WO2007003346A2 - Fungicidal agents - Google Patents

Abstract

The strongly negative effect of farnesol and its derivatives on the development of rust fungi is used to provide effective plant protection against pathogenic rust fungi.

Description

Fungicidal Agents

The present invention relates to fungicidal composition comprising as an active compound famesol or a derivative thereof for protecting plants against rust fungi. It also relates to the use of polynucleotides for producing transgenic plants with an increased resistance to rust fungi as well as to methods for the production of such transgenic plants.

Rust fungi are a species-rich group of highly aggressive plant parasites that have plagued farmers since the beginning of agriculture. Unlike many other pathogenic fungi they do not kill their host, but they have evolved effective ways to exploit living cells as feeding sources. The hallmark of this obligate biotrophic lifestyle is a highly specialized cell, the haustorium (from Latin haurire, "to suck"), first described by DeBary in 1863 (A. DeBary, Annals des Sciences naturelles/Botanique 20, 5 (1863)). It differentiates only in the interior of the leaf to complete a developmental program that starts with the germination of uredospores on the leaf surface and is followed by penetration through a stomatal opening. Within the leaf, the rust grows towards mesophyll cells where a haustorial mother cell (HMC) is formed, which begins host cell penetration and haustorium differentiation. The mature haustorium is inserted into a living mesophyll cell and effectively absorbs nutrients without promoting host defenses (R. T. Voegele, C. Struck, M. Hahn, K. Mendgen, Proc. Natl. Acad. ScL U.S.A. 98, 8133 (2001); K. Mendgen, M. Hahn, Trends Plant Sd. 7, 352 (2002); P. Schulze-Lefert, R. Panstruga, Annu. Rev. Phytopathol. 41, 641 (2003); R. C. Staples, Annu. Rev. Phytopathol. 38, 49 (2000). That this developmental program depends on host-derived signals has been shown for cell types differentiated prior to haustoria (K. Mendgen, M. Hahn, H. Deising, Annu. Rev. Phytopathol. 34, 367 (1996); M. C. Heath, Ann. Bot. (London) 80, 713 (1997). However, attempts to identify the signals regulating the differentiation of the haustorium within the plant remained unsuccessful.

It was now found that famesol and its derivates are regulators of the last developmental step needed to establish biotrophy. These compounds may thus be used to provide effective plant protection against pathogenic rust fungi. Thus, the present invention relates to fungicidal formulations, comprising at least one active compound selected from farnesol and farnesol derivatives and at least one carrier.

In a further aspect, the invention relates to a method of controlling rust fungi, comprising the application of the active compound selected from farnesol or farnesol derivatives to plants, seeds, or to the locus of plants. Moreover, the present invention relates to the use of an active compound selected from farnesol or farnesol derivatives for controlling rust fungi.

The active compound to be used in the context of the present invention comprises a farnesyl (3,7,1 l-trimethyl-2,6,10-dodecatrien-l-yl) moiety. The compound is selected from farnesol, or from its derivatives, such as ethers or esters thereof. The farnesyl moiety has two double bonds in the position C2 and C6, respectively, which may independently take a cis- or transconfiguration. Preferred is a trans-configuration for both double bonds, i.e. an all-trans configuration.

Particularly preferred as an active compound in the context of the present invention is a compound of the following structural formula (I)

(I) wherein R is H, optionally substituted alkyl, optionally substituted aryl or -C(O)R¹, and R¹ is H, optionally substituted alkyl or optionally substituted alkoxy.

The alkyl groups which are represented by R and R¹ in the compound of formula (I) may be branched or linear or may comprise cyclic moieties. They are preferably selected from C₁-C₈ alkyl, particularly from methyl, ethyl, n-propyl, or i-propyl. The same applies to the alkyl moieties in the alkoxy groups represented by R¹. An aryl group represented by R is preferably phenyl or naphtyl, particularly preferably phenyl.

To the optionally substituted alkyl or alkoxy group, one to three substituents may be bound such as -OH. To the optionally substituted phenyl group, one to three substituents may be bound, such as -OH, Cl, Br, methyl, ethyl, n-propyl or i-propyl.

Particularly preferred as R are H, methyl, ethyl and acetyl.

The active compounds to be used in the context of the invention are either commercially available, or may be obtained from commercially available starting compounds via standard routes of synthesis, such as (trans-)esterification or (trans-)etherification reactions known to the skilled practitioner.

Alternatively, the active compounds may be obtained/extracted from plants.

The fungicidal formulation according to the invention, comprising at least one of the above active compounds, is adapted for the application to any part of a plant including foliage, stems, branches, roots or fruits, the seed before it is planted or to the soil or any other plant growth medium. To that extent, it comprises a solid or liquid carrier which should be acceptable for the use in a fungicidal formulation.

The term "carrier"; in the context of the present invention, relates to a natural or synthetic organic or inorganic substance with which the active compound is formulated to facilitate its application to plants. This carrier is generally inert and should be acceptable in the agri- foodstuffs sector. The carrier may be solid or liquid. Examples of solid or semi-solid carriers include clays, natural or synthetic silicates, silica, resins, natural or synthetic waxes such as lanolin or solid fertilizers. Examples of liquid carriers include water and organic solvents. As specific organic solvents, mention may be made of ketones, such as cyclohexanone and methylcyclohexanone, hydrocarbons, alcohols such as bezyl alcohol, furfuryl alcohol, butanol, glycol ethers and natural oils, such as olive oil. As solid or semi-solid formulations, there may be mentioned powders and granules which may be impregnated with the active compound and waxes. As liquid formulations, there may be mentioned solutions, emulsions, suspension concentrates or wettable powders.

Moreover, the active compounds may be combined with any solid or liquid additives corresponding to the usual formulation techniques, such as a surfactant. For example, the presence of at least one surfactant is generally desirable when the carrier includes water. The surfactant may be an emulsifying agent, a dispersing agent or a wetting agent of the ionic or nonionic type or a mixture of such surfactants.

The fungicidal formulations cover not only formulations ready to be applied to the plant or plant growth medium to be treated, but also commercial concentrated compositions which are diluted before application. Accordingly, the compositions according to the invention may contain the active compound in very broad limits, ranging e.g. from 0.05% to 95%. Unless otherwise stated, the percentages given in this description are percentages by weight, based on the total weight of the fungicidal formulation. The active compounds used in the context of the present invention have proven to be effective in concentrations as low as 0.05 %, and formulations ready to be applied usually have concentrations of 0.05% or above, preferably 1%, 2%, 3% or 5% or above. While the active compounds may even be applied in neat form, the formulations usually comprise concentrations of 95% or less, such as 80 or 70 %, and they are usually applied in diluted form with a content of the active compound of 40 % or 30 % or less, or even 20 or 10 % or less.

