WO1995003695A1 - Novel fungicidal properties of metabolites, culture broth, stilbene derivatives and indole derivatives produced by the bacteria xenorhabdus and photorhabdus spp. - Google Patents

Novel fungicidal properties of metabolites, culture broth, stilbene derivatives and indole derivatives produced by the bacteria xenorhabdus and photorhabdus spp. Download PDF

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Publication number
WO1995003695A1
WO1995003695A1 PCT/US1994/008216 US9408216W WO9503695A1 WO 1995003695 A1 WO1995003695 A1 WO 1995003695A1 US 9408216 W US9408216 W US 9408216W WO 9503695 A1 WO9503695 A1 WO 9503695A1
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Prior art keywords
bacteria
composition according
effective amount
indole
xenorhabdus
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PCT/US1994/008216
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French (fr)
Inventor
John M. Webster
Genhui Chen
Jianxiong Li
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Agro-Biotech Corporation
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Priority to AU75139/94A priority Critical patent/AU7513994A/en
Publication of WO1995003695A1 publication Critical patent/WO1995003695A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/08Oxygen or sulfur directly attached to an aromatic ring system
    • A01N31/16Oxygen or sulfur directly attached to an aromatic ring system with two or more oxygen or sulfur atoms directly attached to the same aromatic ring system
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A01N43/38Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20Bacteria; Substances produced thereby or obtained therefrom

Definitions

  • This invention relates to fungus disease control employing biological agents especially for the benefit of agriculture, food storage, garden,
  • This invention futher relates to employing the stilbene derivatives of the form 3,5-dihydroxy-4-R- trans-stilbene; where R is an alkyl group of carbon length 1 to 6 with either a straight chain or branched configuration, such as 3,5-dihydroxy-4-isopropyl- trans-stilbene or 3,5-dihydroxy-4-ethyl- trans-stilbene.
  • the latter compounds are produced by the bacterial symbiont Photorhabdus luminescens and other Xenorhabdus species, or are produced by chemical methods, and are useful for fungus disease control for the benefit of agriculture, food storage, garden, ornamental plants, trees and wood products, humans, animals, and other materials and organisms mitigated by fungal organisms.
  • it relates to the preventative, protective and
  • This invention also relates to employing the indole derivatives 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole, and 3- (2'-hydroxy-4-methyl-3'-oxopentyl)-indole, and their stereoisomers, produced either by the bacterial symbiont Xenorhabdus bovienii and other Xenorhabdus and Photorhabdus species and or by chemical method for fungus disease control for the benefit of
  • indole derivatives 3-(2'-acetoxy-4'-methyl-3'- oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'- oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'- oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole) produced by Xenorhabdus bovienii and other Xenorhabdus and Photorhabdus species.
  • Fungi are eucaryotic micro-organisms characterized by parasitic and saprophytic habits. Many derive nutritional benefit through invasion of hosts and deprivation of essential materials. Fungi may affect plants and animals by causing reduced vigor, inhibition of growth, physical damage and
  • fungi are recognized as human pathogens among the 100,000 or so species known. About 20 generate systemic infections, another 20 are
  • albicans can be endogenous to humans.
  • AIDS are found to be very susceptible to secondary infections of fungi, especially after antibiotic treatment for bacterial infections.
  • Histoplasma capsulatum and Coccidioides immitis are the more common secondary infections following AIDS. With the predicted worldwide increase in AIDS cases in the 1990's, prevention and treatment of these secondary infections by effective fungicides becomes increasingly important, especially as the infective fungi develop resistance to antifungal agents.
  • Desenex, Vioform antifungal synthetic agents (e.g., Tolnaftate, Tinactin, Loprox), antifungal imidazole derivatives (e.g., Miconazole, Micatin, Lotrimin), antifungal allylamine derivatives (e.g., Naftifine, Naftin) or antifungal and steroid combinations (e.g., Clotrimazole + Betamethasone, Lotrisone).
  • a new class of antimycotics, the triazoles show some promise as broad-spectrum treatments for fungus infection and they can be given orally. They are effective against some of the yeasts, such as Candida sp, and against aspergillosis. Whereas species of virus, bacteria and nematodes that cause plant disease are numbered in the hundreds, plant-pathogenic fungi number in the thousands.
  • Fungi probably cause more than 100,000 diseases in green plants alone. Protection of agricultural crops, stored foods, gardens, ornamental plants, trees and wood products, fish and animals from fungal disease requires applications of materials to:
  • fungicides for treatment of plant disease must have little or no
  • pesticides have proved to be more effective and easier to handle. Thus, very few useful fungicidal agents of natural origin have been discovered. Those biological control agents whose effect is of benefit to man are of limited scope (i.e. the eradication of grey molds on stored foods) and have yet to be generally commercialized.
  • Bacteria of the genera Xenorhabdus and Photorhabdus (Enterobacteriacea) were first discovered inside microscopic worms called nematodes. The nematodes act as vectors which carry the bacteria into insects. In particular, bacteria of these genera are carried within these entomopathogenic nematodes which inject them into insect hosts, thereby killing the host.
  • This nematode-bacteria complex was recognized as a biological insecticide and subsequently
  • the infective juvenile stage of the nematode carries the living bacteria in a non-reproducing stasis inside its gut while the nematode travels freely through the soils seeking insect hosts. As such, the bacteria are protected from many outside biotic and abiotic influences. The bacteria have thereby avoided co-evolution with other soil inhabitants.
  • the nematode upon finding a host, enters the insect through oral, anal and other orifices and then penetrates into the body cavity.
  • the nematode releases factors which activate the insect's immune system. This response kills any unprotected microbes present inside the insect, including those accidentally introduced by the invading nematode.
  • the nematode expels the Xenorhabdus or Photorhabdus spp. bacteria from its gut, into the insect haemocoel (internal body space) and the bacteria grow and multiply slowly.
  • the bacteria cause septicemia and produce metabolites which kill the insect.
  • Such metabolites are known to be antibiotics which kill other bacteria that may have invaded the insect host during the interim.
  • the nematode feeds on the multiplying Xenorhabdus bacteria and then enters a reproductive state.
  • the host fills with infective juvenile nematodes , each of which carries live
  • luminescens and other closely related species
  • a bacterial symbiont carried by nematodes of the genus Heterorhabdi tis and other Xenorhabdus species carried by nematodes of the genus Steinernema were found to be active against many fungi of agricultural and medicinal importance (Chen, G. and Webster, J.M., U.S. Patent Application No. 08/097,968, Filed
  • fungistatic activities occur only at and after a key point in the life cycle of the bacteria; within the dead or dying insect.
  • phase change In artificial culture, it has been found that both Xenorhabdus and Photorhabdus bacteria undergo a phase change from their primary form.
  • the primary form is found in the nematode or within infested insects. These primary forms produce lecithinase, absorb dyes, contain paracrystalline inclusions and produce antibiotics and pigments, whereas secondary forms produce little or no antibiotics and pigments.
  • the precise cause of the phase change is unknown, but it can be induced under certain culture conditions, and can occur spontaneously in artificial culture.
  • antibiotics varies with the species and strain of bacteria. Four types of antibiotics produced by the bacterial species have been described: (1) indole derivatives from X. bovienii (Paul et al.,
  • Antibiotics in Microbial Ecology Isolation and Structure Assignment of Several New Antibacterial Compounds from the Insect-Symbiotic Bacteria
  • Xenorhabdus and P. luminescens in artificial culture and identified active chemicals. They identified two dihydroxy stilbene derivatives from X. luminescens (Strain Hb) by mass spectrometry and NMR as 3,5-dihydroxy-4-is ⁇ propyl-trans-stilbene and 3,5- dihydroxy-4-ethyl-trans-stilbene. They also
  • fungistatic rather than the fungicidal properties. They postulated potential "antifungal” properties on the basis of chemical similarity with another group of compounds, without demonstration of fungicidal activity. Furthermore, they used only refined materials of low molecular weight without recognition of the potent complex of low and high molecular weight factors which forms during the natural growth cycle within host insects. There is no teaching on the use of raw bacteria and raw or partially refined bacterial products as fungistatic and fungicidal materials. Richardson et al. (1988) identified one dihydroxy stilbene from X. luminescens (Strain HK) by NMR and mass spectrometry as 3,5-dihydroxy-4-isopropyl- stilbene.
  • a group of 3-substituted indole compounds contain an ester at the 3-indole position, such ester extended with an alkyl, benzyl or indolyl carbonyl group
  • hydroxytryptamine receptors which are useful for the treatment of psychotic disorders, neurotic diseases, gastric stasis symptoms, gastrointestinal disorders, nausea and vomiting.
  • a Photorhabdus sp. (previous known as X. luminescens) has been found in association with human wounds
  • fungicides with broad-spectrum capabilities to kill fungal diseases of many classes and in many forms, such as reproductive, vegetative or resting stages.
  • An additional objective is to produce such a fungicide which is easy to formulate and apply using
  • a further objective of this invention to produce naturally-based fungicides which have protectant, preventative and eradicant capabilities in
  • An additional objective of this invention to utilize stilbene derivatives of 3,5-dihydroxy-4-substituted- trans-stilbene where the substituent is an alkyl group of carbon length 1 to 6 with either a straight chain or branched configuration, such as 3,5- dihydroxy-4-isopropyl- trans-stilbene or 3,5- dihydroxy-4-ethyl-trans-stilbene as fungicides which have protectant, preventative and eradicant
  • Another objective of this invention to utilize indole derivatives of the formulas: 3-(2'-acetoxy-4'-methyl- 3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'- oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'- oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole and their stereoisomers, as fungicides which have protectant, preventative and eradicant capabilities.
  • Photorhabdus spp. Enterobacteriaceae
  • their raw or partially refined metabolites, as well as specific metabolites have a beneficial effect by destroying many pathogenic fungi.
  • Xenorhabdus nematophilus Further characterization of Xenorhabdus is provided by Akhurst and Boemare (1988), where both phases and 21 strains were examined for 240 characters.
  • Xenorhabdus luminescens has recently been placed into a separate genus and is now classified as
  • Candidate fungal pathogens used in bioassays are readily available from many sources, including the American Type Culture Collection, Rockville, MD.
  • ml tryptic soy broth
  • TSB tryptic soy broth
  • Cultures were shaken at 120 rpm on an Eberbach gyrorotary shaker for 24 hours at 25°C.
  • Bacterial fermentation was initiated by adding 100 ml of the bacterial culture (OD 600 at ⁇ 2.0) to 900 ml of TSB in a 2,000 ml flask. The flask is
  • the active fungicidal ingredients of the composition of the present invention include a culture medium containing nonviable bacteria, the culture medium from which the bacterial cells have been removed, the nonviable bacteria per se, or filtrates of the culture medium.
  • a preferred filtrate of the culture medium will be a filtrate from which compounds of molecular weight of lower than about 750 gm/mole have been excluded.
