METHOD TO PRODUCE FR901228, FR901465, TEMPLAMID A, TEMPLAMID B, TEMPLAZOL A, OR TEMPLAZOL B AND METH

专利摘要:
pesticide compound, composition, method for producing a compound, method for isolating a compound from a burkholderia strain, combination, method for modulating pest infestation in a plant, method for modulating weed emergence and / or growth and use of a compound. the present invention relates to a species of burkholderia sp without known pathogenicity for vertebrates, but with pesticidal activity (for example, plants, insects, fungi, weeds and nematodes) which is provided. natural products derived from a culture of said species and methods of controlling pests using said natural products are still provided.
公开号:BR112012021715B1
申请号:R112012021715-1
申请日:2011-02-24
公开日:2020-08-11
发明作者:Ratnakar Asolkar；Marja Koivunen；Pamela Marrone；Huazhang Huang；Ana Lucia Cordova-Kreylos
申请人:Marrone Bio Innovations, Inc；
IPC主号:

专利说明:

TECHNICAL FIELD
[0001] The present invention relates to a species of Burkholderia sp. no known pathogenicity for vertebrates, such as mammals, fish and birds, but pesticidal activity against plants, insects, fungi and nematodes. Natural products derived from a culture of these species and methods of controlling the germination and growth of dicotyledonous, monocotyledon and weed, modulation of fungal growth and control of parasites such as insects and nematodes are also provided using these natural products. BACKGROUND
[0002] Natural products are substances produced by microorganisms, plants and other organisms. Microbial natural products offer an abundant source of chemical diversity, and there is no long history of using natural products for pharmaceutical purposes. Said compound is FR901228 isolated from Chromobacteriume and has been found to be useful as an antibacterial agent and an anti-tumor agent (see, for example, Ueda et al., US Patent 7,396,665).
[0003] However, secondary metabolites produced by microbes have also been successfully considered 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 7,393,812). Natural microbial products have also been successfully developed in agricultural insecticides (see, for example, Salama et al. 1981; Thompson et al., 2000; Krieg et al. 1983). Sometimes, these natural products have been combined with chemical pesticides (see, for example, Gottlieb, US Patent 4,808,207). Burkholderia
[0004] The genus Burkholderia, β subdivision of proteobacteria, comprises more than 40 species that inhabit different ecological niches (Compant et al., 2008). The bacterial species in the Burkholderia genus are ubiquitous organisms in the soil and in the rhizosphere (Coenye and Vandamme, 2003; Parke and Gurian-Sherman, 2001). Traditionally, they have been known as plant pathogens, B. cepacias, the first being discovered and identified as a pathogen causing disease in onions (Burkholder, 1950). Several species of Burkholderia have developed beneficial interactions with their host plants (see, for example, Cabballero-Mellado et al., 2004, Chen et al., 2007). Some species of Burkholderia have also been shown to be opportunistic human pathogens (see, for example, Cheng and Currie, 2005 and Nierman et al., 2004). In addition, some species of Burkholderia have been shown to have potential as biocontrol products (see, for example, Burkhead et al., 1994; Knudsen et al., 1987; Jansiewicz et al., 1988; Gouge et al., US Patent Application 2003/0082147; Parke et al., US Patent 6,077,505; Casida et al., US Patent 6,689,357; Jeddeloh et al., W02001055398; Zhang et al., US Patent 7,141,407). Some species of this genus have been effective in bioremediation to decontaminate polluted soil or groundwater (see, for example, Leahy et al. 1996). In addition, some species of Burkholderia have been shown to secrete a variety of extracellular enzymes with proteolytic, lipolytic and hemolytic activities, as well as toxins, antibiotics and siderophores (see, for example, Ludovic et al., 2007; Nagamatsu, 2001). Oxazoles, Thiazoles and Indols
[0005] Oxazois, thiazoles and indoles are widely distributed in plants, algae, sponges and microorganisms. A large number of natural products contain one or more of the five-membered oxazole, thiazole and indole nuclei / fractions. These natural products have a broad spectrum of biological activity of demonstrable therapeutic value. For example, bleomycin A (Tomohisa et al.), A widely prescribed antineoplastic drug, performs oxidative DNA degradation and uses a bitiazole moiety to link its target DNA sequences (Vanderwall et al., 1997). Bacitracin (Ming et al., 2002), a peptide antibiotic containing thiazoline, prohibits new bacterial cell wall biosynthesis by complexing with C55 bactoprenolpyrophosphate. Thiangazol (Kunze et al., 1993) contains a tandem matrix of one oxazole and three thiazolines and has antiviral activity (Jansen et al., 1992). Still other natural products having oxazole / thiazole, such as thiostreptone (Anderson et al., 1970) and GE2270A (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 indole is derived from tryptophan. The structural variety of the remaining two-thirds is greater and its biological activity appears to cover a broader range, including antimicrobials, antivirals, cytotoxics, insecticides, antithrombotics or enzyme inhibitory activity. BRIEF SUMMARY
[0006] An isolated strain of a non-Burkholderia cepacia, not Burkholderia plantari, not Burkholderia gladioli, Burkholderia sp. which has the following characteristics: a. has a 16S rRNA gene sequence that comprises a forward sequence having at least 99.0% identity to the sequences set out in SEQ ID NO: 8, 11 and 12 and a reverse sequence having at least 99.0% identity to SEQ ID NO: 9, 10, 13-15; B. it has pesticidal activity, in particular, herbicide, insecticide, fungicide and nematicide; ç. produces at least one of the compounds selected from the group consisting of: (i) a compound having the following properties: (a) a molecular weight of about 525 to 555, as determined by Liquid Chromatography / Mass Spectrometry (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, 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 (c) a high pressure liquid chromatography (HPLC) retention time of about 10 to 15 minutes, on a C-18 reverse phase HPLC column using a water gradient: acetonitrile (CH3CN); (ii) a compound having an oxazolyl-indole structure comprising at least an indole fraction, at least an oxazole fraction, at least one substituted alkyl group and at least one carboxylic ester group; at least 17 carbons and at least 3 oxygen and 2 nitrogen; (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 nitrogen; (iv) a compound having at least one ester, at least one amide, at least three methylene groups, at least one tetrahydropyranose fraction and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least minus twenty-five carbons and at least eight oxygen and one nitrogen and d. it is non-pathogenic (non-infectious) to vertebrate animals, such as mammals, birds and fish; and. is susceptible to kanamycin, chloramphenicol, ciprofloxacin, piperacillin, imipenem, and a combination of sulfamethoxazole and trimethoprim and f. contains 16: 0 fatty acids, 17: 0 cycle, 16: 0 3- OH, 14: 0, 19: 0 ωδc cycle, 18: 0.
[0007] In a particular modality, the strain has the identifying characteristics of a strain of Burkholderia A396 (NRRL No. of Accession. B-50319).
[0008] Disclosed here are isolated compounds that are optionally obtained or derived from Burkholderia species, or alternatively, organisms capable of producing these compounds that can be used to control various pests, particularly phytopathogenic plant pests, examples of which include, among others, insects, nematodes, bacteria and fungi. These compounds can also be used as herbicides.
[0009] In particular, isolated pesticidal compounds may include, among others: (A) a compound having the following properties: (i) a molecular weight of about 525 to 555 as determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (ii) 1H NMR values Õ6.22, 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 ÕD172.99, 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, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12 , 51 and (iv) a high pressure liquid chromatography (HPLC) retention time of about 10 to 15 minutes, on a C-18 reverse phase HPLC column using a water gradient: acetonitrile (CH3CN); (B) a compound having an oxazolyl-indole structure comprising at least an indole fraction, at least an oxazole fraction, at least one substituted alkyl group and at least one carboxylic ester group; at least 17 carbons and at least 3 oxygen and 2 nitrogen; (C) 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 nitrogen; (D) a compound having at least one ester, at least one amide, at least three methylene groups, at least one tetrahydropyranose fraction and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least 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, a methylene epoxide group, at least a tetrahydropyranose fraction, at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least 25 carbons, at least 8 oxygen and at least 1 nitrogen.
[00010] In a particular embodiment, the isolated compounds may include, among others: (A) a compound having an oxazolyl-indole structure comprising at least one indole moiety, at least one oxazole moiety, at least one substituted alkyl group, at least a carboxylic ester group, at least 17 carbons, at least 3 oxygen and at least 2 nitrogen; and which has at least one of the following: (i) a molecular weight of about 275-435; (ii) 1H NMR δ values at 8.44, 8.74, 8.19, 7.47, 7.31, 3.98, 2.82, 2.33, 1.08; (iii) 13C NMR Dδ values 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) a High Pressure Liquid Chromatography (HPLC) retention time of about 10 to 20 minutes on a C-18 reverse phase HPLC column using a water: acetonitrile (CH3CN) gradient with a gradient solvent system and 210 nm UV detection; (v) UV absorption bands at about 226, 275, 327 nm; (B) 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, at least 2 nitrogen; and at least one of the following characteristics: (i) a molecular weight of about 240 to 290 as determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (ii) 1H NMR δ values at about 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93; (iii) 13C NMR δ values of 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 to 15 minutes, on a C-18 reverse phase HPLC column using a water gradient: acetonitrile (CH3CN) and (v) bands of UV absorption 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 fraction and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups , at least twenty-five carbons, at least eight oxygen and one nitrogen and at least one of the following characteristics: (i) a molecular weight of about 530 to 580 as determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (ii) 1H NMR δ values of 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; (iii) 13C NMR δ values of 173.92, 166.06, 145.06, 138.76, 135.71, 129.99, 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 to 12 minutes, on a C-18 reverse phase HPLC column using water: acetonitrile (CH3CN) with a gradient solvent system and UV detection 210 nm; (v) a molecular formula of C28H45NO10 that was determined by interpretation of ESI MS and NMR data analysis; (vi) UV absorption bands between about 210 to 450 nm; (D) a compound comprising (i) at least one ester, at least one amide, one methylene epoxide group, at least one tetrahydropyranose fraction and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups , at least 25 carbons, at least 8 oxygen and at least 1 nitrogen, (ii) 13C NMR δ values of 174.03, 166.12, 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, (iii) a molecular formula of C28H43NO9 and at least one of: (i) 1H NMR δ 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; (ii) a High Pressure Liquid Chromatography (HPLC) retention time of about 6 to 15 minutes, on a C-18 reverse phase HPLC column using a water: acetonitrile (CH3CN) gradient; (iii) UV absorption band between about 210 to 450 nm and more particularly about 234 nm.
[00011] In a more particular embodiment, compounds are provided including, among others: (A) a compound having the structure ## STR001 ##
or a pesticidably acceptable salt or stereoisomers thereof, where M is 1, 2, 3 or 4; n is 0, 1, 2, or 3; p and q are independently 1 or 2; X is O, NH or NR; R1, R2 and R3 are the same or different and independently an amino acid side chain fraction or an amino acid side chain derivative and R is a lower alkyl, aryl or arylalkyl fraction; (B) a compound having the structure ## STR002 ##
## STR002 ## where X, Y and Z are each independently --O, --NRi, or --S, where Ri is --H or C1-C10 alkyl; Ri, R2 and 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, alkoxy substituted, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, starch, carboxyl, --C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl and "m" can be located anywhere on the oxazole ring ; (C) a compound having the structure ## STR002a ##
wherein R1 is --H or C1-C10 alkyl; R2 is an alkyl ester; (D) a compound having the structure ## STR003 ##
where: X and Y are each independently --OH, --NR1, or --S, where Ri is -H or C1-C10alkyl; Ri, R2 in, a substituent on the oxazole ring, 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, starch, carboxyl, --C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; (E) a compound having the structure ## STR003a ##
wherein R1 is --H or C1-C10 alkyl; (F) a compound having the structure ## STR004a ##
where X, Y and Z are each independently -O, -NR, or -S, where R is H or C1-C10 alkyl; Ri, R2, R3, FU, Rs, Re, R7, Rs, RΘ, R10, R11, R12, and R13 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, aryl substituted, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, starch, carboxyl, -C (O) H, acyl, oxiacil, carbama, carbama sulfonyl, sulfonamide, or sulfuryl. (G) a compound having the structure ## STR004b ##
where Ri, R2, Rs, R4, Rs, Re, R7, Rs, RΘ, RIO- RL1>RI2> AND RL3 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, starch, carboxyl, -C (O) H, acyl, oxiacila carbamate, sulfonyl, sulfonamide, or sulfuryl; (H) a compound having the structure ## STR004c ##
where Ri, R2, Rs, R4, Rs, Re, R7, Rs, R11, 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, starch, carboxyl, -C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl ; (I) a compound having the structure ## STR005 ##
where X and Y are each independently --OH, --NRi, or --S, where Ri, R2 are each independently -H, alkyl (e.g., C1-C10 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-starch, carboxy (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; (J) a compound having the structure ## STR006a ##
where X, Y and Z are each independently -O, -NR, or -S, where R is H or C1-C10alkyl; Ri, R2, R3, R4, Rs, Re, R7, Rs, R11, R12, and 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, starch, carboxyl, -C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonyl, sulfonyl, sulfonyl or sulfuryl.
[00012] In a more particular embodiment, the compounds may include, among others: (i) templazole A; (ii) templazole B; (iii) templamide A; (iv) templamide B; (v) FR90128; (saw)
(vii)
(viii)
(xi)
(x)
(xi)
(xii)
(xiii)
(xiv)
(xv)
(xvi)
(xvii)
(xviii)
(xix)
(xx)
(xxi)
(xxii)
(xxiii)

