AU2008200758A1 - A method of controlling fungal pathogens and agents useful for same - Google Patents

Description

S&F Ref: 736811AUD2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: The Australian National University, a body established under the Australian National University Act 1991 (Cwth), of Acton, Australian Capital Territory, 0200, Australia Actual Inventor(s): Address for Service: Invention Title: Not Given Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) A method of controlling fungal pathogens and agents useful for same The following statement is a full description of this invention, including the best method of performing it known to me/us: 5845c(1131980_1) 00 A METHOD FOR CONTROLLING FUNGAL PATHOGENS AND AGENTS USEFUL FOR SAME This application is a divisional application of Australian Patent Application No. 2004201516

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filed on 8 April 2004, the contents of which are herein incorporated by reference.

FIELD OF THE INVENTION 00 I The present invention relates generally to the use of a sugar acid to inhibit, retard or otherwise control the growth and/or viability of plant pathogens, such as, for example, Sthose prokaryotic and eukaryotic organisms which infect or otherwise infest plants or 00 10 parts of plants. In particular, the present invention relates to the use of a sugar acid 0selected from the group consisting of gluconic acid, mannonic acid, malic acid, ascorbic acid, glucaric acid, glutaric acid, glucuronic acid, galacturonic acid and galactonic acid to inhibit, retard or otherwise control the growth and/or viability of a plant pathogen, in particular a fungal pathogen. The invention further extends to the use of a sugar acid in the manufacture of a phytoprotective agent for the treatment of fungal infestations of plants. As disclosed herein, the sugar acid are applied to a plant or plant part in crude or pure form, and, as a consequence, the invention further extends to a microorganism which is capable of producing a sugar acid when provided with an appropriate substrate, such as, for example, a species or sub-species of the genus Pseudomonas which is capable of metabolizing D-glucose and other carbon sources to a corresponding sugar acid. The present invention further relates to nucleotide sequences of Pseudomonas sp. which encode and/or otherwise facilitate the synthesis of sugar acids in prokaryotic and eukaryotic organisms, in particular bacteria and/or plants. The present invention further contemplates transgenic plants and microorganisms which have been genetically engineered to produce sugar acids at levels sufficient to control the growth and/or viability of one or more plant pathogens.

BACKGROUND OF THE INVENTION 1. General As used in the specification and claims, the singular form "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a fungal pathogen" includes a plurality of fungal pathogens, including mixtures thereof.

As used herein the term "derived from" shall be taken to indicate that a specified integer are obtained from a particular source albeit not necessarily directly from that source.

00 The terms 'fungus" or "fungi" include a wide variety of nucleated spore-bearing d organisms that are devoid of chlorophyll. Examples of fungi include yeast, moulds, mildews, rusts, and mushrooms.

"Fungicidal" means the ability of a substance to increase mortality of fungi.

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r "Antifungal" includes any substance that is able to kill, inhibit or slow the growth of 0 fungi.

00 SAs used herein, an acaricide, a bactericide, an insecticide, a herbicide, a molluscide, a nematicide, a rodenticide or vermin animal or noxious plant growth regulant are herein understood to be collectively referred to as "pesticides" and the term "pesticide" shall be understood to refer to any one or more of the afore-mentioned.

A "composition" is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

The embodiments of the invention described herein with respect to any single embodiment shall be taken to apply mutatis mutandis to any other embodiment of the invention described herein.

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

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be 00

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O understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred Sto or indicated in this specification, individually or collectively, and any and all t combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific examples described 0t herein. Functionally equivalent products, compositions and methods are clearly within t the scope of the invention, as described herein.