  • a particularly preferred filtrate has compounds of molecular weight lower than about 500 gm/mole excluded therefrom.
  • Heat treatment of the culture medium or bacteria is also an alternative. Typically, treatments above about 50oC up to about 121oC at 15 psi may be utilized from periods of about 10 to 30 minutes.
  • the relative simplicity of the individual organic molecules of the present instance lend themselves to organic synthetic methods, in addition to microbial production methods. Such standard synthetic
  • Stilbenes Can J. Chem. 48:1554 (1970) and Krow et al . "Synthesis of Antibiotic Stilbenes Using
  • the fungicidal compositions may be formed using one of the active ingredients in an inert carrier. If formulated as a solid, the ingredient (s) may be mixed with typical carriers such as Fuller's earth, kaolin clays, silicas or other wettable inorganic diluents. Free-flowing dust formulations may also be utilized by combining the dry active ingredient with finely divided solids such as talc, kieselguhr,
  • the powders may also be applied as a suspension or solution, depending on the solubility in the liquid carrier.
  • Pressurized sprays typically aerosols with the active ingredient dispersed in a low-boiling dispersant solvent carrier, may be used. Percentages of weight may vary according to the manner in which the composition is to be applied, and formulation used. In general, the active ingredient will comprise 0.005% to 95% of the active ingredient by weight in the fungicidal composition.
  • fungicidal composition may be applied with other ingredients, including growth regulators,
  • Formulation of the active ingredients to assist applicability, ease handling, maintain chemical stability and increase effectiveness may require addition of various materials.
  • Solvents may be chosen on the basis of affecting the solubility of the active ingredient, fire hazard and flash point, emulsifiability, specific gravity and economic considerations.
  • Adjuvants may be added to enhance the active ingredients, and can include surfactants which are anionic, cationic or nonionic. Stabilizers and antifreeze compounds will prolong storage.
  • synergists, stickers, spreaders and deodorant compounds can be added to improve the handling characteristics of the commercial
  • the active ingredient can be combined with an inert carrier, such as calcium carbonate, and formed into a pill or other consumable delivery device, including controlled-release devices intended to deliver metered doses of the active ingredient.
  • an inert carrier such as calcium carbonate
  • the fungicidal compositions are useful against fungi which are harmful to agricultural crops, animals and desirable botanical species, such as ornamental and flowering plants. Generally, application doses will be in the range of 100 to 1,000 ppm of active
  • Xenorhabdus e.g., X. nematophilus, ATCC No. 19061, American Type Culture Collection, Rockville, MD.
  • Galleria larvae were injected with 10 microliters ( ⁇ l) of phosphate-buffered saline containing either (i) X. nematophilus (e.g. ATCC No. 19061), (ii) surface-disinfected IJ nematodes or (iii) nematodes containing no bacterial symbiont. Larvae were incubated at 27 degrees Celsius (°C) and extracted for antibiotics at death and every 24 h thereafter until 144 h post-injection.
  • X. nematophilus e.g. ATCC No. 19061
  • surface-disinfected IJ nematodes e.g. ATCC No. 19061
  • nematodes e.g. ATCC No. 19061
  • Larvae were incubated at 27 degrees Celsius (°C) and extracted for antibiotics at death and every 24 h thereafter until 144 h post-injection.
  • Xenorhabdus spp. e.g., X. nematophilus, ATCC No. 19061, ATCC No. 39497, ATCC No. 53200, X. bovienii , ATCC No. 35271
  • Photorhabdus sp. e.g. P.
  • hydrochloric acid HCl
  • concentrations from which no hyphal growth occurred in any one of six replicate discs were defined as fungicidal and those from which hyphal growth occurred in any one of the replicated discs were defined as fungistatic.
  • oxysporum and the conidia of B. cinerea were prepared by growing the fungi on PDA at 24°C and these plates were flooded with sterile water when the conidia were ready to be harvested. The conidia were washed twice with sterile distilled water, centrifuged, resuspended in 15% sterile potato dextrose broth, diluted to give concentrations of 6 ⁇ 10 3
  • Example 3 The methods of Example 3 were used in this study.
  • the culture filtrates of the X. nematophilus were either heated in a water bath at 60°, 100°C or autoclaved at 121oC and 15 pounds per square inch
  • the fungi to be tested were maintained on potato dextrose agar (PDA) in the dark at room temperature. Fifty microliters of the TSB culture of each
  • Xenorhabdus or Photorhabdus isolate were transferred into the center of a 9 cm Petri dish containing 10 ml TSB plus 1.5% agar, and incubated in the dark at 24oC for 4 days. Each plate was subsequently inoculated in a triangular fashion with the test fungal mycelial plug (0.5 cm) cut from cultures freshly grown on PDA plates. Each inoculation was replicated three times using three plates. Control plates were inoculated with TSB minus Xenorhabdus. The growth of the fungi was observed and recorded over a period of 4 to 80 days after inoculation, depending on the species of fungus. The longer incubation times were required for slow-growing fungi. The Xenorhabdus and Photorhabdus isolates used are from the laboratory of Dr. J.M. Webster (Biological Sciences Department, Simon Fraser University,
  • cryocoetidis Mucor piriformis, Pythium coloratum, P. ultimum and Trichoderma pseudokoningii .
  • Considerable resistance was demonstrated by Aspergillus niger, Cephaloascus fragrans, Beauveria bassiana and
  • Metarhizium anisopliae both insect pathogens
  • brasiliensis brasiliensis, occidioides immitis, Cryptococcus neoformans, Aspergillus fumigatus, Mucor sp, Absidia sp., Rhizopus sp., Rhizomurcor sp., Candida albicans, Pseudallesscheria boydii , Wangiella dermatitidis, Phialophora sp., Paelcilomyces sp., Beauveria sp., Scopulariopsis sp., Prototheca sp., Schizophyllum commune and Coprinus sp.
  • the cell-free culture broth of P. luminescens (SFU strain C9) was extracted by combining an equivalent volume of acetyl acetate in a separatory funnel and collecting the organic phase. The process was repeated three times. The organic-phase extracts were combined, dried over anhydrous sodium sulfate and filtered through glass wool to remove the
  • EIMS 256 (M + +2,3%), 255 (M + +1,8%), 254 (M + 38%), 240 (18%), 211 (4%), 239 (100%), 211 (4%), 205 (9%), 179 (5%), 178 (9%), 165 (12%), 149 (18%), 129 (12%), 125 (9%), 123 (9%), 111 (16%), 109 (12%), 105 (8%), 97 (23%), 95 (18%), 91 (11% 0), 85 (16%), 84 (8%), 83 (26%), 82 (11%), 81 (26%), 73 (9%), 71 (22%), 70 (10%), 69 (44%), 67 (11%), 57 (8%), 56 (8%), 55
  • IR (KBr) 3548, 3427, 1610, 1579, 1570, 1449, 1428, 1348, 1348, 1278, 1238, 1436, 1069, 1015, 994, 968, 817, 751, 690, 628 cm -1 .
  • DMSO dimethylsulfoxide
  • PDB potato dextrose broth
  • the active compound was serially diluted by twofold to produce culture media containing from 100 ⁇ g/ml to 0.1 ⁇ g/ml of active ingredient (i.e., 100, 50, 25, 12.5, 6.3, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 ⁇ g/ml) in order to determine the minimum
  • MIC inhibitory concentration
  • Botrytis cinerea were grown for 7 days at 25oC before the conidia were harvested by flooding the plate with sterile, distilled water and diluted to make the final inocula of 2.5 to 3.0 ⁇ 10 6 conidia per ml. The inocula were then added to treated culture media. Replicates were incubated at 35oC (B. cinerea was incubated at 24°C) and the MIC visually determined after 24 hours (48 hours for B. cinerea) . The MIC is defined as the lowest concentration of active compound which
  • Organisms MICs ( ⁇ g/ml)
  • the cell-free culture broth of Xenorhabdus bovienii (e.g., SFU Strain A2 or ATCC No. 35271) was
  • NMR spectra of each of ID1, ID2, ID3 and ID4 were recorded on a Bruker WM400 spectrometer in CDCl 3 , using residual CHC1 3 ( ⁇ 7.25) as internal standard. Low resolution mass spectra were obtained on a
  • ID4 identified as 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole: mass spectrum, El, m/z (relative intensity): 231 (M+, 15), 130 (100), 103 (5), 77 (10).
  • Active compounds ID1, ID2, ID3 and ID4 were dissolved in dimethylsulfoxide (DMSO), filter sterilized with a 0.22 micron filter and diluted into 2 ml of potato dextrose broth (PDB) in a 5 ml flask, resulting a final DMSO concentration of ⁇ 0.2% (v/v).
  • DMSO dimethylsulfoxide
  • PDB potato dextrose broth
  • the active compounds were serially diluted twofold to produce culture media containing from 100 ⁇ g/ml to 0.1 ⁇ g/ml of active ingredient (i.e., 100, 50, 25, 12.5, 6.3, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 ⁇ g/ml) in order to determine the minimum inhibitory concentration (MIC).
  • Candida tropicales and Cryptococcus neoformans were grown on potato dextrose agar (PDA) for 24 hours at 25oC, then scraped from the plate by flooding the plate with 0.8% saline and diluted with the saline to make inocula containing 2.5 to 2.8 ⁇ 10 7 Colony Forming Units (CFU) per ml. Aspergillus spp.
  • Botrytus cinerea were grown on PDA for 7 days at 25oC before the conidia were harvested by flooding the plate with sterile, distilled water and diluted to make the final inocula of 2.5 to 3.0 ⁇ 10 6 conidia per ml. The inocula were then added to treated culture media. Replicates were incubated at 35°C (B. cinerea was incubated at 24°C) and the MIC visually determined after 24 hours (48 hours for B. cinerea). The MIC is defined as the lowest
  • Organisms MICs ( ⁇ g/ml)
  • Botrytis cinerea >100 >100 12.5 12.5
  • ID1 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole ID2: 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole ID3: 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole ID4: 3-(2'-hydroxy-4-methyl-3'-oxopentyl)-indole
  • Xenorhabdus nematophilus (e.g, SFU Strain D1 or ATCC 19061) was cultured in broth medium at 25oC on an oscillating shaker. After 5 days of growth, the spent culture was neutralized with 6 normal
  • the dialization treatment separated these effects from known, lower-molecular weight substances of specified molecular structure.
  • the use of raw, unrefined culture media, partially refined culture media, bacteria and bacterial extracts were found to be highly potent.
  • the compound 3,5-dihydroxy-4-isopropyl-trans-stilbene shows potent antifungal properties, and that differential effects against fungal pathogens occur with each compound. Additionally, it is shown that these compounds 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'-oxopentyl)-indole show potent antifungal properties, and that differential effects against fungal pathogens occur with each compound.
  • oligonucleotide probes for molecular identification of Xenorhabdus species Appl . Environ . Microbiol .