(XL) FR901465
[00013] Methods of obtaining the compounds set out above are still provided. In particular, the method comprises cultivating the Burkholderia strain disclosed here and producing the compound. There is also provided a method for isolating these compounds by isolating the compounds produced by a Burkholderia strain comprising isolating the compounds produced from a supernatant from a culture of said Burkholderia strain.
[00014] A combination is also provided comprising (a) a first established substance of the group consisting of (i) a pure culture, cell fraction or supernatant derived from the Burkholderia strain established above or extract thereof for use optionally as a pesticide; (ii) one or more of the compounds set out above (b) optionally a second substance, wherein said second substance is a chemical or biological pesticide and (c) optionally at least one of a carrier, diluent, surfactant, adjuvant, or pesticide. In a particular embodiment, the combination is a composition. In a related aspect, a seed coated with said composition is provided here.
[00015] In a related aspect, a method is revealed to modulate the pest infestation in a plant comprising applying to the plant and / or seeds thereof and / or substrate used to grow said plant and / or a method to modulate the emergence and / or growth of monocots, sedge or dicots weeds comprising applying to said weed or soil an amount of (I) (a) isolated compounds set out above and (b) optionally another substance, wherein said substance is a pesticide (for example , nematocide, herbicide, fungicide, insecticide) or (II) composition or combination set out above in an effective amount to modulate pest infestation and / or emergence or growths of monocots, sedge or dicot weeds.
[00016] In a related aspect, the use of strains, cultures, extracts, supernatants, combinations, the compounds set out above for modulation of pest infestation in a plant comprising applying to the plant and / or seeds thereof and / or substrate is provided used for the growth of said plant and / or a method to modulate emergence and / or growth of monocots, sedge or dicots weeds. BRIEF DESCRIPTION OF THE FIGURES
[00017] Figure 1 shows the comparison of the growth rate of Burkholderia A396 with Burkholderia multivorans ATCC 17616.
[00018] Figure 2 shows the effect of Burkholderia A396 extract on climbing plants.
[00019] Figure 3 shows the effect of Burkholderia A396 extract on anthill.
[00020] Figure 4 shows the effect of Burkholderia A396 extract on cabbage caterpillar (Tricoplusia ni).
[00021] Figure 5 shows the effect of Burkholderia A396 culture broth on beet cereal caterpillar (Spodoptera exigua).
[00022] Figure 6 shows the effect of Burkholderia A396 broth on the motility of juvenile gall nematodes (Meloidogyne incognita).
[00023] Figure 7 is a schematic representation of the purification scheme for obtaining templazole and templamide compounds.
[00024] Figure 8 shows the results of an in vitro test to test the fungicidal effect of FR90128 on Botrytis cinerea (left) and Phytophtora sp. (right).
[00025] Figure 9 shows the effect of Burkholderia A396 broth on the average gall index (% of control) of cucumber roots cv. Toschka inoculated with 3000 eggs of Meloidogyne sp. 14 days after inoculation and application.
[00026] Figure 10: Effect of Burkholderia A396 broth on the average gall index of cucumber roots cv. Toschka inoculated with 3000 eggs of Meloidogyne sp. 14 days after inoculation and application. DETAILED DESCRIPTION OF MODALITIES
[00027] Although compositions and methods up to this point are susceptible to various modifications and alternative forms, the exemplary modalities will be described in detail here. It should be understood, however, that there is no intention to limit the invention to the particular forms disclosed, on the contrary, the intention is to cover all modifications, equivalents and alternatives that fall within the spirit and scope of the invention as defined by the appended claims.
[00028] Where a range of values is provided, it is understood that each intervening value, of the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of the range and any other declared or intervening value in the declared range, is included in this. Smaller ranges are even included. The upper and lower limits of these lower values are still included in these, subject to any limit specifically excluded in the declared range.
[00029] Unless otherwise defined, all technical and scientific terms used here have the same meanings as commonly understood by a person skilled in the art to which this invention belongs. Although any method and material similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
[00030] It should be noted as used here and in the appended claims, the singular forms "one," "and" and "o / a" include references in the plural unless the context clearly indicates otherwise.
[00031] As defined here, "derived 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.
[00032] As defined herein, an "isolated compound" is essentially free of other compounds or substances, for example, at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, still more preferably about 80% pure, more preferably about 90% pure, and even more preferably about 95% pure, as determined by analytical methods, including, but not limited to, chromatographic methods, electrophoretic methods.
[00033] As used herein, the term "alkyl" refers to a straight or branched monovalent chain hydrocarbon group having from one to about 12 carbon atoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl , isobutyl, tert-butyl, n-hexyl, and the like.
[00034] As used herein, "substituted alkyl" refers to alkyl groups still having one or more substituents selected from hydroxy, alkoxy, mercapto, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl , aryloxy, substituted aryloxy, halogen, cyano, nitro, amino, starch, --C (O) H, acyl, oxycyl, carboxyl, sulfonyl, sulfonamide, sulfuryl, and the like.
[00035] As used herein, "alkenyl" refers to straight or branched chain hydrocarbyl groups having one or more carbon-carbon double bonds, and having in the range of about 2 to 12 carbon atoms, and "substituted alkenyl" refers to alkenyl groups still having one or more substituents as set out above.
[00036] 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 to 12 carbon atoms, and "substituted alkynyl" refers to alkynyl groups still having one or more substituents as set out above.
[00037] As used here, "aryl" refers to aromatic groups having in the range of 6 to 14 carbon atoms and "substituted aryl" refers to aryl groups still having one or more substituents as stated above.
[00038] As used herein, "heteroaryl" refers to aromatic rings having one or more heteroatoms (for example, N, O, S, or the like) as part of the ring structure, and having in the range of 3 to 14 atoms carbon and "substituted heteroaryl" refers to heteroaryl groups still having one or more substituents as set forth above.
[00039] As used herein, "alkoxy" refers to the -O-alkyl- moiety, where alkyl is as defined above, and "substituted alkoxy" refers to alkoxy groups still having one or more substituents as set out above.
[00040] As used herein, "thioalkyl" refers to the --S- alkyl- moiety, where alkyl is as defined above, and "substituted thioalkyl" refers to thioalkyl groups still having one or more substituents as stated above .
[00041] As used herein, "cycloalkyl" refers to alkyl groups having a ring having in the range of about 3 to 8 carbon atoms, and "substituted cycloalkyl" refers to cycloalkyl groups still having one or more substituents as established above.
[00042] As used herein, "heterocyclic" refers to cyclic groups (i.e., having a ring) having one or more hetero atoms (for example, N, O, S, or the like) as part of the structure ring, and having in the range of 3 to 14 carbon atoms and "substituted heterocyclic" refers to heterocyclic groups still having one or more substituents as set out above. The Burkholderia strain
[00043] The Burkholderia strain established here is a complex not Burkholderia cepacia, not Burkholderia plantari, not Burkholderia gladioli, Burkholderia sp and not pathogenic to vertebrates, such as birds, mammals and fish. This strain can be isolated from a soil sample using procedures known in the art and described by Lorch et al., 1995. The Burkholderia strain can 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 they form round, opaque cream-colored colonies that change to pink and pinkish-brown in color and mucoid or viscous over time.
[00044] Colonies are isolated from potato dextrose agar plates and triads for those that have biological, genetic, biochemical and / or enzymatic characteristics of the Burkholderia strain of the present invention set out in the Examples below. In particular, the Burkholderia strain has a 16S rRNA gene comprising a direct sequence that is at least about 99.0%, preferably about 99.5%, more preferably about 99.9% and most preferably about 100% identical to the sequence defined in SEQ ID NO: 8, 11 and 12 and a direct sequence which is at least about 99.0%, preferably about 99.5%, more preferably about 99.9% and most preferably about 100% identical to the sequence defined in SEQ ID NO: 9, 10, 13, 14 and 15 as determined by clustal analysis. In addition, as set out below, this strain of Burkholderia may, as defined below, have pesticidal activity, particularly virucide, herbicide, germicide, fungicide, nematicide, bactericide and insecticide and more particularly, herbicide, insecticide, fungicide and nematicide activity. It is not pathogenic for vertebrate animals, such as mammals, birds and fish.
[00045] In addition, the Burkholderia strain produces at least the pesticidal compounds defined in this description.
[00046] The Burkholderia strain is susceptible to kanamycin, chloramphenicol, ciprofloxacin, piperacillin, imipenem, and a combination of sulfamethoxazole and trimethoprim and contains 16: 0, 17: 0, 16: 0 3- OH, 14: 0 fatty acids , cycle 19: 0, 18: 0.
[00047] This strain of Burkholderia can be obtained by cultivating a microorganism having the identification characteristics of Burkholderia A396 (Accession No. NRRL B-50319) on Potato Dextrose Agar (PDA) or in a fermentation medium having sources of carbon defined as glucose, maltose, fructose, galactose, and undefined nitrogen sources like peptone, tryptone, soitone, and NZ amine. Pesticide Compounds
[00048] The pesticidal compound disclosed here has the following properties: (a) it is obtained from a new Burkholderia species, for example, A396; (b) is, in particular, toxic to the most common insect pests in agriculture; (c) has a molecular weight of about 525 to 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 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, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93 , 12.51 (e) has a retention time of High Pressure Liquid Chromatography (HPLC) of about 10 to 15 minutes, more specifically about 12 minutes and even more specifically about 12.14 min on a phase column reverse C-18 HPLC (Phenomenex, Luna 5p C18 (2) 100A, 100 x 4.60 mm) using a water: acetonitrile gradient (CH3CN) with a gradient solvent system (0 to 20 min 90 to 0% aqueous CH3CN, 20 to 24 min 100% CH3CN, 24 to 27 min, 0 to 90% aqueous CH3CN, 27 to 30 min 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection of 210 nm (f) has a formula molecular, C24H36N4O6S2, which is determined by data interpretation 1H, 13C NMR and LC / MS (g) a 13C NMR spectrum with signals for all 24 carbons, including 5 methyl, 4 methylene, 9 methyl, and 6 quaternary carbons and ( g) spectrum 1 H NMR showing characteristics of a typical depsipeptide, illustrating three amino protons [4.63, 4.31, 3.93], and a carbinol ester proton [5.69]. In a particular embodiment, the compound has the structure ## STR001 ##:
or a pesticidably acceptable salt or stereoisomers thereof, where M is 1, 2, 3 or 4; n is 0, 1, 2, or 3; p and q are independently 1 or 2; X is O, NH or NR; R1, R2 and R3 are the same or different and independently an amino acid side chain fraction or an amino acid side chain derivative and R is an alkyl, aryl or arylalkyl lower chain fraction.
[00049] In a more particular embodiment, the compound has the structure of FR90128:

[00050] With this, compounds defined in ## STR002 ## are provided:
where: X, Y and Z are each independently --O, --NRi, or --S, where Ri is —H or C1-C10 alkyl; Ri, R2 and 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, alkoxy substituted, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, starch, carboxyl, --C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.
[00051] In an even more particular modality, compounds from the Family ## STR002 ## can be the compounds defined in (vi) - (xix).
(vii)
(viii)
(ix)
(x)
(xi)
(xii)
(xiii)
(xiv)
(xv)
(xvi)
(xvii)
(xviii)
(xix)

[00052] These are from natural materials or compounds obtained from commercial sources or by chemical synthesis. Natural sources of compounds from the Family ## STR002 ## include, but are not limited to, microorganisms, algae, and sponges. In a more particular embodiment, microorganisms that include compounds from the Family ## STR002 ## include, but are not limited to, or alternatively, compounds from the Family ## STR002 ## can be derived from species such as Streptoverticillium waksmanii (compound vi) (Umehara, et al ., 1984), Streptomyces pimprína (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), Pseudomonas syringae (compounds xiii, xiv) (Pettit et al., 2002). Compounds of the Family ## STR002 ## can also be derived from algae including, but not limited to, red algae (compound xv) (N'Diaye, et al., 1996), Martensia fragilis red algae (compound xvi) (Takahashi S. et al., 1998), Diazona chinensis (compounds xvii & xviii) (Lindquist N. et al., 1991), Rhodophycota haraldiophyllum sp (compound xix) (Guella et al., 1994).
[00053] ## STR003 ## is still provided:
where: X and Y are each independently --OH, --NRi, or -S, where Ri is --H or C1-C10alkyl; Ri, R2 in, a substituent on the oxazole ring, 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, starch, carboxyl, --C (O) H, acyl, oxyalkyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.
[00054] ## STR005 ## is still provided:
where X and Y are each independently --OH, --NR1, or --S, where Ri, R2 are each independently --H, alkyl (e.g., C1-C10 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 C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.
[00055] In a particular embodiment, compounds from the Family ## STR005 ## as compounds of xx-xxiii defined below can be derived from natural or commercial sources or by chemical synthesis: (xx)
(xxi)
(xxii)
(xxiii)

[00056] Natural sources of compounds from the Family ## STR005 ## include, but are not limited to, plants, corals, microorganisms, and sponges. Microorganisms include, among others, Streptomyces griseus (compound xx) (Hirota et al., 1978), Streptomyces albus (compound xxi) (Werner et al., 1980). Compounds of the STR004 Family can also be derived from algae including, among others, Haraldiophyllum sp (compound xxii (Guella et al., 2006), and red algae (compound xxiii) (N'Diaye et al., 1994).
[00057] In one embodiment, the compound can be derived from or obtained 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 fraction 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. The compound further comprises at least one of the following characteristics: (a) pesticide properties and in particular, nematicide, fungicide, insecticide and herbicide properties; (b) a molecular weight of about 530 to 580 and more particularly, 555 as determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (c) 1H NMR values of δ 6.40, 6.39, 6.00, 5.97, 5.67.54, 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; (d) 13C NMR values of δ 173.92, 166.06, 145.06, 138.76, 135.71, 129.99, 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; (e) a High Pressure Liquid Chromatography (HPLC) retention time of about 7 to 12 minutes, more specifically about 10 minutes and even more specifically about 10.98 min on a C-18 HPLC reverse phase column (Phenomenex, Luna 5p C18 (2) 100 A, 100 x 4.60 mm) using water: acetonitrile (CH3CN) with a gradient solvent system (0 to 20 min; 90 to 0% aqueous CH3CN, 20 to 24 min; 100% CH3CN, 24 to 27 min; 0 to 90% aqueous CH3CN, 27 to 30 min; 90% aqueous CH3CN) in 0.5 mL / min flow and 210 nm UV detection; (f) 13C NMR spectrum showing 28 discrete carbon signals that can be attributed to six methyls, four methylene carbons, and thirteen methods including five sp2, four quaternary carbons; (g) a molecular formula of C28H45NO10 that was determined by interpretation of ESIMS and NMR data analysis; (h) UV absorption bands between about 210 to 450 nm and more particularly about 234 nm.
[00058] Compounds are still supplied having the structure ## STR004a ##:
where X, Y and Z are each independently -O, -NR, or -S, where R is H or C1-C10 alkyl; Ri, R2, Rs, R4, Rs, Re, Rz, Rs- R9 'R10' Rn, R12, and R13 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, aryl substituted, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, starch, carboxyl, -C (O) H, acyl, oxiacila, carbamate sulfonyl, sulfonamide, or sulfuryl.
[00059] In a particular modality, the compound has the structure defined in ## STR004b ##:
where Ri, R2, Rs, R4, Rs, Re, R7, Re, R9, R10, R11, R12, and Rn are as previously defined for ## STR004a ##.
[00060] In a more particular embodiment, the compound is Templamide A with the following structure:
Templamide A
[00061] In another embodiment, a compound having the formula ## STR004c ## is provided:
where Ri, R2, R3, R4, Rs, RΘ, RZ, RS, and Rn are as previously defined for ## STR004a ##.
[00062] In another embodiment, a compound is provided that can be derived from a Burkholdθride species characterized as having a structure comprising at least one ester, at least one amide, a methylene epoxide group, at least a tetrahydropyranose fraction and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least 25 carbons and at least 8 oxygen and 1 nitrogen, and pesticidal activity. The compound further comprises at least one of the following characteristics: (a) pesticidal properties and in particular, insecticidal, fungicidal, nematicidal and herbicidal properties; (b) a molecular weight of about 520 to 560 and particularly 537 as determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (c) 1H NMR δ 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 δ 174.03, 166.12, 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 Pressure Liquid Chromatography (HPLC) retention time of about 6 to 15 minutes, more specifically about 8 minutes on a reverse phase C-18 HPLC column using water gradient: acetonitrile (CH3CN), particularly , a retention time of High Pressure Liquid Chromatography (HPLC) of about 8-15 minutes, more specifically about 11 minutes and even more specifically about 11.73 min on a C-18 HPLC reverse phase column (Phenomenex , Luna 5p C18 (2) 100 A, 100 x 4.60 mm) using a water: acetonitrile gradient (CH3CN) with a gradient solvent system (0 to 20 min; 90 to 0% aqueous CH3CN, 20 to 24 min; 100% CH3CN, 24 to 27 min; 0 to 90% aqueous CH3CN, 27 to 30 min; 90% aqueous CH3CN) in 0.5 mL / min flow and 210 nm UV detection; (f) a molecular formula of C28H43NO9 that was determined by interpreting the analysis of ESIMS and NMR data; (g) UV absorption bands at about 210 to 450 nm and more particularly at about 234 nm.
[00063] In a particular modality, the compound has the structure ## STR006a ##:
where X, Y and Z are each independently -O, -NR, or -S, where R is H or C1-C10alkyl; Ri, R2, R3, R4, Rs, RΘ, R7, Rs, 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 heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, starch, carboxyl, -C (O) H, acyl, oxyacyl, carbamate, sulfonyl, sulfonyl, sulfonyl, sulfonyl or sulfuryl.
[00064] In a particular modality, the compound has the structure:
Templamide A
[00065] In another modality, a compound having formula ## STR006b ## is provided:
where R1, R2, Ra, R4, Rs, Re, Rz, Rs, and Rn are as previously defined for ## STR006a ##.
[00066] In a more particular embodiment, the compound is Templamide B with the following structure:
Templamide B
[00067] In yet another particular embodiment, the compound can be derived from species of Burkholderia and characterized as having a structure comprising at least one ester, at least one amide, a methylene epoxide group, at least a tetrahydropyranose fraction and at least three olefinic double bonds, at least six methyl groups, at least three hydroxyl groups, at least 25 carbons and at least 8 oxygen and at least 1 nitrogen. The compound further comprises at least one of the following characteristics: (a) pesticidal properties and in particular, insecticidal, fungicidal, nematicidal 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 δ 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 δ 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; (e) a High Pressure Liquid Chromatography (HPLC) retention time of about 6 to 15 minutes, more specifically about 8 minutes on a C-18 reverse phase HPLC column using a water gradient: acetonitrile (CH3CN) , in particular, a retention time of High Pressure Liquid Chromatography (HPLC) of about 8-15 minutes, more specifically about 10 minutes and even more specifically about 10.98 min on a C-18 HPLC reverse phase column (Phenomenex, Luna 5p C18 (2) 100 A, 100 x 4.60 mm) using a water: acetonitrile gradient (CH3CN) with a gradient solvent system (0 to 20 min; 90 to 0% aqueous CH3CN, 20 to 24 min; 100% CH3CN, 24 to 27 min; 0 to 90% aqueous CH3CN, 27 to 30 min; 90% aqueous CH3CN) at 0.5 mL / min flow and 210 nm UV detection; (f) a molecular formula of C27H41NO9 that was determined by interpreting the analysis of ESIMS and NMR data; (g) UV absorption bands at about 210 to 450 nm and more particularly at about 234 nm.
[00068] In a more particular embodiment, the compound is a known compound FR901465 that was previously isolated from the culture broth of a Pseudomonassp bacterium. No. 2663 (Nakajima et al. 1996) and has been reported to have anti-cancer activity with the following structure:

