A METHOD OF IDENTIFYING AND RECOVERING PRODUCTS EXUDED FROM A PLANT

Abstract: This invention provides a method of identifying biologically active or otherwise valuable substances exuded from or onto a plant surface, specifically the plant cuticle. This invention also provides a method of identifying and recovering substances exuded from or onto the roots of a plant. The invention further comprises libraries of substances exuded or secreted from various plant species, which may be elicited or induced to produce one or more of such substances.

Description: 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 non-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) colonization. Strigol, a germination stimulant for the parasitic plant Striga asi tica, has been found in the root exudates of many cereals. In addition, root- secreted 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, e.g., 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 activities, 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 (e.g., a leaf surface), the method comprising: (a) 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; (b) assaying the solvent containing the cuticular material for the identification of agents of the cuticular material which have biological activity; and (c) 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 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, Vi tis labrissa, Gratiola officinalis, Symphi tium officinalis, Hosta for tuna, Casia hebecarpa, Thai ic turn flavum, Scutellarian al tissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geum fanieri , Gentiana tibeti ca, Linum hirsutum, Aconi tum napellus, Podophyllum amodii , Thymus cretaceus, Hosta fortunaea, Carlina acaulis, Chamaechrista f sciculata, Pinus pinea, Pegamun hamalis, Tamarindus india, Carica papaya, Cistus incanus, Capparis spinosa inemis, Cupress lusi tanica, Diopiros kaka, Erungiurn campestre, Aesculus woerli tzenis, Aesculus hippocastanum, Cupressus sempervirens and Cel t is occidental is . 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: (a) obtaining a sample of a medium (e.g., an aqueous medium such as water buffer or liquid growth medium) which contacted plant roots; ( b) assaying the sample for biological activity; and (c) analyzing the sample so as to identify the agents 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 under- illumination 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 recover 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 Aconi tum napellus (1881) against Escheri chia 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 al tissima (1671) against Staphylococcus aureus . Figure 7. Agar plate showing antimicrobial activity of the root exudate of Scuttellaria creticola (1691) against Staphylococcus aureus . 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 ci triodorus "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 capi tata (216) against Staphylococcus aureus . Figure 15. Agar plate showing antimicrobial activity of leaf surface compounds of Oreopanax capi tata (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. Figure 18. HPLC profile of the diversity of compounds recovered from root exudates of various plants. Figure 19. HPLC profile of the diversity of compounds recovered from Lupinus luteus . Figure 20. HPLC profile of the diversity of compounds recovered from root exudates of Brassica juncea . Figure 21. HPLC profile of the diversity of compounds recovered from root exudates of Datura metel . Figure 22. HPLC profile of the diversity of compounds recovered from root exudates of Lupinus polyphyl lus . Figure 23. HPLC profile of the diversity of compounds recovered from root exudates of Melil totus 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 spectrum of organisms against which they are active, whether they kill the organism or merely slow its growth and reproduction (i.e., cidal or static) and the biochemical system on which they exert their major biochemical action (e.g., 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, Vi tis labrissa, Gratiola officinalis, Symphi tium officinalis, Hosta fortuna, Casia hebecarpa, Thalictum flavum, Scutellarian al tissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geu fanieri , Gentiana tibetica, Linium hirsutum, Aconi turn napellus , Podophyllum amodii , Thymus cretaceus , Hosta fortunaea, Carlina acaulis, Charnaechrista fasciculata, Pinus pinea, Pegamun hamalis, Tamarindus India, Carica papaya, Cistus ine anus , Capparis spinosa inemis, Cupress lusi tanica, Diopiros, kaka, Erungium campestre, Aesculus woerli tzenis , Aesculus hippocastanum, Cupressus sempervirens and Cel tis occidentali s . Further