in two replicates and the solvent used as a negative control. The plate was closed with a lid and incubated for 72 hours under constant light at room temperature. After 72 hours the fluorescence (at 680 nm) ofthe suspension in each well was measured using a SpectraMax M2 plate reader, and the reduction in fluorescence compared with the negative l was converted into percent control of algal growth. Each sample was visually compared to the negative control; a well that was visually clearer than the negative control was scored as active. Results presented in Table 12 below shows control ofthe specified algae in samples ning templamide B (MW 537), FR901228 (MW 540), templazole A (MW 298), and F8H18 (MW 1080). Tests were run in two replicates and%. Control was calculated as a reduction of scence at 680 nm ed with the negative control. Each sample was visually compared to the negative control; a well that was visually r than the negative control was scored as active. wo 2013/032693 Table 12: Control of Chlamydomonas reinhardtii by purified compounds from lderia sp. fermentation broth (MBI 206). 1-1L of Sample Sample per 750 1-1L of %Control Visual Algae 22.5 0 Not Active Solvent Blank 11 0 Not Active 0 Not Active 22.5 98.29620264 Active Templamide B 11 99.34438783 Active (MW 537) 95.05204335 Active 22.5 -3.887169203 Not Active 65 (MW 11 -33.58351827 Not Active 523) -86.58233289 Not Active 22.5 -151.6466844 Not Active Templamide A 11 -21.16166036 Not Active (MW 555) -67.61183948 Not Active 22.5 98.71299647 Active FR901228 (MW 11 99.35532773 Active 540) 89.48079462 Active 22.5 -30.78693813 Not Active Templazole B 11 52.94712906 Not Active (MW 258) -102.0883867 Not Active 22.5 98.1523303 Active zole A 11 98.72823743 Active (MW 298) 99.18429591 Active 22.5 95.71173214 Active Templazole A 11 98.31330291 Active (MW 298) 98.69251947 Active 22.5 94.98474386 Active F8H18 (MW 11 82.90378804 Active 1080) -21.38764258 Not Active Templazole A was tested twice in this bioassay.
Example 10: Control ofScenedesmus quadricauda by heat-treated lderia sp. fermentation atant.
Burkholderia sp. was grown in a fermentation broth as previously described. The broth was heat treated at the end ofthe fermentation to inactivate all cells. The cell free supernatant was tested for ide activity against Scenedesmus quadricauda. An increasing volume of supernatant was added to a clear 48 well polystyrene plate with 750 micro liters ofthe specified algae growing. Each treatment is done in two replicates and the blank growth medium used as a negative l. The plate is closed with a lid and incubated for 72 hours under constant light at room temperature. After 72 hours the fluorescence (at 680 nm) ofthe suspension in each well wo 2013/032693 is measured using a SpectraMax M2 plate reader, and the reduction in fluorescence compared with the ted control is converted into percent control of algal growth. Results presented in Table 13 below shows control ofthe specified algae. Tests were run in two replicates and% Control was calculated as a reduction of fluorescence at 680 nm compared with the untreated control.
Table 13: Control of Scenedesmus quadricauda by supernatant of heat kill Burkholderia sp. (MBI 206).
Material Volume (~-tL) % Inhibition MBI 206 0 0 120522ST HK 10 97.21347952 TGAI 20 99.36167161 99.42844203 40 99.50798231 50 98.90136045 100 95.9474484 Example 11: Control of Oscillatoria tenius by heat kill lderia sp. fermentation supernatant Burkholderia sp. was grown in a fermentation broth as previously described. The broth was heat d at the end ofthe fermentation to vate all cells. The cell free atant was tested for algaecide ty against Oscillatoria tenius. An increasing volume of supernatant was added to a clear 48 well polystyrene plate with 750 1-1L of the specified algae growing. Each treatment is done in two replicates and the blank growth medium used as a negative control. The plate is closed with a lid and ted for 72 hours under constant light at room temperature. After 72 hours the absorbance at 680 nm is measured in each well using a SpectraMax M2 plate , and the reduction in absorbance compared with the untreated control is converted into percent control of algal growth. Results presented in Table 14 below shows control ofthe specified algae. Tests were run in two replicates and% control was calculated as a reduction of absorbance at 680 nm ed with the untreated control.
Table 14: l of Oscillatoria tenius by supernatant of heat kill Burkholderia sp. (MBI 206).
Material Volume (11L) %Control MBI 206 0 0 120522ST HK 10 6.177042802 TGAI 20 25.12413108 10.56583534 40 37.70086527 50 45.47313627 100 36.96205601 Example 12: Efficacy of Burkholderia sp. against two-spotted spidermites infesting marigold plants e 13: Efficacy of Burkholderia sp. fermentation supernatant against twospotted spidermites infesting Marigold plants AH26(10414908_1):JIN low temperature of 72F during trial dates. Average humidity levels ranged from 40% to 75%. Test plants received natural ng for duration of trial. Test plants were soil d every twenty—four (24) hours as needed. Plants were evaluated prior to application (pre—count), 3, 5, 7 and 14 days after application. Evaluations were taken on a 6cm square total area per replicate. Actual count was ed on live/dead two—spotted spidermite nymph and live/dead two—spotted spidermite adult.
