AU771575B2 - A method of identifying and recovering products exuded from a plant - Google Patents

Description

WO 00/07437 PCT/US99/17893 A METHOD OF IDENTIFYING AND RECOVERING PRODUCTS EXUDED FROM A PLANT This application claims priority to U.S.

Application No. 09/130,185, filed August 6, 1999, the entirety of which is incorporated by reference herein.

This application is related to commonly-owned, co-pending U.S. Application Serial Nos. 09/067,836, filed April 28, 1998, and 09/203,772, filed June 23, 1998, the entireties of which are incorporated by reference herein.

FIELD OF THE INVENTION The present invention relates to a method of identifying and recovering biologically active agents that are exuded from or onto a plant or plant part. The methods disclosed herein will aid in the discovery of new agents, compounds or drugs having diverse biological activities and properties, for treatments of various diseases or conditions, as well as serving as flavors, fragrances, and additives.

BACKGROUND OF THE INVENTION Plants are recognized as being a potential source of chemical compounds (phytochemicals) having biological activity or other properties of interest to the medical, agricultural and food processing communities, among others. A variety of such compounds have been isolated from plants and used either as a crude extract or as purified compounds.

For example, the leaf surfaces of plants are a rich source of phytochemicals. In addition to their internal components, leaf surfaces of higher plants are covered with non-cellular cuticular materials, which are WO 00/07437 PCT/US99/17893 -2non-living, and heterogeneous in chemical composition such as lipids, wax and cutin (biopolymer composing fatty and hydroxy fatty acids), as well as proteins and many secondary metabolites associated with leaf surface or present in hairs or trichomes covering leaf surfaces.

These cuticular compounds can be assessed or removed by rapid immersion of intact leaves in organic solvents or by running the solvents over the leaf surface. It is generally agreed that these techniques avoid contamination by substances within the leaf making the process significantly different from total tissue extraction. The amounts of cuticular compounds present on the leaf surfaces of different species are variable, but normally lie in the range 0.01-0.5mg/cm 2 More cuticular compounds are usually obtained from the lower than the upper leaf surface. Waxes and other cuticular compounds are deposited in early stage of leaf growth and continues throughout the period of leaf expansion. Leaf waxes are known to inhibit spore germination of pathogenic fungi.

In addition to leaves, plant roots continuously produce and secrete a characteristically unique set of compounds into their immediate environment (rhizosphere) While up to 10% of photosynthetically fixed carbon is secreted from the roots in the form of biologically active compounds, the systematic study of compounds present in root exudates of diverse plant species and their biological activity has not been undertaken.

However, certain compounds present in root exudates have been shown to play an important role in several biological processes, including activation of the Rhizobium genes responsible for the nodulation process and, possibly, for vesicular-arbuscular mycorrhiza (VAM) WO 00/07437 PCT/US99/17893 -3colonization. Strigol, a germination stimulant for the parasitic plant Striga asitica, has been found in the root exudates of many cereals. In addition, rootsecreted compounds called phytosiderophores may be involved in the acquisition of essential plant nutrients from soils and in defense against such toxic metals as aluminum. While many biologically active compounds were isolated from extracts of plant roots, screening the root exudates for compounds and isolation of biologically active or anti-microbial compounds from exudates have not been investigated.

In general, compounds have been recovered from roots and leaves and used either as a crude root exudate extract or as purified compounds which require the use of complex extraction and purification procedures.

Heretofore, it was not known that one could identify and recover collections (libraries) novel agents which have biologic activity which are exuded from a plant part, onto the surface of the leaf or in the media in which roots are contained.

SUMMARY OF THE INVENTION This invention provides a method of identifying and recovering chemical compounds or other substances exuded from a plant or plant part. In a preferred embodiment, the invention is directed to recovering compounds from plants which have been subjected to treatments or conditions to induce and increase production of such compounds in the plant. The compounds recovered and identified preferably possess one or more biological activities, including but not limited to such activities as antibacterial, antifungal, anti-herbicidal, insecticidal, anti-cancer, sporicidal cytotoxic WO 00/07437 PCT/US99/17893 -4activities, smell, taste, scents, and flavor enhancement.

According to one aspect of the invention, a method is provided for recovering and identifying biologically active compounds exuded from a portion of a plant covered with a cuticle a leaf surface), the method comprising: removing cuticular material located on the surface of the portion of the plant by contacting the leaf surface with a solvent capable of partially or completely dissolving the cuticular material; assaying the solvent containing the cuticular material for the identification of agents of the cuticular material which have biological activity; and analyzing the solvent containing the cuticular material so as to identify the agent or agents which have the biological activity.

In one embodiment the biological activity is anti-microbial, herbicidal, or one which provides smell, taste, or flavor enhancement. Examples of anti-microbial activity includes antibacterial or antifungal activity.

In another embodiment, the step of assaying the solvent solution comprises contacting the solution or its components with a medium containing a suspension of a microorganism, wherein the inhibition of the growth of the suspension of the microorganism is indicative of an agent in the solvent solution having biological activity.

The medium may be a liquid medium or an agar medium.

In another embodiment the cuticular material is a lipid, wax, cutin, protein, primary or secondary metabolite. In another embodiment the solvent is an organic solvent. Examples of solvents include, but are not limited to, methylene chloride and chloroform. In another embodiment the suspension of microorganism is a bacterium, fungus, or virus. Examples of microorganism WO 00/07437 PCT/US99/17893 include but are not limited to the following: Escherichia coli K-12. F, prototropic Str., Staphylococcus aureus subsp. Aureus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus flavus and Penicillium nigra.

In another embodiment the plant is selected from a group consisting of: Atropa belladonna, Erythrinia glabeliferus, Ipomea tricolor, Erythrinia crista, Celosia cristata, Gallium sporium, Laurus nobilis, Vitis labrissa, Gratiola officinalis, Symphitium officinalis, Hosta fortuna, Casia hebecarpa, Thalictum flavum, Scutellarian altissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geum fanieri, Gentiana tibetica, Linum hirsutum, Aconitum napellus, Podophyllum amodii, Thymus cretaceus, Hosta fortunaea, Carlina acaulis, Chamaechrista fasciculata, Pinus pinea, Pegamun hamalis, Tamarindus india, Carica papaya, Cistus incanus, Capparis spinosa inemis, Cupress lusitanica, Diopiros kaka, Erungiurn campestre, Aesculus woerlitzenis, Aesculus hippocastanum, Cupressus sempervirens and Celtis occidentalis.

In another embodiment, the step of analyzing the cuticle-containing solvent furthers comprises fractioning the agents identified in the solvent. In another embodiment, subsequent to fractioning the solution, the resulting agent is identified.

This invention also provides a method of recovering and identifying agents, preferably biologically active compounds, exuded from or onto the roots of a plant, the method comprising: obtaining a sample of a medium an aqueous medium such as water buffer or liquid growth medium) which contacted plant roots;( b) assaying the sample for biological activity; and analyzing the sample so as to identify the agents WO 00/07437 PCTUS99/17893 which have the biological activity. This method preferably is performed on a living plant, the roots of which are maintained in contact with the medium in order to recover from the medium one or more compounds produced by the plant.

In one embodiment, the method comprises adding an elicitor to the aqueous media prior to removal of the roots from the media. In another embodiment the method further comprises adding an elicitor to the aqueous media during the growth of the roots. In another embodiment the elicitor is an abiotic or biotic elicitor. Examples of biotic elicitors include but are not limited to the following: chitosan, or fungal and bacterial cell wall fragments, methyl salicylate and methyl jasmonate.

Examples of abiotic elicitors include, but are not limited to, silver nitrate and acetic acid.

In another embodiment, the plant is subjected to conditions known to induce production of novel compounds, or to increase production of compounds of interest, after which such compounds are recovered from the medium contacting the roots. Such conditions include heat stress, drought stress, salt stress, over- or underillumination and nutrient deprivation, among others.

In accordance with a further aspect of the invention, there is generated a chemical compound library which may be used for screening for a desired compound or activity. In one embodiment, plants or plant parts which are specifically grown or maintained for the purpose of recovering compounds therefrom are contacted with water or another aqueous medium while alive in order to recover a variety of compounds for potential screening. In another embodiment, cuticle covered plant parts are treated with a cuticle-dissolving solvent in order to WO 00/07437 PCT/US99/17893 -7recover compounds from cuticular material. The plants or plant parts are preferably contacted with an elicitor, or subjected to the growth conditions summarized above, to increase the amount and/or diversity of compounds which can be recovered in the plant exudates.

Other features and advantages of the present invention will be appreciated from the drawings, detailed description of the invention, and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Agar plate showing antimicrobial activity of the root exudate of Laurus nobilis (1881) against Escherichia coli.

Figure 2. Agar plate showing antimicrobial activity of the root exudate of Gentiana tibetica (1881) against Escherichia coli.

Figure 3. Agar plate showing antimicrobial of the root exudate of Aconitum napellus (1881) against Escherichia coli.

Figure 4. Agar plate showing antimicrobial activity of the leaf surface compounds (identified on the Figure as samples) of Erythrina christagalli (1363) against Staphylococcus aureus.

Figure 5. Agar plate showing antimicrobial activity of the leaf surface compounds (identified on the Figure as samples) of Laurus nobilis (1513) against Staphylococcus aureus.