[00069] In an even more particular way, compounds from the Family ## STR006a ## can be the compounds defined in xxiv to xxxix These are natural materials or compounds obtained from commercial sources or by chemical synthesis. Natural sources of compounds from the Family ## STR006a ## include, but are not limited to, microorganisms, algae and sponges. In a more particular embodiment, microorganisms that include compounds from the Family ## STR006a ## that can be derived from species such as Pseudomonassp. No. 2663 (compounds xxiv-xxvi) (Nakajima et al., 1996). The synthetic analogues of FR901464 (xxvii-xxxix) that have been synthesized and patented as anticancer compounds (see Koide et al., US Patent Application 2008/0096879 A1).
Compositions
[00070] A substantially pure culture, cell fraction or supernatant and compounds produced by the Burkholderia strain of the present invention, can be formulated in pesticidal compositions.
[00071] The substances defined above can be formulated in any way. Non-limiting examples of formulation include, but are not limited to, emulsified concentrates (EC), wettable powders (WP), soluble liquids (SL), aerosols, ultra low volume concentrate solutions (ULV), soluble powders (SP), microencapsulation, granules dispersed in water, fluid compositions (FL), microemulsions (ME), nanoemulsions (NE), etc. In particular, the concentrate, powders, granules and emulsions can be lyophilized. In any formulation described here, the percentage of the active ingredient is within the range of 0.01% to 99.99%.
[00072] The compositions can be in the form of a liquid, gel or solid. Liquid compositions comprise pesticidal compounds derived from said Burkholderia strain, for example, a strain having the identification characteristics of Burkholderia A396 (Accession No. NRRL B-50319).
[00073] A solid composition can be prepared by suspending a solid carrier in a solution of pesticidal compounds and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65 ° C or below.
[00074] A composition of the invention may comprise gel encapsulated compounds derived from the Burkholderia strain of the present invention. Said gel-encapsulated materials can be prepared by mixing a gel-forming agent (for example, gelatin, cellulose, or lignin) with a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.
[00075] The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients and improvement of fluidity or inhibition of oxidation. In a particular modality, the surfactant is a non-phytotoxic nonionic surfactant that preferably belongs to the EPA List 4B. In another particular modality, the nonionic surfactant is polyoxyethylene monolaurate (20). The concentration of surfactants can vary between 0.1-35% of the total formulation, the preferred range is 5 to 25%. The choice of dispersing and emulsifying agents, such as nonionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of these compositions.
[00076] The composition may further comprise other microorganisms and / or pesticides (for example, nematocide, fungicide, insecticide). The microorganism may include, but is not limited to, an agent derived from Bacillussp., Pseudomonassp., Brevabacillus sp., Lecanicillium sp., Not Ampelomyces sp., Pseudozyma sp., Streptomyces sp, Burkholderia sp, Trichodermasp, Gliocladium sp. Alternatively, the agent may be a natural oil or oil product having fungicidal and / or insecticidal activity (for example, paraffinic oil, tea tree oil, citronella oil, clove oil, cinnamon oil, citrus oil, Rosemary).
[00077] The composition, in particular, may further comprise an insecticide. The insecticide may include, but is not limited to, avermectin, Bacillus thuringiensis, neem and azadiractin oil, spinosades, Chromobacterium subtsugae, eucalyptus extract, entomopathogenic bacteria or fungi such as Beauveria bassiana, and Metarrhizium anisopliae and chemical insecticides including, among other, chemical insecticides including , organophosphate compounds, carbamates, pyrethroids, and neonicotinoids.
[00078] The composition may further comprise a nematicide. The nematicide can include, among others, chemical nematicides such as fenamiphos, aldicarb, oxamyl, carbofuran, natural product nematicide, avermectin, the fungi Paecilomyces lilacinas and Muscodor spp., The bacteria Bacillus firmus and other Bacillus spp. and Pasteuria penetrans.
[00079] The composition may also comprise a biofungicide as an extract of R. sachalinensis (Regalia) or a fungicide. Said fungicides include, but are not limited to, a unique local antifungal agent which may include, but are not limited to, a benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate , external quinone inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine). In yet another embodiment, the antifungal agent is a demethylation inhibitor selected from the group consisting of imidazole (for example, triflumizole), piperazine, pyrimidine and triazole (for example, bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromoconglazazole, cyproconazole, biol diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole, propiconazole).
[00080] The antimicrobial agent may also be a multi-local, non-inorganic chemical fungicide, selected from the group consisting of nitrile (for example, chloronitrile or fludioxonil), quinoxaline, sulfamide, phosphonate, phosphite, dithiocarbamate, chloralquitios, phenylpyridinamine, cyanoacetamide oxime.
[00081] The compositions can be applied using methods known in the art. Specifically, these compositions can be applied to plants or parts of plants. Plants are understood to mean in the present context all plants and plant populations as desired and unwanted wild plants or crop plants (including naturally occurring crop plants). Harvesting plants are plants that can be obtained by conventional plant breeding and optimization methods or biotechnological and genetic engineering methods or by combinations of these methods, including transgenic plants and including plant cultivars protected or not protected by plant breeders' rights . Plant parts should be understood as all plant parts and organs above and below ground, such as bud, leaf, flower and root, examples that can be mentioned being leaves, needles, stems, stems, flowers, fruit bodies, fruits , seeds, roots, tubers and rhizomes. The plant parts still include harvested material, and material of vegetative and germinative propagation, for example, cuttings, tubers, rhizomes, branches and seeds.
[00082] The treatment of plants and plant parts with the compositions defined above can be carried out directly or letting the compositions act in their surroundings, habitat or storage space in, for example, immersion, dispersion, evaporation, spraying, spreading, injection . In case the composition is applied to a seed, the composition can be applied to the seed as one or more coatings prior to planting the seed using one or more coatings using methods known in the art.
[00083] As noted above, the compositions can be herbicidal compositions. The composition may further comprise one or more herbicides. These may include, but are not limited to, a bioherbicide and / or a chemical herbicide. The bioherbicide can be selected from the group consisting of cloves, cinnamon, lemongrass, citrus oil, orange peel oil, tentoxine, cornexistin, AAL-toxin, leptospermone, taxtomine, sarmentine, momylactone B, sorgoleone, ascaulatoxin and ascaulatoxia aglicona . 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, tifensulfuron-methyl, asulam, metribuzin, diclofopmethyl, fluazifop, fenoxaprop-p-ethyl, asulam, oxifluorfen, rimsulfuron, mecoprop, and quinclorac, thiobencarb, clomazone, cihalofop, propanyl, bensulfuron-methyl, penoxsulam, triclopir, triclopyrin, triclopir -sodium, ethyl carfentrazone, sodium bentazone / sodium acifluorfen, glyphosate, glufosinate and orthosulfamuron.
[00084] Herbicidal compositions can be applied in liquid or solid form as pre-emergence or post-emergence formulations.
[00085] For dry pre-emergence formulations, the vehicle granule size is typically 1 to 2 mm (diameter), but the granules can be smaller or larger depending on the required ground cover. The granules can comprise porous or non-porous particles.
[00086] For post-emergence formulations, the components of the formulation used may contain smectite clays, atapulgite clays and similar swelling clays, thickeners such as xanthan gums, arabic gum and other polysaccharide thickeners as well as dispersion stabilizers such as non-ionic surfactants (for example , polyoxyethylene monolaurate (20)). Uses
[00087] The pesticide compositions and compounds derived from the Burkholderia strain defined here can be used as pesticides, particularly as insecticides, nematocides, fungicides and herbicides.
[00088] Specifically, nematodes that can be controlled using the method defined above include, among others, parasitic nematodes such as galls, ring, stinger, spear, cyst, and lesion nematodes, including but not limited to Meloidogyne, Heterodera and Globodera spp; particularly Meloidogyne incognita (gall nematodes), as well as Globodera rostochiensis and globodera pailida (potato cyst nematodes); Heterodera glycines (soy cyst nematodes); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode).
[00089] Phytopathogenic insects controlled by the method of the present invention include, among others, insects of the origin (a) Lepidoptera, for example, Aderis spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp. , Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp. ., Hell ui a undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Maestra brassicae, Manduca sexta, Operophtera spil. spp., Pandemis spp ., Panolis fiam mea, Pectinophora gossypiella, Ftorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopo 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 decemlinea spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga spp., Tribolium and Trogoderma spp .; (c) Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp .; (d) Isoptera, for example, Reticulitermes spp .; (e) Psocoptera, for example, 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., Hercinotnrips 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. and Tniatoma spp .; (j) Homoptera, for example, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp. , Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratori a spp., Pemphigus spp., Pianoc spp., Pseudococcus spp., Psylla spp., Pu Ivin aria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp. (k) Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia politoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. and Vespa spp .; (I) Diptera, for example, Aedes spp., Antherigona soccata, Bi bio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxis spp., Tabanus spp., Tannia spp. and Tipula spp .; (m) Siphonaptera, for example, Ceratophyllus spp. and Xenopsylla cheopis and (n) of the order of Thysanura, for example, Lepisma saccharina. The active ingredients according to the invention can also be used to control cruciferous flea beetles (Phyllotreta spp.), Root larvae (Delia spp.), Cabbage capsule weevils (Ceutorhynchus spp.) And aphids in oilseeds such as canola (rapeseed), mustard seed, and hybrids thereof, and rice and corn.
[00090] In a particular embodiment, the insect may be a member of Spodoptera, more particularly, Spodoptera exigua, Myzus persicae, Plutella xylostella or Euschistus sp.
[00091] Substances and compositions can also be used to modulate emergencies in pre-emergent or post-emergent monocots, junctions or dicots weeds. In a particular embodiment, weeds may be Chenopodium album, Abutilon theophrasti, Helianthus annuus, Ambrosia artemesifolia, Amaranthus retroflexus, Convolvulus arvensis, Brassica kaber, Taraxacum officinale, Solanum nigrum, Malva neglect ,, Setaria lutescens, Brom usum, Brom us , Poa pratensis, Lolium perenne L. var. Pace, Festuca arundinaceae Schreb. var. Aztec II, Anthem II, LS1100, Echinochloa crus-galli, Lactuca sativa. The Burkholderia strain, compounds and compositions defined above can still be used as a fungicide. The targeted fungus can be Fusariumsp., Botrytis 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 more particular embodiment, the bacteria are Xanthomonas.
[00092] The invention will now be described in detail with reference to the following non-limiting examples. EXAMPLES
[00093] The compositions and methods defined above will be better illustrated in the following non-limiting Examples. The examples are illustrative of various modalities only and do not limit the claimed invention with respect to the materials, conditions, weight ratios, process parameters and the like mentioned here. 1. Example 1. Isolation and identification of the microbe 1.1 Isolation of microorganism
[00094] The microbe is isolated using established techniques known in the art from a soil sample under an evergreen tree at Rinnoji Temple, Nikko, Japan. Isolation is done using potato dextrose agar (PDA) using a procedure described in details by Lorch et al., 1995. In this procedure, the soil sample is first diluted in sterile water, after which it is planted on a solid agar medium such as potato dextrose agar (PDA). The plates are grown at 25 ° C for five days, after which the microbial colonies are isolated on separate PDA plates. The isolated bacteria are gram negative, and their colonies are round, opaque cream in color that change to pink and brown-pink in color and mucoid or viscous over time. 1.2. Identification in the microorganism
[00095] The microbe is identified in gene sequencing using universal bacterial primers to amplify the 16S rRNA region. The following protocol is used: Burkholderia sp A396 is grown on potato dextrose agar plates. Growth from a 24-hour old plate is grooved with a sterile loop in DNA extraction buffer. DNA is extracted using the Ultra Clean Microbial DNA extraction kit. The DNA extract is checked for quality / quantity by running 5pl on a 1% agarose gel.
[00096] PCR reactions are adjusted as follows: 2 pl DNA extract, 5 pl PCR buffer, 1 pl dNTPs (10 mM each), 1.25 pl direct primer (27F; 5-AGAGTTTGATCCTGGCTCAG-3 '( SEQ ID NO: 1), 1.25 pl reverse primer (907R; 5'- CCGTCAATTCCTTTGAGTTT-3 '(SEQ ID NO: 2)) and 0.25 pl Taq enzyme. The reaction volume is prepared up to 50 pl using water sterile, free of nuclease.The PCR reaction includes an initial denaturation step at 95 ° C for 10 minutes, followed by 30 cycles of 94 ° C / 30 s, 57 ° C / 20 s, 72 ° C / 30 s, and a final extension step at 72 ° C for 10 minutes.
[00097] The approximate product concentration and size is calculated by running a volume of 5 pl in a 1% agarose gel and comparing the product band on a mass ladder.
[00098] Excess primers, dNTPs and enzyme are removed from the PCR product with the MoBio PCR clean up kit. The PCR product cleaned as directly sequenced using primers 27F (as above), 530F (5'-GTGCCAGCCGCCGCGG-3 '(SEQ ID NO: 3)), 1114F (5-GCAACGAGCGCA ACCC (SEQ ID NO: 4)) and 1525R (5'- AAGGAGGTGWTCCARCC-3 '(SEQ ID NO: 5)), 1100R (5'-GGGTTGCGCTCGTTG-3' (SEQ ID NO: 6)), 519R (5'- GWATTACCGCGGCKGCTG-3 '(SEQ ID NO: 7) ).
[00099] The 16S rRNA gene sequence of the A396 strain is compared to the 16s rRNA gene sequences available from representatives of β-proteobacteria using BLAST. The A395 A396 strain is closely related to members of the Burkholderia cepacia complex, with 99% or greater similarity for several Burkholderia multivorans isolates, Burkholderia vietnamensis, and Burkholderia cepacia. A BLAST survey excluding the B. cepacia complex, showed 98% similarity B. plantarii, B. gladiolie Burkholderia sp. isolated.
[000100] A distant results tree using the neighborhood joining method, showed that A396 is related to Burkholderia multivorans and other isolates of the Burkholderia cepacia complex. Burkholderia plantarii and Burkholderia glumae grouped on a separate branch of the tree.
[000101] The isolated strain of Burkholderia has been shown to contain the following sequences: direct sequence, DNA sequence with 27F primer, 815 nucleotides (SEQ ID NO: 8); reverse sequence, 1453 bp, using primers 1525R, 1100R, 519R (SEQ ID NO: 9); 824 bp reverse sequence using primer 907R (SEQ NO: 10); direct sequence 1152 bp using primer 530F (SEQ ID NO: 11); 1067 bp direct sequence using the 1114F primer (SEQ ID NO: 12); reverse sequence 1223 bp using primer 1525R (SEQ NO: 13); 1216 bp reverse sequence using 1100R primer (SEQ ID NO: 14); 1194 bp reverse sequence using 519R primer (SEQ ID NO: 15). 1.3. Proof that Burkholderia A396 does not belong to the Burkholderia cepacia complex 1.3.1 Molecular Biology work using specific PCR primers
[000102] To confirm the identification of Burkholderia A396 as Burkholderia multivorans, additional sequencing of housekeeping genes is performed. Burkholderia multivorans is a known member of the Burkholderia cepacia complex. Efforts are focused on PCR of recA genes, as described in Mahenthiralingam et al., 2000. The following primers are used: (a) BCR1 and BCR2 defined in Mahenthiralingam et al., 2000 to confirm combination of B. cepacia complex and (b) BCRBM1 and BCRBM2 defined in Mahenthiralingam et al, 2000 to conform the combination to B. multivorans. A PCR reaction generating product for the first primer could confirm that the microbe belongs to the B. cepacia complex. A PCR reaction generating product for the second primer could confirm that the microbe is in fact B. multivorans.
[000103] No PCR product is obtained for one of the primer pairs. The performance of the PCR reaction and primers is tested using Burkholderia multivorans ATCC 17616 (positive control) and Pseudomonas fluorescens (negative control). Strong bands are observed for both B. multivorans using both sets of primers. No band is observed for Pseudomonas fluorescens. The results indicate that A396 is a Burkholderia, but not a member of the B. cepacia complex, and not Burkholderia multivorans. This is further demonstrated in a comparative culture experiment in which both A396 and a type of B. multivorans culture are grown side by side in a shaking culture, and growth is monitored daily using optical density measurements at 600 nm. Under the defined conditions, the new A396 species grew much faster than the type B. multivorans strain (figure 1). 1.3.2 DNA-DNA hybridization
[000104] To confirm that the A396 isolate is a new species of Burkholderia, a DNA-DNA hybridization experiment with Burkholderia multivorans (the closest sequence combines 16S rRNA) is conducted. The biomass for both A396 and B. multivorans is produced in ISP2 broth, grown in 48 hours at 200 rpm / 25 ° C in a Fernbach flask. The biomass is harvested aseptically by centrifugation. The broth is decanted and the cell pellet is resuspended in a 1: 1 solution of water: isopropanol. DNA-DNA hybridization experiments are carried out by 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 hydroxyapatite chromatography as described in Cashion et al., 1977. DNA-DNA hybridization is conducted as described in De Ley et al., 1970 under consideration of the modifications described by Huss et al., 1983 using a Cary 100 Bio UV / VIS spectrophotometer model equipped with a 6x6 multicell heat exchanger Peltier and a temperature controller with in-situ temperature probe (Varian). DSMZ reported% DNA-DNA similarity between A396 and Burkholderia multivorans of 37.4%. The results indicate that the strain of Burkholderia sp A396 does not belong to the species Burkholderia multivorans when the recommendations of a threshold value of 70% DNA-DNA similarity for the definition of bacterial species by the ad hoc committee (Wayne et al., 1987) are considered . 1. 4. Biochemical profile using Biolog GN2 plates
[000105] For the carbon source utilization profile, A396 is grown overnight on potato dextrose agar (PDA). The culture is transferred to BUG agar to produce a culture suitable for Biolog experiments as recommended by the manufacturer (Biolog, Hayward, CA).
[000106] The biochemical profile of microorganism is determined by inoculation on a Biolog GN2 plate and reading the plate after a 24-hour incubation using the MicroLog 4 automated microstation system. The identification of unknown bacteria is attempted by comparing their carbon usage pattern with the negative Microlog 4 Gram database.
[000107] No clear definitive combination is found for the Biolog profile. The closest combinations all had less than 35% similarity with A396: Pseudomonas spinosa (Burkholderia), Burkholderia cepacia, and Burkholderia pseudomallei. The results are shown in Table I. Table 1. Biochemical profile of A396