Example 14: Efficacy of Burkholderia sp. formulation (MBI 206) for control of two spotted spidermite (TSM) in strawberry — field data.
The efficacies of five traditional chemistry-derived and MBI 206 were evaluated for TSM control under field conditions. berry al’ transplants were set in the field in plastic mulched beds, 13 inches high and 27 inches across the top, and with 4 ft bed spacing.
Overhead irrigation was applied for 10 days after setting to aid in establishment of the transplants. Trickle irrigation was used for the remainder of the experiment. Each 12.5-ft. plot consisted of 20 plants in two ten-plant rows per bed. Plots were infested from a tory colony in four sessions with 10 to 20 motile TSM, per plant. Each session accomplished the infestation of one block of the experiment. The experiment consisted of treatments of various rates and schedules of application of miticides, some combined with an adjuvant, and a non- treated check. Treatments were replicated four times in a RCB design. Savey and Acramite treatments were applied before TSM densities reached threshold levels (6 Jan); the remainder of the treatment programs began 2 wks later. Treatments were applied using a hand-held sprayer with a spray wand outfitted with a nozzle containing a 45-degree core and a number four disc.
The sprayer was rized by C02, to 40 psi, and calibrated to deliver 100 gal per acre. Pre- treatment samples were taken on Day 1 and sampling ued weekly through 2 wks after the last application of treatments. Samples consisted of ten randomly selected leaflets per plot and were collected from the middle one-third stratum of the plants. Samples were transported to the tory where motile and egg TSM were brushed from the s onto ng sticky discs and counted on 1/ 10 of the disc surface to estimate average numbers per leaflet. Distinctions could not be made between viable and non-viable eggs, thus total eggs were recorded. MBI 206 at the highest rate (3 gal/acre) shows decrease in the number of eggs at a level comparable to at least two of the chemical controls.
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NH ku NM H HH mH.m mm Nmm 03 mo 369.93: Murm om.m QHQ mirm who Emu woo HNN ONE mg Hm§\HowHHOH NM Mam me.m wow Haw HHNH m HmH.m 0313 Hmw new NmH.m wa new me mm qu 03‘ mon REM mama cum NHm mwmm mom mmc ONE SUBSTITUTE SHEET (RULE 26) e 15: Control of citrus rust mites (Phyllocoptruta oleivom) on citrus under filed conditions MBI 206 (formulated broth of Burkhoderia Sp.) was sprayed on Valencia Sweet Orange at l, 2, and 3 gal/acre in combination with 0.25% v/v/ of LI-700 (surfactant) and delivered in a volume of 100 GPA. A single treatment was delivered and compared to an untreated sample.
Mite counts were performed pre-treatment, and then at l, 7, 10 and 14 days after treatment.
Mite counts were an average of 10 fruits per treatment per sampling point. A reduction in the number of mites present in the MBI 206 treatments was observed at 14 days after ents with 1 and 2 gal/acre MBI 206 (approximately 6-8 mites per count), when compared to the untreated control (approx. 16 mites per count).
Example 16: Insecticidal (sucking contact) activity of Templamide, FR901465 and FR901228 against ed bugs.
The insecticidal activity of the pure compounds templamide B (MBI 206; MW 537), FR 901465 (MBI 206; MW 523) and FR901228 (MBI 206; MW 540) were tested in a laboratory assay using a sucking contact bioassay system. The compounds were dissolved in 100% l to concentrations of 1mg/mL. Individual 4th instar ed bugs, penultimate nymph, larvae were placed in 5C maid container with 2 sunflower seeds in each tub and 1 water cup (water in contact cup with cotton wick) into each tub. A Hamilton Micropipette was used to apply 1 ML (1 drop) of nd onto abdomen of ed bugs (MWB) of each larvae. Tubs were place into the Rubbermaid container and cap with mesh lid. Eight larvae per sample were treated. The assay was incubated at 25°C, 12h light/12h dark. Larvae were scored at 4 and 7 day after application. All the three nds exhibited contact activity against MWB, while not all insects died but many were clearly affected and unable to move. Most of the MWB on day 7 had molted which suggests that the compounds may inhibit molting or affect normal MWB development.
Thus, FR 901465 provided a better (87.5 %) control of milkweed bugs, than FR 901228 (MW 540) and templamide B (Figure 4).