Figure 6. Agar plate showing antimicrobial activity of the root exudate of Scutellaria altissima (1671) against Staphylococcus aureus.

Figure 7. Agar plate showing antimicrobial activity of the root exudate of Scuttellaria creticola (1691) against Staphylococcus aureus.

WO 00/07437 PCT/US99/17893 -8- Figure 8. Agar plate showing antimicrobial activity of the root exudate of Hosta fortuna (1645) against Saccharomyces cerevisiae.

Figure 9. Agar plate showing antimicrobial activity of the root exudate of Cunninghamia lancelota (2489) against Aspergillus flavus.

Figure 10. Agar plate showing antimicrobial activity of leaf surface compounds (identified on the Figure as samples) from leaves of Thymus citriodorus "aureus" (59) and Hydrocotyle asiatica (32a) against Staphylococcus aureus.

Figure 11. Agar plate showing antimicrobial activity of leaf surface compounds (identified on the Figure as samples) from leaves of Betula pendula (24) against Staphylococcus aureus.

Figure 12. Agar plate showing antimicrobial activity of leaf surface compounds of Eucalyptus radus (229) against Staphylococcus aureus.

Figure 13. Agar plate showing antimicrobial activity of leaf surface compounds of Eucalyptus radus (229)against Saccharomyces cerevisiae.

Figure 14. Agar plate showing antimicrobial activity of leaf surface compounds of Oreopanax capitata (216) against Staphylococcus aureus.

Figure 15. Agar plate showing antimicrobial activity of leaf surface compounds of Oreopanax capitata (216) against Escherichia coli.

Figure 16. Graphical presentation of the amount of daidzein recovered from root exudates produced by soybean plants treated with different elicitors.

Figure 17. Graphical presentation of the amount of genistein recovered from root exudates produced by soybean plants treated with different elicitors.

WO 00/07437 PCT/US99/17893 -9compounds compounds compounds juncea.

compounds compounds polyphyllu Figure 18. HPLC profile of the diversity of recovered from root exudates of various plants.

Figure 19. HPLC profile of the diversity of recovered from Lupinus luteus.

Figure 20. HPLC profile of the diversity of recovered from root exudates of Brassica Figure 21. HPLC profile of the diversity of recovered from root exudates of Datura metel.

Figure 22. HPLC profile of the diversity of recovered from root exudates of Lupinus Is.

Figure 23. HPLC profile of the diversity of compounds recovered from root exudates of Meliltotus medicaginoides.

DETAILED DESCRIPTION OF THE INVENTION The plant or plant portion that is subjected to one or more of the processes of the present invention is a living plant, or obtained from a living plant. The plant may be any plant as described in greater detail herein. Any portion of the plant may be utilized, including but not limited to, leaves, shoots, seeds, seedlings, stems, flowers and roots.

As defined herein "cuticular material" means cutin and other materials located in the cuticle covering the leaf surface or leaf hairs or trichomes of a plant.

As defined herein "exudate" refers to substances which are exuded, secreted, released or deposited from a plant or plant part, either onto the surface of the plant or plant part, or into the surrounding medium.

As defined herein, "antimicrobials" means the WO 00/07437 PCT/US99/17893 spectrum of organisms against which they are active, whether they kill the organism or merely slow its growth and reproduction cidal or static) and the biochemical system on which they exert their major biochemical action inhibit protein synthesis or cell wall synthesis). Antimicrobials include agents acting against any microorganism, including gram positive or gram negative bacteria, Rickettsia, fungi or protozoans.

The plant preferably is a higher plant. It is contemplated by this invention that any plant may be employed in the method. For example, the following plants may be employed in order to identify agents which are exuded: Atropa Belladonna, Erythrinia glabeliferus, Ipomea tricolor, Erythrinia crista, Celosia cristata, Gallium sporium, Laurus nobilis, Vitis labrissa, Gratiola officinalis, Symphitium officinalis, Hosta fortuna, Casia hebecarpa, Thalictum flavum, Scutellarian altissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geum fanieri, Gentiana tibetica, Linium hirsutum, Aconitum napellus, Podophyllum amodii, Thymus cretaceus, Hosta fortunaea, Carlina acaulis, Charnaechrista fasciculata, Pinus pinea, Pegamun hamalis, Tamarindus india, Carica papaya, Cistus incanus, Capparis spinosa inemis, Cupress lusitanica, Diopiros,kaka, Erungium campestre, Aesculus woerlitzenis, Aesculus hippocastanum, Cupressus sempervirens and Celtis occidentalis.

Further plants species used for screening exudates are as follows: Polygonum cuspidatum, Eleagnus angustifolia, Eleagnus cemutata, Gentiana macrophilla, Brassica napa, Sesbania exaltata, Sesbania speciosa, Spartina potentiflora, Brassica juncea, Helianthus annus, WO 00/07437 WO 0907437PCTIUS99II 7893 -11- Puansetia sp., Pelargoniurn zonale, Sundapsis spp., Leon topodiun alpinun, Lupinus luteaus, Buxus microphilla "japonica", Liatris spinata, Rimula japonica, Betula nigra, Filipendula vuigrais, Lobelia siphitica, Gra villa robusta,. Reseda luteola, Gentiana littorala, Campanula carpa ti ca, Aesculus hypocastanum, Aesculus waertilensis, Agera turn conizoides, Psidiun guajava, Ailantus altissina, Buxus microphylla "japonica"l, Hydrocotile asiatica, Gravilea robusta, Brugmansia suaveolens, Thyrnus puliglodes, Thymus lemabarona, Thyrnus serphylum (wild), Gaultheria procumbens, Thymnus serphylun, Thymus camosus, Thymnus thrasicus, Calicatus floridus, Zingiber off icinalis, Lapia dulcis, Thymus vulgaris "argenteus", Thumus praecox "arcticus", Thymus puleglodes "lemons", Thymnus speciosa, Thymus carnosus, Thymus pseudol arginosus, Thymus praecox, Thymus vulgari s "ore gano", Ficus religiosa, Forsi thsia suspensa, Chelidonium majus, Thymus woolyv, Thymus portugalense, Nicotiana tabacum, Thymus cytridorus "aureus", Thymnus vulgaris, Cactus off icinailis, Lal lab purpurea, Juglands regia, Actinidia chinensis, Hernerocalis spp., Betula pendul a, Gardenia j asminoi des, Taxodiurn dixti cum, Magnolia loebheril, Crataegus praegophyrum, Larix dedidua, Tuja orientalis "eligantissima"l, Tula ocidentalis "columbia"l, Xeupressocyparis deylandii, Pseudotsuga menzisia, Abies firma, Fautenousus qualiqualia, Alium cernum (wild), Juniperus "blue pacific", Taraxacum off icinalis, Juca sp., Ilex agnifolium, Tsuga canadensis "penola", Tsuga canadensis "penola", Ilex cornuta, Taxus xiksii, Taxus media, Metasequoia glyptotrobioldes, Pinus bungiaria, Boxus sempervirens, Stevartia coreana, Prunus xocane, Betula daurica, Plan tago minor, Acer palma turn "burgundy", Acer campestre, Cotynus cogygria, Quercus robur "fastigiata"l, WO 00/07437 WO 0007437PCTIUS99/1 7893 -12- Acer trunca turn, Archirantus bidentata, Alum japonica, Carun capsicumn, Agastache mexuicana, Prunella -Vulgaris, Tagetes rninuta, Nepeta cataria, Ratibiunda columnus-Fera, Aster-Nova anglicae, Mi ri ca certif era, Pit tisporum tibica, Taxodium dixticum (H20), Taxodiun dixticun (Acetic acid), Plantago major, Scotch pine, Asorum canadiensis, Pieras japonica, Pinus sirtrobus, Trifolium pra tense, Prunus serotica, Darura strarnonium, Geranium maculata, Hydrocotile asiatica, Astragulus sinicus, Centauria maculata, Ruschia indurata, Myrthus comunis, Platanus acidentalis, Liclun barbatum, Lavandula off icinalis, Gravilea robusta, Hyppoach rhamnoides, Filipendula ulmaria, Betula pendula Polygonium odora turn, Brugnansa.