1.5. Fatty acid composition
[000108] After incubation for 24 hours at 28 ° C, a range of well-grown cells are harvested and fatty acid methyl esters are prepared, separated and identified using the Sherlock Microbial Identification System (MIDI) as described (see Vandamme et al ., 1992). The predominant fatty acids present in Burkholderia A396 are as follows: 16: 0 (24.4%), cycle 17: 0 (7.1%), 16: 0 3- OH (4.4%), 14: 0 (3.6%), 19: 0 ωδc (2.6%) cycle, 18: 0 (1.0%). Added feature 8 (comprising 18: 1 ω7c) and added feature 3 (comprising 16: 1 ω7c and 16: 1 ω6c) corresponded to 26.2% and 20.2% of the total peak area, respectively. Added feature 2 comprising 12: 0 ALDE, 16: 1 iso I, and 14: 0 3-OH) corresponded to 5.8% of the total peak area while added feature 5 comprising 18: 0 ANTE and 18: 2 ω6 , 9c corresponded to 0.4%. Other fatty acids detected in A396 in smaller amounts included: 13: 1 to 12-13 (0.2%), 14: 1 ω5c (0.2%), 15: 0 3-OH (0.13%), 17 : 1 ω7c (0.14%), 17: 0 (0.15%), 16: 0 iso 3-OH (0.2%), 16: 0 2-OH (0.8%), 18: 1 ω7c 11-methyl (0.15%), and 18: 1 2-OH (0.4%).
[000109] A comparison of the fatty acid composition of A396 with those of the microbial strains known in the MIDI database that the fatty acids in the new strain A396 were the most similar with those of Burkholderia cenocepacia. 1.6. Antibiotic Resistance
[000110] The antibiotic susceptibility of Burkholderia A396 is tested using antibiotic discs in Muller-Hinton medium as described in PML # 535 Microbiological data sheets. The results obtained after 72 hours of incubation at 25 ° C are shown in Table 2 below. Table 2: Susceptibility of MBI-206 to various antibiotics. +++ very susceptible, ++ susceptible, - resistant

[000111] The results indicate that the susceptibility to the antibiotic spectrum of Burkholderia A396 is slightly different from the pathogenic strains of the B. cepacia complex. The Burkholderia A396 strain is susceptible to kanamycin, chloramphenicol, ciprofloxacin, piperacillin, imipenem, and a combination of sulfamethoxazole and trimethoprim. As a comparison, Zhou et al., 2007 tested the susceptibility of 2,621 different strains in the B. cepacia complex isolated from cystic fibrosis patients, and demonstrated that only 7% and 5% of all strains were susceptible to imipenem or ciprofloxacin, respectively . They also demonstrated that 85% of all strains are resistant to chloramphenicol (15% susceptible), and 95% resistant (5% susceptible) to the combination of sulfamethoxazole and trimethoprim. Results from Zhou et al., 2007 are similar to those from Pitt et al., 1996 who determined antibiotic resistance among 366 isolates of B. cepaciae reported that most are resistant to ciprofloxacin, cefuroxime, imipenem, chloramphenicol, tetracycline, and sulfamethoxazole . 2. Example 2. Burkholderia sp. as a herbicide 2. 1. Study # 1
[000112] To confirm the activity found in initial herbicide screening, an in vivo study is conducted using the Amberlite 7 XAD resin extract derived from a 5-day-old total cell broth of the new Burkholderia species. The dry crude extract is resuspended in 4% ethanol and 0.2% non-ionic surfactant (glycosperse) at a concentration of 10mg / mL, and further diluted to a concentration of 5.0mg / mL. The two samples are dispersed in 4-week-old climbing plants (Convolvulus arvensis), and the plants are kept under light growth at 25 ° C for 2 weeks, at which point phytotoxicity assessments are performed. In the same study, 2-week-old amaranth plants are dispersed with increased concentrations of the crude extract derived from the bacterial culture. The test concentrations are 1.25, 2.5, 5.0 and 10.0 mg / mL, and the plants are incubated as described above prior to phytotoxicity assessments.
[000113] Results presented in figures 2 (creeper) and 3 (anthill) show the phytotoxic effect of crude Burkholderia extract in different concentrations, and show good herbicidal effect in anthill even at low treatment concentrations. Both extract treatments (5 and 10 mg / mL) result in climbing dwarfism. 2.2. Study # 2
[000114] A new strain of Burkholderia sp. A396 is grown in an undefined mineral medium for 5 days (25 ° C, 200 rpm). The total cell broth is extracted using XAD7 resin. The dry crude extract is resuspended in 4% ethanol and 0.2% non-ionic surfactant at a concentration of 10 mg / mL, and further diluted to a concentration of 5.0, 0.25 and 1.25 mg / mL. All four test solutions are then tested on the following broadleaf and grass weed species listed in Table 3: Table 3. Species tested for Broadleaf and Grass weeds

[000115] A 0.2% glycosperse solution and rounding rate at 6 fl oz per gallon is used as negative and positive controls, respectively.
[000116] All plant species are tested in 4 "x4" plastic pots in three replicates. The untreated control plates are spread with the carrier solution (4% Ethanol, 0.2% glycoside) and the positive control plants rounded up at a rate corresponding to 6 fl. oz / acre. The treated plates are kept in a greenhouse under 12h light / 12h dark conditions. Phytotoxicity data taken 22 days after treatment for species # 1 to 8 and 12 days for species # 9 to 12 are shown in Tables 5 and 6, respectively. The rating scale for both tables is shown in table 4: Table 4. Rating scale
Table 5. Phytotoxicity data for Species # 1 to 8

* developmental delay which resulted in plants approximately half the size of untreated plants. Table 6. Phytotoxicity data for Species # 9 to 12