In the method for controlling rust fungi according to the present invention, the fungicidal formulations or the unformulated active compounds can be applied in a number of ways. For example, they can be applied to any part of a plant including foliage, stems, branches, roots or fruits, the seed before it is planted or to locus of the plant, e.g. the soil or any other plant growth medium. They can be sprayed on or applied as a cream or paste formulation, or they can be applied as a vapour or in the form of impregnated solid or liquid formulations for sustained release. The compounds may also be applied in a microencapsulated form, e.g. in cyclodextrines. A fungicidal formulation in the context of the present application is meant to be a formulation which confers protection of plants against one or more rust fungi. In connection with the present invention, the term "protection" or "protected" refers to the property of the formulation to make plants less susceptible to an attack by a rust fungus. In a preferred embodiment this protection may range from a delay of the infection or infestation of the plant by the rust fungus to a complete inhibition of infection or infestation of the plant by the rust fungus or the development of the rust fungus. Thus, protection may range from a delay to a complete inhibition of disease development. Preferably, protection means a block of rust fungus development on or in a plant or plant species so that the fungus is not able to successfully colonize the plant or plant species. Protection also includes delaying or inhibiting the further spreading of an existing rust fungus infection on a plant or in a plant population.

In connection with the present invention, protection is preferably exerted at the level of development of the haustorium by the rust fungus. In particular, the protective capacity of the formulation according to the present invention resides in the fact that the formulation can inhibit haustorium formation by the rust fungus. This property can be determined, e.g., by light or electron microscopy, preferably in plants treated as described in the attached example. Protection preferably also means a reduction of pustule formation by the rust fungus, e.g. of the number of rust fungus pustules formed on the plant and/or the size of the pustules. The determination of the number of pustules can be achieved by counting the pustules on the plant surface.

Protection in the context of the present invention preferably means a reduction of haustorium development and/or a reduction of pustule formation as compared to corresponding untreated plants by at least 10%, more preferably by at least 20%, still more preferably by at least 50%, even more preferably by at least 80% and most preferably to approximately 100%. A reduction of haustorium development preferably means that the development is severely delayed or hampered, most preferably that the haustorium is not developed. Moreover, a reduction of haustorium development means that, when determining whether the haustorium mother cells (HMC) of a given number of germinated spores on a plant develop a haustorium, the number of developed haustoria (in %) is lower in a plant treated with a formulation according to the invention as compared to an untreated cell. A reduction of pustule formation may mean a reduction of the total number of pustules formed per area (e.g. mm² or cm²). Preferably, it can also mean a reduction of the average pustule size.

Rust fungi, against which the fungicidal agents according to the present invention may be used, include all known rust fungi. In the context of the present invention the term "rust fungi" means fungi of the order Uredinales. In a preferred embodiment, the rust fungus belongs to a family selected from the group consisting of Chaconiaceae, Coleosporiaceae, Cronartiaceae, Melampsoraceae, Phakopsoraceae, Phragmidiaceae, Pileolariaceae, Pucciniaceae, Pucciniastraceae, Pucciniosiraceae, Sphaerophragmiaceae, Uropyxidaceae, mitosporic Uridinales or Uridinales incertae sedis.

More preferably, the rust fungus belongs to a genus selected from the group consisting of Marvalia, Ochropsora, Chrysomyxa, Coleosporium, Cronartium, Endocronartium, mitosporic Cronartiaceae, Melamspora, Dasturella, Phakospora, Prospodium, Gerwasia, Gymnoconia, Kuehneola, Phragmidium, Trachyspora, Triphragmium, Pileolaria, Racospermyces, Uromycladium, Cumminsiella, Endophyllum, Gymnosporangium, Miyagia, Puccinia, Uromyces, Hyalopsora, Melampsorella, Melampsoridium, Milesia, Milesina, Naohidemyces, Pucciniastrum, Thekopsora, Uredinopsis, Dietelia, Nyssopsora, Tranzschelia, Aecidium, Caeoma, Hemileia, Physopella and Uredo.

In a particularly preferred embodiment the rust fungus is of the genus Uromyces, Hemileia, Puccinia or Phakopsora and even more preferred of the species Uromyces fabae, Uromyces appendiculatus, Uromyces striatus, Hemileia vastatrix, Puccinia arachidis, Puccinia graminis, Puccinia hordei, Puccinia recondite or Phakopsora pachyrhizi. Most preferred are the following rust fungi: Uromyces appendiculatus (isolate SWBRl), Uromyces striatus (isolate KNl-I), Puccinia hordei (isolate 22), Puccinia recondite f. sp. tritici (race Arak), P. graminis f. sp. tritici (race ANZ) and Phakopsora pachyrhizi (race Thail).

It is not only possible to protect plants from rust fungi by applying the formulation according to the invention to the plants but also by genetically engineering plants so as to synthesize increased levels of farnesyl diphosphate, farnesyl acetate and/or farnesol. This is possible by increasing in transgenic plants the activity of farnesyl diphosphate synthase (FPS) (EC2.5.1.1/EC2.5.1.10). This enzyme, which is a prenyltransferase, catalyses the two sequential l'-4 condensation reactions of isopentenyl diphosphate (IPP) with the ally lie diphosphates, dimethylallyl diphosphate (DMAPP) and the resulting geranyl diphosphate (GPP), to produce farnesyl diphosphate (FPP). The farnesyl diphosphate formed by this reaction can be further converted in the plants into farnesol either spontaneously or by a reaction catalyzed by specific or unspecific phosphatases. Moreover, it can be further converted in the plants into farnesyl acetate by the action of an acyl transferase.

The increase in activity of farnesyl diphosphate synthase can in particular be achieved by introducing into plants nucleotide sequences encoding farnesyl diphosphate synthase. Thus, the present invention also relates to the use of a nucleotide sequence encoding farnesyl diphosphate synthase for the production of transgenic plants having an increased resistance to rust fungi. Nucleotide sequences encoding farnesyl diphosphate synthase are known in the prior art, e.g. for Arabidopsis thaliana (Delouπne et al., Plant MoI. Biol. 26 (1994), 1867- 1873; GenBank accession numbers NM_202836), Ginkgo biloba (Wang et al., MoI. Cells 18 (2004), 150-156 and GenBank accession number AY 389818), Lycopersicon esculentum (GenBank accession number AF 048747), Artemisia annua (GenBank accession numbers AAU 36376 and AF 136602), Lupinus albus (GenBank accession numbers S 66471 and LAU 20771), Parthenium argentatum (GenBank accession numbers O 24241 and O 24242), Phaseolus lunatus (GenBank accession number BAC 53873), Zea mays (GenBank accession number P 49353), Helianthus annuus (GenBank accession number O 64905), Oryza sativa (GenBank accession numbers BAD 81810, BAB92292, NP_917069), Gossypium arboreum (GenBank accession number CAA72793), Hevea brasiliensis (GenBank accession number AY135188), Mentha x piperita (GenBank accession number AAK 63847), Capsicum annuum (GenBank accession number CAA 59170), Malus x domestica (GenBank accession number AAM 08927), Humulus lupulus (GenBank accession number BAB 40665) and Eucommia ulmoides (GenBank accession number AB 052681). In principle, any nucleotide sequence encoding a farnesyl diphosphate synthase can be used, i.e. any nucleotide sequence encoding FPS from any possible organism which express such an enzyme, such as plants, fungi, animals or bacteria. Moreover, the polynucleotide sequence may encode any possible isoform of FPS. For example, in Arabidopsis thaliana three isoforms of FPS are known, i.e. FPSlS, FPSlL and FPS2. FPSlS and FPSlL are both encoded by the same gene, i.e. the FPSl gene, and only differ at their N-terminus (see, Masferrer et al., Plant J. 30 (2002), 123-132). The polynucleotide encoding FPS may be heterologous or homologous with respect to the plant species into which it is introduced.