graveolens (raif), Rhus toxicodenta, Armoraica ristica, Ficus benjarninii, Sluffera sp., Pelagonium zonale, Allium sp. Asirnina triloba, Lippa dulcis, Epilobium augustifolium, Brugmansia suavecolens (old), Brugmansia suaveolens (young), Xanthosona sagittifolium (leaf), Xanthosona sagi ttifolium (stem), Monstera deliciosa, Aglaonena commutatus, Dieffenbachia leopoldii, Anthurium andreanurn, Syngoniurn podophyllun, Dracaena fragrans, Ananas comosus, Strelitzia reglinae, Diffenbachia segiunae, Syngoniurn aurutum, Dracaena sp., Hhaernanthus katharina, Anthuriun altersianum, Spa thiphyllun grandiflorun, Spa thiphyllun cochlearispa turn, Monstera pertusa, Anthurium magnificurn, Anthuriun hookeri, Anthurium elegans, Calathea zebrina, Yucca elephantipes, Bromelia balansae, Musa textilis (Leaf), textilis (Stem), Myrthus communis, Olea olcaster, Olea europaea, Veriurn oleander, Cocculus laurifolius, Microsorium puncta turn, Ficus sp., Senseviera sp., Adansonia digitata, Boechirneria boloba, Piper nigrun, Phyrnatosorus scolopendria, Turnera ulmifolia, Nicodemia diversifolia, Tapeinochilos spectabilis, Rauwolfia tetraphylla, Ficus WO 00/07437 WO 0007437PCT/US99/17893 -13elastica, Cycas cirinalis, Caryota ureus, Cynnarnonum zeylonicum, Aechmea luddemoniana, Foenix zeulonica, Ficus benjamina, Ficus purnila, Murraya exotica, Trevesia sungaica, Clerodendrurn speciossicun, Actinidi coloni cta, Paeonia lactiflora, Paeonia sufifructicisa, Quercus imbricaria, Iris alida, Portulaca olleracea, Poligonurn aviculare, Iris pseudocarpus, Allium nu tans, Allium fistulosum, Antericum ramosun, Vera trum nigrum, Poligornun latifolia, Hosta lancefolia, Hosta zibalda, Echinops sphae, Paeonia daurica, ITnula hilenium, Trarabe pontica, Digitalis lutea, Bactisia australis, Austolachia australis, Hissopus zeraucharicus, Feucrium hamedris, Sedum album, Heraclelum pubescens, Origanurn vulgare, Cachris alpina, Haser trilobum, Matteucia strutioptoris, Sedum teichiun, Bocconia cordata, Hiuga reptans,* Talictrum minus, Anemona japonica, Clematis rectae, Talictrum sp., Aichemilla sp., Potentilla alba, Poterium sangiusorba, Minispermum dauricum, Oxobachus nictogenea, Armoracea rusticana, Craznble cardifolia, Agrimonia eupatora, Uschusa sp., Polymonium ceruleum, Valeriana offocionalis, Pulmonaria molissima, Stachis lanata, Coronolla varia, Platicada grandiflora, Lavandula off icinali s, Vincetoasi cum off icinali s, Acolyvpha hi spida, Gnetum guemon, Psychotria nigropunctata, Psycho tria metbacteriodomasica, Cobiaeum varilaturn, Phyllan thus grandifolium, Pterigota alata, Pachyra affinis, Sterulia elata, Phylidendron speciosus, Pithecelobium unguis, Sanchezia nobilis, Oreopanax capitata, Ficus triangularis, Pigelia pennata, Piper chaba, Laurus nobilis, Erythrinia caffra, Metrosideros excelsa, Osmanthus spp., Cupressus sempervi-rens, Jacobinia sp.., Senecio platifilla, Livistona fragrans, Tetraclinis articula ta hinensi s, Eucaliptus rudis, Podocarpus spinulosus, Eriobotria japonica, Gingko biloba, WO 00/07437 WO 007437PT/US99I17893 -14- Rhododendron spp., Thuja occidentalis, Fagopyrum sufifruticosun, Geum macrophullun., Magnolia cobus, Vinca minor, Convalaria majalis, Cor~ylus avelana, Barbaric sp., Rosa inultiflora, Ostrea carpinifolia, Ostrea connote, Quercus rubra, Tulip tree, Sorbus aucuparia, Betula nigra (leaf), Betula nigra. (flower), Castanea sativa Bergenia crassifolia, Arternisia dracunculus, Ruta gra veol ens, Quercus nigra, Schisandra chinensis, Betula alba, Sambucus niora, Gentiana cruciata, Encephalaris horridun, Phebodium aureun, Microlepia platphylla, Ceratorania mexicana, Stepochlaena tenuifolia, Adianthum trapezi eformi s, Adi an thum radi aturn, Lycodiurn j aponi cum, Aessopteria crasifolia, Aspleniun australasicum, Agatis robusta, Osmunda regalis, Osrnundastrum claytonionum, Phyllitis scolopendriun, Polyschium braunii, Crytomiun fortunei, Dryopteris filis-max, Equisetum variega turn, Anthyrium nopponicum, Anthyriun filis-fenina, Parthenosicus tricuspidata, Ligus turn vulgare, Charnaeciparis pisif era, Rosa cocanica, Citinis coggriaria, Pinus strobus, Celtis occidentalis, Picea schrenkiana, Cydonia oblonga, Ulrnus pumila, Euonornus verrucosa, Deutria scabra, Mespilus germanica, Quercus castanufolia, Euonornus europea, Seruginea suffruticisa, Keyleiteria paniculata, Seringa josiceae, Zelcova, carpinifolia, Abies cephalonica, Taccus bacata, Taxus cuspidata, Salis babilonics, Thuja occidentalis, Actinidia colornicta, Magonia agrifolia, Aralis mandshurica, Luglands nigra, Euonirnus elata, Princepia sp., Forsitsia europea, Sorbocotoneaster sp., Morus alba, Crategus macrophyllun, Eucornia ulurifolia, Sorbus corninicta, Philodendron arnurense, Cornus mass, Korria japonica, Parrotia persica, Jasrninum frutocarus, Sulda sanganea, Pen taphylloides fruticosa, Sibirea al taiensis, Cerasus japonica, Kolkwitzia arnabilis, Amigdalus nana, WO 00/07437 PCT/US99/17893 Acer mandshurica, Salix tamarisifolia, Amelanchier spicata, Cerasus maghabab, Prunus cerasifera, Coryllus avelana, Acer tataricum, Viburnum opulus, Siringa vulgaris, Fraxinus exelsior, Quercus trojana, Chaernomelis superba, Pinus salinifolia, Berberis vulgaris, Cotoneaster horisontalis, Cotoneaster fangianus, Fagus silvatica, Pinus pumila, Pinus silvestris and Berberis thungergi.

Recovery of substances exuded into aqueous media: In one aspect of the invention, compounds exuded from the plant into a surrounding aqueous medium are recovered and thereafter identified or otherwise analyzed. The aqueous medium may be water, which may or may not contain other constituents, such as nutrients, elicitors and similar substances commonly found in hydroponic plant culture media.

In accordance with a preferred aspect, the chemical compounds are those which are exuded, secreted or leached from the plant, preferably from the root or plant seedling or shoot. In the case where seedlings or shoots are used, the plant seed, shoot or root may be treated with an elicitor or inducer to increase production in the plant of one or more produces, as described in greater detail below. The inducer or elicitor may be in the aqueous solution that contacts the plant for recovery of the exuded compounds, or may be separately applied to the plant.

The contact with the aqueous medium may be effected by placing at least the plant roots in water, or by "aeroponics", which involves contacting the plant, particularly the roots, with water droplets from which chemical compounds are recovered.

WO 00/07437 PCTIUS99/17893 -16- In a preferred embodiment, secreted substances are recovered from a plant in a continuous process by having the plant roots (or rooted shoot) of the plant or shoot in contact with the aqueous medium, which may be continuously or periodically passed over the roots to recover the substances secreted into the medium. The aqueous medium is then assayed for biological activity, or further subjected to processes for recovering the substances contained therein.

In another embodiment, seedlings are employed as a source of the phytochemicals. In this embodiment, seeds are germinated in aerated aqueous medium, which is recovered on a continuous or semi-continuous basis in order to obtain and identify the secreted substances.

Thus, a preferred embodiment of the invention provides a method of identifying an agent from root exudates of a plant having biological activity, the method comprising: obtaining a sample of media which contacted plant roots; assaying the sample for biological activity; and analyzing the sample so as to identify the agent which has the biological activity.

In any of the embodiments set forth above, the method may further comprise treatment with an elicitor or inducer. The elicitor may be an abiotic or biotic elicitor. Examples of biotic elicitors include but are not limited to the following: chitosan, fungal or bacterial cell walls or fragments thereof, cultivation medium, methyl salicylate and methyl jasmonate. Examples of abiotic elicitors include but are not limited to silver nitrate, or acetic acid. The plants, in particular roots thereof, may be contacted with an elicitor or inducer, which is a chemical compound, for example, organic and inorganic acids, fatty acids, WO 00/07437 PCT/US99/17893 -17glycerides, phospholipids, glycolipids, organic solvents, amino acids and peptides, monosaccharides, oligosaccharides, polysaccharides and lipopolysaccharides, phenolics, alkaloids, terpenes and terpenoids, antibiotics, detergents, polvamines, peroxides, ionophores. etc., or subjected to a physical treatment, such as ultra-violet radiation, low and high temperature stress, osmotic stress induced by salt or sugars, nutritional stress defined as depriving the plant of essential nutrients N, P or in order to induce or elicit increased production of one or more chemicals. Such chemical compound or physical treatment may be applied continuously or intermittently to the plant or plant part. In one embodiment, such treatment may be accomplished by contacting the plant roots with a solution containing the elicitor or by irradiating the plant as temperature stresses. However, the invention is not limited to such an embodiment in that other portions of a plant or seedlings may be contacted with an elicitor. For example, a glycopeptide elicitor may be prepared from germ tubes of the rust fungus Puccinia graminis Pers. f. sp. tritici Erikss. Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (E.