[000117] Based on the results obtained in these studies, the compounds extracted from fermentation broths of the isolated species of Burkholderia had herbicidal activity against various weed species are tested. Of the twelve species tested, Ançarinha branco and mustard are the most susceptible, followed by mallow and clove. The concentration of the extract as low as 1.25 mg / mL is capable of providing almost complete control of Ançarinha branco and mustard, while the higher concentration is required for mallow and mallow.
[000118] In a separate experiment, using the same design as the one described above, systemic activity is tested. 10 mg / ml of supernatant from the crude extract of Burkholderia sp. A396 is painted on the first real leaves of Tasneira, Mustard, Herb, Grassland, Wheat and Barn. Seedlings are evaluated 7 days after treatment. The symptoms observed include: burning, deformation, bleaching, herbicidal activity is observed on the next leaf above the treated leaf in Tasneira, Mustard and nightshade. No systemic activity is observed in the tested grams. In a second experiment. Five fractions of the same crude extract (10 mg / ml) are evaluated using the same experimental design as described above. Mustard, Wheat and Capim-da-roça seedlings are treated. Seven and 20 days after treatment, symptoms of herbicide activity are observed in Mustard from four of the five fractions (091113B4F6, 091113B4F7, 091113B4F8 and 091113B4F9) using a C-18 column (Fenomenex Sepra C18-E, 50 pm, 65Â) . Symptoms are seen on the leaf following the treated leaf. No systemic activity is observed in the tested grams. 3. Example 3. Burkholderia sp. as an insecticide 3.1. Contact Activity Studies
[000119] The following test is used in the initial screening phase to determine whether compounds derived from a culture of the new Burkholderia species have contact activity against a Lepidopteran pest (larva). It is also used as a tool for fractionation guided by the bioassay to determine the active fractions and peaks derived from the full cell broth extract. The test is conducted in individual 1.25 oz plastic cups using late cabbage caterpillar (Tricoplusia ni) third stage larva or early third stage beet cereal caterpillar (Spodoptera exigua). A 1cm x 1cm piece of solid beet cereal caterpillar diet is placed in the center of each cup along with a larva. A 1DX 1cm aliquot of solid beet cereal caterpillar diet is placed in the center of each cup (Burkholderia A396) is injected into each larva's thorax (dorsal side) using a Hamilton precision syringe. Each treatment is replicated ten times. Water is used as a negative control treatment and malation as a positive control treatment. After injection, each cup is covered with parafilm with an air hole, and the cups are incubated for three days at 26 ° C. Mortality assessments are done daily, starting at 24 after treatment.
[000120] Figures 4 and 5 show the results of tests of contact activity. According to the results, the filter sterilized broth from a culture of Burkholderia sp killed about 40% of all insects within 3 days. The diluted broth (50%) has less activity, resulting in about 10% control in both tested insects. 3.2. Activity Against Larvae Through Feeding
[000121] Direct toxicity through feeding is tested using the diet overlay tests following the 96-well plate assay using microtiter plates with 200 pl of direct toxicity through beet cereal caterpillar in each well. One hundred (100) microliters of each test sample is pipetted into the upper part of the diet (one sample in each well), and the sample is allowed to dry under air flow until the surface is dry. Each sample (filter sterilized through a 0.2 micron filter) is tested in six replicates, and water and a commercial Bt product (B. thuringiensis) are used as negative and positive controls, respectively. A third stage larva of the test insect (cabbage caterpillar - Trichoplusia nr, beet cereal caterpillar - Spodoptera exiqua; Nosema- Plutella xylostella) is placed in each well, and the plate is covered with plastic cover with air holes. The insect plates are incubated at 26 ° C for 6 days with daily mortality assessments.
[000122] Figure 5 represents data from a direct overlapping study with larvae of beet cereal caterpillar (Spodoptera exigua) in an early third stage treated in four different broth concentrations: 1x (100%), 1 / 4x (25 %), 1 / 8x (12.5%), 1 / 16x (6.125%). The data show that the undiluted broth sterilized in a filter is capable of generating 100% control at the end of the 7-day incubation period. Similar control is achieved with a 4-fold dilution of the broth, and at the end of the study, both undiluted and 4-fold broths are comparable to Bt used as a positive control. However, no Bt effect is significantly faster than Burkholderia broths. The effectiveness against cartridge caterpillar larvae is dependent on the broth concentration, and the two lowest broth concentrations (12.5% and 6.125%) provided less control than the two largest. However, the performance of the 12.5% dilution is not much less than the 25% dilution. The 16-fold dilution of broth is clearly not efficient enough, and it only provided partial control (33%) of the caterpillar larva during this 7-day study. The corresponding mortality rates for the same dilution used in cabbage caterpillar and caterpillar larva are slightly higher with 6.125% broth killing 80% and 50% of the larvae, respectively. 3.3. In vitro activity against sucking insects
[000123] Five adult bedbugs (Euschistussp.) Are placed in each 16 oz plastic container lined with a piece of paper towel. A microcentrifuge tube containing 2 ml_ of each test sample (complete sterile filter broth) is covered with a cotton ball and placed on the bottom of the plastic container. A sunflower seed is placed next to the tube as bait. Water and a commercial product with a mixture of pyrethrin and PBO at a recommended rate are used as negative and positive controls, respectively. Each container is closed with a lid, and incubated at 25 ° C for 7 days with daily mortality checks.
[000124] The results are shown below in Table 7 and showed about 80% of sucking insect control (bedbugs) by day 7 in this in vitro system with 50% diluted broth. In this study, the diluted fermentation broth of Burkholderia A396 is more effective in controlling bed bugs than the commercial product used as a positive control. Interestingly, the undiluted broth resulted in inferior insect control, which could be an indication of anti-food properties (food inhibition) of the secondary active metabolic produced by these new species of Burkholderia. Table 7. Effect of A396 on bedbugs
4. Example 4. Insect sucking test in vivo
[000125] The in vivo efficacy of the filtered full cell broth is tested in a plant assay with mustard and green aphid plants (Myzus persicae) with the test insect. Florida Broadleaf mustard plants (Brassicasp.) Approximately one month old are sprayed with two different concentrations (1x and 0.5x) of the full cell broth sterilized by Burkholderia sp. using a Paasche air brush. Water and a commercial avermectin product (Avid) are used as negative and positive controls, respectively. The plants are allowed to dry on the bench, after which they are placed in a 6-cup plastic container with a lid with air holes. Ten aphids at various stages of development are placed in each test plant, and the plants are incubated under growth lamps for 7 days at 25 ° C. Daily assessments for the number of aphids on each plant (summarized in Table 8 below) are made and recorded in a notebook. Table 8. In vivo efficacy of A396 on Green Peach Aphids

[000126] According to the results, both concentrations of the sterile filter broth derived from a culture of a new species of Burkholderia are able to control the population growth of a sucking insect, M. persicae. 5. Example 5. Nematicidal activity 5.1 Study # 1
[000127] To evaluate the effect of sterile culture broth in Burkholderia sp A396 filter on the motility (and subsequent recovery) of juvenile gall nematodes (J2) (Meloidogyne incognita VW6), the following test is conducted on plastic culture plates of 24-well cells:
[000128] A 300 ul aliquot of each test solution (broth sterilized in a 1x or 0.5x filter) is added in appropriate wells, after which fifteen nematodes dispensed in 10 µm aliquot of 300 ul of each test solution (sterile broth filter at 1x or 0.5x) 25 ° C for 24 hours. Water and Avid at 20,000x dilution are used as negative and positive controls, respectively. The effect of each compound on nematode mobility is verified after 24 hours by probing each nematode with a needle, and the proportion of immobile nematodes in each treatment is recorded in a notebook using a% scale. To assess the recovery of mobility in each treatment, a volume of 200 for each treatment, a volume of 200 for the nematode with a needle, and the proportion of immobile nematodes in the plates are again incubated for 24 hours as described above, after which the second mobility assessment is carried out.
[000129] The results presented in figure 6 show the filter sterilized broth in both test concentrations can immobilize the free-living nematodes of juvenile galls. This effect lasts at least 24 hours, which suggests that Burkholderia A396 broth can be used to prevent plants from nematode infections. 5.2 Study # 2 Materials and methods
[000130] Mini Dose Test: Total cell broth of Burkholderia A396 is tested in an oven test in 45 ml pots. Cucumber seeds cv. Toshka are planted directly in pots filled with sandy soil. Ten days later, the pots were each treated with 5 ml of a suspension. The specific quantities are shown in Table 9: Table 9.

[000131] As indicated in Table 9, the pots are inoculated with 3000 eggs of M. incognita. Four replicates were prepared for each treatment and rate. The assay was collected fourteen days after application and inoculation of the assay. Root antlers were evaluated according to the Zeck gall index (Zeck, 1971). Phytotoxicity was measured as a reduction of root antlers compared to the control. The results are shown in figures 9 and 10.
[000132] In the Mini Potion Test no. 1 (see figure 9), the treatment activity was very high and a reduction of almost 100% observed when applied at a concentration of 100 ml / L Burkholderia A396. Fostiazato performed as usual (100% control at 20 ppm). [000133] In the Minipoção Test no. 2 (see figure 10) 100% reduction of root antlers was obtained at the highest concentration of 100 ml / L dropping to approximately 50% at 1.5 ml / L.Postiazate performed as usual (100% control at 20 ppm) . 5.3 Study # 3
[000134] To demonstrate the nematicidal activity of Burkholderia A396, a greenhouse study on cucumber (Cucumis sativus) is performed using a full cell broth of Burkholderia A396 as the test product to control gall nematodes (Meloidogyne incognita). of cucumber per pot is planted in soil and grown in a greenhouse under artificial light at 28 ° C. Each pot with a plant is treated with an aliquot (about 80 ml) of undiluted test product or a test product diluted to 5% with water. Each treatment with Burkholderia A396 as well as a treatment with positive control with Temik (at a label rate) and a negative control without additions consisted of five replicates. The plants are grown in a greenhouse for 60 days, after which each plant has been harvested and evaluated for fresh weights and roots. The number of nematode eggs in each pot was recorded and a parameter indicating the number of eggs per gram of root mass was calculated. Statistical analysis (ANOVA) is performed and the statistical differences between treatment averages at p <0.1 were calculated. The data presented in Table 10 below show that even though not statistically different from the untreated control, pots treated with A396 full cell broth contained fewer nematode eggs than pots from untreated controls. The effect calculated as number of eggs per root mass is clearest when the undiluted broth is used as a treatment. Table 10.
[000135] Effect of total cell broth of A396 on the weight of bud and root of cucumber, total number of eggs of M. incognit by pot and the number of eggs per gram of root mass.
6. Examples 6. Isolation of Templazole A and B Methods and materials
[000136] The following procedure is used for the purification of Templazole A and B extracted from the cell culture of Burkholderia sp (see figure 7):
[000137] The culture broth derived from the fermentation of 10 L of Burkholderia (A396) in Hy soy growth medium is extracted with Amberlite XAD-7 resin (Asolkar et al., 2006) by stirring the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell mass are collected by filtration through a swab and washed with Dl water to remove salts. The resin, cell mass and dough are then immersed for 2 h in acetone after which the acetone is filtered and dried in vacuo using a rotary evaporator to generate the crude extract. The crude extract is then fractionated using C18 reverse phase liquid chromatography in vacuum (H2O / CH3OH; gradient 90:20 to 0: 100%) to generate 10 fractions. These fractions are then concentrated to dryness using a rotary evaporator and the resulting dry residues are screened for biological activity using 96-plate lettuce seeding test plates. The active fractions are then subjected to reverse phase HPLC (Spectra P4000 system (Thermo Scientific)) to generate pure compounds, which are then screened in the aforementioned bioassays to locate / identify the active compounds. To confirm the identity of the compound, additional spectroscopy data such as LC / MS and NMR are recorded.
[000138] The active fraction 4 is further purified using HPLC column C-18 (Phenomenex, Luna 10u C18 (2) 100 A, 250 x 30), water: acetonitrile gradient solvent system (0 to 10 min; 80% aqueous CH3CN , 10 to 25 min; 80 - 65% aqueous CH3CN, 25 to 50 min; 65 to 50% aqueous CH3CN, 50 to 60 min; 50 to 70% CH3CN, 60 to 80 min; 70 to 0% aqueous CH3CN, 80 to 85 min; 0 to 20% aqueous CH3CN) in flow 8 mL / min and UV detection of 210 nm, to generate templazole B, retention time 46.65 min. The other active fraction 6 is further purified using HPLC column C-18 (Phenomenex, Luna 10u C18 (2) 100 A, 250 x 30), water: acetonitrile gradient solvent system (0 to 10 min; 80% aqueous CH3CN, 10 at 25 min; 80 to 60% aqueous CH3CN, 25 to 50 min; 60 to 40% aqueous CH3CN, 50 to 60 min; 40% CH3CN, 60 to 80 min; 40 to 0% aqueous CH3CN, 80 to 85 min; 0 20% aqueous CH3CN) in a flow rate of 8 mL / min and 210 nm UV detection, to generate templazole A, retention time 70.82 min.
[000139] Mass spectroscopy analysis of pure compounds is performed on a Thermo Finnigan LCQ Deca XP Plus (ESI) electrospray instrument using both positive and negative ionization modes in a full scan mode (m / z 100-1500 Da) on a DECA XPplus LCQ mass spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) instrument with Finnigan Surveyor PDA plus detector, self-sampling plus, MS pump and a 4.6 mm x 100 mm Luna C18 5 pm column (Fenomenex). The solvent system consists of water (solvent A) and acetonitrile (solvent B). The mobile phase starts at 10% solvent B and is linearly increased to 100% solvent B for 20 min and then maintained for 4 min, and finally returning to 10% solvent B for 3 min and maintained for 3 min, the flow is 0, 5 ml / min. The injection volume was 10 pL and the samples are kept at room temperature in an autosampler. The compounds are analyzed by LC-MS using LC and reverse phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the following conditions: Flow of nitrogen gas was fixed at 30 and 15 arb for the casing and flow of aux / drag gas, respectively. Electrospray ionization was performed with a spray voltage set to 5000 V and a capillary voltage to 35.0 V. The capillary temperature was adjusted to 400 ° C. The data were analyzed using Xcalibur software. The active compound templazole A has a molecular mass of 298 and showed m / z ion in 297.34 in negative ionization mode. The LC-MS chromatogram for templazole B suggests a molecular mass of 258 and showed m / z ion in 257.74 negative ionization mode.
[000140] 1H, 13C and 2D NMR spectra were measured on a 500 MHz & 600 MHz Bruker gradient field spectrometer. The reference is adjusted to the internal standard tetramethylsilane (TMS, 0.00 ppm).
[000141] To elucidate the structure of templazole A, the purified compound with a molecular weight 298 is further analyzed using a 500 MHz NMR instrument, and has 1H NMR δ values at 8.44, 8.74, 8.19, 7, 47, 7.31, 3.98, 2.82, 2.33, 1.08 and has 13C NMR δ values of 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 suggests the presence of indole and oxazo rings. The molecular formula, C17H18N2O3, was determined by interpreting data from 1H, 13C NMR and HRESI MS m / z 299.1396 (M + H) + (Calculated for C17H19N2O3, 299.1397), which implies a high degree of unsaturation shown for 10 equivalent double bonds. The 13C NMR spectrum revealed signals for all 17 carbons, including two methyls, a methoxy, a methylene carbon, an aliphatic methyl, a carbonyl ester, and eleven aromatic carbons. The presence of 3'-substituted indole was revealed from 1H-1H COZY and HMBC spectral data. 1H-1H COZY and HMBC also indicated the presence of a methyl ester group of carboxylic acid and a side chain - CH2-CH- (CH3) 2. From the detailed analysis of 1H-1H COZY, 13C, and HMBC data it was derived that the compound contained an oxazole core. From 2D analysis it was demonstrated that the iso-butyl side chain was joined at the C-2 position, a methyl ester of carboxylic acid at the C-4 position and the indole unit at the C-5 position to generate templazole A.
[000142] The second herbicidally active compound, templazole B, with a molecular weight of 258 is further analyzed using a 500 MHz NMR instrument, and has 1H NMR δ values at 7.08, 7.06, 6.75, 3.75, 2.56, 2.15, 0.93, 0.93 and 13C NMR values of δ 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 molecular formula is designated as C15H18N2O2, which is determined by interpreting 1H, 13C NMR and mass data. The 13C NMR spectrum revealed signals for all 15 carbons, including two methyl, two methylene carbons, an aliphatic methyl, a carbonyl amide, and nine aromatic carbons. The general nature of a structure was deduced from 1H and 13C NMR spectra that showed a para-substituted aromatic ring [δ 7.08 (2H, d, J = 8.8 Hz), 6.75 (2H, d, J = 8.8 Hz), and 132.7, 129.5, 115.2, 127.3, 115.2, 129.5], The 1H NMR spectrum of this structure along with the 1H-1H COZY and HSQC spectra , showed characteristic signs for an isobutyl fraction [δ 0.93 (6H, d, J = 6.9 Hz), 2.15 (1H, sept, J = 6.9 Hz), 2.57 (2H, d, J = 6.9 Hz). In addition, an olefinic / aromatic proton (δ 7.06, s), and a carbonyl carbon group (δ 158.9) were also found in the 1H and 13C NMR spectrum. On inspection of the HMBC spectra, the HT signal in the isobutyl fraction correlated with the olefinic carbon (C-2, δ 156.3), and the H-4 olefinic proton correlated with (C-5, δ 155.5; C-2 , 156.3 and C-1 ", 41.2). The methylene signal at δ 3.75 correlates with C-5, C-4 as well as C-2" of the para-substituted aromatic fraction. All of these observed correlations suggested the connectivity between isobutyl, and the para-substituted benzyl fractions for the skeleton of the structure as shown. In addition, the carboxamide group is designated in the para position of the benzyl fraction based on the HMBC correlation of the aromatic proton in the H-4 "and H-6" position. Thus, based on the above data, the structure was designated as templazole B. 7. Example 7. FR90128 insulation
[000143] The complete fermentation cell broth of Burkholderia sp. in an undefined growth medium with Amberlite XAD-7 resin (Asolkar et al., 2006) by stirring the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell mass are collected by filtration through a swab and washed with Dl water to remove salts. The resin, cell mass and dough are then immersed for 2 h in acetone after which the acetone is filtered and dried in vacuo using a rotary evaporator to generate the crude extract. The crude extract is then fractionated using C18 reverse phase liquid chromatography in vacuum (H2O / CH3OH; gradient 90:20 to 0: 100%) to generate 10 fractions. These fractions are then concentrated to dryness using a rotary evaporator and the resulting dry residues are screened for biological activity using insert bioassay as well as herbicidal bioassay. The active fractions are then subjected to reverse / normal phase HPLC (Spectra P4000 system (Thermo Scientific)) to generate pure compounds, which are then screened in the herbicide, insecticide and nematicide bioassays described below to locate / identify the active compounds. To confirm the identity of the compound, additional spectroscopy data such as LC / MS and NMR are recorded.
[000144] Mass spectroscopy analysis of active peaks is performed on a Thermo Finnigan LCQ Deca XP Plus (ESI) electrospray instrument using both positive and negative ionization modes in a full scan mode (m / z 100 to 1500 Da) on a DECA XPplus LCQ mass spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) instrument with Finnigan Surveyor PDA plus detector, self-sampling plus, MS pump and a 4.6 mm x 100 mm Luna C18 5 pm column (Fenomenex). The solvent system consists of water (solvent A) and acetonitrile (solvent B). The mobile phase starts at 10% solvent B and is linearly increased to 100% solvent B for 20 min and then maintained for 4 min, and finally returned to 10% solvent B for 3 min and maintained for 3 min. The flow is 0.5 ml / min. The injection volume is 10 pL and the samples are kept at room temperature in an autosampler. The compounds are analyzed by LC-MS using LC and reverse phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the following conditions: Flow of nitrogen gas was fixed at 30 and 15 arb for the casing and flow of aux / drag gas, respectively. Electrospray ionization is carried out with a spray voltage set to 5000 V and a capillary voltage to 35.0 V. The capillary temperature has been adjusted to 400 ° C. The data is analyzed using Xcalibur software. Based on the LC-MS analysis, the active insecticidal compound of fraction 5 has a molecular mass of 540 in negative ionization mode.
[000145] For clarification of the structure, the purified insecticidal compound of fraction 5 with molecular weight 540 is further analyzed using a 500 MHz NMR instrument and has 1H NMR values in 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, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12, 51. NMR data indicates that the compound contains amino, ester, carboxylic acid, aliphatic methyl, ethyl, methylene, oxymethylene, methyl, oxymethyl and sulfur groups. Detailed analysis of 1D and 2D NMR confirms the structure for the compound as FR90128 as a known compound. 8. Example 8. Herbicidal activity of FR90128
[000146] The herbicidal activity of the active compound FR90128 (MW 540) is tested in a laboratory trial using one-week-old barn grass seedlings (Echinochloa crus-gallí) on a 96-well plate platform. A grass seedling was placed in each well having 99 microliters of Dl water. A one microliter aliquot of the pure compound in ethanol (10 mg / mL) is added to each well, and the plate is closed with a lid. One microliter of ethanol in 99 microliters of water is used as a negative control. The treatments were carried out in eight replicates, and the closed plate is incubated in a greenhouse under artificial light (12 hours of light / dark cycle). After five days, the results are read. Grass seedlings in all eight wells that received the active compost are killed without green tissue on the left, while seedlings in the negative control wells have been actively grown. 9. Example 9. FR90128 Insecticidal Activity
[000147] The insecticidal activity of the active compound FR90128 (MW 540) is tested in a laboratory test using a contact bioassay system. The compound is dissolved in 100% ethanol in concentrations of 0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 pg / pL. individual beet cereal caterpillar in the early third stage, Spodoptera exigua, the larvae are placed in (1.25 oz) plastic cups with a 1 cm2 piece of artificial diet (Bio-Serv). A Hamilton micropipette is used to apply 1 pL of compound to each larva's chest. The cups are covered with stretched parafilm and a single hole is cut in the parafilm for aeration. Ten larvae per concentration are treated. The assay is incubated at 25 ° C, 12h light / 12h dark. The larvae are marked at 48 and 72 hours after application. Probit analysis is performed to evaluate the LCso value that is found for the compound (MW 540) as 0.213. 10. Example 10. Isolation of Templamide A, B, FR901465 and FR90128 Methods and materials
[000148] The following procedure is used for the purification of compounds extracted from the cell culture of Burkholderia sp (see figure 7):
[000149] The culture broth derived from the fermentation of 10 L of Burkholderia (A396) in Hy soy growth medium is extracted with Amberlite XAD-7 resin (Asolkar et al., 2006) by stirring the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell mass are collected by filtration through a swab and washed with Dl water to remove salts. The resin, cell mass and dough are then immersed for 2 h in acetone after which the acetone is filtered and dried in vacuo using a rotary evaporator to generate the crude extract. The crude extract is then fractionated using C18 reverse phase liquid chromatography in vacuum (H2O / CH3OH; gradient 90:20 to 0: 100%) to generate 10 fractions. These fractions are then concentrated to dryness using a rotary evaporator and the resulting dry residues are screened for biological activity using 96-plate lettuce seeding test plates (herbicide) and beet cartridge caterpillar test in the third stage (insecticide). The active fractions are then repeatedly subjected to reverse phase HPLC (Spectra P4000 system (Thermo Scientific)) to generate pure compounds, which are then screened in the aforementioned bioassays to locate / identify the active compounds. To confirm the identity of the compound, additional spectroscopy data such as LC / MS and HRMS, NMR are recorded.
[000150] The active fraction 5 is further purified using HPLC column C-18 (Phenomenex, Luna 10u C18 (2) 100 A, 250 x 30), solvent gradient system water: acetonitrile (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% aqueous CH3CN, 60-80 min; 70 - 0% aqueous CH3CN, 80 -85 min; 0 - 20% aqueous CH3CN) in 8 mL / min flow and 210 nm UV detection, to generate templamide A, retention time 55.64 min and FR901465, retention time 63.59 min and FR90128, retention time 66.65 min respectively. The other active fraction 6 is further purified using HPLC column C-18 (Phenomenex, Luna 10u C18 (2) 100 A, 250 x 30), water: acetonitrile gradient solvent system (0 to 10 min; 70 to 60% aqueous CH3CN , 10 to 20 min; 60 to 40% aqueous CH3CN, 20 to 50 min; 40 to 15% aqueous CH3CN, 50 to 75 min; 15 to 0% CH3CN, 75 to 85 min; 0 to 70% aqueous CH3CN) in flow 8 mL / min and 210 nm UV detection, to generate templamide B, retention time 38.55 min.
[000151] Mass spectroscopy analysis of pure compounds is performed on a Thermo Finnigan LCQ Deca XP Plus (ESI) electrospray instrument using both positive and negative ionization modes in a full scan mode (m / z 100 to 1500 Da) on a DECA XPpIus LCQ mass spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) instrument with Finnigan Surveyor PDA plus detector, self-sampling plus, MS pump and a 4.6 mm x 100 mm Luna C18 5 pm column (Fenomenex). The solvent system consists of water (solvent A) and acetonitrile (solvent B). The mobile phase starts at 10% solvent B and is linearly increased to 100% solvent B for 20 min and then maintained for 4 min, and finally returned to 10% solvent B for 3 min and maintained for 3 min. The flow is 0.5 ml / min. The injection volume is 10 pL and the samples are kept at room temperature in an autosampler. The compounds are analyzed by LC-MS using LC and reverse phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the following conditions: Flow of nitrogen gas was fixed at 30 and 15 arb for the casing and flow of aux / drag gas, respectively. Electrospray ionization is carried out with a spray voltage set to 5000 V and a capillary voltage to 45.0 V. The capillary temperature has been adjusted to 300 ° C. The data is analyzed using Xcalibur software. The active compound templamide A has a molecular mass of 555 based on the peak m / z at 556.41 [M + H] + and 578.34 [M + Na] + in a positive ionization mode. LC-MS analysis in positive ionization mode for templamide B suggests a molecular mass of 537 based on m / z ions at 538.47 [M + H] + and 560.65 [M + Na] +. The molecular weight for compounds FR901465 and FR90128 are designated as 523 and 540 respectively based on LCMS analysis.
[000152] 1H, 13C and 2D NMR spectra are measured on a 600 MHz Bruker gradient field spectrometer. The reference is adjusted to the internal standard tetramethylsilane (TMS, 0.00 ppm).
[000153] To elucidate the structure of templamide A, the purified compound with molecular weight 555 is further analyzed using a 600 MHz NMR instrument, and has 1H NMR δ 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 δ 173.92, 166.06, 145.06, 138 , 76, 135.71, 129.99, 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. The 13C NMR spectrum shows 28 discrete carbon signals that are assigned to six methyls, four methylene carbons, and thirteen methods including five sp2, four quaternary carbons. The molecular formula, C28H45NO10, is determined by interpreting 1H, 13C NMR and HRESI MS data. Detailed analysis of 1H-1H COZY, HMBC and HMQC spectral data reveals the following substructures (I - IV) and two isolated methylene & methyl singlet groups. These substructures are subsequently connected using the main HMBC correlations to generate the planar structure for the compound, which has not yet been reported in the literature and designated as templamide A. This polyketide molecule contains two tetrahydropyranose rings, and a conjugated amide.