plants species used for screening exudates are as follows: Polygonum cuspidatum, Eleagnus angusti folia , Eleagnus cemutata, Gentiana macrophilla, Brassica napa, Sesbania exal tata, Sesbania speciosa, Spartina potenti flora , Brassica juncea, Helianthus annus, Puansetia sp. , Pelargonium zonale, Sundapsis spp. , Leontopodium alpinum, Lupinus luteaus, Buxus microphilla " japonic a " , Liatris spinata, Rimula japonica, Betula nigra, Filipendula vulgrais, Lobelia siphi tica, Gravilia robusta, Reseda luteola, Gentiana li ttorala, Campanula carpatica, Aesculus hypocastanum, Aesculus waertilensis , Ageratum conizoides , Psidium guajava, Ailantus al tissima, Buxus microphylla "japonica" , Hydrocotile asiatica, Gravilea robusta, Brugmansia suaveolens, Thymus puliglodes, Thymus lemabarona, Thymus serphylum (wild) , Gaul theria procumbens , Thymus serphylum, Thymus camosus, Thymus thrasicus, Calicatus floridus, Zingiber officinalis, Lapia dulcis, Thymus vulgaris "argenteus " , Thumus praecox "arcticus " , Thymus puleglodes "lemons " , Thymus speciosa, Thymus carnosus, Thymus pseudolamginosus , Thymus praecox, Thymus vulgaris "oregano" , Ficus religiosa, Forsi thsia suspensa, Chelidonium ma jus, Thymus wooly, Thymus portugalense , Nicotiana tabacum, Thymus cytridorus "aureus " , Thymus vulgaris, Cactus officinailis, Lal lab purpurea, Juglands regia, Actinidia chinensis, Hernerocalis spp . , Betula pendula, Gardenia jasminoides, Taxodium dixticum, Magnolia loebheril , Crataegus praegophyrum , Larix dedidua, Tuja orientalis "eligantissima " , Tula ocidentalis "Columbia " , Xeupressocyparis deylandii , Pseudotsuga menzisia, Abies firma, Fautenousus qualiqualia, Alium cernu (wild) , Juniperus "blue pacific" , Taraxacum officinalis, Juca sp . , Ilex agnifoliu , Tsuga canadensis "penola " , Tsuga canadensis "penola " , Ilex cornuta, Taxus xiksii , Taxus media, Metasequoia glyptotrobioldes , Pinus bungiana, Boxus sempervirens, Stevartia coreana, Prunus xocane, Betula daurica, Plantago minor, Acer palmaturn "burgundy" , Acer campestre, Cotynus cogygria, Quercus robur " fastigiata " , Acer truncatum, Archirantus bidentata, Alum japonica, Carum capsicum, Agastache mexuicana, Prunella vulgaris, Tagetes minuta, Nepeta cataria, Ratibiunda columnus-Fera, Aster-Nova anglicae, Ulrica certifera, Pi ttisporum tibica, Taxodium dixticum (H 2 0) , Taxodium dixticum (Acetic acid) , Plantago major, Scotch pine, Asorum canadiensis, Pieras japonica, Pinus sirtrobus, Trifolium pratense, Prunus serotica, Darura stramonium, Geranium maculata, Hydrocotile asiatica, Astragulus sinicus, Centauria maculata, Ruschia indurata, Myrthus comunis, Platanus acidentalis, Liclu barbatum, Lavandula officinalis , Gravilea robusta, Hyppoach rhamnoides, Filipendula ulmaria, Betula pendula Polygonium odoratum, Brugmansi graveolens (ralf) , Rhus toxicodenta, Armoraica ristica, Ficus benjaminii , Sluffera sp . , Pelagonium zonale, Allium sp. Asimina triloba, Lippa dulcis, Epilobium augusti folium, Brugmansia suavecolens (old) , Brugmansia suaveolens (young) , Xanthoso a sag i tti folium (leaf) , Xanthosoma sagi ttifolium (stem) , Monstera deliciosa, Aglaonema commutatus, Dieffenbachia leopoldii , Anthurium andreanum, Syngoniurn podophyllum, Dracaena fragrans, Ananas comosus, Streli tzia reglinae, Diffenbachia segiunae, Syngoniurn aurutum, Dracaena sp . , Hhaemanthus katharina, Anthurium al tersianum, Spathiphyllum grandiflorum, Spathiphyllum cochlearispaturn, Monstera pertusa, Anthurium magnificurn, Anthurium hookeri , Anthurium elegans, Calathea zebrina, Yucca elephant ipe , Bromelia balansae, Musa textilis (Leaf) , textilis (Stem) , Myrthus communis, Olea oleaster, Olea europaea, Verium oleander, Cocculus laurifolius, Microsorium punctatum, Ficus sp. , Senseviera sp. , Adansonia digi tata, Boechimeria boloba, Piper nigrum, Phymatosorus scolopendria, Turnera ulmifolia, Nicodemia diversifolia, Tapeinochilos spectabilis, Rauwolfia tetraphylla, Ficus elastica, Cycas cirinalis, Caryota ureus, Cynnamonum zeylonicum, Aechmea luddemoniana, Foenix zeulonica, Ficus benjamina, Ficus purnila, Murraya exotica, Trevesia sungaica, Clerodendrurn spec ios si cum, Actinidi colonicta, Paeonia lactiflora, Paeonia suffructicisa, Quercus imbricaria, Iris alida, Portulaca olleracea, Poligonum aviculare, Iris pseudocarpus , Allium nutans , Allium fistulosum, Antericum ramosum, Veratrum nigrum, Poligornun latifolia, Hosta lancefolia, Hosta zibalda, Echinops sphae, Paeonia daurica, Inula hilenium, Trambe pontica, Digi talis lutea, Bactisia australis, Austolachia australis, Hissopus zeraucharicus , Feucrium hamedris, Sedum album, Heraclelum pubescens, Origanum vulgare, Cachris alpina, Haser