C. 1. B. 11. 12) in wheat (Triticum aestivum) leaves.

The effects of elicitor concentration and exposure time on growth and levels of biologically active compounds vary. For example, transient studies at the same level demonstrated possible catabolism as serpentine, tabersonine, and lochnericine levels decreased immediately after elicitation. The levels of these compounds recovered back to control levels or were higher than the control levels after some time. Jasmonic acid WO 00/07437 PCT/US99/17893 -18was found to be a unique elicitor leading to an enhancement in flux to several branches in the indole alkaloid pathway. Jasmonic acid addition caused an increase in the specific yields of ajmalicine serpentine lochnericine (15 and horhammericine (500%) in dosage studies. Tabersonine, the likely precursor of lochnericine and horhammericine, decreased at lower levels of jasmonic acid and then increased with increasing jasmonic acid concentration.

Transient studies showed that lochnericine and tabersonine levels go through a maximum, then decrease back to control levels and reduce below control levels, respectively. The yields of ajmalicine, serpentine, and horharnmericine increased continuously after the addition of jasmonic acid.

Recovery of compounds from cuticular material: In another aspect of the invention, compounds exuded from the plant onto or into the cuticle of the plant are recovered by contacting the plant part containing the cuticular material with a solvent that partially or completely dissolves the cuticular material, thereby removing that material from the plant for subsequent identification of compounds of interest disposed therein.

The cuticular material may comprise lipid, wax, cutin, protein, primary or secondary metabolite.

Solvents that dissolve such materials generally are composed at least in part of organic solvents. Examples of suitable solvents include, but are not limited to, methylene chloride and chloroform.

Elicitors and other treatments to induce production of novel compounds, or to increase production WO 00/07437 PCT/US99/17893 -19of a compound of interest, may be used in this aspect of the invention in a manner similar to their use described above. The plant is treated as described above, prior to contacting the plant part with the cuticle-removing solvent.

Identification and analysis of compounds of interest in plant exudates: The results presented herein demonstrate that agents having biological activity can be identified by employing the cuticular exudate and root exudate assay.

Specifically, a total of 400 plant species cuticular washings has been prepared and tested against microbial/fungal cultures. A significant percentage of cuticular washings shows antimicrobial activity. Figures 4, 5, 10, 11, 12, 13, 14 and 15 demonstrate the results of this activity.

In one embodiment, the step of assaying the various solutions containing the exudates comprises contacting the solution or its components with a medium containing a suspension of a microorganism, wherein the inhibition of the growth of the suspension of the microorganism is indicative of an agent in the exudate having biological activity. The medium may be a liquid media or an agar media. Microorganisms including but not limited to bacteria or fungi, may grow within, or develop in or on the medium. Inhibition of the growth is detected by standard means known to those skilled in the art. For example, the growth inhibition on agar may be measured in terms of zone of inhibition which is known to those skilled in the art.

Two common applications of agar diffusion assays are potency testing of new production lots in the WO 00/07437 PCT/US99/17893 pharmaceutical industry and bacterial susceptibility testing. They are based on the same principles, but susceptibility assays use unknown bacterial strains, and potency assays use bacterial strains with well characterized performance against the test drug. Agar diffusion potency assays are relatively comparable in sensitivity and accuracy to radiometric enzyme, fluorescent (FIA) ELISA. For example, single-plate assay is based on having all standard and unknown concentrations on one single plate. This eliminates plate to plate variation, and facilitates layout and reading. Alternatively, 2 or 3 identical plates may be laid out, examined and averaged. Usually NUNC (Denmark) large 243 mm square plates are used, which allow up to 64 samples that easily fit on one plate. It thus provides for up to 6 standards, and up to 10 samples, with 4 replicates of each standard and unknown sample concentrations, on one plate. Multiple-Plate format uses many 90-100 mm petri dishes, and conforms strictly to US- FDA, US-CFR and US-USP published methodology. Zone diameters are measured by Video or Caliper directly into the software.

The suspension of microorganism may comprise a gram positive or a gram negative bacterium, protozoan, fungus, or virus. Examples of gram positive and gram negative bacteria are known to those skilled in the art.

Microorganism include but are not limited to the following: Escherichia coli K-12. F, prototropic Str., Staphylococcus aureus subsp. Aureus, Pseudomonas aeruginosa, Saccharomyces Cerevisiae, Aspergillus flavus and Penicillium nigra.

In another embodiment, the step of analyzing the solution furthers comprises fractioning the solvent WO 00/07437 PCT/US99/17893 -21solution directly or following drying and resuspension step. In another embodiment, subsequent to fractioning the solution, the resulting agent is identified.

Fractionation methods are known to those skilled in the art. For example, chromatographic methods, for example HPLC, may be employed to identify the compound. The chromatography separation of extracted products may be employed with an HPLC-system consisting of Waters 996 Photodiode Array Detector (PDA) with usable UV range from 190 to 800 nm; a Waters 717 plus autosampler; two Beckmanll0B solvent Delivery Modules, connected with a Beckman System Organizer (mixer) and a Beckman System Gold Analog Interface Module 406. The Beckman solvent delivery system is controlled by a NEC PC-8300 computer.

Chromatography and spectral data is managed by Waters Millennium chromatography software, version 2.10, using a NEC Image 466es computer. All hardware components, except the solvent delivery system, are connected through a standard IEEE communication system. Compounds are separated Son a Waters Nova Pak6 C-18 reverse 25 phase column, 3.9 x 150 mm, 60A pore size, and 4pm particle size. Prior to use, each batch of solvent A is digested under vacuum and ultrasonications for 5 minutes. The mobile phase flow is adjusted. to Iml/min, and a gradient mode of separation is used for all separations.

Compounds are detected with PDA detector or with Waters Thermabeam T m Mass Detector.

Further, one may fractionate the sample by chromatography techniques followed by the chemical structure analysis using mass spectroscopy; infra red spectroscopy; or 1D or 2D nuclear magnetic resonance spectroscopy (proton or 13C. Fractionation and analysis methods are known to those skilled in the art. As WO 00/07437 PCT/US99/17893 -22demonstrated herein, cuticular washings from Betula pendula showed very high activity. After 10 repeat experiments the sample was fractionated by HPLC. From fractions, fraction No. 3 showed very high antibacterial activity for Staphylococcus aureus. After analyzing this fraction on LC-MS, a fragmentation pattern of peak A66(the main component in all active fractions) with many steroids as cholestane derivatives and lanostane derivatives resulted.

The methods described in this invention could generally be used in devising strategies for enhancement in productivity of secondary metabolites and for probing and studying the complex secondary metabolite pathways in plant tissue cultures. The methods are considered to be of great utility in recovering novel compounds for use in medicine, agriculture and the food industry, among others.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1 Bioassay of Leaf and Root Exudates Methods Preparation of cuticular washings: Leaves from plants were contacted with 5 ml of solvent (methylene chloride) contained in a plastic sandwich bags (quart size, 7in x 8in)as containers. To standardize the cuticular wash concentration by relating it to the used leaf surface from which it was taken, approximately cm2 of leaf surface were used. Sandwich bags with zippers were used to guarantee that the surface of leaf WO 00/07437 PCT/US99/17893 -23is totally moisturized with solvent. To facilitate the removal of cuticular compounds the bag containing a leaf and the solvent was shaken for approximately 20-40 seconds. The end of bag was cut and the content removed into 20ml scintillation vials,and closed with Teflon or foil faced liner screw caps and stored in refrigerator.

Alternatively, the solvent containing the cuticular washings can be dried inside the scintillation vial before cold storage.

Preparation of bacteria and fungus suspensions: 6 different organisms were used for antibacterial and antifungal screening: 1. Escherichia coli K-12. F, prototropic Str.; 2. Staphylococcus aureus subsp.

Aureus; 3. Pseudomonas aeruginosa; 4. Saccharomyces cerevisiae; 5. Aspergillus flavus; 6. Penicillium nigra.

Bacteria (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) were maintained on solid agar media (LB Agar, Miller). Before screening, bacteria was transferred into liquid media and cultivated for 12 hours at +3 7oC on shaker with a cell density 1056.

Saccharomyces cerevisiae (yeast), Aspergillus flavus and Penicillium nigra were cultivated on Potato dextrose media. Before treatment yeast cells were transferred into liquid media and cultivated for 48 hours at +300C on a shaker. The spores of Aspergillus flavus were washed with distilled water from fungus surface grown in Petri dish and resuspended in fresh distilled water.

The standard method used to determine in vitro antibacterial and antifungal activity of leaf surface cuticular washings consisted of testing suspension of microorganisms and spores of fungus for growth inhibition in the presence of washings. The antibacterial and antifungal activity was indicated by 30% or more, growth reduction of cells/spores in the presence of cuticular WO 00/07437 PCT/US99/17893 -24washings. All samples were plated in 3 replicates. One ml of nutrient media was put in each well in 24 well plates. Thereafter, 10 ul of methylene chloride solution containing cuticular washings were placed on agar surface and after the drop dried (2-3 min) 30 ul of microorganism suspension or fungus spores were plated on top of agar and equally distributed throughout the surface. After 24 hours of incubation at +300C, the plates were examined for the presence/absence of activity. To test antimicrobial/antifungal activity of root exudates, cell suspension was plated and spread on the agar surface into each of 24 well plates. Using a 5ml Eppendorf pipet tip attached to a vacuum line, a hole was made in the center of each well and 20A1 of exudate dissolved in water 5mg/300ml) gently poured into the hole. The following elicitors were employed: methyl salicylate, methyl jasmonate, silver nitrate, acetic acid and chitosan.