[000154] Substructures l-IV designated by analysis of 1D and 2D NMR spectroscopic data.
[000155] The analysis of (+) for 0 second herbicidal compound, shows m / z ions in 538.47 [M + H] + and 560.65 [M + Na] + corresponding to the molecular weight of 537. The molecular formula of C28H43NO9 is determined by interpretation of ESIMS and NMR data analysis. The 1H and 13C NMR of this compound is similar to that of templamide A except that a new-CH2- isolate appears in the unbound methylene group in templamide A. The small 4.3 Hz germline coupling constant is characteristic of the presence of a group methylene epoxide. The presence of this epoxide is further confirmed by the 13C NMR shift from 60.98 in templamide A to 41.07 in the compound with MW 537. The differences in molecular formulas between these two compounds are reasonably explained by elimination of the water molecule followed by epoxide formation. Thus, based on NMR and MS analysis, the structure for the new compound was assigned and was designated as templamide B.
[000156] For clarification of the structure, the purified compound of fraction 5 with molecular weight 523 is further analyzed using a 600 MHz NMR instrument, and has values 1H NMR δ 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 δ values at 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. Detailed analysis of the 1H and 13C NMR of the compound suggested that this compound was almost similar to the compound templamide B; the only difference was in the ester side chain; an acetate fraction was present in place of a propionate fraction in the side chain. Detailed analysis of 1D and 2D NMR confirms the structure for the compound as FR901465 as a known compound.
[000157] Based on the LC-MS analysis, the other active compound of fraction 5 has a molecular mass of 540 in negative ionization mode. To elucidate the structure, the purified compound of fraction 5 with molecular weight 540 is further analyzed using a 500 MHz NMR instrument, and has 1H NMR δ values of 6.22, 5.81, 5.69, 5.65, 4.64 , 4.31, 3.93, 3.22, 3.21, 3.15, 3.10, 2.69, 2.62, 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, 30.36, 29.26, 18.59, 18.38, 18.09, 17.93, 12, 51. NMR data indicates that the compound contains amino, ester, carboxylic acid, aliphatic methyl, ethyl, methylene, oxymethylene, methyl, oxymethyl and sulfur groups. Detailed analysis of 1D and 2D NMR confirms the structure for the compound as FR90128 as a known compound. 11. Example 11. Herbicidal activity of Templamide A, Templamide B, FR901465e FR90128
[000158] The herbicidal activity of templamide A, B, FR901465 and FR90128 are tested in a laboratory trial using week-old barn grass seedlings (Echinochloa crus-gallí) and lettuce (Lactuca sativa L.) on a platform 96 plates. A seedling is placed in each well having 99 microliters of Dl water. In each well, a microliter aliquot of the pure compound in ethanol (10 mg / mL) is added to each well, and the plate is closed with a lid. One microliter of ethanol in 99 microliters of water is used as a negative control. The treatments are done in eight replicates, and the closed plate is incubated in a greenhouse under artificial light (12 hours of light / dark cycle). After five days, the results are read. Grass seedlings in all eight wells that received the active compost are killed without green tissue on the left, while seedlings in the negative control wells have been actively grown. The herbicidal activity of templamide A against lettuce seedlings is slightly less than for grass. On the other hand, templamide B provides better control (100%) of lettuce seedlings (used as a model system for broadleaf weeds) than templamide A (Table 11). Table 11: Herbicide bioassay data for Templamide A, B, FR901465 and FR90128
110 pg / mL concentration per well. 12. Examples 12. Insecticidal activity of active compounds
[000159] The insecticidal activities of templamide A, B, FR901465 and FR90128 are tested in a laboratory trial using a 96-well diet overlay trial with first stage beet cartridge caterpillar larvae using 200 pl microtiter plates of solid artificial beet cartridge caterpillar diet in each well. One hundred (100) microliters of each test sample is pipetted into the upper part of the diet (one sample in each well), and the sample is allowed to dry under air flow until the surface is dry. Each sample was tested in six replicates, and water and a commercial Bt product (B. thuringiensis) are used as negative and positive controls, respectively. A first stage larva of the test insect (beet cereal caterpillar - Spodoptera exiqua) was placed in each well, and the plate was covered with plastic cover with air holes. The insect plates were incubated at 26 ° C for 6 days with daily mortality assessments. Based on the results presented in Table 12, templamide A and B results in 40% and 80% mortality, respectively. Table 12:
[000160] Insecticide Bioassay Data for Templamide A, B, FR901465 and FR90128 against first stage beet cereal caterpillar (Spodoptera exigua).
110 pg / mL concentration per well. Example 11. FR90128 Fungicidal Activity (MW 540)
[000161] FR90128 (MW 540) fungicidal activity against three plant pathogenic fungi (Botrytis cinerea, Phytophtora sp., Monilinia fructicolá) is tested in an in vitro assay of PDA plate (potato dextrose agar). The plates are inoculated with the fungi using a buffer method. After the fungi have established and begin to grow in the growth medium, eight sterile paper discs are placed on each plate about 1 cm from the edge in a circle. Ten microliters of ethanol solution having 20, 15, 10, 7.5, 5, 2.5 1.25 mg FR90128 / mL is added on filter paper discs, and the solution is allowed to evaporate. A disk soaked with 10 pl of pure ethanol is used as a negative control. The test is carried out with three replicates. The plates are incubated at room temperature for 5 days, after which the fungicidal activity is recorded by measuring the zone of inhibition around each disc of filter paper corresponding to the different concentrations of FR90128. According to the results, FR90128 has no effect on the growth of Monilinia, but it is effective in controlling the growth of hyphae of both Botrytis and Phytophtora. There appears to be a clear dose-response relationship in inhibition with threshold concentrations of 10 mg / mL and 1.25 mg / mL for Botrytis and Phytophtora, respectively (figure 8). BIOLOGICAL MATERL DEPOSIT
[000162] The following biological material was deposited under the Budapest Treaty with the Agriculture Research Culture Collection (NRRL), 1815 N. University Street, Peoria, Illinois 61604 USA, and received the following numbers:
Burkholderia sp. A396 NRRL B-50319
[000163] The strain was deposited under conditions that guarantee that access to the crop will be available pending this patent application to someone determined by the Patent and Trademark Commissioner entitled to it in accordance with 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by international patent laws in countries where the counterparties to the subject order, or their descendants, are deposited. However, it must be understood that the availability of a deposit does not constitute a license to practice the object object by way of derogation from patent rights granted by government action.
[000164] The invention described and the claims herein are not limited in scope by the specific aspects disclosed herein, since these aspects are intended to be illustrations of the various aspects of the invention. Any equivalent aspect is intended to be within the scope of this invention. In fact, various modifications of the invention in addition to those shown and described here will become apparent to those skilled in the art of the above description. These modifications are also intended to fall within the scope of the attached claims. In the event of a conflict, the present disclosure including definitions will prevail.
[000165] Several references are cited here, whose descriptions are incorporated by reference in their entirety. Literature cited:
[000166] Anderson, et al. "The structure of thiostrepton," Nature 225: 233-235. 1970.
[000167] Andra, "Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarir. Immune cell stimulation and biophysical characterization." Biol. Chem. 387: 301-310. 2006.
[000168] Arena, et al. "The mechanism of action of avermectins in Caenorhabditis elegans - correlation between activation of glutamatesensitive chloride current, membrane binding and biological activity." J Parasitol. 81: 286-294. 1995.
[000169] Asolkar, et al., "Weakly cytotoxic polyketides from a marine-derived Actinomycete of the genus Streptomyces strain CNQ-085." J. Nat. Prod. 69: 1756-1759. 2006.
[000170] Burkhead, et al., "Pyrrolnitrin production by biological control agent Pseudomonas cepacia B37w in culture and in colonized wounds of potatoes." Appl. Environ. Microbiol. 60: 2031-2039. 1994.
[000171] Burkholder, W. H "Sour skin, a bacterial rot of onion bulbs." Phytopathology 40: 115-117. 1950.
[000172] Caballero-Mellado et al., "Burkholderia unamae sp. Nov., An N2-fixing rhizospheric and endophytic species." Int. J. Syst. Evol. Microbiol. 54: 1165-1172. 2004.
[000173] Cashion et al. "Rapid method for base ratio determination of bacterial DNA." Anal. Biochem. 81: 461-466. 1977.
[000174] Casida, et al., US Patent 6,689,357.
[000175] Chen et al., "Burkholderia nodosa sp. Nov., Isolated from root nodules of the woody Brazilian vegetables Mimosa bimucronata and Mimosa scabrella" Int. J. Syst. Evol. Microbiol. 57: 1055-1059. 2007.
[000176] Cheng, A. C. and Currie, B. J. "Melioidosis: epidemiology, pathophysiology, and management." Clin. Microbiol. 18: 383-416. 2005.
[000177] Coenye, T. and P. Vandamme, P. "Diversity and significance of Burkholderia species occupying diverse ecological niches." Environ. Microbiol. 5: 719-729. 2003.
[000178] Compant, et al. "Diversity and occurence of Burkholderia spp. In the natural environment." FEMS Microbiol. Rev. 32: 607-626. 2008.
[000179] De Ley et al. "The quantitative measurement of DNA hybridization from renaturation rates." Eur. J. Biochem. 12: 133-142. 1970.
[000180] Duke et al. "Natural products as sources for herbicides: current status and future trends." Weed Res 40: 99-111.2000.
[000181] Gerwick et al., US Patent 7,393,812.
[000182] Gottlieb et al., US Patent 4,808,207.
[000183] Gouge et al., US Patent Application Publication 2003/0082147.
[000184] Guella et al. "Almazole C, a new indole alkaloid bearing an unusually 2,5-disubstituted oxazole moiety and its putative biogenetic precursors, from a Senegalese Delesseriacean sea weed." Helv. Chim. Minutes 77: 1999-2006. 1994.
[000185] Guella et al. "Isolation, synthesis and photochemical properties of almazolone, a new indole alkaloid from a red alga from Senegal." Tetrahedron. 62: 1165-1170. 2006.
[000186] Henderson, P.J. and Lardy H. A. "Bongkrekic acid. An inhibitor of the adenine nucleotide translocase of mitochondria." J. Biol. Chem. 245: 1319-1326. 1970.
[000187] Hirota et al. "Isolation of indolmycin and its derivatives as antagonists of L-tryptophan." Agri. Biol Chem. 42: 147-151. 1978.
[000188] Hu, F.-P. and Young, J. M. "Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herbaspirillum, Ralstonia, and Xanthomonas spp." J. Appl. Microbiol. 84: 263-271. 1998.
[000189] Huss et al. "Studies of the spectrophotometric determination of DNA hybridization from renaturation rates." System. Appl. Microbiol. 4: 184-192. 1983.
[000190] Jansiewicz, W. J. and Roitman J. "Biological control of blue mold and gray mold on apple and pear with Pseudomonas cepacia." Phytopathology 78: 1697-1700. 1988.
[000191] Jeddeloh et al., WO2001 / 055398.
[000192] Jansen et al. "Thiangazole: a novel inhibitor of HIV-1 from Polyangium Spec." Liebigs Ann. Chem. 4: 357-3359. 1992.
[000193] Jeong et al. "Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops." Plant Disease 87: 890-895. 2003.
[000194] Knudsen, G. R. and Spurr, J. "Field persistence and efficacy of five bacterial preparations for control of peanut leaf spot." Plant Disease 71: 442-445. 1987.
[000195] Koga-Ban et al. "cDNA sequences of three kinds of betatubulins from rice." DNA Research 2: 21-26. 1995.
[000196] Koide et al., US Patent Application Publication 2008/0096879.
[000197] Koyama et al. "Isolation, characterization, and synthesis of pimprinine, pimrinrthine, and pimprinaphine, metabolites of Streptoverticillium olivoreticuli." Agri. Biol. Chem. 45: 1285-1287. 1981.
[000198] Krieg et al. "Bacillus thuringiensis var. Tenebrionis: Ein neuer, gegenuber Larven von Coleopteren wirksamer Pathotyp." Z. Angew. Entomol._96: 500-508. 1983.
[000199] Kunze et al. "Thiangazole, a new thiazoline antibiotic from Polyangium sp (Myxobacteria Production, antimicrobial activity and mechanism of action." J. Antibiot., 46: 1752-1755. 1993.
[000200] Leahy et al. "Comparison of factors influencing trichlorethylene degradation by toluene-oxidizing bacteria." Appl. Environ. Microbiol. 62: 825-833. 1996.
[000201] Lessie et al. "Genomic complexity and plasticity of Burkholderia cepacia." FEMS Microbiol. Lett. 144: 117 to 128. 1996.
[000202] Lindquist, N. et al. "Isolation and structure determination of diazonamides A and B, unusual cytotoxic metabolites from the marine ascidian Diazona chinensis." J. Am Chem. Soc. 113: 2303-2304. 1991.
[000203] Lorch, H et al. "Basic methods for counting microoganisms in soil and water. In Methods in applied soil microbiology and biochemistry. K. Alef and P. Nannipieri. Eds. San Diego, CA, Academic Press: pp. 146-161. 1995.
[000204] Ludovic et al. "Burkholderia diveristy and versatility: An inventory of the extracellular products." J. Microbiol. Biotechnol. 17: 1407-1429. 2007.
[000205] Lydon, J. and Duke, S. "Inhibitors of glutamine biosynthesis." in Plant amino acids: Biochemistry and Biotechnology. B. Singh., Ed. New York, USA, Marcel Decker, pp. 445-464. 1999.
[000206] Mahenthiralingam et al. "DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III." J.Clin. Microbiol. 38: 3165-3173. 2000.
[000207] Ming, L.-J. and Epperson. "Metal binding and structureactivity relationship of the metalloantibiotic peptide bacitracin." Biochemistry 91: 46-58. 2002.
[000208] Morita et al. "Biological activity of tropolone." Biol. Pharm. Bull. 26: 1487-1490. 2003.
[000209] Nagamatsu, T. "Syntheses, transformation, and biological activities of 7-azapteridine antibiotics: toxoflavin, fervenulin, reumycin, and their analogs". Recent Res. Devel. Org. Bioorg. Chem. 4: 97-121. 2001.
[000210] Naik et al., "Pimprine, an extracellular alkaloid produced by Streptomyces CDRIL-312: fermentation, isolation and pharmacological activity", J. Biotech. 88: 1-10. 2001.
[000211] Nakajima et al., "Antitumor Substances, FR901463, FR901464 and FR901465. I. Taxonomy, Fermentation, Isolation, Physico-chemical Properties and Biological Activities." J. Antibiot. 49: 1196-1203. 1996.
[000212] Nakajima et al., US Patent 5,545,542.
[000213] Nakajima et al., "Hydantocidin: a new compound with herbicidal activity." J Antibiot. 44: 293-300. 1991.
[000214] N'Diaye, I. et al., "Almazole A and amazole B, unusual marine alkaloids of an unidentified red seaweed of the family Delesseriaceae from the coasts of Senegal." Tet Lett. 35: 4827-4830. 1994.
[000215] N'Diaye, I. et al., "Almazole D, a new type of antibacterial 2,5-disubstituted oxazolic dipeptide from a red alga from the coast of Senegal." Tet Lett. 37: 3049-3050. 1996.
[000216] Nierman et al., "Structural flexibility in the Burkholderia mallei genome." Proc. Natl. Acad. Sci.JJSA 101: 14246-14251.2004.
[000217] Okazaki et al., "Rhizobial strategies to enhance symbiotic interaction: Rhizobitoxine and 1-aminocyclopropane-1-carboxylate deaminase." Environ microbes. 19: 99-111.2004.
[000218] Parke, J. L. and D. Gurian-Sherman, D. 2001. "Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains." Annual Reviews in Phytopathology 39: 225-258. 2001.
[000219] Parke, et al., US Patent 6,077,505.
[000220] Pettit, G. et al. "Isolation of Labradorins 1 and 2 from Pseudomonas syringae." J. Nat. Prod. 65: 1793-1797. 2002.
[000221] Pitt, et al., "Type characterization and antibiotic susceptibility of Burkholderia (Pseudomonas') cepacia isolates from patients with cystic fibrosis in the United Kingdom and the Republic of Ireland." J. Med. Microbiol. 44: 203-210. 1996.
[000222] Ramette et al., "Species abundance and diversity of Burkholderia cepacia complex in the environment." Appl. Environ. Microbiol. 71: 1193-1201.2005.
[000223] Resi et al., "Burkholderia tropica sp. Nov., A novel nitrogenfixing, plant-associated bacterium." Int. J. Syst. Evol. Microbiol. 54: 2155-2162. 2004.
[000224] Salama et al. "Potency of spore-gamma-endotoxin complexes of Bacillus thuringiensis against some cotton pests." Z. Angew. Entomol. 91: 388-398. 1981.
[000225] Selva et al., "Targeted screening for elongation factor Tu binding antibiotics." J. Antibiot. 50: 22-26. 1997.
[000226] Takahashi, S. et al. "Martefragin A, a novel indole alkaloid isolated from a red alga, inhibits lipid peroxidation." Chem Pharm. Bull. 46: 1527-1529. 1998.
[000227] Thompson et al. "Spinosad - a case study: an example from a natural products discovery program." Pest Management Science 56: 696-702. 2000.
[000228] Takita et al., "Chemistry of Bleomycin. XIX Revised structures of bleomycin and phleomycin." J. Antibiot. 31: 801-804. 1978.
[000229] Tran Van et al., "Repeated beneficial effects of rice inoculation with a strain of Burkholderia vietnamiensis on early and late yield component in low fertility sulphate acid soils of Vietnam." Plant and Soil 218: 273-284. 2000.
[000230] Tsuruo et al., "Rhizoxin, a macrocyclic lactone antibiotic, as a new antitumor agent against human and murine tumor cells and their vincristine-resistant sublines." Cancer Res. 46: 381-385. 1986.
[000231] Ueda et al., US Patent 7,396,665.
[000232] Umehara, K. et al. "Studies of new antiplatelet agents WS- 30581 A and B." J. Antibiot. 37: 1153-1160. 1984.
[000233] Vandamme et al. Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. "Int. J. Syst. Bacteriol. 42: 344-356. 1992.
[000234] Vanderwall et al., "A model of the structure of HOO- Co« bleomycin bound to d (CCAGTACTGG): recognition at the d (GpT) site and implications for double-stranded DNA cleavage, Chem. Biol. 4: 373-387. 1997.
[000235] Vermis K., et al. "Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex." J. Med. Microbiol 51: 937-940. 2002.
[000236] Watanabe, H. et al. "A new antibiotic SF2583A, 4-chloro-5- (3'indoly) oxazole, produced by Streptomyces." Meiji Seika Kenkyu Nenpo 27: 55-62. 1988.
[000237] Wayne et al., "Report of the Ad Hoc committee on reconciliation of approaches to bacterial systematics." Int. J. Syst. Evol. Microbiol. 37: 463-464. 1987.
[000238] Werner et al., "Uptake of indolmycin in gram-positive bacteria." Antimicrob Agents Chemotherapy 18: 858-862. 1980.
[000239] Wilson et al. "Toxicity of rhizonin A, isolated from Rhizopus microsporus, in laboratory animals." Food Chem. Toxicol. 22: 275-281. 1984.
[000240] Zeck W.M. "Ein Bonitierungsschema zur Feldauswertung von Wurzelgallenbefall. Pflanzenschutznachrichten." Bayer 24.1: 144-117. 1971.
[000241] Zhang et al., US Patent 7,141,407.
[000242] Zhou et al., "Antimicrobial susceptibility and synergy studies of Burkholderia cepacia complex isolated from patients with cystic fibrosis." Antimicrobial Agents and Chemotherapy 51: 1085-088. 2007.