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Abstract

The present invention relates to the use of microbes of the genera Xenorhabdus and Photorhabdus and the refined or raw metabolites of same from culture media for use as a fungicide for agricultural, horticultural, veterinary or human use. The present invention further relates to the use of dihydroxyalkyl-substituted stilbene derivatives of the formula: 3,5-dihydroxy-4-R-trans-stilbene; where R is an alkyl group of carbon length 1 to 6 with either a straight chain or branched configuration, such as 3,5-dihydroxy-4-isopropyl-trans-stilbene or 3,5-dihydroxy-4-ethyl-trans-stilbene, as fungicides which have protectant, preventative and eradicant capabilities. The invention also relates to the use of 3-substituted indole derivatives of the formulas: 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'-oxopentyl)-indole and their stereoisomer as fungicides which have protectant, preventative and eradicant capabilities.

Description

NOVEL FTJNGICIDAL PROPERTIES OF METABOLITES, CULTURE BROTH, STILBENE DERIVATIVES AND
INDOLE DERIVATIVES PRODUCED BY THE BACTERIA XENORHABDUS and PHOTORHABDUS spp. FIELD OF THE INVENTION
This invention relates to fungus disease control employing biological agents especially for the benefit of agriculture, food storage, garden,
ornamental plants, trees and wood products, humans, animals, and other materials and organisms mitigated by fungal organisms. In particular, it relates to the preventative, protective and eradicative
treatment of fungal diseases of all types through the use of biological agents and the raw and partially refined metabolites found from the bacterial genera Xenorhabdus and Photorhabdus.
This invention futher relates to employing the stilbene derivatives of the form 3,5-dihydroxy-4-R- trans-stilbene; where R is an alkyl group of carbon length 1 to 6 with either a straight chain or branched configuration, such as 3,5-dihydroxy-4-isopropyl- trans-stilbene or 3,5-dihydroxy-4-ethyl- trans-stilbene. The latter compounds are produced by the bacterial symbiont Photorhabdus luminescens and other Xenorhabdus species, or are produced by chemical methods, and are useful for fungus disease control for the benefit of agriculture, food storage, garden, ornamental plants, trees and wood products, humans, animals, and other materials and organisms mitigated by fungal organisms. In particular, it relates to the preventative, protective and
eradicative treatment of fungal diseases of all types through the use of these stilbene derivatives.
This invention also relates to employing the indole derivatives 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole, and 3- (2'-hydroxy-4-methyl-3'-oxopentyl)-indole, and their stereoisomers, produced either by the bacterial symbiont Xenorhabdus bovienii and other Xenorhabdus and Photorhabdus species and or by chemical method for fungus disease control for the benefit of
agriculture, food storage, garden, ornamental plants, trees and wood products, humans, animals, and other materials and organisms mitigated by fungal
organisms. In particular, it relates to the to the preventative, protective and eradicative treatment of fungal diseases of all types through the use of these indole derivatives (3-(2'-acetoxy-4'-methyl-3'- oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'- oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'- oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole) produced by Xenorhabdus bovienii and other Xenorhabdus and Photorhabdus species.
BACKGROUND TO THE INVENTION
Fungi are eucaryotic micro-organisms characterized by parasitic and saprophytic habits. Many derive nutritional benefit through invasion of hosts and deprivation of essential materials. Fungi may affect plants and animals by causing reduced vigor, inhibition of growth, physical damage and
deformation, reduced fecundity or even host death.
Over 200 fungi are recognized as human pathogens among the 100,000 or so species known. About 20 generate systemic infections, another 20 are
regularly found as cutaneous infections, and a dozen or more are identified with severe, localized
subcutaneous diseases. In addition, there is a long list of opportunistic fungi that cause diseases in debilitated patients, and these, like Candida
albicans, can be endogenous to humans. Debilitated persons with acquired immunodeficiency syndrome
(AIDS) are found to be very susceptible to secondary infections of fungi, especially after antibiotic treatment for bacterial infections. Candida
albicans, C. krusei , Cryptococcus neoformans,
Histoplasma capsulatum and Coccidioides immitis are the more common secondary infections following AIDS. With the predicted worldwide increase in AIDS cases in the 1990's, prevention and treatment of these secondary infections by effective fungicides becomes increasingly important, especially as the infective fungi develop resistance to antifungal agents.
Currently, dermal infections by fungi are treated topically with chemical agents (e.g., Castederm,
Desenex, Vioform), antifungal synthetic agents (e.g., Tolnaftate, Tinactin, Loprox), antifungal imidazole derivatives (e.g., Miconazole, Micatin, Lotrimin), antifungal allylamine derivatives (e.g., Naftifine, Naftin) or antifungal and steroid combinations (e.g., Clotrimazole + Betamethasone, Lotrisone). A new class of antimycotics, the triazoles, show some promise as broad-spectrum treatments for fungus infection and they can be given orally. They are effective against some of the yeasts, such as Candida sp, and against aspergillosis. Whereas species of virus, bacteria and nematodes that cause plant disease are numbered in the hundreds, plant-pathogenic fungi number in the thousands.
Fungi probably cause more than 100,000 diseases in green plants alone. Protection of agricultural crops, stored foods, gardens, ornamental plants, trees and wood products, fish and animals from fungal disease requires applications of materials to:
(a) prevent attack from germinating reproductive stages such as spores, conidia, and conidospores at times when the host is particularly sensitive (e.g. new seedlings), or when climatic conditions or other stresses are particularly conducive to new fungal growth (e.g. high humidity);
(b) protect hosts from invasion by the actively growing fungal tissues, such as mycelial mats or hyphal extensions; and
(c) eradicate growing fungal disease organisms after the host has been attacked, thus mitigating damage and preserving the host from further harm and possible destruction.
Conventional, petroleum-based fungicidal pesticides are limited in their scope and abilities to fulfill these requirements for all potential pest situations. Some products are limited by the types of disease organisms they kill, while others are limited by their effectiveness as preventatives or eradicants. Many have very short-lived effects in the environment because of degradation by ultra-violet light, chemical effects (e.g., ozonolysis), adsorption onto soil particles and, in many cases, through
degradation by other microbes, especially bacteria which occur naturally in the soils. Synthetic pesticides also have detrimental effects on the environment and its inhabitants. Regulatory
pressures are reducing the future usefulness of such conventional pesticides. An important attribute of a useful fungicide is its differential toxicity to the fungi and the host. Thus, fungicides for treatment of plant disease must have little or no
phytotoxicity. Low water solubility of the substance may minimize the phytotoxic components to the host plant, but allow it to remain available to affect pathogenic fungi. Materials used in treatments also must have low toxicity to animals. For a fungicide to be useful it must not be carcinogenic, teratogenic or cause sub-toxic effects.
Many natural interactions exist between the
microscopic flora and fauna of soils. Attempts have been made to select beneficial soil microbes
antagonistic to fungal pathogens, thus providing biological control systems for the benefit of crop production and food storage. Unfortunately, due to the complexity of the inter-relationships of large numbers of these soil organisms, it has proved frustrating to isolate single microbes of beneficial action. It has been postulated that this poor result is because these soil inhabitants, through existence in a common environment and through continued
interaction, have faced similar evolutionary
pressures, and through their mutual interactions, evolved to a mutual, albeit antagonistic, existence. It is further a disadvantage of other known
biological control agents that conventional
pesticides have proved to be more effective and easier to handle. Thus, very few useful fungicidal agents of natural origin have been discovered. Those biological control agents whose effect is of benefit to man are of limited scope (i.e. the eradication of grey molds on stored foods) and have yet to be generally commercialized. Bacteria of the genera Xenorhabdus and Photorhabdus (Enterobacteriacea) were first discovered inside microscopic worms called nematodes. The nematodes act as vectors which carry the bacteria into insects. In particular, bacteria of these genera are carried within these entomopathogenic nematodes which inject them into insect hosts, thereby killing the host.
This nematode-bacteria complex was recognized as a biological insecticide and subsequently
commercialized.
The infective juvenile stage of the nematode carries the living bacteria in a non-reproducing stasis inside its gut while the nematode travels freely through the soils seeking insect hosts. As such, the bacteria are protected from many outside biotic and abiotic influences. The bacteria have thereby avoided co-evolution with other soil inhabitants.
The nematode, upon finding a host, enters the insect through oral, anal and other orifices and then penetrates into the body cavity.
Once in the body cavity, the nematode releases factors which activate the insect's immune system. This response kills any unprotected microbes present inside the insect, including those accidentally introduced by the invading nematode. After the insect response subsides, the nematode expels the Xenorhabdus or Photorhabdus spp. bacteria from its gut, into the insect haemocoel (internal body space) and the bacteria grow and multiply slowly. The bacteria cause septicemia and produce metabolites which kill the insect. Such metabolites are known to be antibiotics which kill other bacteria that may have invaded the insect host during the interim.
Once the insect host is dead, the nematode feeds on the multiplying Xenorhabdus bacteria and then enters a reproductive state. The host fills with infective juvenile nematodes , each of which carries live
Xenorhabdus or Photorhabdus cells. The insect cadaver bursts, thus releasing tens of thousands of questing nematodes ready to seek new insect hosts. Thus, it was presumed that the insecticidal and anti-bacterial activities were the key roles of the genera Xenorhabdus and Photorhabdus.
Different Xenorhabdus species and a Photorhabdus species are characteristically associated in
monoculture with different species of
entomopathogenic nematodes.
The cell-free culture broths of Photorhabdus
luminescens (and other closely related species), a bacterial symbiont carried by nematodes of the genus Heterorhabdi tis and other Xenorhabdus species carried by nematodes of the genus Steinernema, were found to be active against many fungi of agricultural and medicinal importance (Chen, G. and Webster, J.M., U.S. Patent Application No. 08/097,968, Filed
07/27/93 and Chen, G., G. Dunphy and J.M. Webster, "Antifungal Activity of Two Xenorhabdus Species and Photorhabdus luminescens, Bacteria Associated with the Nematodes Steinernema Species and Heterorhabditis megidis. " Biological Control 4:157 (1994)). In order to understand the nature of this bioactivity and to develop more bioactive products, the isolation and identification of the bioactive components from this bacteria were pursued. Although there is a limited number of publications on these genera of bacteria, it has been recognized that active, anti-microbial substances are produced by Xenorhabdus species and the closely-related
Photorhabdus species. Some of these specific compounds have been isolated, identified and their structures elucidated. However, the importance of these specific purified metabolites as extremely potent fungicides or fungistatic agents has
heretofore been unrecognized.