00 10 The present invention is performed without undue experimentation using, unless 0otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombining DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated herein by reference: 1. Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; 2. DNA Cloning: A Practical Approach, Vols. I and II N. Glover, ed., 1985), IRL Press, Oxford, whole of text; 3. Oligonucleotide Synthesis: A Practical Approach J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135- 151; 4. Nucleic Acid Hybridization: A Practical Approach D. Hames S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Perbal, A Practical Guide to Molecular Cloning (1984); 6. Wiinsch, ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Miiler, vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

7. Handbook of Experimental Immunology, Vols. I-IV M. Weir and C. C.

Blackwell, eds., 1986, Blackwell Scientific Publications).

Bibliographic details of the publications referred to in this specification are collected at the end of the description.

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O This specification contains nucleotide and amino acid sequence information prepared Susing PatentIn Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed t^ by the sequence identifier <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, 0 0 <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID followed by the sequence identifier (eg. SEQ O ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).

00 The designation of nucleotide residues referred to herein are those recommended by the c IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

For the purposes of nomenclature, the nucleotide sequences set forth in the Sequence Listing are as follows: SEQ ID NO: 1 is the terminal 757 bp of a sugar oxidase-encoding gene contained in cosmid clone pMN M53; The nucleotide sequences set forth in SEQ ID NOs: 2 to 6 relate to fragments of the PQQ operon of Pseudomonas strain AN5. In particular, SEQ ID NO: 2 comprises at least a fragment of the PQQA gene and a fragment of the PQQB gene of Pseudomonas strain AN5; SEQ ID NO: 3 comprisese a fragment of the PQQC gene of Pseudomonas strain AN5; SEQ ID NO: 4 comprises a fragment of the PQQD gene of Pseudomonas strain AN5; SEQ ID NO: 5 comprises a fragment of the PQQE gene of Pseudomonas strain AN5; and SEQ ID NO: 6 comprises a fragment of the PQQF gene of Pseudomonas strain

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O SEQ ID NO: 7 relates to the nucleotide sequence of the full-length sugar oxidase gene of Pseudomonas strain AN5. The amino acid sequence of the polpeptide encoded by SSEQ ID NO: 7 is set forth in SEQ ID NO: 8.

SEQ ID NOs: 9, 11, 13, 15, 17 and 19 provide the nucleotide sequence of the PQQ operon of Pseudomonas strain AN5, including the PQQA, PQQB, PQQC, PQQD, 0 0 PQQE and PQQF genes.

O SEQ ID NO: 10 comprises the amino acid sequence of a protein involved in the 00 10 synthesis of PQQ and encoded by the PQQF gene of the PQQ operon of Pseudomonas Sstrain SEQ ID NO: 12 comprises the amino acid sequence of a protein involved in the synthesis of PQQ and encoded by the PQQA gene of the PQQ operon of Pseudomonas strain SEQ ID NO: 14 comprises the amino acid sequence of a protein involved in the synthesis of PQQ and encoded by the PQQB gene of the PQQ operon of Pseudomonas strain SEQ ID NO: 16 comprises the amino acid sequence of a protein involved in the synthesis of PQQ and encoded by the PQQC gene of the PQQ operon of Pseudomonas strain SEQ ID NO: 18 comprises the amino acid sequence of a protein involved in the synthesis of PQQ and encoded by the PQQD gene of the PQQ operon of Pseudomonas strain SEQ ID NO: 20 comprises the amino acid sequence of a protein involved in the synthesis of PQQ and encoded by the PQQE gene of the PQQ operon of Pseudomonas strain 2. Description of the related art Pathogenic infections, in particular fungal infections, of animals (including humans and agricultural and domestic animals), plants, (in particular, agriculturally-, 00

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O horticulturally- and silviculturally significant plants) or products of animals and/or plants produce significant losses in productive capacity worldwide.

True fungal pathogens are classified into at least three major classes, the Phycomycetes, the Ascomycetes, and the Basidiomycetes.