In connection with the present invention, the term "resistant" or "resistance" refers to the property of a given plant or plant species to protect itself against an attack by a rust fungus, whereby said protection may range from a delay to a complete inhibition of disease development. Preferably, "resistance" refers to an effective block of rust fungus growth on or in said plant or plant species so that the rust fungus is not able to successfully colonize the plant or plant species. Generally, resistance involves an interplay of various means that aim at blocking penetration of the rust fungus into the plant. In a preferred embodiment of the present application resistance means that the plant produces increased levels of farnesyl diphosphate, farnesyl acetate and/or farnesol in comparison to wild-type plants. Such increased levels of farnesyl diphosphate, farnesyl acetate and/or farnesol are either constitutively present in the plant, i.e. independent of whether there is a rust fungus attack or not. On the other hand, such increased levels of farnesyl diphosphate, farnesyl acetate and/or farnesol may also be achieved by inducible mechanisms with which a plant reacts to a rust fungus attack. An increased level of farnesyl diphosphate, farnesyl acetate and/or farnesol production in comparison to wild-type plants preferably means a farnesyl diphosphate, farnesyl acetate and/or farnesol production which is at least 10% higher, more preferably at least 20% higher, even more preferably at least 50% higher and particularly preferred at least 100% higher than in wild-type plants. The level of farnesyl diphosphate, farnesyl acetate and/or farnesol is preferably measured after infection with a rust fungus. The level of farnesyl diphosphate, farnesyl acetate and/or farnesol can be measured according to methods known to the person skilled in the art. Preferably, it is measured by gas chromatography/mass spectrometry (GC/MS) and most preferably in an assay as described in Materials and Methods and in the Example section further below, hi connection with the present invention, resistance is preferably exerted at the level of development of the haustorium by the rust fungus. This means that the provisions of the invention preferably improve or establish resistance against a rust fungus by decreasing its capacity to develop a haustorium from the haustorium mother cell.

Furthermore, the term "improved resistance" refers to a significant reduction of susceptibility to a rust fungus in plants treated according to the provisions of the present invention as compared to corresponding untreated plants, hi particular, such a reduction of susceptibility may be evident from a significant reduction of haustorium development and/or a significant reduction of pustule formation. Preferably, such a reduction of susceptibility of a plant so- treated is by at least 10%, more preferably by at least 20%, still more preferably by at least 50%, even more preferably by at least 80% and most preferably to approximately 100% as compared to an untreated plant and with respect to the number of haustoria developed and/or formed pustules. The term "treated according to the provisions of the invention" refers to any means of increasing farnesyl diphosphate, farnesyl acetate and/or farnesol in or on the plant such as by overexpressing a farnesyl diphosphate synthase in transgenic plants or by administering a formulation according to the invention to a plant.

Plants overexpressing a farnesyl diphosphate synthase can be produced by methods well known in the art, e.g., by the introduction of a farnesyl diphosphate synthase encoding nucleotide sequence into plants/plant cells by genetic engineering. For example, such plants can be produced by a method comprising the steps of

(a) introducing a polynucleotide encoding farnesyl diphosphate synthase into the genome of a plant cell; and

(b) regenerating the cell of (a) to a transgenic plant.

Optionally, the method may further comprise step (c) producing progeny from the plants produced in step (b). Such a method is also an object of the present invention.

The method according to the invention allows to produce to transgenic plants or plant tissue comprising plant cells which are genetically engineered with a polynucleotide encoding farnesyl diphosphate synthase.

Preferably, in such transgenic plants, the polynucleotide encoding farnesyl diphosphate synthase is expressed at least in one part, i.e. organ, tissue or cell type, of the plant. Preferably, this expression leads to an increase of farnesyl diphosphate synthase activity in the cells which express said polynucleotide or in the environment of such cells, e.g. in the apoplast, in particular in the cell wall, or in the mesophyll. Increase of activity can be detected for instance by measuring the amount of transcript and/or protein in the transformed cell, tissue or plant in comparison to corresponding measurements at non-transformed plant cells, tissue or plants. According to the teachings of the present invention, an increase of the activity of farnesyl diphosphate synthase in transgenic plants leads to an increase of resistance against a rust fungus to which a corresponding wild-type plant is susceptible or at least more susceptible.

The polynucleotide encoding farnesyl diphosphate synthase introduced into the transgenic plant can in principle be expressed in all or substantially all cells of the plant. However, it is also possible that it is only expressed in certain parts, organs, cell types, tissues etc. Moreover, it is possible that expression of the polynucleotide only takes place upon induction, at a certain developmental stage or, as it may be preferred in some embodiments, upon rust fungus attack. In a preferred embodiment, the polynucleotide encoding farnesyl diphosphate synthase is expressed in those parts of the plant that are exposed to rust fungus attack, for example the epidermis or the rhizodermis, preferably in the mesophyll.

In order to be expressed, the polynucleotide that is introduced into a plant cell is preferably operatively linked to one or more expression control sequences, e.g. a promoter, active in this plant cell.