trilobum, Matteucia strutioptoris , Sedum telchium, Bocconia cordata, Hiuga reptans, Talictrum minus, Anemona japonica, Clematis rectae, Talictrum sp . , Alchemilla sp. , Potentilla alba, Poterium sangiusorba, Minispermum dauricum, Oxobachus nictogenea, Armoracea rusticana, Cramble cardifolia, Agrimonia eupatora, Uschusa sp . , Polymonium ceruleum, Valeriana offocionalis, Pulmonaria molissima, Stachis lanata, Coronolla varia, Platicada grandiflora, Lavandula officinalis, Vincetocsicum officinalis, Acolypha hispida, Gnetum guemon, Psychotria nigropunctata, Psychotria metbacteriodomasica, Cobiaeum varilaturn, Phyllanthus grandifolium, Pterigota alata, Pachyra affinis, Sterulia elata, Phylidendron speciosus, Pi thecelobium unguis, Sanchezia nobilis, Oreopanax capi tata, Ficus triangularis, Pigelia pennata, Piper chaba, Laurus nobilis, Erythrinia caffra, Metrosideros excel sa, Osmanthus spp. , Cupressus sempervirens , Jacobinia sp. , Senecio platifilla, Livistona fragrans, Tetraclinis articulata hinensis, Eucaliptus rudis, Podocarpus spinulosus, Eriobotria japonica, Gingko biloba, Rhododendron spp . , Thuja occidental is , Fagopyrum suffruticosum, Geum macrophullum . , Magnolia cobus, Vinca minor, Convalaria majalis, Corylus avelana, Barbaric sp . , Rosa mul ti flora, Ostrea carpini folia, Ostrea connote, Quercus rubra, Tulip tree, Sorbus aucuparia, Betula nigra (leaf) , Betula nigra (flower) , Castanea sativa Bergenia crassifolia, Artemisia dracunculus, Ruta graveolens, Quercus nigra, Schisandra chinensis, Betula alba, Sambucus niora, Gentiana cruciata, Encephalaris horridum, Phebodium aureu , Microlepia platphylla, Ceratoramia mexicana, Stepochlaena tenuifolia, Adianthum trapezieformis, Adianthum radiatum, Lycodium japonicum, Aessopteria crasifolia, Asplenium australasicum, Agatis robusta, Osmunda regalis, Osmundastrum claytonionum, Phylli tis scolopendrium, Polyschium braunii , Crytomium fortunei , Dryopteris filis-max, Equisetum variegatum, Anthyrium nopponicum, Anthyrium filis -femina, Parthenosicus tricuspidata, Ligustum vulgare, Charnaeciparis pisifera, Rosa cocani ca, Ci tinis coggriaria, Pinus strobus, Cel tis occidentali s, Picea schrenkiana, Cydonia oblonga, Ul us pu ila, Euonomus verrucosa, Deutria scabra, Mespilus germanica, Quercus castanufolia, Euonomus europea, Seruginea suffruticisa, Keylei teria paniculata, Seringa josiceae, Zelcova, carpinifolia, Abies cephalonica, Taccus bacata, Taxus cuspidata, Sails babilonics, Thuja occidentalis , Actinidia colomicta, Magonia agrifolia, Aral is mandshuri ca, Luglands nigra, Euonimus elata , Princepia sp . , Forsi tsia europea, Sorbocotoneaster sp . , Morus alba, Crategus macrophyllum, Eucomia ulurifolia, Sorbus cominicta, Philodendron amurense, Comus mass, Korria japonica, Parrotia persica, Jasminum frutocarus , Sulda sanganea, Pentaphylloides fruti cosa, Sibirea al taiensis, Cerasus japoni ca, Kolkwi tzia amabili s , Amigdalus nana, Acer mandshurica, Salix tamari si folia, Amelanchier spicata, Cerasus maghabab, Prunus cerasifera, Coryllus avelana, Acer tataricum, Viburnum opulus, Siringa vulgaris, Fraxinus exelsior, Quercus trojana, Chaernomelis superba, Pinus salinifolia, Berber is 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. 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: (a) obtaining a sample of media which contacted plant roots; (b) assaying the sample for biological activity; and (c) 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, glycerides, 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 (e.g., N, P or K) , 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 . tri tici 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 ( Tri ticum 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 was 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 80%) , serpentine (60%) , lochnericine (15 0%) , 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 horhammericine 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 of 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 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 solution 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 BeckmanllOB 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 5on a Waters Nova Pak6 C-18 reverse 25 phase column, 3.9 x 150 mm, 60A pore size, and 4μm 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 lml/min, and a gradient mode of separation is used for all separations. Compounds are detected with PDA detector or with Waters Thermabeam Tm Mass Detector. Further, one may fractionate the sample by chromatography techniques followed by the chemical structure analysis using mass spectroscopy; infra red spectroscopy; or ID or 2D nuclear magnetic resonance spectroscopy (proton or 13 C . Fractionation and analysis methods are known to those skilled in the art. As demonstrated herein, cuticular washings from Betula pendula showed very high activity. After 10 repeat experiments the sample was fractionated by HPLC. From 35 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 Bioassav 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 60 cm2 of leaf surface were used. Sandwich bags with zippers were used to guarantee that the surface of leaf is 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 7°c on shaker with a cell density 10 5"6 . 