0 0 With particular regard to fungal pathogens which infect humans and other mammals, over 40 species are known which attack epithelial tissues (eg. hair, skin, and nails), Scausing diseases and discomforts of an annoying nature, such as, for example, tinea 00 10 pedis (athlete's foot), tinea cruris, tinea corporis (ringworm) due to infection by the dermatophytes Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum, c and Microsporum canis; candidiasis due to Candida albicans, including cutaneous candidiasis (thrush), onychia, paronychia, external genital candidiasis, candidal balanitis; pityriasis versicolor due to Pityrosporum orbiculare (Malasseziafurfur); and jock-strap itch. Additionally, certain species of fungi infect sub-cutaneous tissues, including those of major internal organs, to produce serious systemic diseases, including blastomycosis, coccidiomycosis, histoplasmosis, and sporotrichosis. These more serious fungal infections are generally soil-borne, easily aerosolized, spread by air currents, and, as a consequence, contracted by inhalation. The fungal pathogens are mostly dimorphic, occurring as a mycelium in the non-pathogenic state, which bears infectious spores.

Many more fungal pathogens produce a variety of diseases in plants, wherein different species of fungi tend to invade particular species, and particular tissues, of plants. Crop damage from fungal infection accounts for many millions of dollars in lost profits annually. The Basidiomycetes are of particular importance in agriculture and horticulture, and include the rust fungi, such as, for example, Puccinia spp., Cronartium ribicola, and Gymnosporangiumjuniperi-virginianae; the smut fungi, such as, for example, Ustilago spp., which infect corn and oats, amongst others, causing up to 30% losses annually. Additionally, take-all disease, which is caused by infection of wheat plants by the fungal pathogen Gaeumannomyces graminis var. tritici (or commonly known as the "take-all" fungus) is the most significant root disease of wheat around the world and currently leads to 10% loss of the annual wheat crop in Australia (Murray and Brown, 1987). Other important fungal diseases in plants include crown wart of alfalfa disease, which is caused by Physoderma alfalfae; bitter rot of apple disease, caused by Glomerella cingulata; apple rust, caused by Gymnosporangium 00

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Sjuniperi-virginianae; apple scab, caused by Venturia inaequalis; banana wilt, caused by Fusarium oxysporum f. cubense; loose smut of barley, caused by Ustilago nuda Rostr.; Searly and late blight in celery, caused by Septoria apiicola; Fusarium yellow in celery, -n caused by Fusarium oxysporum f. apii Snyder Hansen; ergot in grain crops, and grasses, caused by Claviceps purpurea; stem rusts, caused by Puccinia spp., in particular P. graminis; late blight in potato, caused by Phytopthera infestans; and citrus 00 root rot diseases caused by Armillaria mellae. This list is, however, not exhaustive.

0Post-harvest spoilage of agricultural commodities such as fruits and vegetables has 00 10 been estimated to result in losses of up to 50% of the crop worldwide.

c N Much of the post-harvest loss is due to fungal diseases, such as moulds and rots. Often infection is initiated by injuries made at harvest or by mechanical wounds to the surface of the agricultural, horticultural or silvicultural products during processing. Chemical fungicides are the principal means of controlling post-harvest losses due to fungal disease. The traditional method of fungicide use is to treat the product, e.g. fruit, vegetable, bloom or wood, after harvest and before storage with chemical fungicides.

Fungicide-tolerant strains are, however, present in most packing and processing facilities, rendering chemical fungicides less effective or totally ineffective. For this reason, multiple fungicides are sometimes used simultaneously to improve control of fungal pathogens.

An additional problem with the use of chemical fungicides is the fact that many are carcinogenic or environmentally hazardous. At a time when synthetic pesticide use is being curtailed, there is clearly an urgent need to develop safe, new and effective methods of controlling post-harvest diseases of agricultural, horticultural or silvicultural products that are safe, environmentally benign, and effective. Biological control offers an attractive alternative to synthetic chemical fungicides. Biopesticides (living organisms and the naturally produced compounds produced by these organisms) can be safer, more biodegradable, and less expensive to develop.

