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 _Xenor.ha_ba.L-S 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 neofonαans , Histoplasma capsulatum and Coccidioides iπtmitis 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 PhotorhaJbdus 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 Heterorhabdi tis 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 (Mclnerney 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- rans-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.

Mclnerney 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- rans- 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-HT ₃ (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 - 18'C 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 (OD ₆₀₀ at «2.0) to 900 ml of TSB in a 2,000 ml flask. The flask is incubated in the dark at 25'C 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 50'C up to about 121'C 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 .

95 _ _{1 Q} _

"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,

95 _ _ιg _

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 x 10 ⁶ 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, 24 ^* C) to remove insect tissue and fat droplets. The supernatants were adjusted to pH 7.0, centrifuged (12,000 g; 20 minutes, 4 ^* C), filter sterilized (0.22μ) and stored at 4°C. Fifty ml of filtrate was pipetted into each well (0 = 0.5 cm), cut on TSA (10 ml in each 9-cm plate) freshly inoculated with Bacillus subtilis spore (6 x 10 ⁹ CFU) . The diameter of inhibition zone size was determined after 24 hours of incubation at 25"C.

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)

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 25 ^* 0 for 5 days. The spent culture

media were then neutralized with 6 Normal (N) hydrochloric acid (HCI), 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 HCI, 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 24'C. 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, re- suspended in 15% sterile potato dextrose broth, diluted to give concentrations of 6xl0 ³ macroconidia/ml, 7xl0 ³ macroconidia/ml, and 5xl0 ³ 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 121"C 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 Bύchi 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 Pythiiun 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 24 ^* C 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 IS6) , as follows: Dl X. nematophilus Dl from Steinernema carpocapsae nematode

DS X. nematophilus Dl secondary form as above BC1 X. nematophilus BC1 from Steinernema carpocapsae nematode A2 X. bovienii A2 from __>. 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 Heterorhabdi ts 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, Ceratocγstis 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 Mclnerney 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, Rhino sporidium seeberi , Loboa loboi , Sporothrix schenchii , Histoplasma capsulatum,

695 __

"" _____ / "

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.

TABLE 2 Spectrum of Antimycotic Activity of Species and Strains of Xenorhabdus spp. and Photorhabdus luminescens

Fungal Species Activity of Xenorhabdus Species

Dl DS BC1 BC2 A3 A2 C9

Alternaria sp. A ++ 0 ++ ++ ++ ++ + Alternaria sp. B ++ 0 ++ ++ ++ ++ + Aspergillus niger 0 + + + Beauveria Jbassiana + + + + Botrytis cinerea +++ ++ +++ +++ +++ +++ Cephaloascus fragrans + 0 + + + + Ceratocystis ulmi +++ 0 +++ +++ +++ +++ +++ C. dryocoetidis +++ 0 +++ +++ +++ +++ +++ Fusarium oxysporum ++ ++ ++ ++ ++ ++ ++ F. solani ++ ++ ++ ++ ++ ++ ++

Geotrichum candidum ++ + ++ ++ ++ ++ ++ Gleosporium perannans ++ 0 ++ ++ ++ ++ Metarhizium anisopliae + Monilina fructicola ++ ++ ++ + Mucor pirifor is +++ +++ +++ +++ +++ Oidiodendron griseum + + - - Ophiostoma piceae ++ ++ ++ ++ ++ Pythium coloratum +++ +++ +++ +++ +++ P. ultimum +++ +++ +++ +++ +++ Penicillium expansum ++ ++ ++ + + P. notatum +++ ++ ++ + ++ Penicillium sp. +++ ++ ++ +++ + Rhizoctonia cerealis +++ +++ +++ +++ ++ R . solani +++ +++ +++ +++ ++ Rhizopus stonifer ++ ++ ++ ++ ++ Sclerotinia minor +++ +++ ++ ++ ++ Suillus pseudobrevipes Thielaviopsis basicola +++ +++ ++ +++ ++ Trichoderma pseudokingii +++ +++ +++ +++ +++ Venturia inaequalis + + + + + Verticillium albo-atrum +++ ++ ++ ++ ++ V. dahliae +++ • - +++ ++ +++

KEY: +++ clear inhibition zone that persisted for at least 1 week. ++ clear inhibition zone that was subsequently colonized by aerial hyphae or small clusters of hyphae after a few days. + a zone of poor fungal growth surrounding the Xenorhabdus inoculum no inhibition 0 not tested

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 (Bϋchi) held under 30'C 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

¹ HNMR spectrum of ST1 was recorded on a Bruker WM400 spectrometer in CDC1 ₃ , using residual CHC1 ₃ (~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. ¹³ CNMR datum was recorded on the same instrument in CD ₃ 0D. The IR spectrum was recorded on a Perkin-Elmer S99B spectrometer. (Abbreviations used as follows: El = 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%) .

^J HNMR (CDC1 ₃ ) : 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) .