The usefulness of the Xenorhabdus and Photorhabdus species, their unrefined cells, culture media and raw or partially refined metabolic products have been undiscovered until now because of certain aspects newly recognized, that are the subjects of this invention. It is believed that it has not heretofore been shown that operable aspects of Xenorhabdus or Photorhabdus exist for use in its raw or partially refined or cultured form as fungicide or fungistatic agents. Furthermore, it has been unrecognized until now that, through the complex life cycle of the subject bacteria of the genus Xenorhabdus and
Photorhabdus, the most potent antifungal and
fungistatic activities occur only at and after a key point in the life cycle of the bacteria; within the dead or dying insect.
The usefulness of both the stilbene derivative compounds and the indole derivative compounds found in these bacteria have been undiscovered until now because of certain aspects newly recognized, that are the subjects of this invention. Prior art references have not shown use of these specific compounds or any operable aspects as fungicide or fungistatic agents.
In artificial culture, it has been found that both Xenorhabdus and Photorhabdus bacteria undergo a phase change from their primary form. The primary form is found in the nematode or within infested insects. These primary forms produce lecithinase, absorb dyes, contain paracrystalline inclusions and produce antibiotics and pigments, whereas secondary forms produce little or no antibiotics and pigments. The precise cause of the phase change is unknown, but it can be induced under certain culture conditions, and can occur spontaneously in artificial culture.
Antibiotic production has been reported from
artificial culture of phase one of Xenorhabdus nematophilus, X. bovienii and Photorhabdus
luminescens. The activity spectrum of the
antibiotics varies with the species and strain of bacteria. Four types of antibiotics produced by the bacterial species have been described: (1) indole derivatives from X. bovienii (Paul et al.,
"Antibiotics in Microbial Ecology: Isolation and Structure Assignment of Several New Antibacterial Compounds from the Insect-Symbiotic Bacteria
Xenorhabdus spp." J. Chem. Ecol . Vol. 7, pp. 589-597 (1981)), (2) stilbene derivatives from P. luminescens (Ibid; Richardson et al . , "Identification of an anthraquinone pigment and a hydroxystilbene
antibiotic from Xenorhabdus [Photorhabdus]" App.
Environ. Microbiol . Vol. 54, pp. 1602-1605 (1988)); (3) the organically-soluble dithiolopyrrolones, xenorhabdins, for Xenorhabdus sp. strain Q, X.
bovienii and strains of X. nematophilus (McInerney et al . "Biologically Active Metabolites from Xenorhabdus spp., Part 1. Dithiolopyrrolone derivatives with antibiotic activity" J. Nat. Prod. Vol. 54, pp. 774- 784 (1991)); and (4) the water-soluble benzopyran derivatives, xenocoumacins, for Xenorhabdus strain Q and the All strain of X. nematophilus (Mclnerney et al . "Biologically Active Metabolites from Xenorhabdus spp., Part 2. Benzopyran-1-one derivatives with gastroprotective activity" J. Nat. Prod. Vol. 54, pp. 785-795 (1991)). Akhurst, "Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect
pathogenic nematodes of the Families
Heterorhabditidae and Steinernematidae" J. Gen.
Microbiol . Vol. 128, pp. 3061 - 3065 (1982)
demonstrated anti-bacterial inhibition by killed Xenorhabdus colonies grown on artificial media. He also showed inhibition of three species of yeast, including the human pathogen, Candida albicans, with extracts containing low-molecular-weight factors from Xenorhabdus, and stated there was no activity from factors of molecular weight greater than 14,000 when Xenorhabdus spp. were artificially cultured.
Akhurst, however, did not examine the bacterial response within the insect, thereby failing to cause the bacteria to produce its most potent response.
Paul et al. (1981) examined nine strains of
Xenorhabdus (and P. luminescens) in artificial culture and identified active chemicals. They identified two dihydroxy stilbene derivatives from X. luminescens (Strain Hb) by mass spectrometry and NMR as 3,5-dihydroxy-4-isσpropyl-trans-stilbene and 3,5- dihydroxy-4-ethyl-trans-stilbene. They also
identified four 3-substituted indoles from X.
nematophilus (Strain R) by mass spectrometry and NMR as 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole, 3- (2'-acetoxy-4'-methyl-3'-oxopentyl)-indole, 3-(2'- hydroxy-4'-methyl-3'-oxohexyl)-indole, and 3-(2'- hydroxy-4-methyl-3'-oxopentyl)-indole, but they did not examine the bacteria in vivo. Paul et al. did not test for fungicidal activities of these stilbene derivatives.
McInerney et al . , J. Nat. Prod., 54 , 774-784 (1991); 54 , 785-795 (1991); and their corresponding patents Rhodes et al . "Process for Making Xenorhabdin Antibiotics", U.S. Patent 4,672,130 (June 9, 1987) and Gregson et al." Xenocoumacins", U.S. Patent
4,837,222 (June 6, 1989), respectively, elucidated the structures of various low-molecular-weight active chemicals from Xenorhabdus. While they demonstrate an anti-bacterial activity for xenorhabdins and xenocoumacins their technique evaluated the
fungistatic rather than the fungicidal properties. They postulated potential "antifungal" properties on the basis of chemical similarity with another group of compounds, without demonstration of fungicidal activity. Furthermore, they used only refined materials of low molecular weight without recognition of the potent complex of low and high molecular weight factors which forms during the natural growth cycle within host insects. There is no teaching on the use of raw bacteria and raw or partially refined bacterial products as fungistatic and fungicidal materials. Richardson et al. (1988) identified one dihydroxy stilbene from X. luminescens (Strain HK) by NMR and mass spectrometry as 3,5-dihydroxy-4-isopropyl- stilbene.
Sundar, L. and F.N. Chang "The role of Guanosine-3'- 5'-Bis-Pyrophosphate in Mediating Antimicrobial
Activity of the Antibiotic 3,5-Dihydroxy-4-Ethyl- trans-Stilbene" Antimicrobial Agents and Chemotherapy 36:2645-2651 (1992) examined the mode of action of 3,5-dihydroxy-4-ethyl-trans-stilbene as an
antimicrobial. However, Sundar and Chang and the references therein revealed no prior knowledge of the antimycotic nature of 3,5-dihydroxy-4-ethyl-trans- stilbene or of 3,5-dihydroxy-4-isopropyl-trans- stilbene. The stilbenes found in nature which are fungicidal are referred to as phytoalexins, and are produced by host plants in response to fungal infections. These compounds are found in various unrelated plants, such as peanut, grape, sugar cane and pine. The compounds of the instant invention arise from a unique source in nature: bacteria. Their specific 4-substitution is also unique in nature.
Hart, "Role of Phytostilbenes in Decay and Disease Resistance" Ann. Rev. Phytopathol . 19:437-458 (1981) and references therein recognize that 14-carbon phenolic stilbenes have (page 437) "long been known for their antifungal properties," but noted that chemical analogues of phytoalexins are not
necessarily anti-fungal.
Sundar, L. and F.N. Chang "Antimicrobial Activity and Biosynthesis of Indole Antibiotics Produced by
Xenorhabdus nematophilus. " J. Gen. Microbiol . 139: 3139-3148 (1993), (p. 3139): "investigated the mechanism of action and physiology of production of the indole derivative antibiotics." They report additional effectiveness of the derivatives against both Gram-positive and Gram-negative bacteria by "causing a severe inhibition of RNA synthesis, accompanied by a less severe effect on protein synthesis." They include no fungal organisms in their activity survey.
A group of 3-substituted indole compounds contain an ester at the 3-indole position, such ester extended with an alkyl, benzyl or indolyl carbonyl group
(Kikuchi et al . "Indole Derivatives." U.S. Patent 5,124,324 (Jun. 23, 1992). These compounds are claimed as selective antagonists of 5-HT3
(hydroxytryptamine) receptors, which are useful for the treatment of psychotic disorders, neurotic diseases, gastric stasis symptoms, gastrointestinal disorders, nausea and vomiting.
A Photorhabdus sp. (previous known as X. luminescens) has been found in association with human wounds
(Farmer et al . 1989). Although they are similar to Xenorhabdus and Photorhabdus found in host nematodes, the bacteria associated with human wounds have a different range of adaptation; as indicated by their optimal temperature being the same as that for warm-blooded animals. Colepicolo et al . (1989)
characterized these bacteria according to the effects of ionic strength, temperature, oxygen, and iron on the bacteria's growth and development. In addition, the culture medium after growth of this species exhibited antibiotic activity.
Cochrum et al . (1990), Meighen (1991) and Xi et al . (1992) cloned the luminescence genes of Photorhabdus luminescens and found that the enzymes were
structurally very similar to luminescence enzymes of other terrestrial bacteria and marine bacteria.
Colepicolo et al . (1992) found that, unlike several species of marine bioluminescent bacteria, human wound Xenorhabdus could grow in an atmosphere of pure oxygen, indicating a significant functional
difference from other luminescent bacteria at physiological levels.
SUMMARY OF THE INVENTION
It is therefore an objective of this invention to produce naturally-based fungicides which have protectant, preventative and eradicant capabilities It is further an objective to produce low cost material, through organic synthesis methods or by the use of bacteria (alive and killed), and raw,
unrefined or partially refined, or purified
metabolites from within the bacteria and found in its culture media, separately and in combination, that is an effective fungicide.
Furthermore, it is an objective to produce fungicides with broad-spectrum capabilities to kill fungal diseases of many classes and in many forms, such as reproductive, vegetative or resting stages. An additional objective is to produce such a fungicide which is easy to formulate and apply using
conventional pesticide-application equipment and methods. A further objective of this invention to produce naturally-based fungicides which have protectant, preventative and eradicant capabilities in
controlling fungi which are human pathogens.
An additional objective of this invention to utilize stilbene derivatives of 3,5-dihydroxy-4-substituted- trans-stilbene where the substituent is an alkyl group of carbon length 1 to 6 with either a straight chain or branched configuration, such as 3,5- dihydroxy-4-isopropyl- trans-stilbene or 3,5- dihydroxy-4-ethyl-trans-stilbene as fungicides which have protectant, preventative and eradicant
capabilities.
Another objective of this invention to utilize indole derivatives of the formulas: 3-(2'-acetoxy-4'-methyl- 3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'- oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'- oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole and their stereoisomers, as fungicides which have protectant, preventative and eradicant capabilities.
Still further objects and advantages shall become apparent from consideration of the ensuing
descriptions and preferred embodiments.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
It has been discovered that Xenorhabdus and
Photorhabdus spp. (Enterobacteriaceae) and their raw or partially refined metabolites, as well as specific metabolites, have a beneficial effect by destroying many pathogenic fungi.
Bioassays of the antifungal activity of Xenorhabdus spp. and Photorhabdus sp., and bioassays of the antifungal activity of stilbene derivatives from Photorhabdus luminescens and indole derivatives from Xenorhabdus spp., were done by the methods based on the standard procedure of the American Society of Phytopathology for testing chemical fungicides.