Methods for the prophylactic and/or therapeutic treatment of fungal and bacterial infections in animals and plants generally involve the application of anti-fungal and anti-bacterial chemicals; the use of biocontrol agents; and, more recently, and particularly in the case of plants, the genetic engineering of crops to express disease tolerance or disease-resistance genes therein.

00 Fungicidal and fungistatic chemical compounds: r Anti-fungal chemicals are varied in composition and designed to either eradicate the fungal pathogen, such as, for example, by acting against the fungal spores, alternatively, to prevent the germination of fungal spores once they have infected their host. Most chemicals, however, do not fall exclusively into a single category. For 00 00 example, elemental sulfur has been used to protect apple crops against apple scab, whereas the same chemical is eradicative when used against a rust fungus.

00 10 A wide variety of pyridine compounds and derivatives thereof having varying, and 0often multiple, action, are used in agrochemicals and pharmaceuticals against fungal pathogens, such as, for example, 3-(2-methylpiperidino) propyl-3,4-dichlorobenzoate; cephalosporin C; cephapirin sodium; pyrithione zinc 2-mercaptopyridine N-oxide); and 2-sulfanylamidopyridine.

Many of the compounds used commonly to treat fungal infections in humans interfere with fungal sterol biosynthesis. For example, the imidazoles (including bifonazole [i.e.

l-(a-biphenyl-4-ylbenzyl)-imidazole], clotrimazole, econazole nitrate, and miconazole nitrate 1-[2,4-dichloro-P-(2, 4-dichlorobenzyloxy)phenethyl] imidazole nitrate]), which have broad spectrum antifungal activity against dermatophytes and yeasts alter fungal cell membranes by interfering with ergosterol production. The allylamines, such as, for example, terbafine hydrochloride (C 21

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25 N. HC1), are also of use in the treatment of infections by dermatophytes and also interfere with ergosterol production.

Terbafine also results in the accumulation of squalene in the fungus, resulting in fungal cell death. Amorolfine hydrochloride -dimethylpropyl) phenyl]-2methylpropyl]-2,6-dimethyl morpholine hydrochloride) is in a relatively new class of compounds active against a wide range of yeasts, dermatophytes, moulds, and dimorphic fungi. The fungicidal/fungistatic effect of amorolfine hydrochloride is based upon modification to the fungal cell membrane which is produced by reducing the ergosterol content and increasing the levels of sterically-nonplanar sterols in the fungal cell membrane.

Agricultural fungicides and their modes of application are reviewed in detail by Gennaro et al. (1990), and Kirk-Othmer (1980). These compounds are generally of the class of polysulfides; heavy-metal fungicides; and the organic fungicides, such as, for example, the quinones (in particular, chloranil and dichlone), organic sulfur-containing

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0 compounds (in particular, the dithiocarbamates), imidazolines and guanines (in particular heptadecyl-2-imidozolinium acetate; and dodecylguanidium acetate), r trichloromethylthiocarboximides [in particular, N-trichloromethylthio)-4-cyclohexeneit 1,2-dicarboximide (captan); and N-(trichloromethylthio) phthalimide (folpet)], the chlorinated and/or nitrated benzene derivatives [in particular, 2,3,5,6tetrachloronitrobenzene; pentachloronitrobenzene (PCNB); 1,3,5- trichloro-2,4,6- 00 0t trinitrobenzene;1,2,4-trichloro-3,5-dinitrobenzene; hexachlorobenzene; 2,6-dichloro-4nitroaniline (dichloran); 1,4-dichloro-2,5-dimethoxybenzene; and Otetrachloroisophthalonitrile (chlorothalonil)]. Various other compounds having 00 10 systemic anti-fungal activity in agricultural and horticultural applications systemic 0fungicides) include the oxathiins (in particular, carboxin); benzimidazoles [in particular, methyl-2-benzimidazolylcarbamate pyrimidines [in particular, 5-butyl-2-dimethylamino-6-methyl-4(1 H)-pyrimidinone (cimethirimol); the 2-ethylamino analogue, ethirimol; and a-(2,4-dichlorophenyl)a-phenyl-5-pyrimidinemethanol (triarimol)]. The antibiotics, including cycloheximide, bastocidin S, kasugamycin, and the polyoxins, are also used extensively.