The promoter may be homologous or heterologous with regard to its origin and/or with regard to the gene to be expressed. Suitable promoters are for instance the promoter of the 35S RNA of the Cauliflower Mosaic Virus (see for instance US-A 5,352,605) and the ubiquitin- promoter (see for instance US-A 5,614,399) which lend themselves to constitutive expression, the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) which lends itself to a tuber-specific expression in potatoes or a promoter ensuring expression in photosynthetically active tissues only, for instance the ST-LSl promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO, J. 8 (1989) 2445- 2451), the Ca/b-promoter (see for instance US-A-5 ,656,496, US-A-5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (see for instance US-A-5,034,322; US-A-4,962,028) or the glutelin promoter from wheat which lends itself to endosperm-specific expression (HMW promoter) (Anderson, Theoretical and Applied Genetics 96, (1998), 568-576, Thomas, Plant Cell 2 (12), (1990), 1171-1180), the glutelin promoter from rice (Takaiwa, Plant MoI. Biol. 30(6) (1996), 1207-1221, Yoshihara, FEBS Lett. 383 (1996), 213-218, Yoshihara, Plant and Cell Physiology 37 (1996), 107-111), the shrunken promoter from maize (Maas, EMBO J. 8 (11) (1990), 3447-3452, Werr, MoI. Gen. Genet. 202(3) (1986), 471-475, Werr, MoI. Gen. Genet. 212(2), (1988), 342-350), the USP promoter, the phaseolin promoter (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA 82 (1985), 3320-3324, Bustos, Plant Cell 1 (9) (1989), 839-853) or promoters of zein genes from maize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant MoI. Biol. 15 (1990), 81- 93). However, promoters which are only activated at a point in time determined by external influences can also be used (see for instance WO 93/07279). In this connection, promoters of heat shock proteins which permit simple induction may be of particular interest. Likewise, artificial and/or chemically inducible promoters may be used in this context. Moreover, seed- specific promoters such as the USP promoter from Vicia faba which ensures a seed-specific expression in Vicia faba and other plants may be used (Fiedler et al., Plant MoI. Biol. 22 (1993), 669-679; Baumlein et al., MoI. Gen. Genet. 225 (1991), 459-467). Moreover, fruit- specific promoters, such as described in WO 91/01373 may be used too. In one embodiment, promoters which ensure constitutive expression are preferred. However, in another preferred embodiment, the polynucleotide may be operatively linked to a promoter which is inducible upon pathogen attack, in particular rust fungus attack, as described, e.g. in Rushton et al., (Plant Cell 14 (2002), 749-762) or in Foley and Singh (FEBS Letters 563 (2004), 141-145) and Zhang and Singh (Proc. Natl. Acad. Sci. USA 91 (1994), 2507-2511). Moreover, the polynucleotide may be linked to a termination sequence which serves to terminate transcription correctly and to add a poly-A-tail to the transcript which is believed to have a function in the stabilization of the transcripts. Such elements are described in the literature (see for instance Gi el en et al., EMBO J. 8 (1989), 23-29) and can be replaced at will.

Furthermore, if needed, polypeptide expression can in principle be targeted to any sub- localization of plant cells (e.g. cytosol, plastids, vacuole, mitochondria, ER) or the plant (e.g. apoplast). Localization of the polypeptide in the cytosol is preferred. In order to achieve the localization in a particular compartment, the coding region to be expressed may be linked to DNA sequences encoding a signal sequence (also called "transit peptide") ensuring localization in the respective compartment. It is evident that these DNA sequences are to be arranged in the same reading frame as the coding region to be expressed. Preferably, signal sequences directing expression into the apoplast are used in connection with the present invention.

In order to ensure the location in the plastids it is conceivable to use one of the following transit peptides: of the plastidic Ferredoxin: NADP+ oxidoreductase (FNR) of spinach which is enclosed in Jansen et al. (Current Genetics 13 (1988), 517-522). In particular, the sequence ranging from nucleotides -171 to 165 of the cDNA sequence disclosed therein can be used which comprises the 5' non-translated region as well as the sequence encoding the transit peptide. Another example is the transit peptide of the waxy protein of maize including the first 34 amino acid residues of the mature waxy protein (Klδsgen et al., MoI. Gen. Genet. 217 (1989), 155-161). It is also possible to use this transit peptide without the first 34 amino acids of the mature protein. Furthermore, the signal peptides of the ribulose bisphosphate carboxylase small subunit (Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Nawrath et al., Proc. Natl. Acad. Sci. USA 91 (1994), 12760-12764), of the NADP malat dehydrogenase (Gallardo et al., Planta 197 (1995), 324-332), of the glutathione reductase (Creissen et al., Plant J. 8 (1995), 167-175) or of the Rl protein (Lorberth et al. Nature Biotechnology 16, (1998), 473-477) can be used.

In order to ensure the location in the vacuole, it is conceivable to use one of the following transit peptides: the N-terminal sequence (146 amino acids) of the patatin protein (Sonnewald et al., Plant J. 1 (1991), 95-106) or the signal sequences described by Matsuoka and Neuhaus (Journal of Experimental Botany 50 (1999), 165-174); Chrispeels and Raikhel (Cell 68 (1992), 613-616); Matsuoka and Nakamura (Proc. Natl. Acad. Sci. USA 88 (1991), 834-838); Bednarek and Raikhel (Plant Cell 3 (1991), 1195-1206); and Nakamura and Matsuoka (Plant Phys. 101 (1993), 1-5).

In order to ensure the localization in the mitochondria, it is for example conceivable to use the transit peptide described by Braun (EMBO J. 11, (1992), 3219-3227).

In order to ensure the localization in the apoplast, it is conceivable to use one of the following transit peptides: signal sequence of the proteinase inhibitor II-gene (Keil et al., Nucleic Acid Res. 14 (1986), 5641-5650; von Schaewen et al., EMBO J. 9 (1990), 30-33), of the levansucrase gene from Erwinia amylovora (Geier and Geider, Phys. MoI. Plant Pathol. 42 (1993), 387-404), of a fragment of the patatin gene B33 from Solanum tuberosum, which encodes the first 33 amino acids (Rosahl et al., MoI Gen. Genet. 203 (1986), 214-220) or of the one described by Oshima et al. (Nucleic Acid Res. 18 (1990), 181). The transgenic plants prepared according to the method of the invention may, in principle, be plants of any plant species. They may be both monocotyledonous and dicotyledonous plants. Preferably, the plants are plants which are naturally susceptible to rust fungus infection. More preferably, they are useful plants, i.e. commercially important plants, cultivated by man for nutrition or for technical, in particular industrial, purposes. They may be sugar storing and/or starch-storing plants, for instance cereal species (rye, barley, oat, wheat, maize, millet, sago etc.), rice, pea, marrow pea, cassava, sugar cane, sugar beet and potato; tomato, rape, soybean, hemp, flax, sunflower, cow pea or arrowroot, fiber-forming plants (e.g. flax, hemp, cotton, linseed), oil-storing plants (e.g. rape, sunflower, soybean) and protein-storing plants (e.g. legumes, cereals, soybeans). The plants include fruit trees, like apple or pear trees, palms and other trees, e.g. poplar, or wooden plants being of economical value such as in forestry. Moreover, the method of the invention relates to forage plants (e.g. forage and pasture grasses, such as alfalfa, clover, ryegrass), vegetable plants (e.g. tomato, lettuce, chicory), bean, asparagus, carrot, onion, fruit plants (e.g. gooseberry) ornamental plants (e.g. roses, tulips, hyacinths). The transgenic plants in particular also include plants belonging to the family of Poaceae.

According to the provisions of the invention, transgenic plants can be prepared by introducing a polynucleotide encoding a farnesyl diphosphate synthase into plant cells and regenerating the transformed cells to plants by methods well known to the person skilled in the art. Methods for the introduction of foreign genes into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, vacuum infiltration, biolistic methods like particle bombardment, pollen-mediated transformation, plant RNA virus- mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods known in the art. The vectors used in the method of the invention may contain further functional elements, for example "left border"- and "right border"-sequences of the T-DNA of Agrobacterium which allow stable integration into the plant genome. Furthermore, methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e. the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example co-transformation (Lyznik, Plant MoI. Biol. 13 (1989), 151-161; Peng, Plant MoI. Biol. 27 (1995), 91-104) and/or by using systems which utilize enzymes capable of promoting homologous recombination in plants (see, e.g., WO97/08331; Bayley, Plant MoI. Biol. 18 (1992), 353-361); Lloyd, MoI. Gen. Genet. 242 (1994), 653-657; Maeser, MoI. Gen. Genet. 230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378). Methods for the preparation of appropriate vectors are described by, e.g., Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA. Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (pMK90RK), Koncz, MoI. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471; Koncz, Plant MoI. Biol. 20 (1992), 963-976; Koncz, Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual VoI 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A- 120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example if a phenotype conferred by said strain is desired.