Sources and depositories of Xenorhabdus and
Photorhabdus species and strains are noted in Akhurst and Boemare "A numerical taxonomic study of the genus Xenorhabdus (Enterobacteriaceae) and proposed
elevation of the subspecies of X. nematophilus to species" J. Gen. Microbiol . Vol 134, pp. 1835-1845 (1988). Putz et al. "Development and application of oligonucleotide probes for molecular identification of Xenorhabdus species" Appl . Environ. Microbiol . Vol. 56, pp. 181-186 (1990) notes additional sources and depositories, including the American Type Culture Collection, Rockville, MD. They identify and
separate 35 strains of Xenorhabdus into one of five species:
Xenorhabdus beddingii Xenorhabdus bovienii
Xenorhabdus poinarii
Xenorhabdus luminescens
Xenorhabdus nematophilus Further characterization of Xenorhabdus is provided by Akhurst and Boemare (1988), where both phases and 21 strains were examined for 240 characters.
Xenorhabdus luminescens has recently been placed into a separate genus and is now classified as
Photorhabdus luminescens.
Candidate fungal pathogens used in bioassays are readily available from many sources, including the American Type Culture Collection, Rockville, MD.
The species of Xenorhabdus and their nematode
symbionts used in all studies were collected and maintained in culture. Last instar larvae of the greater wax moth, Galleria mellonella, were infected with infective juvenile (IJ) nematodes, carrying the respective Xenorhabdus spp. at a rate of 25
IJs/larvae. After 24 to 48 hours (h) the dead insect larvae were surface disinfected by dipping them into 95% ethanol and igniting them. The cadavers were aseptically opened with sterile forceps, haemolymph (fluid plasma and blood cells) from the haemocoel was streaked onto an NBTA medium (nutrient agar
supplemented with 0.025 grams (g) of bromothymol blue and 0.04 g of 2,3,5-triphenyltetrazolium chloride per liter) and incubated in the dark at room temperature. The resulting primary form Xenorhabdus spp. of each isolate was maintained and subcultured at 14 day intervals. For consistency, glycerinated
(17%/nutrient broth) stocks of bacteria frozen at - 18ºC were frequently used as starting materials for cultures. Inocula of the primary form were prepared by adding one loop-full of the culture to 50
milliliters (ml) of tryptic soy broth (TSB) in a 100 ml Erlenmeyer flasks. Cultures were shaken at 120 rpm on an Eberbach gyrorotary shaker for 24 hours at 25°C. Bacterial fermentation was initiated by adding 100 ml of the bacterial culture (OD600 at ≈2.0) to 900 ml of TSB in a 2,000 ml flask. The flask is
incubated in the dark at 25ºC on an Eberbach
gyrorotary shaker. After 96 hours, the culture is centrifuged (12,000 xg, 20 minutes, 4°C) to separate the bacterial cells. The cell-free material is then extracted with ethyl acetate. The organic phase is then separated into fractions, from which the active stilbene or indole derivatives can be isolated. The active fungicidal ingredients of the composition of the present invention include a culture medium containing nonviable bacteria, the culture medium from which the bacterial cells have been removed, the nonviable bacteria per se, or filtrates of the culture medium. A preferred filtrate of the culture medium will be a filtrate from which compounds of molecular weight of lower than about 750 gm/mole have been excluded. A particularly preferred filtrate has compounds of molecular weight lower than about 500 gm/mole excluded therefrom. Heat treatment of the culture medium or bacteria is also an alternative. Typically, treatments above about 50ºC up to about 121ºC at 15 psi may be utilized from periods of about 10 to 30 minutes. The relative simplicity of the individual organic molecules of the present instance lend themselves to organic synthetic methods, in addition to microbial production methods. Such standard synthetic
processes are described in the literature, such as, for the stilbene derivatives, Bachelor et al . "Synthesis of Pinosylvin and Related Heartwood
Stilbenes" Can J. Chem. 48:1554 (1970) and Krow et al . "Synthesis of Antibiotic Stilbenes Using
Organomanganese Arene Complexes" J. Org. Chem.
57:4040 (1992) and the references therein.
The fungicidal compositions may be formed using one of the active ingredients in an inert carrier. If formulated as a solid, the ingredient (s) may be mixed with typical carriers such as Fuller's earth, kaolin clays, silicas or other wettable inorganic diluents. Free-flowing dust formulations may also be utilized by combining the dry active ingredient with finely divided solids such as talc, kieselguhr,
pyrophyllite, clays, diatomaceous earth and the like. The powders may also be applied as a suspension or solution, depending on the solubility in the liquid carrier. Pressurized sprays, typically aerosols with the active ingredient dispersed in a low-boiling dispersant solvent carrier, may be used. Percentages of weight may vary according to the manner in which the composition is to be applied, and formulation used. In general, the active ingredient will comprise 0.005% to 95% of the active ingredient by weight in the fungicidal composition. The
fungicidal composition may be applied with other ingredients, including growth regulators,
insecticides, fertilizers, etc.
Formulation of the active ingredients to assist applicability, ease handling, maintain chemical stability and increase effectiveness may require addition of various materials. Solvents may be chosen on the basis of affecting the solubility of the active ingredient, fire hazard and flash point, emulsifiability, specific gravity and economic considerations. Adjuvants may be added to enhance the active ingredients, and can include surfactants which are anionic, cationic or nonionic. Stabilizers and antifreeze compounds will prolong storage.
Additionally, synergists, stickers, spreaders and deodorant compounds can be added to improve the handling characteristics of the commercial
formulation. Alternatively, the active ingredient can be combined with an inert carrier, such as calcium carbonate, and formed into a pill or other consumable delivery device, including controlled-release devices intended to deliver metered doses of the active ingredient. The fungicidal compositions are useful against fungi which are harmful to agricultural crops, animals and desirable botanical species, such as ornamental and flowering plants. Generally, application doses will be in the range of 100 to 1,000 ppm of active
ingredient to inert carrier. However, dosages will vary based on the particular fungi, host and
environment.
EXAMPLE 1
Effect of Xenorhabdus-killed insect cadavers
on soil microbes.
Galleria larvae killed by either injection of 5 μl of Xenorhabdus (e.g., X. nematophilus, ATCC No. 19061, American Type Culture Collection, Rockville, MD.) bacterial suspension (5.0 × 106 bacteria/ml) or by traumatic insult through repeated needle punctures were placed for comparison into soils collected from natural sites. After 8 days, the Xenorhabdus-treated replicates contained significantly fewer soil microbes than the untreated samples. The test was originally intended to measure effects on soil bacteria, however it was noted also that the fungal populations were unexpectedly affected. This led to further investigations of these results.
EXAMPLE 2
Timing of production of active metabolites
by Xenorhabdus spp.
under natural conditions. To assess the natural production of active materials by Xenorhabdus, insect larvae were artificially infested and antibiotic production measured.
Galleria larvae were injected with 10 microliters (μl) of phosphate-buffered saline containing either (i) X. nematophilus (e.g. ATCC No. 19061), (ii) surface-disinfected IJ nematodes or (iii) nematodes containing no bacterial symbiont. Larvae were incubated at 27 degrees Celsius (°C) and extracted for antibiotics at death and every 24 h thereafter until 144 h post-injection.
Antibiotics were extracted by homogenizing the insects in distilled water at a ratio of 1 ml/larva and the suspension stirred on a magnetic plate for 20 minutes. The resulting mixtures were centrifuged (6000 g., 20 minutes, 24ºC) to remove insect tissue and fat droplets. The supernatants were adjusted to pH 7.0, centrifuged (12,000 g; 20 minutes, 4ºC), filter sterilized (0.22μ) and stored at 4°C. Fifty ml of filtrate was pipetted into each well (∅ = 0.5 cm), cut on TSA (10 ml in each 9 -cm plate) freshly
inoculated with Bacillus subtilis spore (6 × 109 CFU). The diameter of inhibition zone size was determined after 24 hours of incubation at 25ºC. Antibiotic activity was expressed as the radius of the annular clearing around the antibiotic disk.
Antibiotic activity was detected after the insect was killed. TABLE 1 shows that Xenorhabdus alone, and Xenorhabdus with the nematode symbiont produced highest antibiotic titres after 24 hours, while the bacteria-free treatment produced no antibiotics.
Antibiotic levels dropped when the nematode was present, but were sustained when only the bacteria were introduced. This study showed that a key factor activated the Xenorhabdus under natural conditions.
TABLE 1: Antibiotic Activity of Xenorhabdus under natural conditions in the insect host (diameter inhibition zone, cm ± standard error)
Figure imgf000023_0001
EXAMPLE 3
Fungicidal activity of Xenorhabdus filtrates
on mycelium growth of Pythium spp.
Xenorhabdus spp. (e.g., X. nematophilus, ATCC No. 19061, ATCC No. 39497, ATCC No. 53200, X. bovienii , ATCC No. 35271) and Photorhabdus sp. (e.g. P.
(Xenorhabdus) luminescens, ATCC No. 29999) were cultured separately in agitated broth medium
incubated at 25º0 for 5 days. The spent culture media were then neutralized with 6 Normal (N)
hydrochloric acid (HCl), centrifuged at 11,000 xg for 20 m to remove bacterial cells, precipitated, and the filtrates were collected and filter sterilized with an effective pore size of 0.2 micron (μ) before bioassay.
The filtrates of X. nematophilus and X. bovienii, prepared as above without filtration, were further adjusted to pH 5.8 with HCl, centrifuged at 11,000 xg for 10 m filter sterilized and lypholized
(lypholization has no effect on the efficacy of the filtrates) to yield a concentrated filtrate powder. The powder was added to potato dextrose agar (PDA) at 49°C and mixed thoroughly to give the final filtrate concentration required for bioassay.
To determine the fungicidal or fungistatic property of the filtrate, fungal discs of the test fungus were placed in the center of Petri dishes containing the filtrate-supplemented PDA. Those discs which had mycelium growth totally inhibited by Xenorhabdus were removed from the filtrate-supplemented plates and placed into new filtrate-free PDA plates, incubated at 24ºC. The growth of those discs in the filtrate- free plates was further investigated 4 days after the removal and incubation. Those filtrate
concentrations from which no hyphal growth occurred in any one of six replicate discs were defined as fungicidal and those from which hyphal growth occurred in any one of the replicated discs were defined as fungistatic.
Results: Xenorhabdus spp. filtrates were fungicidal to Pythium splendens, P. sulcatum, P. sylvaticum and P. ultimum when they were supplemented in PDA at a concentration of 30%. EXAMPLE 4
Fungicidal activity of Xenorhabdus filtrates on macroconidal and conidial germinations of
Fusarium solani, F. oxysporum and Botrytis cinerea. The methods of Example 3 above were used for this study. The macroconidia of F. solani and F.
oxysporum and the conidia of B. cinerea were prepared by growing the fungi on PDA at 24°C and these plates were flooded with sterile water when the conidia were ready to be harvested. The conidia were washed twice with sterile distilled water, centrifuged, resuspended in 15% sterile potato dextrose broth, diluted to give concentrations of 6×103
macroconidia/ml, 7×103 macroconidia/ml, and 5×103 conidia/ml for F. solani, F. oxysporum and B. cinerea respectively, and bio-assayed for germination.