In most cases the mode of action of known agricultural, horticultural or silvicultural anti-fungal compounds remains elusive, and, as with antifungal compounds that are effective against fungal pathogens of humans, many compounds alter fungal cell membrane metabolism. For example, triarimol and related compounds inhibit steps in sterol biosynthesis. Carboxin is, however, known to block mitochondrial respiration by inhibiting succinate dehydrogenase, whilst the benzimidazoles disrupt cell structures, such as those required for mitosis.

Anti-fungal biocontrol agents: Biological control protection refers to the introduction of living organisms, such as, for example, bacteria, fungi, and insects, to control plant pathogens. Three properties make biocontrol agents a desirable option in plant and post-harvest product protection against fungal pathogens. First, biocontrol agents are generally natural products and, as such, are less likely to have a detrimental effect on the environment than synthetic chemicals.

Second, biocontrol agents are relatively inexpensive compared to synthetic chemicals.

Third, biocontrol agents represent an inexhaustible source of protectant, because microorganisms can be maintained in culture, and expanded rapidly and inexpensively.

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O Biocontrol agents either act by occupying the site of infection of a pathogen, and N competing with the pathogen for nutrients derived from the plant or post-harvest Sproduct, alternatively, the biocontrol agent produces fungicidal and/or fungistatic t compounds that specifically attack the fungal pathogen. Significant factors important in biological control (such as colonization, antibiosis, siderophore production etc.) have been identified. The influence of these factors, however, varies with environmental 00 conditions so it is difficult to assign a universal mechanism of action to biocontrol Sagents (Weller, 1988).

00 10 The universal feature of all biocontrol agents is their capacity to multiply rapidly in the cells and tissues that are infected by the pathogenic agent and to form an association c therewith, a process which has been termed "colonization" (Suslow, 1982). In fact, Weller (1988) identified three factors as being responsible for the suppressive nature of various bacteria against fungi: colonization of the root; the production of fungicides and/or fungistatic compounds, including antibiotics, HCN (Fravel, 1988), or hydrogen peroxide (Wu et al., 1995; US Patent No. 5, 516, 671), amongst others; and the production of fluorescent siderophores, or high-affinity iron-transport agents. In this regard, the different colonization host range shown by biocontrol bacteria (Weller, 1988) suggests there is some host specificity in colonization, and that a number of factors are involved. As will be known to those skilled in the art, there is also hostspecificity in the action of fungicides, and this might explain why some strains are ineffective against certain pathogens (Schroth and Hancock, 1981). Additionally, not all biocontrol agents act by producing fungicides or fungistatic compounds (Kraus and Loper, 1992).

There are a large number of systems in which biocontrol is effective (Weller, 1988). In general, biocontrol agents have been shown to have a beneficial effect to the plant under controlled conditions in the glasshouse and in a large number of cases in the field (Baker and Cook, 1974) There are currently numerous biocontrol agents used by farmers for disease control (Schroth and Hancock, 1981), showing that it is effective and viable as a method to control plant diseases in the field.

For example, Sclerotinia rot, caused by the fungi Sclerotinia sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum, is one of the most destructive diseases of plants, affecting over 380 ornamentals aster, begonia, calendula, chrysanthemum, fuchsia, gerbera, lupin, pelargonium, and petunia), field crops alfalfa, canola, dry bean, i.