To harness the vast and largely unexplored diversity of biological natural products exuded by plant roots, an efficient method for collecting root exudates from various plants was developed. This method is based on a modified hydroponic technology which allows maintaining plant roots in water or diluted nutrient solution followed by of compounds exuded from roots.

The seeds of cultivated and wild species obtained from the commercial seed companies or botanical gardens were germinated in a greenhouse inside a 0.9 cm in diameter, 0.cm deep well cut into Grodan rockwool cubes (3.4 cm width x 3.4 cm depth x 3.7 cm height).

Rockwool cubes were placed inside standard greenhouse plastic trays (dimensions 52 cm width x 25 cm depth x 7 cm height)and watered with an overhead misting system.

Seeds were allowed to germinate for 3-6 days until the roots started to emerge from the bottom of the rockwool cube.

WO 00/07437 PCTIUS99/17893 After germination, the cubes containing the seedlings were inserted into the center of a Styrofoam ring with an inside diameter 3.2 cm, outside diameter 8.2 cm and 2.5 cm thickness. The ring was floated on the surface of 400-800 mL of hydrophic nutrient solution (2 g/L Hydro-Sol [Scotts-Sierra Horticultureal products Comp., Marysville, OH.] supplemented with 1.2 g/L Ca[N0 3 2 contained inside a light impermeable, high density polyethylene cylinder (9.0 cm in diameter, 16 cm in height) Aeration was provided by shaking the cylinders at 50 rpm on a platform shaker (Labline Orbital Shaker, Model 3590). Seedlings were cultivated hydroponically in this system for 3 to 6 weeks with roots growing in a nutrient solution. Thereafter, the root system (average root dry weight 0.1±0.05g) was removed from the nutrient solution and placed inside a 30 mL glass beaker, containing 10-20 mL of distilled water or distilled water supplemented with an elicitor. To prevent water loss from the plant canopy and drying of the collecting solution, plant shoots were covered with transparent plastic bags. After 24h, unless noted otherwise, a small sample from the root solution was removed and analyzed for the phytosecreted products. This system of hydroponic plant cultivation and exudates collection is referred to as standard exudate collection system. Root exudates may also be freeze-dried and stored in the freezer at -20 0 C. When needed, the exudate powder may be re-dissolved in water and used for screening or chemical analysis.

Results A total of 844 plant species root exudates elicited have been prepared and tested against six-above mentioned microbial/fungal cultures (Table II). The WO 00/07437 PCT/US99/17893 -26final concentration of exudates used in assay was mg of dry exudate diluted in 300 il of distilled water.

It has been found that great majority of material tested at this concentration did not affect adversely growth of the tested organisms. Figures 1, 2, 3, 6, 7, 8, 9 demonstrate the results of the inhibition of the biological activity. A number of primary hits has been identified against all, but one (Aspergillus) microorganisms tested. The hit rate under the conditions used varied between 0% and 7.8% (Table It is noteworthy that majority of the hits come from exudates from elicited roots. The unusually high proportion of hits in the materials elicited by silver may also be partially explained by the toxic effects of silver on a given microorganism.

Table 1. Frequency of antimicrobial effects on root exudates Target Organism Number of hits Hit Rate Escherichia coli 23 (884)* 2.6 Staphylococcus aureus 34(884) 3.8 Pseudomonas aeruginosa 8(102) 7.8 Aspergillus flavus 0(510) 0 Penicillium nigra 4(102) 3.9 Saccharomyces cerevisiae 6(718) 0.8 Number in parenthesis indicate total number of root exudate samples tested for a particular microorganism.

WO 00/07437 PCT/US99/17893 -27- Table II List of plant species from which root exudates show antimicrobial/antifungal activity. The strengths of activity is denoted by the number of with a larger number of II II referring to a greater activity.

Af Ec Sa Sc Pn Pa Aspergillus flavus Escherichia coli Staphylococcus aureus Saccharomyces cerevisiae Penicillium nigra Pseudomonas aeruginosa Plant Name Elicitor Af Ec Sa Sc Pn Pa 1 845 Atropa belladonna Silver Erythrina 2 857 Silver glabelliformis 3 949 Ipomea tricolor Silver 4 1363 Erythrina galli Silver 1475 Celosia cristata Acetic a.

6 1501 Gallium spurium Acetic a. 7 1513 Laurus nobilis Silver 8 1563 Vitis labrusca Control Gratiola 9 1585 Control officinalis Symphytum 1617 Silver officinalis 11 1645 Hosta fortunea Acetic a.

12 1649 Cassia hebecarpa Control 13 1659 Thalictrum flavum Acetic a.

WO 00/07437 PTU9/79 PCT/US99/17893 -28- Scutellaria 14 1671 Silver alt is sima Portulaca 1681 Silver* oleracea Portulaca 16 1683 olrcaChitosan* Portulaca 17 1685 olrcaMeta* Scutellaria 18 1691 ceioaSilver* 19 .1695 Physalis ixocarpa Silver- 11757 Geum fourieri Control 21 1791 Gentiana tibetica Silver- 22 1875 Linum hirsutum Acetic 23 11879 Aconitum napellus Acetic a.

24 1881 Aconitum napellus Silver- 1887 Podophyllum Silver aunodi i 26 1897 Thymus cretaceus Silver* 27 1913 Hosta fortunea Silver *28 1975 Hosta fortunea Chitosan 29 1985 Carlina acaulis Silver-*- Charnaechri sta 003fasciculata Sle 31 2013 Pinus pinea Silver-- 32 2043 Peganum harmala Silver* 33 12053 Tamarindus india Silver 34 12063 Carica papaya Silver* 12111 Cistus incanus Control 36 2161 Capparis inerrnis Silver WO 00/07437 WO 0007437PCTIUS99/1 7893 -29- 37 2117 Cpes Silver lusitanica 38 2133 Diopiros khaki Acetic a. 39 12135 Diopiros khaki Silver-- 2137 Diopiros khaki Chitosan -41 2145 rygimSilver* campestre Aesculus 42 255woerlitzienis Sle Aesculus 43 265hippocastanum Sle 44 2299 Cpes Silver sempervi rens Celtis 2315 Silver occidentalis 46 325Calycanthus Sle fl1oridus Chimonanthus 47 2335 Control** praecox Clematis 48 2345 Silver** ranschurica 49 2377 Liatris spicata Silver* 2379 Liatris spicata Silver* 51 2407 Cladium mariscus Silver 52 2425 Lablab purpureus Silver 53 2455 CmauaSilver* carpatica 54 2443 Chilopsis Silver* linearis 2461 TuaSilver I Loccidentalis WO 00/07437 PCT/US99/17893 56 2479 Cosmos sulphureus Silver Cunninghamia 57 2489 Silver lanceolata Euptelea 58 2503 Silver pleiosperma Acetic 59 2535 Juglands regia a.

A total of 400 plant species cuticular washings has been prepared and tested against six-above mentioned microbial or fungal cultures and the amount of cuticular washings used for each well was 10 pl. It has been found that a majority of materials tested at this concentration did not affect adversely growth of the tested organisms.

However, a significant percentage of cuticular washings showed antimicrobial activity. Figures 4, 5, 10, 11, 12, 13, 14, 15 demonstrate the results of this activity. A number of strong antimicrobial hits have been identified for Escherichia coli, Staphylococcus aureus and Saccharomyces cerevisiae (Table III). The hit rate among cuticular washings from different plant species varies from 0.5 to 5.0% depending on the microorganism.

WO 00/07437 PCT/US99/17893 -31- Table III. List of plants which leaf surface cuticular washings show strong antifungal or antimicrobial activity Sample Plant name Sa Ec Sc Identification 1 125 Taxodium dixticum X 2 133 Gravilea robusta X 3 136 Betula pendula x 4 171 Anthurium elegans x 198 Foenix zeulinica X X 6 216 Oreopanax capitate X X 7 229 Eucaliptus rudis X X X 8 248 Betula nigra X 9 274 Paeonia daurica 276 Betula alba X X 11 294 Talictrum sp. x X 12 302 Agrimonia eupatori X 13 355 Salix babilonics X 14 377 Cerasus janonica EXAMPLE 2 Sniffing Test of Root Exudates and Cuticular Washings The assay was done by thawing a frozen sample to a room temperature, opening a glass vial containing a sample, sniffing it, and immediately marking the results.

Samples are stored frozen at -200C in tightly sealed glass vials (5 ml vials for cuticular washings and 20 ml vials for root exudates). The amount of root exudates in each vial ranges from 5 to 500 mg. The amounts of cuticular compounds in each vial ranges from 5 to 100 mg.

WO 00/07437 PCT/US99/17893 -32- As demonstrated by Table IV and V a significant proportion, 36 out of 100 tested samples of exudates, and out of 100 tested cuticular washings have a strong fragrance. Root exudates were treated with Acetate 03 0.1% acetic acid, AgNO3-20.5 mM Ag(N0 3 2 Chito 02 0.1% chitosan, water, HSL 01 200 yM N-hexanoyl homoerinelactone, and MeJa 03 100 methl jasmonate. In Table IV and V, smell was rated by scientist as follows: 0 no smell; 1 light smell;2 medium smell; 3 strong smell.