The bioassay of the conidial germination was
performed in depression slides incubated in a humid chamber. Conidial germination after incubation was determined under the microscope based on three counts of 100 conidia for each replicate. In the case of no or low germination, at the time of observation, the slides were re-incubated and re-examined after further incubation. Results: The filtrates of Xenorhabdus spp. were fungicidal to the germination of macroconidia of Fusarium solani , F. oxysporum and conidia of Botrytis cinerea. EXAMPLE 5
Heat-stable and large molecular properties and their fungal activity on macroconidial germination of Fusarium solani, F. oxysporum,
and the conidial germination of Botrytis cinerea and the mycelial growth of Pythium ultimum .
The methods of Example 3 were used in this study. The culture filtrates of the X. nematophilus were either heated in a water bath at 60°, 100°C or autoclaved at 121ºC and 15 pounds per square inch
(psi) for 20 m or were dialyzed in dialysis tubing of molecular weight cut-off (MWC) of 8,000 to 9,000 and 12,000 to 14,000 overnight in running distilled water, thus removing materials of lower molecular weights. The dialysates were concentrated using a Rotavapour-R Büchi at room temperature, then
reconstituted with distilled water. The heat-treated and the dialyzed filtrates were then adjusted to pH 5.8 or 7, centrifuged at 11,000 xg at room
temperature for 10 m to remove the precipitates, filter sterilized (0.22μ) and bio-assayed for their effect on the growth of Pythium ultimum and on the macroconidial germination of F. solani , F. oxysporum and the conidial germination of B. cinerea. Results: Heat-treated and the high molecular weight dialyzed filtrates were fungicidal to the
macroconidial germination of Fusarium solani , F.
oxysporum, and conidial germination of Botrytis cinerea and the mycelial growth of Pythium ultimum. EXAMPLE 6
Broad spectrum fungicidal activity of Xenorhabdus and Photorhabdus species.
The fungi to be tested were maintained on potato dextrose agar (PDA) in the dark at room temperature. Fifty microliters of the TSB culture of each
Xenorhabdus or Photorhabdus isolate were transferred into the center of a 9 cm Petri dish containing 10 ml TSB plus 1.5% agar, and incubated in the dark at 24ºC for 4 days. Each plate was subsequently inoculated in a triangular fashion with the test fungal mycelial plug (0.5 cm) cut from cultures freshly grown on PDA plates. Each inoculation was replicated three times using three plates. Control plates were inoculated with TSB minus Xenorhabdus. The growth of the fungi was observed and recorded over a period of 4 to 80 days after inoculation, depending on the species of fungus. The longer incubation times were required for slow-growing fungi. The Xenorhabdus and Photorhabdus isolates used are from the laboratory of Dr. J.M. Webster (Biological Sciences Department, Simon Fraser University,
Burnaby, B.C. Canada, V5A 1S6), as follows:
D1 X. nematophilus D1 from Steinernema
carpocapsae nematode
DS X. nematophilus D1 secondary form as above BC1 X. nematophilus BC1 from Steinernema
carpocapsae nematode
A2 X. bovienii A2 from S. feltiae
A3 X. bovienii A3 from S. feltiae
NC X. bovienii NC from S. feltiae
BC2 X. bovienii BC2 from S. feltiae
C9 P. luminescens from Heterorhabdits megidis TABLE 2 shows the results. Antimycotic substance(s) inhibited fungal growth to an extent that varied with the fungal species and the bacterial isolate. The growth of all fungi except for the mycorrhizal fungus Suillus pseudobrevipes was inhibited to some extent by one or more of the Xenorhabdus or Photorhabdus isolates. Seven species of plant pathogenic fungi had their growth completely inhibited by the primary form of all of the Xenorhabdus and Photorhabdus
(Botrytis cinerea, Ceratocystis ulmi , C.
cryocoetidis, Mucor piriformis, Pythium coloratum, P. ultimum and Trichoderma pseudokoningii . Considerable resistance was demonstrated by Aspergillus niger, Cephaloascus fragrans, Beauveria bassiana and
Metarhizium anisopliae (both insect pathogens) and by the mycorrhizal fungi Oidiodendron griseum and S.
pseudobrevipes. It is notable that the xenocoumacin 1 of McInerney was shown to affect Aspergillus niger. The mycorrhizal fungi are beneficial species existing in commensual relationships with plants. Their resistance to treatment would thus be useful.
Surprisingly, antifungal activity was seen even in the second phase of Xenorhabdus (i.e., SFU Strain DS), where the current teachings state no anti-biotic activity should be present.
In addition to the fungi in TABLE 2, the activity of the compounds of the present invention also
encompasses:
Malassezia furfur, Trichosporon beigelii , Piedraia hortae, Microsporum sp., Trichophyton sp.,
Epidermophyton sp., Candida albicans, Fonsecaea pedrosoi , Pseudallescheria boydii , Madurella
mycetomtis, Basidiobolus ranarum, Conidiobolus coronatus, Rhinosporidium seeberi , Loboa loboi , Sporothrix schenchii , Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides
brasiliensis, occidioides immitis, Cryptococcus neoformans, Aspergillus fumigatus, Mucor sp, Absidia sp., Rhizopus sp., Rhizomurcor sp., Candida albicans, Pseudallesscheria boydii , Wangiella dermatitidis, Phialophora sp., Paelcilomyces sp., Beauveria sp., Scopulariopsis sp., Prototheca sp., Schizophyllum commune and Coprinus sp.
Figure imgf000030_0001
EXAMPLE 7
Isolation of the active stilbene components
From Photorhabdus luminescens
The cell-free culture broth of P. luminescens (SFU strain C9) was extracted by combining an equivalent volume of acetyl acetate in a separatory funnel and collecting the organic phase. The process was repeated three times. The organic-phase extracts were combined, dried over anhydrous sodium sulfate and filtered through glass wool to remove the
inorganic materials. The liquid filtrate was
evaporated by rotatory evaporator (Büchi) held under 30ºC under a vacuum. The resulting concentrated crude material was then separated into different fractions with flush silica gel chromatography
(silica gel 60 in 50 cm X 8 cm glass column) with first 30% ether in hexane, then 60% ether in hexane and finally 100% ether. The most active fraction was found to be present in the middle eluate, which upon concentration gave the active compound ST1.
EXAMPLE 8
Identification of the active stilbene components
From Photorhabdus
1HNMR spectrum of ST1 was recorded on a Bruker WM400 spectrometer in CDCl3, using residual CHCl3 (~7.25) as internal standard. The mass spectrum was obtained on a Hewlett-Packard 5985B GC/MS system operating at 70 eV using a direct probe. 13CNMR datum was recorded on the same instrument in CD3OD. The IR spectrum was recorded on a Perkin-Elmer S99B spectrometer.
(Abbreviations used as follows: EI = Electron
Impact, M+ = Molecular Ion, s = singlet, t = triplet, q=quartet, J = coupling constant, Hz = Hertz, d = doublet, hept = heptet, AB = a pair of protons separated by a small chemical shift).
EIMS: 256 (M+ +2,3%), 255 (M+ +1,8%), 254 (M+ 38%), 240 (18%), 211 (4%), 239 (100%), 211 (4%), 205 (9%), 179 (5%), 178 (9%), 165 (12%), 149 (18%), 129 (12%), 125 (9%), 123 (9%), 111 (16%), 109 (12%), 105 (8%), 97 (23%), 95 (18%), 91 (11% 0), 85 (16%), 84 (8%), 83 (26%), 82 (11%), 81 (26%), 73 (9%), 71 (22%), 70 (10%), 69 (44%), 67 (11%), 57 (8%), 56 (8%), 55
(24%).
1HNMR (CDCl3) : 7.47 (2H, d, J=7 Hz), 7.33 (3H, t, J=8 Hz), 6.94 (2H, AB, J= 16 Hz), 6.50 (2H, s), 4.73 (2H,s), 3.43 (1H, hept, J=7 Hz), 1.37 (6H, d, J=7 Hz). 13CNMR (CD3OD) : 157.7 (s), 139.1 (s), 136.9 (s), 130.0 (d), 129.6 (d), 128.4 (d), 128.3 (d), 127.3 (d), 122.2 (S), 106.6 (d), 25.7 (d), 21.0 (q).
IR (KBr) : 3548, 3427, 1610, 1579, 1570, 1449, 1428, 1348, 1348, 1278, 1238, 1436, 1069, 1015, 994, 968, 817, 751, 690, 628 cm-1.
EXAMPLE 9
Bioassays of the active stilbene components
From Photorhabdus.
Active compound 3,5-dihydroxy-4-isopropyl-trans- stilbene was dissolved in dimethylsulfoxide (DMSO), filter sterilized with a 0.22 μ filter and diluted into 2 mis of potato dextrose broth (PDB) in a 5 ml flask, resulting a final DMSO concentration of
<0.2%(v/v). The active compound was serially diluted by twofold to produce culture media containing from 100 μg/ml to 0.1 μg/ml of active ingredient (i.e., 100, 50, 25, 12.5, 6.3, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 μg/ml) in order to determine the minimum
inhibitory concentration (MIC). Each dilution was replicated 3 times with both liquid media and agar plates. The two yeast test candidates ( Candida tropicales and Cryptococcus neoformans) were grown on potato dextrose agar (PDA) for 24 hours at 25°C, then scraped from the plate by flooding the plate with 8% saline and diluted with the saline to make inocula containing 2.5 to 2.8 × 107 Colony Forming Units (CFU) per ml. Aspergillus spp. and Botrytis cinerea were grown for 7 days at 25ºC before the conidia were harvested by flooding the plate with sterile, distilled water and diluted to make the final inocula of 2.5 to 3.0 × 106 conidia per ml. The inocula were then added to treated culture media. Replicates were incubated at 35ºC (B. cinerea was incubated at 24°C) and the MIC visually determined after 24 hours (48 hours for B. cinerea) . The MIC is defined as the lowest concentration of active compound which
prevents the growth of the test organism within 24 hours.
RESULTS: It was found that similar effects were seen from both liquid and agar culture methods. TABLE 3 shows the MICs determined for the compound against each fungus organism.
TABLE 3 : Minimum Inhibitory Concentrations (MIC) of chemical isolated from Photorhabdus luminescens on candidate fungal
pathogens.