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 +30°C 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 vi tro 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 washings . 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 +30°C, 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 20μl 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. 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°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 final concentration of exudates used in assay was 5 mg of dry exudate diluted in 300 μl 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 1) . 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 * Number in parenthesis indicate total number of root exudate samples tested for a particular microorganism. Table I I 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 referring to a greater activity. Af - Aspergillus flavus Ec - Escherichia coli Sa - Staphylococcus aureus Sc - Saccharomyces cerevisiae Pn - Penicillium nigra Pa - Pseudomonas aeruginosa 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 μl . 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. Table III. List of plants which leaf surface cuticular washings show strong antifungal or antimicrobial activity 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 -20°C 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. As demonstrated by Table IV and V a significant proportion, 36 out of 100 tested samples of exudates, and 20 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 μM N-hexanoyl homoerinelactone, and MeJa 03 100 methl jasmonate. In Table IV and V, smell was rated by scientist as follows: 0 0 no smell; 1 light smell,-2 medium smell; 3 strong smell. Table IV: Sniffing test on root exudates Table V: Sniffing test on cuticular washings 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°C). 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 70% ethyl -Alcohol for 10-15 seconds, then in 2.5% Sodium 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°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 (9.0 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 5 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 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 (N0 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 4.5 L/day. The intake tube of the peristaltic pump was immersed in a 60 L plastic storage container containing nutrient 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 5 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) HOB 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. Isoflavonoid compounds were separated on a Waters Nova Pak* C-18 reverse phase column, 3.9 x 150 mm, 60A pore size, and 4 μm 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; 20 - 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 exudates from unelicited plants grown under the same conditions was below the detection limits - 400 pg, or 4 μg/L for daidzein, and 25 pg, or 250 ng/L for genistein. Elicitor treatments: 3 - Salicylic acid (5 mM) , Tetcyclases (0.2 mM) and 7.5% EtOH 5 - 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 10 - Salicylic acid (1 mM) and Pentafluorobenzoic acid (2 mM) 21 - Silver nitrate (1 mM) in acidic pH (citric acid, pH 2.7) 24 - Silver nitrate (2 mM) in acidic pH (acetic acid, pH 2.7) 25 - Acetic acid (pH 2.7) 37 - Pentafluorobenzoic acid (5 mM) 38 - 2.6 - Dihydroxybenzoic acid (10 mM) 40 - Cinnamic acid (16.5 mM) and 35.5% EtOH 42 - Cinnamic acid (3.3 mM) and 7.1% EtOH 45 - 2-Fluorobenzoic acid (10 mm) and 2% EtOH 47 - UV-light irradiation of the whole plant for 3 hours 55 - 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 higher 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, 1- amino 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 (5-0-methyl-genistein) 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. 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 5-0-Methyl-genistein 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 etel , 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.