Methods for the transformation using biolistic methods are well known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553- 1558 and Christou (1996) Trends in Plant Science 1, 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY (1995).

The transformation of most dicotyledonous plants is possible with the methods described above. But also for the transformation of monocotyledonous plants several successful transformation techniques have been developed. These include the transformation using biolistic methods as, e.g., described above as well as protoplast transformation, electroporation of partially permeabilized cells, introduction of DNA using glass fibers, etc. Also, the transformation of monocotyledonous plants by means of Agrobacterium-based vectors has been described (Chan et al., Plant MoI. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994) 271-282; Deng et al, Science in China 33 (1990), 28-34; Wilmink et al, Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492; Conner and Dormisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al. Transgenic Res. 2 (1993), 252-265). An alternative system for transforming monocotyledonous plants is the transformation by the biolistic approach (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553- 1558; Ritala et al., Plant MoI. Biol. 24 (1994) 317-325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631). The transformation of maize in particular has been repeatedly described in the literature (see for instance WO 95/06128, EP 0 513 849, EP 0 465 875, EP 29 24 35; Fromm et al, Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726). The successful transformation of other types of cereals has also been described for instance of barley (Wan and Lemaux, supra; Ritala et al., supra, Krens et al., Nature 296 (1982), 72-74), wheat (Nehra et al., Plant J. 5 (1994), 285-297) and rice. The resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known by a skilled person.

The transgenic plants prepared according to the method of the invention show an increased activity of farnesyl diphosphate synthase compared to a corresponding wild-type plant. The term "increased activity" refers to a significant increase of the farnesyl diphosphate synthase activity in the transgenic plant compared to a corresponding wild-type plant. Preferably, said activity is increased in the transgenic plant by at least 10%, preferably by at least 20%, more preferably by at least 50%, and even more preferred by at least 100% as compared to the corresponding wild-type plant. Farnesyl diphosphate synthase activity may be determined in enzyme assays using a preparation from a plant sample. Methods for determining farnesyl diphosphate synthase activity have, e.g., been described in Masferrer et al. (Plant J. 30 (2002), 123-132) and in Chambon et al. (Curr. Genet. 18 (1990), 41-46). Advantageously, a substrate compound is used for such assays for which farnesyl diphosphate synthase is specific and which can be detected by suitable methods known in the art. However, an increase of the activity of the farnesyl diphosphate synthase may also be inferred from a significant increase of the amount of corresponding transcript and/or protein present in the transgenic plant. Preferentially, transgenic plants having an increased activity of the farnesyl diphosphate synthase may be characterized by an increase of the amount of farnesyl diphosphate synthase transcript by at least 20%, preferably at least 50% and more preferably at least 100% as compared to the corresponding wild-type plant. Likewise, it is preferred that transgenic plants having an increased farnesyl diphosphate synthase activity may be characterized by an increase of the protein amount of farnesyl diphosphate synthase by at least 20%, preferably at least 50% and more preferably at least 100% as compared to the corresponding wild-type plant.

Corresponding increases of the farnesyl diphosphate synthase activity may for instance be achieved by expressing said polynucleotide in cells of a transgenic plant from a heterologous construct comprising a polynucleotide encoding farnesyl diphosphate synthase and regularory elements allowing expression in plant cells. Moreover, the state of the art provides further methods for achieving a corresponding increased activity. For example, the endogenous gene encoding farnesyl diphosphate synthase may be modified accordingly at its natural location, e.g. by homologous recombination. In particular, the promoter of this gene can for instance be altered in a way that promoter activity is enhanced. In the alternative, the coding region of the gene can be modified so that the encoded polypeptide shows an increased activity, e.g. by specifically substituting amino acid residues in the catalytically active domain of the polypeptide. Applicable homologous recombination techniques (also known as "in vivo mutagenesis") are known to the person skilled in the art and are described in the literature. One such technique involves the use of a hybrid RNA-DNA oligonucleotide ("chimeroplast") which is introduced into cells by transformation (TIBTECH 15 (1997), 441-447; WO 95/15972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273 (1996), 1386-1389). Thereby, part of the DNA component of the RNA-DNA oligonucleotide is homologous with the target gene sequence, however, displays in comparison to this sequence a mutation or a heterologous region which is surrounded by the homologous regions. The term "heterologous region" refers to any sequence that can be introduced and which is different from that to be modified. By means of base pairing of the homologous regions with the target sequence followed by a homologous recombination, the mutation or the heterologous region contained in the DNA component of the RNA-DNA oligonucleotide can be transferred to the corresponding gene. By means of in vivo mutagenesis, any part of the gene encoding the farnesyl diphosphate synthase can be modified as long as it results in an increase of the activity of this protein, hi a particularly preferred embodiment the transgenic plants show an increased farnesyl diphosphate synthase activity after infection with a rust fungus.

hi a preferred embodiment, the above-described transgenic plants show, upon an increased activity of farnesyl diphosphate synthase, an increased resistance against a rust fungus to which a corresponding wild-type plant is susceptible.

The term "increased resistance" may refer both to an enhancement of a resistance already present in the wild-type plant and to the establishment of a resistance that is not present in the wild-type plant.

Preferably, these transgenic plants contain a polynucleotide encoding farnesyl diphosphate synthase that is introduced in a plant cell and the presence of which in the genome of said plant preferably leads to an increased activity of the farnesyl diphosphate synthase of the invention, stably integrated into the genome.

In another preferred embodiment the plants prepared according to the method of the invention are characterized by increased levels of farnesyl diphosphate, farnesyl acetate and/or farnesol. The determination of farnesyl diphosphate, farnesyl acetate and/or farnesol can, e.g., be determined by measuring the concentration of these compounds in plant extracts. A suitable method is GC/MS after hydrolysis of the diphosphates. Preferably, the amount of farnesyl diphosphate, farnesyl acetate and/or farnesol in the transgenic plants is at least 10%, more preferably at least 20%, more preferably at least 50%, even more preferably at least 80% and most preferably at least 100% higher than the amount in corresponding wild-type plants. In a further preferred embodiment the above-described transgenic plants are characterized by the feature that they are capable of releasing higher amounts of farnesol or farnesol acetate after infection with a rust fungus when compared to a corresponding wild-type plant. The release of farnesol or farnesol acetate can, e.g., be determined as described in the attached Example. Preferably, the release is at least 10%, more preferably at least 20%, even more preferably at least 50%, still more preferably at least 80% and particularly preferred at least 100% higher than in corresponding wild-type plants.