Organisms MICs (μg/ml)
3,5-dihydroxy-4-isopropyl-trans-stilbene
Aspergillus fumigatus 12
A. flavus 25
Botrytis cinerea 25
Candida tropicales 12
Cryptococcus neoformans 12
EXAMPLE 10
Isolation of the active indole components
From Xenorhabdus
The cell-free culture broth of Xenorhabdus bovienii (e.g., SFU Strain A2 or ATCC No. 35271) was
extracted by combining an equivalent volume of ethyl acetate in a separatory funnel and collecting the organic phase. The process was repeated three times. The organic-phase extracts were combined, dried over anhydrous sodium sulfate and filtered through glass wool to remove the inorganic materials. The liquid filtrate was evaporated by flash evaporator (Büchi) held under 35ºC under a vacuum. The resulting concentrated crude material was then separated into different fractions with flash silica gel
chromatography (silica gel 60 in 50 cm X 8 cm glass column) with 50% ether in hexane. Pure compound one (ID1) was eluted first, followed by a mixture of ID1 and compound two (ID2), then pure ID2, a mixture of ID2 and compound three (ID3), followed by pure ID3, a mixture of ID3 and compound four (ID4), and finally pure ID4.
EXAMPLE 11
Identification of the active indole components
From Xenorhabdus
NMR spectra of each of ID1, ID2, ID3 and ID4 were recorded on a Bruker WM400 spectrometer in CDCl3, using residual CHC13 (~7.25) as internal standard. Low resolution mass spectra were obtained on a
Hewlett-Packard 5985B GC/MS system operating at 70 eV using a direct probe. High resolution MS spectra were recorded on a Kratos MS80 instrument. IR spectra were recorded as neat film on NaCl using a Perkin-Elmer S99B spectrometer. (Abbreviations used as follows: EI = Electron Impact, M+ = Molecular
Ion, t = triplet, J = coupling constant, Hz = Hertz, d = doublet, m = multiplet, sext = sextet, dd = doublet doublet, q = quartet, bs = broad singlet, hept = heptet). ID1: identified as 3-(2'-acetoxy-4'-methyl-3'- oxohexyl)-indole; mass spectrum, EI, m/z (relative intensity): 287 (M+, 10), 227 (M+, -60, 10), 170 (47), 130 (100), 103 (10), 84 (25), 77 (20), 57 (35); 1H NMR, δ: 0.86(3H, t, J=7 Hz), 0.98 (3H, d, J=6.5 Hz), 1.39 (1H, m), 1.73 (1 H, m), 2.07 (3H, s), 2.63 (1H, sext, J=7 Hz), 3.18 (1H, dd, J=7 Hz, J=15 Hz), 3.30 (1H, dd, J=4.5 Hz, J=15 Hz), 5.46 (1H, q, J=4.5 Hz, J=8 Hz), 7.05 (1H, d, J=2.2 Hz), 7.15 (1H, t, J=7.5 Hz), 7.21 (1H, t, J=7.5 Hz), 7.37 (1H, d,
J=7.5Hz), 7.64 (1H, d, J=7.5 Hz), 8.06 (1H, bs, NH).
ID2, identified as 3-(2'-acetoxy-4'-methyl-3'- oxopentyl)-indole: mass spectrum, EI, m/z (relative intensity): 223 (M+, 25), 213 (M+ -60, 15), 170 (60), 130 (100), 103 (6), 84 (12), 77 (8); 1H NMR, δ: 0.99 (3H, d, J=6 Hz), 1.09 (3H, d, J=6 Hz), 2.07 (3H, s,
COCH3), 2.72 (1H, hept, J=7 Hz), 3.19 (1H, dd, J=7
Hz, J=15 Hz), 3.27 (1H, dd, J=4.5 Hz, J-15 Hz), 5.46 (1H, dd, J=4.5 Hz, J=8 Hz), 7.05 (1H, d, J=2.2 Hz),
7.15 (1H, t, J=7.5 Hz). 7.21 (1H, t, J=7 5 Hz). 7.37 (1H, d, J=7.5 Hz), 7.64 (1H, d, J=7.5 Hz), 8.06 (1H, bs, NH).
ID3 identified as 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole: mass spectrum, El, m/z (relative intensity): 245 (M+, 10), 130 (100), 92 (5), 77 (3), 57 (2); mass spectrum, CI, isobutane: 246 (M+1, 100), 130 (55); IR (film), cm-1: 3410, 2967, 2932, 1705, 1457, 742; 1HNMR δ: 0.875 (3H, t, J=7 Hz), 0.99 (3H, d, J=6.5 Hz), 1.42 (1H, m), 1.65 (1H, m), 2.77 (1H, sext, J= 6.5 Hz), 3.04 (1H, dd, J=7 Hz, J=15 Hz), 3.32 (1H, dd, J=4.5 Hz, J=15 Hz), 3.52 (1H, d, J= 5.5Hz, OH), 4.6 (1H, m), 7.12 (1H, d, J= 2.3Hz), 7.15 (1H, t, J=7.5 Hz), 7.2 (1H, t, J=7.5 Hz), 7.36 (1H, d, J=7.5 Hz), 7.63 (1H, d, J=7.5 Hz), 8.06 (1H, bs, NH). 13C NMR, δ: 11.39, 14.78, 27.07, 29.83, 43.11, 76.23, 110.89, 111.21, 118.64, 119.54, 122.15,
122.81, 127.43, 136.13, 215.82.
High resolution mass spectrum calculated for C15E19NO2 : 245.1416, found: 245.1416. Long-range 1H-13C
correlation spectroscopic result: the carbon with chemical shift at 110.89 ppm was coupled with the proton with chemical shift at 3.04 ppm (1H, dd, J=7 Hz, J=15 Hz). ID4 identified as 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole: mass spectrum, El, m/z (relative intensity): 231 (M+, 15), 130 (100), 103 (5), 77 (10). 1H NMR, δ: 1.02 (3H, d, J=6.5 Hz), 1.13 (3H, d, J=7 Hz), 2.88 (1H, hept, J=7 Hz), 3.06 (1H, dd, J=7 Hz, J=15 Hz), 3 31 (1H, dd, J=4.5 Hz, J=15 Hz), 3.45 (1H, d, J=5.5 Hz, OH), 4 67 (1H, m), 7.11 (1H, d, J=2.3 Hz), 7.14 (1H, t, J=7.5 Hz), 7.2 (1H, t, J=7.5 Hz), 7.36 (1H, d, J=7.5 Hz), 7.63 (1H, d, J=7.5 Hz), 8.05 (1H, bs, NH) EXAMPLE 12
Bioassays of the active indole components
From Xenorhabdus
Active compounds ID1, ID2, ID3 and ID4 were dissolved in dimethylsulfoxide (DMSO), filter sterilized with a 0.22 micron filter and diluted into 2 ml of potato dextrose broth (PDB) in a 5 ml flask, resulting a final DMSO concentration of <0.2% (v/v). The active compounds were serially diluted twofold to produce culture media containing from 100 μg/ml to 0.1 μg/ml of active ingredient (i.e., 100, 50, 25, 12.5, 6.3, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 μg/ml) in order to determine the minimum inhibitory concentration (MIC). Each dilution was replicated three times with both liquid media and agar plates. The two yeast test candidates ( Candida tropicales and Cryptococcus neoformans) were grown on potato dextrose agar (PDA) for 24 hours at 25ºC, then scraped from the plate by flooding the plate with 0.8% saline and diluted with the saline to make inocula containing 2.5 to 2.8 × 107 Colony Forming Units (CFU) per ml. Aspergillus spp. and Botrytus cinerea were grown on PDA for 7 days at 25ºC before the conidia were harvested by flooding the plate with sterile, distilled water and diluted to make the final inocula of 2.5 to 3.0 × 106 conidia per ml. The inocula were then added to treated culture media. Replicates were incubated at 35°C (B. cinerea was incubated at 24°C) and the MIC visually determined after 24 hours (48 hours for B. cinerea). The MIC is defined as the lowest
concentration of active compound which prevents the growth of the test organism at the above conditions.
RESULTS: It was found that similar effects were seen from both liquid and agar culture methods. TABLE 4 shows the MICs determined for each compound against each fungus.
TABLE 4: Minimum Inhibitory Concentrations (MIC) of chemicals isolated from Xenorhabdus bovienii on candidate fungal pathogens.
Organisms MICs (μg/ml)
ID1 ID2 ID3 ID4
Aspergillus fumigatus >100 >100 >100 >100
A . flavus >100 >100 >100 >100
Botrytis cinerea >100 >100 12.5 12.5
Candida tropicales >100 >100 >100 >100
Cryptococcus neoformans 50 25 >100 >100
ID1: 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole ID2: 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole ID3: 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole ID4: 3-(2'-hydroxy-4-methyl-3'-oxopentyl)-indole
EXAMPLE 13
Effect of Xenorhabdus nematophilus on the
opportunistic human fungal pathogen,
Candida albicans.
Xenorhabdus nematophilus (e.g, SFU Strain D1 or ATCC 19061) was cultured in broth medium at 25ºC on an oscillating shaker. After 5 days of growth, the spent culture was neutralized with 6 normal
hydrochloric acid and centrifuged at 11,000xg for 20 minutes at 4°C to remove bacteria cells. The filtrates were sterilized by filtration with a 0.2 micropore filter (Millipore, Bedford, MA). An agar diffusion bioassay was routinely used to test for antibody activity. Bacterial agar medium was
autoclaved for 15 minutes and then held in a water bath at 50°C for one hour. Ten milliliters of medium were poured into a 10 cm petri dish. Once the petri dishes cooled, they were stored at room temperature until utilized in tests. The bacteria and fungi were cultured in their appropriate broth media, shaken at 80 rpm at 25ºC overnight and 100 μl of bacteria or fungi was then spread evenly over the surface of medium of each bioassay petri dish. The dishes were placed into a laminar air-flow hood for 20-30 minutes to dry. Two to four wells (0.5 cm in diameter) were cut into the agar of each inoculated petri dish, using a alcohol-flame sterilized cork borer. Fifty microliters of test solution were added into each well, the dishes were covered with lids and placed at 25ºC. Following 24 hours of incubation, the diameter of the inhibition zone was measured with hand-held calipers.
RESULTS: In all replicates that included the Candida albicans on plates containing the spent culture medium of Xenorhabdus nematophilus a significant clear area was present, indicating that growth of Candida albicans was inhibited by components in the medium.
In conclusion, it is shown that potent antifungal properties of Xenorhabdus species and Photorhabdus species and their raw or partially purified
metabolites affect a broad spectrum of fungal
pathogens. The dialization treatment separated these effects from known, lower-molecular weight substances of specified molecular structure. The use of raw, unrefined culture media, partially refined culture media, bacteria and bacterial extracts were found to be highly potent.
It was further shown that newly-discovered potent antifungal properties of Xenorhabdus nematophilus and its purified, raw or partially purified metabolites affect the growth of an opportunistic human pathogen, Candida albicans.
Furthermore, it is shown that the compound 3,5-dihydroxy-4-isopropyl-trans-stilbene shows potent antifungal properties, and that differential effects against fungal pathogens occur with each compound. Additionally, it is shown that these compounds 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'-oxopentyl)-indole show potent antifungal properties, and that differential effects against fungal pathogens occur with each compound.