Table IV: Sniffing test on root exudates Sample# rating family genus species treatment 1199 3 Solanaceae Hyoscamus niger Acetate 03 1201 3 Solanaceae Hyoscamus niger AgN03 02 1203 2 Solanaceae Hyoscamus niger Chito 02 1205 3 Fabaceae Genista tinctoria Acetate 03 1207 2 Fabaceae Genista tinctoria AgN03 02 1209 2 Fabaceae Cicer arietinum control 01 1211 3 Fabaceae Cicer arietinum Acetate 03 1213 3 Fabaceae Cicer arietinum AgN03 02 1215 3 Fabaceae Cicer arietinum Chito 02 1217 1 Fabaceae Cicer arietinum HSL 01 1219 2 Fabaceae Thermopsis fabacea HSL 01 1221 3 Fabaceae Thermopsis fabacea acetate 03 1223 2 Cucurbitaceae Trichosanthes kirilowii control 01 1225 3 Cucurbitaceae Trichosanthes kirilowii acetate 03 1227 1 Cucurbitaceae Trichosanthes kirilowii HSL 01 1229 2 Cucurbitaceae Trichosanthes kirilowii Chito 02 1233 0 Asteraceae Xanthium sibiricum HSL 01 WO 00/07437 WO 0007437PCT/US99/I 7893 -33- 1235 3 Solanaceae Brugmansia suaevolens AgN03 02 1237 0 Solanaceae Brugmansia suaevolens HSL 01 1239 1 Asteraceae Eclipta alba control 01 1241 3 Asteraceae Eclipta alba Acetate 03 1243 2 Asteraceae Eclipta alba AgNO3 02 1245 0 Asteraceae Eclipta alba Chitc, 1247 3 Asteraceae Aremisia absinthium control 01 1249 3 Asteraceae Arternsia absinthium Acetate 03 1251 2 Asteraceae Arternsia absinthium AgNO3 02 1253 0 Asteraceae Arternsia absinthium Chito 02 1255 1 Asteraceae Silybum marianum control 01 1257 2 Asteraceae Silybum marianum Acetate 03 1259 3 Asteraceae Silybum marianum AgNO3 02 1261 3 Asteraceae Silybum rnarianum Chito 02 1263 2 Asteraceae Silybum marianum MeJa 03 1267 3 Apiaceae Cnidium monnieri Acetate 02 1269 2 Apiaceae Cnidium monnieri AgNO3 02 1271 2 Apiaceae, Cnidium monnieri Chito, 02 1275 2 Apiaceae Cnidium rnonnieri HSLOI 1277 3 Solanaceae B~rugmansia suaevolens control 01 1279 2 Solanaceae Brugmansia suaevolens Acetate 03 1281 1 Clusiaceae Hypercum perforatum control 01 1283 2 Clusiaceae Hypercum perforatum Acetate-03 1285 1 Clusiaceae Hypercum perforatum AcgNO3 02 1287 1 Clusiaceae Hypercum perforatum Chito 02 1289 3 Clusiaceae Hypercum perforatum MeJa 03 1291 3 Boraginaceae Arichusa off icinalis control 01 1293 2 Boraginaceae Anchusa officinalis Acetate 03 1295 11 sp. IHSL 01 WO 00/07437 WO 0007437PCTIUS99/1 7893 -34- 1297 3 Asteraceae Xanthium sibiricum AgNO3 02 1299 3 Zgpil Larrea tridentata control 01 ceae 1301 3 Zgpyl Larrea tridenta Acetate 03 ceae, 1303 2 Zgpyl Larrea tridenta AgNO3 02 ceae Zygophyll1a 1305 0 Larrea tridenta Chito 02 ceae 1307 2 Zgpyl Larrea tridenta MeJa 03 ceae control 1309 2 Lamiaceae Scutellaria baicalensis 01 1311 2 Lamiaceae Scutellaria baicalensis Acetate 03 1313 2 Lamiaceae Scutellaria baicalensis AgNO3 02 1315 0 Lamiaceae Scutellaria baicalensis Chito 02 1317 3 Fabaceae Cytissus scoparius control 02.

1319 1 Fabaceae Cytissus scoparius Acetate 03 1321 3 Apocyraaceae Rauvolfia cat fra Chito 02 1333 1 Cyperaceae Cyperus esculentus control 01 1325 3 Cyperaceae Cyperus esculentus Acetate 03 1327 2 Cyperaceae Cyperus esculentus AgNO3 02 1329 2 Cyperaceae Cyperus esculentus Chito 02 1331 0 Asteraceae Arnica chamissois HSL 01 1333 3 Solanaceae Physalis ixocarpa control 01 1335 3 Solanaceae Physalis ixocarpa Acetate 03 1337 2 'Solanaceae Physalis ixocarpa AgNO3 02 1339 0 Solanaceae Physalis ixocarpa Chito 02 1341 1 Solanaceae Physalis lixocarpa IMeJa 03 WO 00/07437 PTfS9179 PCT/US99/17893 1343 3 Apiaceae Anigelica polym~orh 'control a-sinesis 01 polymorh 1345 2 Apiaceae Angelica A-N03 02 a-sinesis 1347 3 Apiaceae Angelica oyrh a-sinesis 1349 2 Apiaceae Angelica poyoh Chito 02 a- sinesis 1351 1 Apiaceae Angelica poyoh MeJa 03 a- sinesis 1353 2 Rosaceae Agrimonia pilosa control 01 1357 3 Asteraceae Amica Chamissois Acetate 03 christacontrol 1359 0 Fabaceae Erythrina galli 01 christa- Acetate 1361 3 Fabaceae Erythrina 03 christa- 1363 2 Fabaceae Erythrina christa- 1365 1 Fabaceae Erythrina gliChito 02 christa- 1367 0 Fabaceae Erythrina HSL 01 gall i control 01 1369 3 Ranunculaceae Aquilegia vulgaris 1371 1 Ranunculaceae Aquilegia vulgaris Acetate 03 1373 1 Ranunculaceae Aquilegia vulgaris AgNO3 02 1375 1 Ranunculaceae Aquilegia vulgaris Chito 02 1377 2 Ranunculaceae Aquilegia vulgaris MeJa 03 1379 3 Lamiaceae Leonurus sibiricus control 01 1381 13 Lamiaceae Leonurus sibiricus Acetate 03 WO 00/07437 WO 0007437PCT/US99/1 7893 -36- 1383 2 Lamiaceae Leonurus sibiricus AgN03 02 1385 0 Lamiaceae Leonurus sibiricus Chito 02 1387 1 Lamiaceae Leonurus sibiricus MeJa 03 1395 1 Ephedraceae Ephedra nevadensis Acetate 03 Convolvul a- cant rol 1397 3 Ipornoea purpurea ceae 01 Convolvul a- Acetate 1399 3 Ipomoea purpurea.

ceae 03 Convolvula- 1401 2 Ipomoea purpurea AgNO3 02 ceae Convolvula 1403 2 Ipomoea purpurea Chito 02 ceae Convolvula 1405 3 Ipomoea purpurea MeJa 03 ceae Arnarantha 1407 3 Cyathula off icinalis Acetate 03 ceae 1409 3 Asteraceae Xanthium sibiricum, Acetate 03 1413 3 Fabaceae Tephrosia grandiflora Acetate 03 Table V: Sniffing test on cuticular washings sape#rating family genus species wx 201 1 Rutaceae Murrays exotica wx 202 2 Araliaceae Trevesia sungaica wx 203 1 Verbenaceae Clerodendrum speciosissimun wx 204 0 Euphorbiaceae Acalypha hispida wx 205 2 Gnetaceae Gnetum gnemon wx 206 11 Rubiaceae IPsychotria nigropunctata WO 00/07437 WO 0007437PCTIUS99/1 7893 -37me tbac terio wx 207 1 Rubiaceae Psychotria dmsc wx 208 2 Euphorbiaceae Codiaeum variegatum wx 209 2 Euphorbiaceae Phyllanthus grandifolius wx 210 3 Sterculiaceae Pterigota alata wx 2 11 1 Bombacaceae Pachira affinis wx 212 1 Sterculiaceae Sterculia elata wx 213 0 Araceae Philodendron speciosum wx 214 2 Fabaceae Pithecellobium unguiscati wx 215 1 Acanthaceae Sanchezia nobilis wx 216 1 Araliaceae Oreopanax capitata wx 217 0 Moraceae Ficus triangularis wx 218 2 Bignoniaceae Kigelia pinnata wx 219 1 Piperaceae Piper cubeba wx 220 3 Lauraceae Laurus nobilis wx 221 2 Fabaceae Erythrinia cristagalli wx 222 1 Myrataceae Metrosideros excelsa wx 223 3 Oleaceae Osmanthus fragrans wx 224 1 Cupressaceae Tetraclinis articulata wx 225 0 Cupressaceae Cupressus sempervirens wx 226, 0 Acanthaceae Jacobinia sp.

wx 227 0 Asteraceae Senecio platifilla wx 228 1 Arecaceae Livistona chinensis wx 229 3 Myrataceae Eucalyptus rudis wx 230 1 Podocarpaceae Podocarpus spinulosus wx 231 2 Rosaceae Eriobotrya japonica wx 232 2 Ginkgoaceae Gingko biloba wx 233 2 Ericeae Rhododendron sp.