The transgenic plants prepared according to a method of the present invention can preferably be distinguished from wild-type plants inter alia by the fact that they contain integrated into their genome a foreign polynucleotide leading to an increase of activity of farnesyl diphosphate synthase. "Foreign" in this context means that this molecule does either not naturally occur in the genome of a corresponding wild-type plant or, if it does, that it is integrated at a different genomic location, i.e. in a different genomic context. Preferably the polynucleotide encoding farnesyl diphosphate synthase is heterologous with respect to the corresponding wild-type plant. In another preferred embodiment, the polynucleotide encoding farnesyl diphosphate synthase is linked to regulatory regions which are heterologous with respect to the farnesyl diphosphate synthase coding sequence.

Figure 1 shows the morphology of an in vitro differentiated haustorium of Uromyces fabae. Differentiation initiated from uredospores inoculated on a polyethylene membrane and was stimulated by volatiles emitted from a leaf of V. faba. a, Light micrograph of a haustorium inserted into a hypha that had germinated from another uredospore (scale bar, 5 μm). b, Electron micrograph of the haustorial neck (scale bar, 1 μm). c, Electron micrograph of the haustorial body (scale bar, 1 μm).

Figure 2 shows volatiles from Vicia faba affect the differentiation of U. fabae. a, Identification of volatiles by GC/MS from whole plants, control, and infected by U. fabae. Compounds were identified by GC/MS using authentic references. Elution time is given in minutes. IS = internal standard (1-bromodecane), 1 = (3Z)-hexen-3-enyl acetate, 2 = decanal, 3 = nonanal, 4 = farnesyl acetate, (b-f), In vitro differentiation of haustoria on polyethylene membranes after exposure to authentic volatiles. Dilution factors are indicated on the x-axis. Data represent mean ± SD (n= 600 spores/variant). Percentages are based on the uredospores inoculated, g, In planta differentiation of haustoria after exposure to authentic volatiles. V. faba was inoculated with U. fabae and incubated for 10 d in closed glass cylinders. Volatiles evaporated into the gas phase from filter paper. Data represent mean ± SD (n= 300/variant). Percentages are based on HMCs that had formed haustoria within the leaf. Values that differ significantly from controls are indicated by different letters on top of the columns (Dunnett test, P < 0.05).

Figure 3 shows pustule formation by rust fungi on compatible host plants after exposure to volatiles. Incubation and application of volatiles as indicated in the legend to Figure 2 g. For each pathosystem, pustule counts from untreated controls were set to 100%. Data represent mean ± SD (n= 400/variant).

Figure 4 shows pustule formation by Phakopsora pachyrhizi on soybean treated with farnesyl acetate. Farnesyl was mixed with three inert carrier substances and applied 24 h after inoculation with the rust. Data represent mean ± SD (n= 12/variant) and were recorded after 9 d of incubation in a growth chamber. Values that differ significantly from the controls are indicated by different letters on top of the columns (Tukey-Kramer test, P < 0.05).

Figure 5 shows pustule formation by Puccinia graminis on wheat exposed to volatiles. Whole wheat plants were inoculated with P. graminis f.sp. tritici and exposed to volatiles for 6 d in closed glass cylinders. Volatiles evaporated into the gas phase from filter paper, a, Control. Treatment with b, farnesyl acetate; c, nonanal. d, Pustule diameter from the variants a, b and c (data represent mean ± SD, n= 400/variant, ANOVA: P < 0.05).

Figure 6 shows pustule formation by Phakopsora pachyrhizi on soybean after treatment with farnesyl acetate. Whole soybean plants were inoculated with Phakopsora pachyrhizi. 24 h later, the right halves of the leaves were coated with a thin layer of lanolin, the left halves remained untreated, a, lanolin only, b, 10 μl farnesyl acetate added per ml lanolin. Incubation continued then for an additional 9 d in a growth chamber.

Figure 7 shows a schematic representation of Uromyces fabae development on Vicia faba. Aerially dispersed, dicaryotic uredospores are responsible for the build-up of rust epidemics during the summer months. After landing on a leaf, uredospores attach to the cuticle by forming of an adhesion pad. Germination initiates at high humidity and the germ tube grows toward a stoma guided by physical cues on the host surface. An appressorium forms that drives a penetration hypha into the substomatal cavity. Morphogenesis continues with the infection hypha that grows toward a parenchymous cell. Shortly after contacting this cell, the hyphal tip differentiates the haustorial mother cell, which in turn, produces the haustorium within the plant cell. At this point, the pathogen starts to absorb massive amounts of nutrients from the host, thereby establishing biotrophy.

The following Examples serve to illustrate the invention:

Methods

Fungal and plant materials. Broad beans {Vicia faba L. cv ConAmore) and the rust fungus {Uromyces fabae (Pers.) Schroet., isolate 12) were propagated and inoculated as described in Deising, H., Jungblut, P. R. & Mendgen, K. Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae, as revealed by high-resolution two-dimensional polyacrylamide-gel electrophoresis. Arch. Microbiol. 155, 191-198 (1991). Additional host plants used were Phaseolus vulgaris (cv Primel), Medicago sativa (cv Europe), Hordeum vulgare (cv Frankengold), Triticum aestivum (cv Kanzler), Glycine max (cv Erin). Rust inocula included U. appendiculatus (isolate SWBR 1), U. striatus (isolate KNl-I), Puccinia hordei (isolate 22), P. recondita f. sp. tritici (race Arak), P. graminis f. Sp. tritici (race ANZ), and Phakopsora pachyrhizi (race Thai 1).

Analyses of fungal differentiation in vitro and in planta. The infection process was simulated in vitro on scratched polyethylene sheets (Rische and Herfurth GmbH, Hamburg, Germany) (see Deising et al., loc. cit.) that were additionally misted with low gel-strength agar, 1% (Serva GmbH, Heidelberg, Germany) and gelatin, 1% (Serva) in water. This resulted in droplets on the membrane, 200-400 μm in diameter, and improved differentiation of HMCs (from 15±3% to 25±5%, based on the uredospores inoculated) without affecting the differentiation of haustoria. Spores were allowed to -settle on such membranes at a density of about 45 spores/mm². Membranes were then immediately placed into air-tight sealed glass Petri dishes (volume of 100 ml) that provided high humidity through wet pieces of paper. Pure substances (Sigma-Aldrich GmbH, Munich, Germany) were diluted in diethyl ether (Merck KG, Darmstadt, Germany). Ten μl of each dilution were dropped onto filter paper (MN 615, Macherey-Nagel GmbH & Co. KG, Dύren, Germany) which was attached to the upper lid of the Petri dish after evaporating the solvent (10 sec). Incubation was for 24 h. Each experiment was repeated at least three times.