权利要求:
Claims (12)
[0001]
1. Method for producing FR901228, FR901465, Templamide A, Templamide B, Templazol A, or Templazol B, characterized by the fact that it comprises growing a strain of a Burkholderia A396 (NRRL Accession No. B-50319), and producing said compounds, where the compounds have the following chemical structure:
[0002]
2. Method for modulating pest infestation in a plant, characterized by the fact that it comprises applying to the plant and / or seeds of the same and / or substrate used for the growth of said plant an amount of a combination comprising Burkholderia A396 (No. Access Code NR-B-50319), or a composition comprising the isolated compound having pesticidal activity selected from (i) a compound having the structure
[0003]
3. Method, according to claim 2, characterized by the fact that the pest is a fungus.
[0004]
4. Method, according to claim 2, characterized by the fact that the pest is an insect.
[0005]
5. Method according to claim 2, characterized by the fact that the pest is a monocot, weed or dicot weed.
[0006]
6. Method according to claim 5, characterized by the fact that the method modulates the emergence and / or growth of monocotyledonous, junction or dicotyledonous weeds.
[0007]
Method according to any one of claims 2 to 6, characterized in that the composition comprising the isolated strain is applied.
[0008]
Method according to any one of claims 2 to 6, characterized in that the composition comprising the isolated compound having the structure
[0009]
Method according to any one of claims 2 to 6, characterized in that the composition comprising the isolated compound having the structure
[0010]
Method according to any one of claims 2 to 6, characterized in that the composition comprising the isolated compound having the structure
[0011]
11. Method, claims 2 to 6, characterized by the fact that the composition comprising the isolated compound having the structure
[0012]
12. Method according to any one of claims 2 to 6, characterized in that the composition comprising the isolated compound having the structure (Templazole B) is applied, where R1 is isobutyl.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112012021715B1|2020-08-11|METHOD TO PRODUCE FR901228, FR901465, TEMPLAMID A, TEMPLAMID B, TEMPLAZOL A, OR TEMPLAZOL B AND METHOD FOR MODULATING PEST INFESTATION ON A PLANT