wx 234 2 Cupressaceae Thuja occidentalis wx 235 1 Polygonaceae Fagopyrum suffruticosum WO 00/07437PCIS9173 PCT/US99/17893 -38wx 236 1 Rosaceae Geurn macrophyllum wx 237 2 Magnoliaceae Magnolia cobus wx 238 3 Apocynaceae Vinca minor wx 239 0 Liliaceae Convallaria majalis wx 240 2 Betulaceae Corvlus avellana wx 241 2 Berberidaceae Berberis sp.

wx 242 2 Rosaceae Rosa multiflora wx 243 1 Betulaceae Ostrya carpinifolia wx 244 1 Betulaceae Ostrya connogea wx 245 1 Fagaceae Quercus rubra wx 246 2 Magnoliaceae Liriodendron tulipifera wx 247 1 Rosaceae Sorbus aucuparia wx 248 3 Betulaceae Betula nigra wx 249 3 Betulaceae Betula. nigra wx 250 2 Fagaceae Castanea sativa wx 251 0 Saxofragaceae Bergenia crussifolia wx 252 3 Asteraceae Artemisia dracunculus wx 253 3 Rutaceae Ruta graveolens wx 254 3 Fragaceae Quercus nigra wx 255 2 Schisandraceae Schisandra chinensis wx 256 3 Betulaceae Betula alba wx 257 2 Caprifoliaceae Sambucus nigra wx 258 3 Actinidiaceae Actinidia colonicta wx 259 2 Paeoniaceae Paeonia. lactiflora wx 260 1 Paeoniaceae Paeonia suffructicisa wx 261 0 Fragaceae Quercus imbricaria wx 262 1 Iridaceae Iris pallida wx 263 2 Portulacaeae Portulaca oleracea wx 264 2 Polygonaceae Tolygonum aviculare WO 00/07437 WO 0007437PCT/US99/1 7893 -39wx 265 0 Iridaceae Iris pseudacorus wx 266 1 Liliaceae Allium. nutans wx 267 3 Liliaceae Allium, fistulosum wx 268 1 Liliaceae Anthericum ramosum wx 269 1 Liliaceae Veratrum nigrum wx 270 1 Polygonaceae Polygonum latifolia wx 271 0 Liliaceae 1-osta lancifolia wx 272 1 Liliaceae Hosta zibalda wx 273 2 Asteraceae Echinops sphaerocephalus wx 274 3 Paeoniaceae Paeonia daurica wx 275 1 Asteraceae Inula helenium wx 276 2 Fabaceae Crambe pontica Scrophuyl ar wx 277 0 Dgtlslutea iaceaeDgiai wx 278 3 Fabraceae Baptisia australis Aristolochiawx279 2 Aristolochia clernatitis ceae wx 280 3 Lamiaceae Hyssopus zeraucharicus wx 281 2 Lamiaceae Teucrium hamedris wx 282 2 Crassulaceae Sedum album wx 283 3 Apiaceae Heracleum pubescens wx 284 3 Lamiaceae Origanum vulgare wx 285 3 Apiaceae Cachrys alpina wx 286 1 Apiaceae Laser trilobun wx 287 2 Dryopteridaceae Matteucia struthiopteris wx 288 Crassulaceae Sedum telephium wx 289 2 Papaveraceae Bocconia cordata wx 290 1 Lamiaceae Ajuga reptans wx 291 1 Ranunculaceae Thalictrum minus wx 292 12 Ranunculaceae Anemone japonica WO 00/07437 PCT/US99/17893 wx 293 1 Ranunculaceae Clematis recta wx 294 2 Ranunculaceae Thalictrum sp.

wx 295 1 Rosaceae Alchemilla sp.

wx 296 1 Rosaceae Potentilla alba wx 297 1 Rosaeae Poterium sangiusorba wx 298 3 Menispermaceae Menispermum dauricum wx 299 3 Nyctaginaceae Oxybaphus nyctagineus wx 300 2 Brassicaceae Armoracia rusticana The results presented hereinabove, demonstrate that plant exudates and components of cuticular coatings constitute a novel and important source of new biologically active compounds having antimicrobial, antifungal, insecticidal, sporidicidal, cytotoxic activities and herbicidal properties that could be used for treatments of various diseases or conditions. In addition, compounds present in cuticular washings and root exudates can be used as fragrances, flavors, and flavor enhancers.

EXAMPLE 3 Phytosecretion of Genistein and Daidzein by Soybean Roots Soybean (Glycine max) seeds were germinated in a greenhouse equipped with supplementary lighting (16-h photoperiod 24-28 0 Seeds were placed inside 0.9 cm diameter, 0.9 cm deep well drilled in Grodan rockwool cubes (3.4 cm width x 3.4 cm depth x 3.7 cm height) purchased from Grodania A/S, Hedehousene, Denmark.

Depending on the speed of germination, the seeds were either placed directly into the rockwool cubes or sterilized to prevent rotting during the germination process. For sterilization, seeds were immersed first in ethyl-Alcohol for 10-15 seconds, then in 2.5% Sodium WO 00/07437 PCT/US99/17893 -41- Hypochlorite for 10-15 min., and finally rinsed thoroughly with distilled water. The sterilized seeds were placed in a Petri dish lined with no. 1 Wattman paper (Wattman International Ltd., Maidstone, England), soaked in either a sterile water for seeds larger than 1 mm in diameter, or for smaller seeds with mineral salts nutrient solution. The Petri-dishes were sealed with parafilm before being placed in a growth chamber (12-h photoperiod 22-24 0 C) until the seeds germinated.

Rockwool cubes were placed inside standard greenhouse plastic trays (dimensions 52 cm width x 25 cm depth x 7 cm height) and watered with an intermittently operating overhead misting system triggered by a moisture sensor (Mist-A-Matic, E.C. Geiger Inc., Larleysville, PA). Seeds were allowed to germinate for 3-6 days till the roots started to emerge from the bottom of-the rockwool cube.

After germination, the cubes with the seedlings were inserted into a 3.2 cm diameter round opening cut in the center of Styrofoam ring (8.2 cm diameter, 2.5 cm thickness). The ring was floated on the surface of 400- 800 mL of hydroponic nutrient solution (2 g/L Hydro-Sol (Scotts-Sierra Horticultural Products Comp., Marysville, OH] supplemented with 1.2 g/L Ca[N0 3 2 containing inside light impermeable, high-density polyethylene cylinder cm in diameter, 16 cm in height).

Aeration was provided either by shaking the cylinders at 50 rpm on the platform shaker (Model Orbit, Lab-Line Instruments, Inc., Melrose Park, IL) or by bubbling compressed air through the solution. Seedlings were cultivated hydroponically in this system for 3 to weeks with roots growing in a nutrient solution.

Thereafter, the root system (average root dry weight WO 00/07437 PCT/US99/17893 -42- 0.1+0.05g) was removed from the nutrient solution and placed inside a 30 mL glass beaker, containing 10-20 mL of distilled water or distilled water supplemented with the elicitor. To prevent the water loss from the plant canopy and the drying of the collecting solution, shoots of the plants were covered with a plastic bag. After 24 h, unless noted otherwise, a small sample from the root solution was removed and analyzed for the phytosecreted products.

EXAMPLE 4 Flow-Through Phytosecretion System The flow-through phytosecretion system consisted of a stainless steel container (53 cm width x 34 cm depth x 20 cm height) with 15-24 soybean plants supported by the rockwool cubes inserted in the openings in the Styrofoam raft (5.0 cm thickness) which had dimensions slightly smaller than the internal dimensions of the container. This Styrofoam raft was floating on top of approximately 10 L of nutrient solution (2g/L Hydro-Sol supplemented with 1.2 g/L Ca (NO 3 2 aerated with compressed air supplied through an air hose placed on the bottom of the container. After 4-5 weeks, or when the roots reached the appropriate size, the volume of nutrient solution was reduced to 2L. The flow of the nutrient solution, with or without an elicitor, through the flow through system was maintained with a peristaltic pump (Variable Flow Mini-Pump, Fisher Scientific, Pittsburgh, PA), which allowed easy adjustments in the volume of the solution entering the system. Typically, flow rates used in the experiments ranged from 1.5 to L/day. The intake tube of the peristaltic pump was immersed in a 60 L plastic storage container containing WO 00/07437 PCT/US99/17893 -43nutrient solution. Solution from the storage container dripped into the phytosecretion system through the tube attached to its wall. When necessary, elicitors were added to the storage container at the desired concentration. The solution was discharged from the phytosecretion container in the side opposite to the inlet through the opening cut in the bottom of the container. Solution level in the phytosecretion container was adjusted by changing the height of the opening of the outlet tube. Solution samples were taken from the end of the outlet tube at the specified intervals and analyzed for the presence of the phytosecreted compounds.

EXAMPLE High-Pressure Liquid Chromatography (HPLC) Analysis of Phytosecreted Natural Products (Isoflavonoids) An HPLC method for separation and identification of phytosecreted compounds, using isoflavonoids daidzein and genistein is used as an example. The chromatography separation was performed with an HPLC-system consisting of Waters 996 Photodiode Array Detector (PDA) with usable UV range from 190 to 800 nm; a Waters (xxx) 717 plus autosampler; two Beckman (xxx) 110B solvent Delivery Modules, connected with a Beckman System Organizer (mixer) and a Beckman System Gold Analog Interface Module 406. The Beckman solvent delivery system was controlled by a NEC PC-8300 computer.

Chromatography and spectral data was managed by Waters Millennium chromatography software, version 2.10, using a NEC Image 466es computer. All hardware components, except the solvent delivery system, were connected through a standard IEEE communication system.