The impact of volatiles on fungal differentiation in whole plants was initially studied with freshly inoculated, ten-days-old host plants kept in tightly closed 10 L glass cylinders for another 10 days (20°C, 16 h light, 200 μE x m^"2 x s^"1). Every day, 0.5 μl of the pure substance was dropped with a syringe onto a filter paper through an opening in the glass cylinder. Haustoria produced within the leaves were examined by microscopy after clearing the tissue with lactophenol (20%) over-night and staining with cotton blue (0.1%) in water for 2 hours. In an additional experiment, fifteen-days-old whole soybean plants that had been inoculated 24 h ago with Phakopsora pachyrhizi were sprayed with 1 ml of water or olive oil in which 1, 10 or 100 μl of farnesyl acetate were suspended. In an alternative treatment, farnesyl was mixed with 1 g of lanolin which was then distributed as a thin layer on the leaves using a brush. Incubation was for another 9 days in a growth chamber that was adjusted to a light period of 16 h, at 22°C and 120 μE x m^"2 x s^"1 and a dark period of 8 h at 18°C.

Microscopy. Light microscopy was performed with a Zeiss Axioplan microscope using a 63x NA 1.4 objective and a digital camera Zeiss Axiocam (Carl Zeiss AG, Jena, Germany). For electron microscopy, samples were fixed in 2% glutaraldehyde and 1% osmic acid, processed and selected for sectioning as described earlier in Boehm, E. W. A. et al. An ultrastructural pachytene karyotype for Puccinia graminis f. sp. tritici. Can. J. Bot. 70, 401-413 (1992). Sections were stained with lead citrate/uranyl acetate and examined with a Hitachi H7000 electron microscope operated at 50 KV.

Identification of volatiles. Whole broad beans incubated at 20°C and 16 h day length under light of 270 μE x m-² x s-¹ were used for collection of volatiles. The aerial parts of the potted plants were enclosed in a flat-flange glass cylinder (50 cm x 12 cm 0) fitting onto the top of the plastic pot that was tightly wrapped with aluminium foil to prevent contamination with soil-derived volatiles. The cylinder was covered with a single-necked flat-flange top attached to an air-circulation system containing charcoal traps (Donath, J. & Boland, W. Biosynthesis of acyclic homoterpenes: Enzyme selectivity and absolute configuration of the nerolidol precursor. Phytochemistry 39, 785-790 (1995)). Volatile collection was performed for 2 x 48 h followed by desorbtion of the carbon traps with 2 x 20 μl dichloromethane. The samples were analysed by gas chromatography/mass spectrometry (Micromass MassSpec 2, Waters GmbH, Eschborn, Germany; Finnigan MAT Magnum, ThermoElectron GmbH, Bremen, Germany) and the eluting volatiles were identified by authentic references.

Example

Morphogenesis of the rust fungus Uromyces fabae, which is pathogenic to broad bean (Vicia /aba), was simulated ex planta on polyethylene membranes imitating the hydrophobic host surface. At high humidity, uredospores germinated and differentiated appressoria triggered by ridges on the membranes that mimicked the stomatal edges (Hoch, H. C, Staples, R. C, Whitehead, B., Comeau, J. & Wolf, E. D. Signalling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235, 1659-1662 (1987)). Overall 25±5% of the uredospores developed HMCs but failed to continue forming haustoria. Recent reports demonstrated that volatiles mediate the interactions of plants with insect herbivores (Pichersky, E. & Gershenzon, J. The formation and function of plant volatiles: perfumes for pollinator attraction and defense. Curr. Opin. Plant Biol. 5, 237-243 (2002)), with rhizobacteria (Ryu, C. M. et al. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 100, 4927-4932 (2003)), and with fungi causing post-harvest fruit rots (Kulakiotu, E. K., Thanassoulopoulos, C. C. & Sfakiotakis, E. M. Biological control of Botrytis cinerea by volatiles of 'Isabella' grapes. Phytopathology 94, 924-931 (2004); Flaishman, M. A. & Kolattukudy, P. E. Timing of fungal invasion using host's ripening hormone as a signal. Proc. Natl. Acad. Sci. U.S.A. 91, 6579-6583. (1994)). To test whether or not volatiles might also influence interactions between rust fungi and their hosts, the development of rust germlings was studied on polyethylene membranes in the presence of volatiles released from broad bean leaves. In the atmosphere of a closed test system (glass petri dish), HMCs differentiated haustoria (0.8%±0.3%) showing the same phenotype as those in planta (Figure Ia). The effect became more pronounced (6.0±0.5%) when the volatile- emitting leaves in the gas phase were previously inoculated with the rust (6 d.p.i.). Haustorial differentiation occurred almost exclusively where HMCs had contacted neighbouring rust hyphae (Figure Ia). The differentiation inside the hypha paralleled the natural infection process and the differentiation proceeded with formation of a haustorial neck with the typical neckband (Figure Ib) and a structure resembling the extrahaustorial matrix sheathing the haustorial body (Figure Ic) in mesophyll cells of the plant host. The enhanced differentiation in the presence of infested leaves suggested that infection might have indeed elicited the release of volatiles (Holopainen, J. K. Multiple functions of inducible plant volatiles. Trends Plant Sci. 9, 529-533 (2004)), which the fungus then recognized as a cue for morphogenesis. That the haustoria formed in neighbouring hyphae rather than plant mesophyll cells, suggests that once the volatile signal is perceived, a rather unspecific surface is sufficient to initiate haustorial development, independently of additional host-specific signals. The volatiles were identified and then the pure synthetic substance(s) were used to reproduce the observed effects and to assess their significance. Gases emitted from healthy and rust- infected plants were collected from intact plants in semi-closed sampling systems for four days after inoculation and analysed by gas chromatography/mass spectrometry (GC/MS) (Kunert, M., Biedermann, A., Koch, T. & Boland, W. Ultrafast sampling and analysis of plant volatiles by a hand-held miniaturised GC with pre-concentration unit: Kinetic and quantitative aspects of plant volatile production. J. Sep. Sci. 25, 677-684 (2002)). Rust infection did not generally alter the blend of fragrances released by broad bean but rather enhanced emission by a factor of about 10 (Figure 2a). Three of the major constituents of this mix, decanal, nonanal, and (3Z)-hexen-3-enyl acetate, induced haustorial formation in a dose-optimum- correlation when introduced into the gas phase of a glass petri dish (Figure 2b-d). Combinations of these volatiles did not significantly increase the rate of haustorium differentiation (not shown). The fourth major volatile, farnesyl acetate, produced no apparent effect when individually employed (Figure 2e). However, the terpenoid acetate counteracted haustorial development when applied simultaneously with decanal (Figure 2f) or the other inducing compounds (not shown). Morphogenesis prior to haustorial development was not altered by these substances.