¹³ CNMR (CD ₃ OD) : 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 x 10 ⁷ Colony Forming Units (CFU) per ml. Aspergillus spp. and Botrytis cinerea were grown for 7 days at 25'C 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 x 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 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 (Bύchi) held under 35 ^* C 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 Xenorha bdus

NMR spectra of each of ID1, ID2, ID3 and ID4 were recorded on a Bruker WM400 spectrometer in CDC1 ₃ , using residual CHC1 ₃ (-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: El = 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, El, m/z (relative intensity): 287 (M+, 10), 227 (M+, -60, 10), 170 (47), 130 (100), 103 (10), 84 (25), 77 (20), 57 (35); IH NMR, δ: 0.86(3H, t, J=7 Hz), 0.98 (3H, d, J=6.5 Hz), 1.39 (IH, m) , 1.73 (1 H, m) , 2.07 (3H, s) , 2.63 (IH, sext, J=7 Hz), 3.18 (IH, dd, J=7 Hz, J=15 Hz), 3.30 (IH, dd, J=4.5 Hz, J=15 Hz), 5.46 (IH, q, J=4.5 Hz, J=8 Hz), 7.05 (IH, d, J=2.2 Hz), 7.15 (IH, t, J=7.5 Hz), 7.21 (IH, t, J=7.5 Hz), 7.37 UH, d, J=7.5Hz), 7.64 (IH, d, J=7.5 Hz), 8.06 (IH, bs, NH) .

ID2, identified as 3- (2' -acetoxy-4' -methyl-3' - oxopentyl) -indole: mass spectrum, El, m/z (relative intensity): 223 (M+, 25), 213 (M+ -60, 15), 170 (60),

130 (100), 103 (6), 84 (12), 77 (8); IH NMR, δ: 0.99 (3H, d, J=6 Hz), 1.09 (3H, d, J=6 Hz), 2.07 (3H, s,

COCH ₃ ) , 2.72 (IH, hept, J=7 Hz), 3.19 (IH, dd, J=7

Hz, J=15 Hz), 3.27 (IH, dd, J=4.5 Hz, J-15 Hz), 5.46 (IH, dd, J=4.5 Hz, J=8 Hz), 7.05 (IH, d, J=2.2 Hz),

7.15 (IH, t, J=7.5 Hz) . 7.21 (IH, t, J=7 5 Hz) . 7.37 (IH, d, J=7.5 Hz), 7.64 (IH, d, J=7.5 Hz), 8.06 (IH, 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+l, 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 (IH, m) , 1.65 (IH, m) , 2.77 (IH, sext, J= 6.5 Hz), 3.04 (IH, dd, J=7 Hz, J=15 Hz), 3.32 (IH, dd, J=4.5 Hz, J=15 Hz), 3.52 (IH, d, J= 5.5Hz, OH), 4.6 (IH, m) , 7.12 (IH, d, J- 2.3Hz), 7.15 (IH, t, J=7.5 Hz), 7.2 (IH, t, J=7.5 Hz), 7.36 (IH, d, J=7.5 Hz), 7.63 (IH, d, J=7.5 Hz), 8.06 (IH, 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 C _i5 E _l9 N0 ₂ : 245.1416, found: 245.1416. Long-range 'H-^C correlation spectroscopic result: the carbon with chemical shift at 110.89 ppm was coupled with the proton with chemical shift at 3.04 ppm (IH, 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). IH NMR, δ: 1.02 (3H, d, J=6.5 Hz) , 1.13 (3H, d, J=7 Hz), 2.88 (IH, hept, J=7 Hz), 3.06 (IH, dd, J=7 Hz, J=15 Hz), 3 31 (IH, dd, J=4.5 Hz, J=15 Hz),

3.45 (IH, d, J=5.5 Hz, OH) , 4 67 (IH, m) , 7.11 (IH, d, J=2.3 Hz) , 7.14 (IH, t, J=7.5 Hz) , 7.2 (IH, t, J=7.5 Hz) , 7.36 (IH, d, J=7.5 Hz) , 7.63 (IH, d, J=7.5 Hz), 8.05 (IH, 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 25"C, 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 x 10 ⁷ Colony Forming Units (CFU) per ml. Aspergillus spp. and Botrytus cinerea were grown on PDA for 7 days at 25'C 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 x 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 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.

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 Dl or ATCC 19061) was cultured in broth medium at 25 ^* C on an oscillating shaker. After 5 days of growth, the spent culture was neutralized with 6 normal hydrochloric acid and centrifuged at ll,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 25'C 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 25"C. 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.

where R is an alky) group of carbon length 1 to 6 with either a straight or branched chain configuration

IDI: 3-(2 ^l -acetoxy-4'-methyl-3 ^l -oxohexyq-lndole

ID3: S-jΣ'-hydroxy^ethyf-a'-oxohexylHndole

ID4: 3-(2'**hydroxy4-methyl-3'-oxopen1yf)-indole

References Cited

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