While our above description contains many
specificities, these should not be construed as limitations on the scope of the inventions, but rather as examples of preferred embodiments.
Accordingly, the scope of the invention should not be determined by the embodiments presented, but by the appended claims and their legal equivalents.
Figure imgf000041_0001
Figure imgf000042_0001
References Cited
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Claims

WHAT IS CLAIMED IS:
1. A fungicidal composition comprising:
a fungicidally effective amount of culture medium containing bacteria, said bacteria selected from the group consisting of the genus Xenorhabdus, the genus Photorhabdus , and mixtures thereof; and
a biologically inert carrier.
2. A composition according to Claim 1 wherein said bacteria are nonviable.
3. A composition according to Claim 1 wherein said fungicidally effective amount of culture medium is sufficient to kill fungi on desirable agricultural crops and botanical species.
4. A composition according to Claim 1 wherein said bacteria is predominantly in its primary form.
5. A composition according to Claim 1 wherein said bacteria are in a lyophilized form.
6. A composition according to Claim 1 wherein said bacteria are in a spray-dried form.
7. A composition according to Claim 1 wherein said culture medium is heat-treated.
8. A composition according to Claim 1 wherein said culture medium is a filtrate, said filtrate having a lower molecular weight cutoff excluding molecules less than about 500 gm/mole.
9. A fungicidal composition, comprising:
a fungicidally effective amount of culture medium from which bacterial cells have been removed, said medium derived from growing bacteria selected from the group consisting of the genus Xenorhabdus , the genus Photorhabdus , and mixtures thereof; and
a biologically inert carrier.
10. A composition according to Claim 9 wherein said fungicidally effective amount of culture medium is sufficient to kill fungi on desirable agricultural crops and botanical species.
11. A composition according to Claim 9 wherein said bacteria is predominantly in its primary form.
12. A composition according to Claim 9 wherein said culture medium is heat-treated.
13. A composition according to Claim 9 wherein said culture medium is a filtrate, said filtrate having a lower molecular weight cutoff excluding molecules less than about 500 gm/mole.
14. A fungicidal composition, comprising:
a fungicidally effective amount of nonviable bacteria, said bacteria selected from the group consisting of the genus Xenorhabdus , the genus
Photorhabdus , and mixtures thereof; and
a biologically inert carrier.
15. A composition according to Claim 14 wherein:
said fungicidally effective amount of bacteria is sufficient to kill fungi on desirable agricultural crops and botanical species.
16. A composition according to Claim 14 wherein:
said bacteria is predominantly in its primary form.
17. A composition according to Claim 14 wherein said bacteria are in a lyophilized form.
18. A composition according to Claim 14 wherein said bacteria are in a spray-dried form.
19. A composition according to Claim 14 wherein said bacteria is heat-treated.
20. A composition according to Claim 14 wherein:
said bacteria is predominantly in its secondary form.
21. A process for producing a fungicidal composition, comprising:
infecting insect larvae with infective juvenile nematodes, said nematodes carrying bacteria species selected from the group consisting of the genus
Xenorhabdus , the genus Photorhabdus , and mixtures thereof;
allowing said insect larvae to die as a result of said infection;
collecting said Xenorhabdus or Photorhabdus bacteria from cadavers of said insect larvae, when said bacteria exhibits its most potent fungicidal activity;
combining a fungicidally effective amount of said culture medium containing said bacteria with a biologically inert carrier.
22. A process according to Claim 21 further
comprising the step of culturing said bacteria in culture medium after collecting same from said cadavers.
23. A process according to Claim 22 further
comprising the steps of obtaining filtrates of said culture medium excluding molecules having a molecular weight of lower than about 500 gm/mole; and
using a fungicidally effective amount of said filtrate in place of said culture medium to combine with said biologically inert carrier.
24. A process according to Claim 22 further
comprising the step of removing cells of said
bacteria from said culture medium.
25. A method for the control of fungi, comprising: contacting said fungi or their habitats with a fungicidally effective amount of a fungicidal
composition according to any one of Claims 1 through 20.
26. A method according to Claim 25 wherein said fungi is selected form the group consisting essentially of Pythium splendens, P. sulcatum, P. sylvaticum, P.
ultimum, Botrytis cicerea, Ceratocystis ulmi, C.
dryocoetidis, Mucor piriformis, Pythium coloratum, Trichoderma pseudokingii, Fusarium solani, F.
oxysporum, Alternaria sp. , Rhizoctonia cerealis, R. solani, Sclerotinia minor, Thielaviopsis basicola, Verticillium albo-atrum, V. dahliae, Geotrichum candidum, Gleosporium perannans, Monilina fructicola, Ophiostoma piceae, Penicillium expansum, P. notatum, Penicillium sp. , Rhizopus stonifer and Venturia inaequalis.
27. A composition according to Claim 1 wherein said fungicidally effective amount of culture medium is sufficient to kill fungi on humans.
28. A method according to Claim 25 wherein said fungi are selected from the group consisting essentially of, Malassezia furfur, Trichosporon beigelii,
Piedraia hortae , Microsporum sp., Trichophyton sp., Epidermophyton sp., Candida albicans, Fonsecaea pedrosoi , Pseudallescheria boydii , Madurella
mycetomtis, Basidiobolus ranarum, Conidiobolus coronatus, Rhinosporidium seeberi, Loboa loboi,
Sporothrix schenchii , Histoplasma capsulatum,
Blastomyces dermatitidis, Paracoccidioides
brasiliensis, occidioides immitis , Cryptococcus neoformans, Aspergillus fumigatus , Mucor sp, Absidia sp., Rhizopus sp., Rhizomurcor sp., Candida albicans , Pseudallesscheria boydii , Wangiella dermatitidis, Phialophora sp., Paelcilomyces sp., Beauveria sp., Scopulariopsis sp., Prototheca sp., Schizophyllum commune and Coprinus sp.
29. A fungicidal composition comprising:
a fungicidally effective amount of 4-substituted 3,5-dihydroxy-trans-stilbene; where the substituent is an alkyl group of carbon length 1 to 6 with a straight chain or branched configuration and
a biologically inert carrier.
30. A fungicidal composition according to Claim 29 comprising:
a fungicidally effective amount of 3,5-dihydroxy- 4-isopropyl-trans-stilbene; and
.a biologically inert carrier.
31. A fungicidal composition according to Claim 29 comprising:
a fungicidally effective amount of 3,5-dihydroxy- 4-ethyl-trans-stilbene; and
a biologically inert carrier.
32. A composition according to Claims 29, 30, or 31, wherein said effective amount of active material is sufficient to kill fungi on agricultural crops and botanical species.
33. A composition according to Claims 29, 30, or 31 wherein said effective amount of active material is sufficient to kill fungi on agricultural livestock or other animals, humans or fish.
34. A fungicidal composition comprising:
a fungicidally effective amount of 3-(2'-acetoxy-4'-methyl-3'-oxohexyl)-indole; and
a biologically inert carrier.
35. A fungicidal composition comprising:
a fungicidally effective amount of 3-(2'-acetoxy-4'-methyl-3'-oxopentyl)-indole; and
a biologically inert carrier.
36. A fungicidal composition comprising:
a fungicidally effective amount of 3-(2'-hydroxy-4'-methyl-3'-oxohexyl)-indole; and
a biologically inert carrier.
37. A fungicidal composition comprising:
a fungicidally effective amount of 3-(2'-hydroxy- 4-methyl-3'-oxopentyl)-indole; and
a biologically inert carrier.
38. A fungicidal composition comprising:
a fungicidally effective amount of a mixture of at least two of the components 3-(2'-acetoxy-4'- methyl-3'-oxohexyl)-indole, 3-(2'-acetoxy-4'-methyl- 3'-oxopentyl)-indole, 3-(2'-hydroxy-4'-methyl-3'- oxohexyl)-indole, and 3-(2'-hydroxy-4-methyl-3'- oxopentyl)-indole.
39. A composition according to Claims 34, 35, 36, 37 or 38 wherein said effective amount of active material is sufficient to kill fungi on agricultural crops and botanical species.
40. A composition according to Claims 34, 35, 36, 37 or 38 wherein said effective amount of active material is sufficient to kill fungi on agricultural livestock or animals, humans, other animals or fish.
PCT/US1994/008216 1993-07-27 1994-07-27 Novel fungicidal properties of metabolites, culture broth, stilbene derivatives and indole derivatives produced by the bacteria xenorhabdus and photorhabdus spp. WO1995003695A1 (en)

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US7569748B2 (en) 1993-05-18 2009-08-04 Wisconsin Alumni Research Foundation Nucleic acid encoding an insecticidal protein toxin from photorhabdus
EP0797659A1 (en) * 1995-11-06 1997-10-01 Wisconsin Alumni Research Foundation Insecticidal protein toxins from photorhabdus
EP0797659A4 (en) * 1995-11-06 1998-11-11 Wisconsin Alumni Res Found Insecticidal protein toxins from photorhabdus
EP0970185A4 (en) * 1996-08-29 2003-02-26 Dowelanco Insecticidal protein toxins from photorhabdus
EP0970185A1 (en) * 1996-08-29 2000-01-12 Dowelanco Insecticidal protein toxins from $i(photorhabdus)
WO2000021368A1 (en) * 1998-10-09 2000-04-20 Ciba Specialty Chemicals Holding Inc. Hydroxystilbene compounds used as microbicidal active substances
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KR100418771B1 (en) * 2000-12-30 2004-02-18 기아자동차주식회사 Operation controlling method of lean burn engine for a vehicle
US8487009B2 (en) 2001-01-18 2013-07-16 Glaxo Group Limited 1,2-diphenylethene derivatives for treatment of immune diseases
US8609083B1 (en) 2007-03-22 2013-12-17 The United States Of America, As Represented By The Secretary Of Agriculture Method for controlling fungal pathogen with bacterial metabolite
WO2016092493A1 (en) 2014-12-12 2016-06-16 Glaxosmithkline Intellectual Property Development Limited Novel method of use
WO2016185428A1 (en) 2015-05-21 2016-11-24 Glaxosmithkline Intellectual Property Development Limited Topical pharmaceutical compositions
EP4094752A1 (en) 2015-05-21 2022-11-30 Dermavant Sciences GmbH Topical pharmaceutical compositions
WO2020018173A1 (en) * 2018-07-19 2020-01-23 Northeastern University Compounds with antimicrobial properties
US11535648B2 (en) 2018-07-19 2022-12-27 Northeastern University Isolated darobactin A analog compounds and compositions thereof
CN113373099A (en) * 2021-08-04 2021-09-10 南京农业大学 Xenorhabdus carbasus NN7 and application thereof
CN113373099B (en) * 2021-08-04 2023-03-03 南京农业大学 Xenorhabdus carbasus NN7 and application thereof
CN114304146A (en) * 2021-12-07 2022-04-12 西北农林科技大学 Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof

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