WO 00/07437 PCT/US99/17893 -44- Isoflavonoid compounds were separated on a Waters Nova Pak® C-18 reverse phase column, 3.9 x 150 mm, 60A pore size, and 4 um particle size.

The mobile phase consisted of two components: Solvent A 0.5% ACS grade acetic acid in double distilled water, pH 3-3.5; and Solvent B acetonitrile.

Prior to use, each batch of solvent A was degassed under vacuum and ultrasonication for 5 minutes.

The mobile phase flow was adjusted to 1 ml/min, and a gradient mode of separation was used for all separations. The gradient profile was as follows: 0 20 min 0% B 100% B; 22 min 100% B; 22 25 min 100% B 0% B; 25 33 min 0% B (column equilibration for next injection).

Compounds were detected with PDA detector within the wavelength range of 200 to 400 nm. The column temperature was ambient.

Under the above conditions, daidzein had RT 11.725 and UV maxima at 250.9 nm and 302.9 nm and genistein had a RT of 12.94 min and UV maximum at 260.3 nm. Depending on the resolution setting of the PDA detector, a negligible shift of +3 nm in the absorbance maxima was observed. A±0.5 min tolerance, in the retention times with the different batches of solvents was detected.

All plants were grown hydroponically, as previously described, and phytosecreted compounds collected for 24 hours in distilled water containing an elicitor or mixtures of different elicitors, except for treatment 47, where no elicitors were present in the collecting water. Daidzein and genistein content in root WO 00/07437 PCT/US99/17893 exudates from unelicited plants grown under the same conditions was below the detection limits 400 pg, or 4 ,g/L for daidzein, and 25 pg, or 250 ng/L for genistein.

Elicitor treatments: 3 Salicylic acid (5 mM) Tetcyclases (0.2 mM) and EtOH Salicylic acid (5 mM) and 5% EtOH 6 Salicylic acid (2.5 mM) and 2.5% EtOH 7 Salicylic acid (5 mM) and 0.5 g/L SDS Salicylic acid (1 mM) and Pentafluorobenzoic acid(2 mM) 21 Silver nitrate (1 mM) in acidic pH (citric acid, pH 2.7) 24- 2.7) 37 38 40 42 47 Silver nitrate (2 mM) in acidic pH (acetic acid, pH Acetic acid (pH 2.7) Pentafluorobenzoic acid (5 mM) 2.6 Dihydroxybenzoic acid (10 mM) Cinnamic acid (16.5 mM) and 35.5% EtOH Cinnamic acid (3.3 mM) and 7.1% EtOH 2-Fluorobenzoic acid (10 mm) and 2% EtOH UV-light irradiation of the whole plant for 3 hours Sodium,-fluoride (250 mM) and 10% EtOH Fifteen treatments which elicited some of the highest levels of daidzein and genistein are shown in Figures 16 and 17 for simplicity. Some of the above elicitors induced mild to moderate phytotoxicity in the treated plants. All compounds used in the mixtures produced significant levels of daidzein and genistein, when applied alone. However, combinations of various elicitors shown in Figures 16 and 17 usually produced WO 00/07437 PCT/US99/17893 -46higher levels of target compounds. Other compounds used as elicitors, such as yeast extract, laminarin, SDS, jasmonic acid, methyl jasmonate, okadaic acid, polygalacturonic acid, 1-phosphatidic acid, polyethylene glycol, hydrogen peroxide, paraquate, calyculin A, 1amino butyrate, eicosapentaenoic acid, arachidonic acid, glutathione, ascorbic acid, and some heavy metals (Nickel, Copper, Lead) showed lower degree of elicitation of the target compounds.

Various plants were grown hydroponically as previous described and secreted compounds (root exudates) were collected in distilled water with and without an elicitor(s).

Figure 18 is an HPLC profile of compounds recovered from the root exudates with UV detection at 251.8 nm. Most compounds were not identified, however, as shown in Figure 18 the following designations in Figure 18 were positively or putatively identified.

1. Positively identified Daidzein 2. Positively identified Genistein A. Putatively identified as nicotine B. Putatively identified as Lupinus luterus roots were not elicited; Lupinus polyphyllus roots were elicited with 2 mM Salicylic acid in 2% Ethanol; all other species were elicited with 0.3 M Acetic acid, pH 2.7.

Figure 19 demonstrates the diversity of compounds excreted from the roots of one plant species (Lupinus luterus) treated-with different elicitors (note the large differences in the HPLC profiles of root exudates produced by different elicitors), UV detection at 251.8 nm.

9.Feb. 2004 16:19 WRAY&ASSOCIATES No.4424 P. 6 -47- Elicitor treatments: control No treatment I Treatment with 2 mM Salicylic acid in 2% Ethanol II Treatment with 0.3 M Acetic acid, pH 2.7 III Treatment with 2 mM AgN03 IV Treatment with 7.5 mM Arachidonic acid V Treatment with 5 mM Jasmonic acid Most compounds were not identified. Those positively or putatively-identified are: 1 Putatively identified as G Positively identified as Genistein SA Salicylic acid Figures 20, 21, 22 and 23 are HPLC profiles of the diversity of compounds exuded or leached from the roots of Brassica juncea, Datura metel, Lupinus polyphyllus and Melilotus medicaginoides, respectively, treated with different elicitors. UV detection was at 254 nm. No compound was identified.

The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to Imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

*ooo• COMS ID No: SMBI-00609893 Received by IP Australia: Time 18:27 Date 2004-02-09

Claims (6)

1. 9.Feb. 2004 16:20 WRAY&ASSOCIATES No.4424 P. 7 -48- What is Claimed: 1. A method of identifying an agent exuded onto the surface of a leaf of a plant having biological activity, the method comprising: removing cuticular material located on the surface of a leaf of the plant, comprising contacting the leaf surface with a solvent capable of partially or completely dissolving the cuticular material, thereby resulting in a solvent solution; assaying the solvent solution for biological activity; and analyzing the solvent solution so as to identify the agent which has the biological activity. 2. The method of claim 1, wherein the biological activity is antimicrobial, insecticidal, or herbicidal, fragrances, scent, flavors, and flavor enhancers. 3. The method of claim 2, wherein the antimicrobial activity is antibacterial or antifungal activity. 4. The method of claim 1, wherein the step of assaying the solvent solution comprises contacting the solution its components with a media containing a suspension of a microorganism, wherein the inhibition of the growth of the suspension of the microorganism is indicative of an agent in the solvent solution having biological activity. 4. 99 .9* o 9 9 o o ooo 9 9 *9 9 9* 9 9999 9 999 9 9 99 9 *o o 5. The method of claim 4, wherein the media is a liquid medium media. 6. The method of claim 1, wherein the cuticular material is a lipid, protein, primary or secondary metabolite. 7. The method of claim 6, wherein the cuticular material is a wax. or an agar wax, cutin, COMS ID No: SMBI-00609893 Received by IP Australia: Time 18:27 Date 2004-02-09 9.Feb. 2004 16:20 WRAY&ASSOCIATES No.4424 P. 8 -49- 8. The method of claim 1, wherein the solvent is an organic solvent. 9. The method of claim 8, wherein the organic solvent is methylene chloride or chloroform. method of claim 1, wherein the suspension of microorganism is a bacteria, fungus, or virus.
2. 11.The method of claim 10, wherein the suspension of microorganism is selected from the group consisting of Escherichia coil K-12. F, prototropic Str., Staphylococcus aureus subsp. Aureus, Pseudomonas aemrginosa, Saccharomyces cerevisiae, Aspergillus flavus and Penicillium nigra.
3. 12.The method of claim 1, wherein the plant is a higher plant.
4. 13.The method of claim 12, wherein the plant is selected from a group consisting of Atropa belladonna, Erythrinia glabeliferus, Ipomea tricolor, Erythrinia crista, Celosia cristata, Gallium sporium, Laurus nobilis, Vitis labrissa, Gratiola officinalis, Symphitum officinalis, Hosta fortune, Casia hebecarpa, Thalictum flavum, Scutellarian altissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geum fanieri, Gentiana tibetica, Linum hirsutum, Aconitum napellus, Podophyllum amodii, Thymus cretaceus, Carlina acaulis, 20 Chamaechrista fasciculata, Pinus pinea, Pegamun hamalis, Tamarindus india, Cupress lusitanica, Diopiros kaka, Erungiurn campestre, Aesculus 0: 6. 20 woerlitzenis, Aesculus hippocastanum, Cupressus sempervirens, and Celtis occidentalis.
5. 14. The method of claim 1, wherein step of 1(c) further comprises fractioning the solvent solution. method of claim 14, further comprising identifying the agents. 0 9V *999 COMS ID No: SMBI-00609893 Received by IP Australia: Time 18:27 Date 2004-02-09 9.Feb. 2004 16:20 WRAY&ASSOCIATES No.4424 P. 9
6. 16.A method according to claim .1 substantially as hereinbefore described with reference to any one of the examples. Dated this NINTH day of FEBRUARY 2004. Rutgers, The State University of New Jersey Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant ft 0 0* *0 0*0 *00 0 0 0 0 0 0 0 0 0 0 0e 0 COMS ID No: SMBI-00609893 Received by IP Australia: Time 18:27 Date 2004-02-09