---

JP2016183157A|2016-10-20|Isolated bacterial strain of genus burkholderia, and formulations and uses of pesticidal metabolites therefrom

---

US20190313645A1|2019-10-17|Isolated Bacterial Strain of the Genus Burkholderia and Pesticidal Metabolites Therefrom

---

AU2015202421B2|2016-12-15|Isolated bacterial strain of the genus Burkholderia and pesticidal metabolites therefrom

---

ASOLKAR et al.0|Patent 2791141 Summary

---

NZ620640B2|2016-01-06|Isolated bacterial strain of the genus burkholderia and pesticidal metabolites therefrom-formulations and uses

同族专利:

---

公开号 | 公开日

---

NZ602434A|2014-05-30|

---

HUE034377T2|2018-02-28|

---

EP2539432A4|2013-07-03|

---

RU2577970C2|2016-03-20|

---

AU2011220788A1|2012-10-11|

---

GT201200249A|2014-07-18|

---

WO2011106491A3|2012-03-15|

---

PL2539432T3|2017-08-31|

---

CN105325460B|2018-09-25|

---

DK2539432T3|2017-07-03|

---

EP2539432A2|2013-01-02|

---

PE20130558A1|2013-05-19|

---

BR112012021715A2|2015-10-06|

---

US9701673B2|2017-07-11|

---

IL221627A|2016-11-30|

---

CN105325460A|2016-02-17|

---

ECSP12012123A|2012-09-28|

---

CA2791141A1|2011-09-01|

---

TN2012000426A1|2014-01-30|

---

AU2011220788B2|2015-03-12|

---

CL2012002354A1|2013-03-22|

---

NZ621960A|2015-06-26|

---

KR101768735B1|2017-08-30|

---

RU2012140248A|2014-03-27|

---

ES2625607T3|2017-07-20|

---

US20110207604A1|2011-08-25|

---

CR20120442A|2012-11-01|

---

EP2539432B1|2017-04-12|

---

CN103261398A|2013-08-21|

---

WO2011106491A2|2011-09-01|

---

JP2013521227A|2013-06-10|

---

JP5784640B2|2015-09-24|

---

AR080234A1|2012-03-21|

---

KR20130043615A|2013-04-30|

---

MX2012009909A|2012-10-01|

---

PT2539432T|2017-05-25|

---

TW201129318A|2011-09-01|

---

MA34018B1|2013-02-01|

---

CN103261398B|2015-11-25|

---

ZA201206707B|2014-05-28|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4808207A|1985-06-21|1989-02-28|The University Of Vermont And State Agricultural College|Synergistic herbicidal compositions comprising microbial herbicides and plant growth regulators|

---

GB8817743D0|1988-07-26|1988-09-01|Fujisawa Pharmaceutical Co|Fr901228 substance & preparation thereof|

---

US5545542A|1991-06-14|1996-08-13|Fujisawa Pharmaceutical Co., Ltd.|WB2663 substances and method for their production|

---

GB9315796D0|1993-07-30|1993-09-15|Rhone Poulenc Agriculture|New compositions of matter|

---

DE19545463A1|1995-12-06|1997-06-12|Bayer Ag|Organic chemical compound and process for its preparation|

---

US6194194B1|1996-12-23|2001-02-27|Daniel Molloy|Method for controlling dreissena species|

---

US6319497B1|1997-04-23|2001-11-20|Virginia Tech Intellectual Properties, Inc.|Non-obligate predatory bacterium burkholderia casidaeand uses thereof|

---

US6077505A|1997-06-11|2000-06-20|Wisconsin Alumni Research Foundation|Biological seed treatment to improve emergence, vigor, uniformity and yield of sweet corn|

---

AU3310301A|2000-01-26|2001-08-07|U S Army Medical Res Inst Of I|Burkholderia toxins|

---

GB0002033D0|2000-01-28|2000-03-22|Zeneca Ltd|Chemical compounds|

---

EP1313872A1|2000-09-01|2003-05-28|Fujisawa Pharmaceutical Co., Ltd.|A method of producing fr901228|

---

CU23176A1|2001-01-03|2006-09-22|Ct Ingenieria Genetica Biotech|PESTICID AND ANTIPARASITARY COMPOSITIONS|

---

US20030082147A1|2001-08-28|2003-05-01|Xeno Insecticides, Inc.|Bacteria for insect control|

---

CA2421373C|2003-03-07|2013-07-02|Alberta Research Council Inc.|Control of chickweed using burkholderia andropogonis as a bioherbicide|

---

KR100537389B1|2003-10-07|2005-12-29| 엠솔|1404 Biocontrol of plant diseases using novel endophytic isolate of Burkholderia vietnamensis MC1404|

---

WO2005115149A2|2004-05-20|2005-12-08|Dow Agrosciences Llc|Insectidal activity of a cyclic peptide|

---

CA2527439C|2004-11-19|2013-10-01|Dow Agrosciences Llc|Methylidene mevalonates and their use as herbicides|

---

CA2512359A1|2005-07-15|2007-01-15|Alberta Research Council Inc.|Natural herbicide|

---

JP2007091701A|2005-08-31|2007-04-12|Jfe Chemical Corp|Tetracarboxylic acid containing fluorenyl group and ester group, polyester imide precursor containing fluorenyl group, polyester imide containing fluorenyl group and method for producing the same|

---

WO2007094014A1|2006-02-16|2007-08-23|Department Of Biotchnology |Process for producing a bio-pesticide composition containing trichoderma harzianum and pseudomonas fluorescens|

---

BRPI0621476A2|2006-03-15|2011-12-13|Csir|computer-aided method of generating a compound that inhibits glutamine synthetase activity, method of generating a test compound, screening method for a protease inhibitor compound, in vitro method for inhibiting bacterial growth and use of a serine inhibitor protease|

---

US7825267B2|2006-09-08|2010-11-02|University Of Pittsburgh-Of The Commonwealth System Of Higher Education|Synthesis of FR901464 and analogs with antitumor activity|

---

WO2009049378A1|2007-10-17|2009-04-23|Qbiotics Limited|Lactone derivatives|

---

JP2010047532A|2008-08-22|2010-03-04|Meiji Univ|Method and agent for controlling plant disease|

---

JP5441092B2|2008-09-30|2014-03-12|四国電力株式会社|Burkholderia bacteria, plant disease control agent and control method using the same|

---

AR080234A1|2010-02-25|2012-03-21|Marrone Bio Innovations Inc|BACTERIAL CEPA ISOLATED FROM THE BURKHOLDERIA AND METABOLITES PESTICIDES OF THE SAME|

---

TW201225844A|2010-10-25|2012-07-01|Marrone Bio Innovations Inc|Chromobacterium bioactive compositions and metabolites|WO2014149241A1|2013-03-15|2014-09-25|Marrone Bio Innovations, Inc.|Isolated bacterial strain of the genus burkholderia and pesticidal metabolites therefrom|

---

US9526251B2|2010-02-25|2016-12-27|Marrone Bio Innovations, Inc.|Use of Burkholderia formulations, compositions and compounds to modulate crop yield and/or corn rootworm infestation|

---

US8822193B2|2010-02-25|2014-09-02|Marrone Bio Innovations, Inc.|Isolated bacterial strain of the genus Burkholderia and pesticidal metabolites therefrom|

---

AR080234A1|2010-02-25|2012-03-21|Marrone Bio Innovations Inc|BACTERIAL CEPA ISOLATED FROM THE BURKHOLDERIA AND METABOLITES PESTICIDES OF THE SAME|

---

TW201225844A|2010-10-25|2012-07-01|Marrone Bio Innovations Inc|Chromobacterium bioactive compositions and metabolites|

---

KR20160054627A|2011-08-27|2016-05-16|마론 바이오 이노베이션스, 인코포레이티드|Isolated bacterial strain of the genus burkholderia and pesticidal metabolites therefrom-formulations and uses|

---

CN103997895B|2011-10-25|2018-01-12|马罗内生物创新公司|Formula, composition, metabolin and the application method of color bacillus|

---

CN107418911A|2011-12-13|2017-12-01|孟山都技术公司|Plant growth promotes microorganism and application thereof|

---

TW201340874A|2012-03-13|2013-10-16|Marrone Bio Innovations Inc|Pesticidal Flavobacterium strain and bioactive compositions, metabolites and uses|

---

US9125419B2|2012-08-14|2015-09-08|Marrone Bio Innovations, Inc.|Bacillus sp. strain with antifungal, antibacterial and growth promotion activity|

---

US9119401B2|2012-10-19|2015-09-01|Marrone Bio Innovations, Inc.|Plant glutamine synthetase inhibitors and methods for their identification|

---

WO2014068443A1|2012-11-05|2014-05-08|Pfizer Inc.|Spliceostatin analogs|

---

CN105072902B|2012-12-31|2017-08-01|巴斯夫欧洲公司|Herbicidal combinations|

---

CN103149313B|2013-02-06|2014-12-03|中国烟草总公司四川省公司|Method for determining residual amount of dicamba in tobacco|

---

WO2015034629A1|2013-09-07|2015-03-12|Marrone Bio Innovations, Inc.|Methods and compositions for control of mite infestations using a newly discovered species of burkholderia|

---

JP6937578B2|2013-11-19|2021-09-22|パーデュー・リサーチ・ファウンデーションＰｕｒｄｕｅ Ｒｅｓｅａｒｃｈ Ｆｏｕｎｄａｔｉｏｎ|Anti-cancer drug and its preparation method|

---

EP3191594A4|2014-09-11|2018-06-13|Marrone Bio Innovations, Inc.|Chromobacterium subtsugae genome|

---

TW201632516A|2014-12-11|2016-09-16|哈佛大學校長及研究員協會|Inhibitors of cellular necrosis and related methods|

---

CN105319313B|2015-12-09|2017-02-01|山东出入境检验检疫局检验检疫技术中心|Liquid chromatogram-tandem mass spectrum detection method of toxoflavin|

---

CN105368953A|2015-12-10|2016-03-02|山东出入境检验检疫局检验检疫技术中心|Real-time fluorescence PCRdetection kit and detection method for burkholderia gladioli|

---

JPWO2018168582A1|2017-03-14|2020-01-09|イビデン株式会社|Insecticide and method for producing insecticide|

---

US11206832B2|2017-09-08|2021-12-28|Marrone Bio Innovations, Inc.|Herbicidal compound|

---

CN107988100B|2017-12-05|2021-06-22|湖南豫园生物科技股份有限公司|Inorganic phosphate solubilizing bacteria, microbial fertilizer and application|

---

CN108739860B|2018-05-02|2020-10-23|华南农业大学|Microbial quorum sensing signal quenching and sterilizing agent and application thereof as biocontrol bacterium|

---

CN109750077A|2019-01-15|2019-05-14|华南农业大学|The Preparation method and use of Doby Asia cockroach symbiotic effects activity secondary metabolite|

---

EP3701796A1|2019-08-08|2020-09-02|Bayer AG|Active compound combinations|

---

CN110699290B|2019-11-01|2021-01-05|华东师范大学|Stable burkholderia, microbial inoculum and preparation method and application thereof|

---

WO2021099271A1|2019-11-18|2021-05-27|Bayer Aktiengesellschaft|Active compound combinations comprising fatty acids|

---

CN110771631B|2019-12-10|2021-03-26|云南大学|Method for preventing and treating nematode diseases by using composite nematode-killing microorganisms|

---

EP3708565A1|2020-03-04|2020-09-16|Bayer AG|Pyrimidinyloxyphenylamidines and the use thereof as fungicides|

---

WO2021209490A1|2020-04-16|2021-10-21|Bayer Aktiengesellschaft|Cyclaminephenylaminoquinolines as fungicides|

---

WO2021224220A1|2020-05-06|2021-11-11|Bayer Aktiengesellschaft|Pyridine amides as fungicidal compounds|

---

WO2021228734A1|2020-05-12|2021-11-18|Bayer Aktiengesellschaft|Triazine and pyrimidine amides as fungicidal compounds|

---

WO2021233861A1|2020-05-19|2021-11-25|Bayer Aktiengesellschaft|Azabicyclicamides as fungicidal compounds|

---

WO2021245087A1|2020-06-04|2021-12-09|Bayer Aktiengesellschaft|Heterocyclyl pyrimidines and triazines as novel fungicides|

---

WO2021249995A1|2020-06-10|2021-12-16|Bayer Aktiengesellschaft|Azabicyclyl-substituted heterocycles as fungicides|

---

WO2021255071A1|2020-06-18|2021-12-23|Bayer Aktiengesellschaft|3--5,6-dihydro-4h-1,2,4-oxadiazine derivatives as fungicides for crop protection|

---

WO2021255089A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazole pyrimidines and 1,3,4-oxadiazole pyridines as fungicides|

---

WO2021255091A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazoles and their derivatives as fungicides|

---

WO2021255169A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazole pyrimidines as fungicides|

---

WO2021255170A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazole pyrimidines as fungicides|

---

CN111705149B|2020-06-28|2022-02-15|江苏省中国科学院植物研究所|Burkholderia gladioli fluorescence quantitative PCR reference gene and screening and application of primer thereof|

---

EP3915971A4|2020-12-16|2021-12-01|Bayer Ag|Phenyl-sn-phenylamidines and the use thereof as fungicides|

---

CN113185505A|2021-04-07|2021-07-30|中国农业大学|Quinolone oxazolidinone compound and preparation method and application thereof|

法律状态:
2017-10-31| B25G| Requested change of headquarter approved|Owner name: MARRONE BIO INNOVATIONS, INC (US) |

---

2019-07-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-02-18| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

---

2020-03-10| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: C12N 1/20 , A01N 63/02 , A01N 43/74 , A01N 43/14 , A01P 7/00 Ipc: C12N 1/20 (2006.01), A01N 63/02 (1980.01), A01N 43 |

---

2020-06-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2020-08-11| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US30828710P| true| 2010-02-25|2010-02-25|

---

US61/308,287|2010-02-25|

---

US40654110P| true| 2010-10-25|2010-10-25|

---

US61/406,541|2010-10-25|

---

PCT/US2011/026016|WO2011106491A2|2010-02-25|2011-02-24|Isolated bacterial strain of the genus burkholderia and pesticidal metabolites therefrom|

---