Claims: hat 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: (a) removing cuticular material located on the surface of a leaf of the plant, comprising contacting the leaf surface with a solvent, thereby resulting in a solvent solution; (b) assaying the solvent solution for biological activity; and (c) 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. 5. The method of claim 4, wherein the media is a liquid medium or an agar media. 6. The method of claim 1, wherein the cuticular material is a lipid, wax, cutin, protein, primary or secondary metabolite. 7. The method of claim 6, wherein the cuticular material is a wax. 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. 10. The method of claim 1, wherein the suspension of microorganism is a bacteria, fungus, or virus . 11. The method of claim 10, wherein the suspension of microorganism is selected from the group consisting of Escherichia coli K- 12. F, prototropic Str., Staphylococcus aureus subsp . Aureus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus flavus and Penicillium nigra . 12. The method of claim 1, wherein the plant is a higher plant . 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, Vi tis labrissa, Gratiola officinalis, Symphi tum officinalis, Hosta fortuna, Casia hebecarpa, Thai ic turn flavu , Scutellarian al tissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geum fanieri , Gentiana tibetica, Linu hirsutum, Aconi tum napellus, Podophyllum amodii , Thymus cretaceus, Carlina acaulis, Chamaechrista fasciculata, Pinus pinea, Pegamun hamalis, Tamarindus India, Carica papaya, Cistus ine anus , Capparis spinosa ine is, Cupress lusi tanica, Diopiros kaka, Erungiurn campestre, Aesculus woerli tzenis, Aesculus hippocastanum, Cupressus sempervirens, and Cel tis occi den talis . 14. The method of claim 1, wherein step of I (c) furthers comprises fractioning the solvent solution. 15. The method of claim 14, furthers comprising identifying the agents. 16. A method of identifying an agent exuded onto roots of a plant having biological activity, the method comprising: (a) obtaining a sample of a aqueous media after it contacted plant roots; (b) assaying the sample for biological activity; and (c) analyzing the sample so as to identify the agent which has the biological activity. 17. The method of claim 16, wherein the biological activity is antimicrobial or herbicidal, fragrances, scent, flavors, and flavor enhancers. 18. The method of claim 17, wherein the antimicrobial activity is antibacterial or antifungal activity. 19. The method of claim 16, wherein the step of assaying the sample comprises contacting the solution onto a medium having thereon 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. 20. The method of claim 16, wherein the suspension of microorganism is a bacteria, fungus, or virus . 21. The method of claim 20, wherein the suspension of microorganism is selected from the group consisting of Escherichia coli K-12. F, prototropic Str., Staphylococcus aureus subsp . Aureus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Aspergillus flavus and Penicillium nigra . 22. The method of claim 16, wherein the plant is a higher plant. 23. The method of claim 22, wherein the plant is selected from a group consisting of Atropa belladonna, Erythrinia glabeliferus , Ipomea tricolor, Erythrinia crista, Celosia cristata, Gallium sporium, Laurus nobilis, Vi tis labrissa, Gratiola officinalis, Symphi tum officinalis, Hosta fortuna, Casia hebecarpa, Thai ic turn flavum, Scutellarian al tissima, Portulaca oleacea, Scutellaria certicola, Physalis creticola, Geum fanieri , Gentiana tibetica, Linum hirsutum, Aconi tum napellus, Podophyllum amodii , Thymus cretaceus, Carlina acaulis, Chamaechrista fasciculata, Pinus pinea, Pegamun hamalis, Tamarindus india, Carica papaya, Cistus incanus, Capparis spinosa inemis, Cupress lusi tanica, Diopiros kaka, Erungiurn campestre, Aesculus woerli tzenis , Aesculus hippocastanum, Cupressus sempervirens, and Cel tis occidentalis . 24. The method of claim 16, wherein the method further comprises adding an elicitor to the aqueous media contacting the roots prior to the removal of the roots from the media. 25. The method of claim 24, wherein the method further comprises adding an elicitor to the aqueous media which contact the roots during the growth of the roots. 26. The method of claim 24, wherein the elicitor is an abiotic or biotic elicitor. 27. The method of claim 26, wherein the biotic elicitor is chitosan, fungal, bacterial cell wall, methyl salicylate, or methyl jasmonate. 28. The method of claim 26, wherein the abiotic elicitor is silver nitrate or acetic acid. 29. A method for recovering substances exuded or secreted from a plant or plant part, which comprises contacting a living plant or plant part with an aqueous medium to extract into the medium the substances exuded by the plant or plant part, and thereafter recovering the exuded substances from the aqueous medium. 30. The method of claim 29, wherein the plant is subjected to a physical or chemical treatment to induce production of novel substances or to increase production of a pre-determined substance, prior to or during the contacting with the aqueous medium. 31. The method of claim 29, wherein the aqueous medium is continuously or semi-continuously brought into contact with the plant or plant part . 32. A method for generating a library of plant-exuded substances for subsequent screening, which comprises contacting a living plant or plant part with an aqueous medium to extract into the medium the substances exuded by the plant or plant part, recovering the exuded substances from the aqueous medium and forming a library of the substances recovered from the aqueous medium.

Inventors: RASKIN ILYA, RASKIN, ILYA
Applicants: RASKIN ILYA, UNIV RUTGERS, RASKIN, ILYA, RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
Application year: 1999
Main classification: A01K65
IPC codes: C12Q1, G01N33