Example 17: Insecticidal activity of pure compounds against Lygus hesperus late 2nd/early 3rd instar The insecticidal ty of the four compounds, templamide A, templamide B, FR901465 & FR901228 isolated from Burkholderia were tested in a tory assay using a 12 well plate with treated green beans bioassay system. The compound was dissolved in 100% ethanol to concentrations of 1 mg/mL and 500 ML of this sample was added to 3.5 mL of water to make a total volume of 4 mL containing 0.25 mg/mL concentration of the compound. Green beans were washed earlier in bleach on and then sat in water to rinse. Beans were dried before using and then were cut with rs to fit into wells of 12 — well plate. With the help of forceps the beans were dunked into a 15 mL plastic falcon tube containing each treatment and then submerged in treatment for exactly one min. one bean part was put into each well and then individual late 2nd/early 3rd instar Lygus us, were placed in wells with help of brush. Plate sealer was used to cover tray and hole poked into the plate sealer for aeration. The numbers of Lygus/well were counted and plates were placed on brench top. Larvae were scored at 24, 48 and 120 hours after application. Based on the results presented in Figure 5, compound FR 901465, was found to be the most potent with mortality of 91.2%, followed by templamide with B 69.2%, and FR901228 with 51.7%. The templamide A was inactive in the Lygus feeding bioassay. The positive control used in this testing was Avid (Avemectin) at the rate of 13 uL/10 mL.
Example 18: Nematicidal Activity of 28 The pure sample of FR 901228 was tested using an in vitro 96-well plastic cell- culture plate bioassay. 15-20 des in a 50 ul water solution were exposed to 3 ul of a mg/ml solution of FR 901228 for a 24 hour period at 25C. Once the incubation period was completed, results were recorded based on a visual grading of immobility of the juvenile des (J2’s) in each well treated with compounds; each treatment was tested in replicate of 4 wells. Three controls are included in each trial; 1 positive (1% Avid) & 2 negative (DMSO & water). Trials (T1) was carried out using Free living nematodes (FLN) and trail (T2) was carried out using M. incognita des, the samples were dissolved in 100% DMSO. FR 901228 (MW 540) showed the ent control with immobility of 75% against free living nematodes as compared to M. incognita with 75% immobility.
MICROORGANISM DEPOSIT The following biological material has been deposited under the terms of the st Treaty with the Agricultural Research Culture Collection (NRRL), 1815 N.
University Street, Peoria, Illinois 61604 USA, and given the ing 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 e will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be ed thereto under 37 C.F.R. §l.l4 and U.S.C. §122. The deposit represents a substantially pure culture of the deposited .
The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a e to practice the subject invention in tion of patent rights granted by government action.
Although this invention has been described with reference to specific embodiments, the details thereof are not to be construed as limiting, as it is obvious that one can use various equivalents, changes and modifications and still be within the scope of the present invention.
Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.
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I/WE

Claims (16)

CLAIM :
1. An insecticidal or herbicidal composition comprising: (A) an ed strain of Burkholderia sp. A 396 (NRRL Accession No. B-50319); (B) a C1-C8 paraben, and (C) a C2-C17 alcohol, wherein said C1-C8 paraben is formed by incubating (A) and (C) at a temperature sufficient to produce said C1-C8 paraben.
2. The ition according to claim 1, wherein said C1-8 paraben is present in the amount of about 0.01–5%, the C2-C17 alcohol is present in the amount of about 0.001–10%.
3. A method for obtaining a C1-C8 paraben comprising (A) providing a composition comprising an isolated strain of Burkholderia sp. A 396 (NRRL Accession No. B-50319); (B) providing a C2-C17 alcohol; (C) ting the composition of (A) and the l of (B) for a time and at a temperature ient to produce said C1-C8 paraben; and (D) isolating said C1-C8 paraben.
4. The composition of claim 1 or claim 2, wherein said C1-C8 paraben is selected from butyl, hexyl and octyl paraben.
5. A method of modulating pest infestation, and/or monocotyledonous, sedge, or dicotyledonous weeds, comprising applying the composition of claim 1 to a location where modulation is desired in an amount effective to modulate said pest infestation, and/or monocotyledonous, sedge, or dicotyledonous weeds.
6. A method for making an icidal or herbicidal composition comprising (A) ing a composition comprising an isolated strain of lderia sp. A 396 (NRRL Accsession No. B-50319); (B) providing a C2-C17 alcohol; and (C) incubating the composition of (A) and the alcohol of (B) for a time at a temperature sufficient to produce C1-C8 paraben.
7. The method of claim 3 or 6, wherein the composition of (A) is a whole cell broth AH26(10414908_1):JIN comprising the isolated strain of lderia sp. A396.
8. The method of claim 3 or 6, n the composition of (A) is a cell-free supernatant.
9. The method of claim 3 or 6, wherein the composition of (A) is a crude extract.
10. The method of claim 3 or 6, n the composition of (A) is a cell fraction.
11. The method of claim 5, wherein the pest is an arachnid.
12. The method of claim 5, wherein the pest is a de.
13. The method of claim 5, wherein the pest is an insect.
14. The method of any one of claims 3, 5 or 6, wherein the C1-C8 paraben is hexyl paraben.
15. The method of any one of claims 3, 5 or 6, wherein the C1-C8 paraben is butyl paraben.
16. The composition of claim 1, wherein the insecticidal or herbicidal composition further comprises additional synthetic C1-C8 parabens. Marrone Bio Innovations, Inc. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: AH26(10414908_1):JIN
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