To examine the impact of these volatiles on fungal development in planta, intact broad bean plants were incubated immediately after inoculation with U. fabae in closed glass cylinders and the volatiles were provided through evaporation from filter paper. In comparison to the control using only the solvent, exposure to decanal and nonanal significantly increased the number of haustoria observed by light microscopy of cleared leaf tissue samples (Figure 2g). hi contrast, farnesyl acetate significantly decreased haustorial development. Microscopy gave no evidence for hypersensitive responses or other defence reactions caused by the treatments. Concurrent with the effects on haustorial development, the number of rust pustules rose by 76% and 132% for decanal and nonanal treatments, but declined by 48% for farnesyl acetate when compared to an untreated control (Figure 3). This discovery was further evaluated in seven additional rust pathosystems, including three cereal rusts that cause severe epidemics. Not only the closely related species U. appendiculatus and U. striatus, but also Phakopsora pachyrhizi infecting soybean, and even the cereal rusts Puccinia graminis, P. hordei and P. recondita responded to volatile treatment in the same way as U. fabae (Figure 3). This important observation suggests that the volatiles involved in the signalling between U. fabae and broad bean may be generally controlling the interactions between plants and rust fungi, hi addition to the number of pustules, their size was also affected by volatiles. This is demonstrated for Puccinia graminis f. sp. tritici infecting wheat (Figure 5). Larger pustules resulted from nonanal, smaller pustules from farnesyl acetate (Figure 5d), which is notable since pustule size also affects the number of spores produced (Robert, C, Bancal, M. O. & Lannou, C. Wheat leaf rust uredospore production on adult plants: influence of leaf nitrogen content and Septoria tritici blotch. Phytopathology 94, 712-721 (2004)). hi a next step it was investigated if farnesyl acetate when directly applied onto the leaves of a soybean plant, which was inoculated with Phakopsora pachyrhizi one day earlier, might also inhibit the pathogen. Three carrier substances were used, i.e. water, olive oil and lanolin, in which farnesyl acetate was suspended to reduce its evaporation. Spray application of farnesyl mixed in water and in olive oil reduced the pustule numbers by up to 67% and 69%, respectively (Figure 4). Application of farnesyl as a thin lanolin coating caused a reduction of up to 98% (Figure 6).

This study uncovered the impact of host-derived volatile signals on fungal morphogenesis within the host. For several species of Colletotrichum it has been shown earlier that ethylene released from ripening fruits induced germination and appressorium formation on the host (Flaishman, M. A. & Kolattukudy, P. E. Timing of fungal invasion using host's ripening hormone as a signal. Proc. Natl. Acad. Sci. U.S.A. 91, 6579-6583. (1994)). The present results indicate that a more complex network of volatile signals between rusts and their host plants may have evolved, which, along with other (macro)molecular signals, controls the differentiation of specialized fungal cell types in order to establish the biotrophic interaction. Fatty-acid-derived aldehydes, known to be involved in the plant's defence against pathogens (Feussner, I. & Wasternack, C. The lipoxygenase pathway. Annu. Rev. Plant Biol. 53, 275- 297 (2002)), serve as positive signals for the differentiation of haustoria of the invading fungus. On the other hand, farnesyl acetate obviously acts as an antagonistic signal and strongly reduces the rate of differentiation. Investigations on the other obligate biotrophic plant pathogens, i.e. the powdery and the downy mildews, will reveal if these taxonomically unrelated groups also respond to volatiles in the development of haustoria. The present results allow to design novel approaches to control rust diseases in crops. Volatiles might either be externally applied as demonstrated here. Alternatively, crop plants may be genetically engineered to release altered volatile profiles. The feasibility of such an approach has been demonstrated (Lewinsohn, E. et al. Enhanced levels of the aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol. 121, 1256-1265 (2001); Kessler, A., Halitschke, R. & Baldwin, I. T. Silencing the jasmonate cascade: induced plant defenses and insect populations. Science 305, 665-668 (2004); Vancanneyt, G. et al. Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance. Proc. Natl. Acad. Sci. U.S.A. 98, 8139-8144 (2001)). Transgenics producing increased levels of farnesyl acetate or derivatives (e.g. farnesyl diphosphat or farnesol) thereof could provide more durable field resistance against rusts than is currently available (Kolmer, J. A. Genetics of resistance to wheat leaf rust. Annu. Rev. Phytopathol. 34, 435-455 (1996); Eversmeyer, M. G. & Kramer, C. L. Epidemiology of wheat leaf and stem rust in the central great plains of the USA. Annu. Rev. Phytopathol. 38, 491-513 (2000)). Such a defence is highly needed especially against the soybean rust Phakopsora pachyrhizi, which has recently spread beyond its original range in eastern Asia and Australia into Africa, Central America, and South America where it had caused losses of $2 billion just in Brazil in 2003 (Stokstad, E. Plant pathologists gear up for battle with dread fungus. Science 306, 1672-1673 (2004); Pivonia, S. & Yang, X. B. Assessment of the potential year-round establishment of soybean rust throughout the world. Plant Disease 88, 523-529 (2004)). This rust, which also represents a major threat to soybean production in the USA has just arrived in five southern states (Stokstad, E., loc. cit; Miles, M. R., Frederick, R. D. & Hartmann, G. L. Soybean rust: Is the U.S. soybean crop at risk? http://www.apsnet.org/online/feature/rust (2003); Rogers, J. & Redding, J. USDA confirms soybean rust in United States. http://www.usda.g0v/wps/p0rtal/Iut/p/j5.7_0_A/7_0_lOB?contentidonly=true&contentid=20 04/ll/0498.xml (2004)). Given that several rust species attack other important crops like wheat and barley, cultivars that carry a trait for general resistance against rusts would be extremely desirable. The present data suggest that host volatiles perceived by rust fungi represent such traits.

Claims

CLAIMS:

1. A fungicidal formulation, comprising at least one active compound selected from farnesol and farnesol derivatives and at least one carrier therefore.

2. The fungicidal formulation according to claim 1, wherein the active compound is at least one farnesol derivative selected from farnesyl ethers and farnesyl esters.

3. The fungicidal formulation according to claim 1 or 2, wherein the farnesol or the farnesol moiety of the farnesol derivatives has an all-trans structure.

4. The fungicidal formulation according to claim 1 , wherein the active compound has a structure represented by formula (I)

(I) wherein R is H, optionally substituted alkyl or -C(O)R¹, and R¹ is H, optionally substituted alkyl or optionally substituted alkoxy.

5. The fungicidal composition according to claim 4, wherein R is selected from H, methyl, ethyl or acetyl.

6. The fungicidal composition according to any of claims 1 to 5, wherein the carrier selected from resins, natural or synthetic waxes water and organic solvents.

7. A method of controlling rust fungi, comprising the application of at least one active compound as defined in any of claims 1 to 5 to plants, seeds, or to the locus of plants.

8. Use of an active compound as defined in any of claims 1 to 5 for controlling rust fungi associated with plants.

9. Use of a nucleotide sequence encoding farnesyl diphosphate synthase for the production of transgenic plants having an increased resistance to a rust fungus.

10. A method for producing transgenic plants with an increased resistance to a rust fungus comprising the steps of:

(a) introducing a polynucleotide encoding farnesyl diphosphate synthase into the genome of a plant cell; and

(b) regenerating the cell of (a) to a transgenic plant.