00 O hemp, lentil, oilseed rape, peanut, potato, red clover, safflower, soybean, sunflower, sweetclover, and tobacco), vegetables and fruits artichoke, asparagus, avocado, Sbean, broccoli, cabbage, carrot, celery, chickpea, chicory, cucumber, eggplant, endive, t fennel, kiwi fruit, leek, lettuce, parsley, pea, pepper (chilli, red or sweet), snap bean, tomato, watermelon, garlic and onion) and herbs coriander, chives, dill, fennel, and wintercress). A biological plant-protection agent, containing as an active ingredient 0n viable spores of the soil fungus Coniothyrium minitans, has been developed recently which has specific antagonistic action against the survival structures (sclerotia) of these 0 fungal pathogenic agents. Once applied and incorporated into the soil, C. minitans 00 10 germinates and attacks the sclerotia (resting survival structures) of the pathogens within Sthe soil, thereby reducing recurrences of the disease in the soil.

In the case of take-all disease in wheat, Pseudomonas sp., have been identified which produce a low molecular weight siderophore, sometimes fluorescent, capable of complexing and actively-transporting iron inside the cell, to produce an iron deficiency in the soil (Buyer and Leong, 1986; Leong, 1986), thereby effectively starving the fungal pathogen of soil-derived iron preferred for germination and growth. Siderophore production is now, however, thought only to be important in the biocontrol of take-all disease in alkaline soils which have low iron concentration, wherein iron are a limiting nutrient for the fungus (Kloepper et al., 1980). Moreover, Fravel, (1988); Hamdan et al.

(1991); and Thomashow et al., (1990), have suggested that the dominant important factor in disease suppression is the production of fungicides and/or fungistatic compounds by Pseudomonas sp. in particular phenazine -1-carboxylic acid (Thomashow et al., 1993); and 2,4-diacetylphloroglucinol, a normal intermediary in a pathway in bacteria which inhibits a range of fungal pathogens, and is found in a wide range of Pseudomonads (Keel et al., 1992; 1996). Phenazine has been extensively characterised in take-all biological control protection, whereas the effectiveness of 2,4-diacetylphloroglucinol against take-all has only been partially characterised (Raaijmakers and Weller, 1998). Pseudomonasfluorescens strain CHAO has been also extensively studied and shown to produce an effective amount of 2, 4-diacetylphloroglucinol for the suppression of black rot disease of tobacco and take-all disease of wheat (Laville et al., 1992).

Additionally, the isolated Pseudomonas strain AN5, has been shown to have a wide host range in so far as it is able to colonize the roots of a number of plant species, and is an effective biocontrol agent against take-all disease in agar plate assays, pot 00 experiments, and in field trials (Nayudu et al. 1994b). Those authors concurred with Thomashow et al. (1993) in concluding that the dominant effects of this bacterium Sappeared to reside in the production of fungicides and/or fungistatic compounds, rather than in the production of siderophores.

Although other biocontrol agents have been tested for their ability to control take-all 00 disease, such as, for example, non-pathogenic strains of G. graminis var. graminis (Wong et al., 1996), they are not a feasible method for large scale control of take-all as there is no current technology available to grow such fungi on a large scale. The cost of 00 10 producing such a fungal agent and to apply same in the field is prohibitively high Scompared to bacterial biocontrol agents.

Genetically-manipulated plants expressing disease resistance or tolerance: In certain instances of disease in plants, genes which encode particular enzymes that are involved in the production of anti-fungal fungicidal and/or fungistatic) compounds have been identified and expressed in plants to introduce tolerance or resistance thereto. In such cases, there is a requirement for the plant to be capable of expressing the introduced gene(s) and to produce the anti-fungal product from either endogenous plant metabolites, alternatively, in addition, from exogenous substrates. As will be known to those skilled in the art, once synthesized, the antifungal compound must be capable of diffusion to an appropriate site of action, or be actively transported, to exert its action against the invading pathogen.

One example of such protection is the expression of a gene encoding the Aspergillus niger or Talaromyces flavus glucose oxidase enzymes (EC 1.1.3.4) to control Phytopthera infestans, or Verticillium dahliae (Murray et al., 1997; Stosz et al., 1996; Wu et al., 1995; US Patent No. 5,516,671) in plants. The efficacy of this approach was based upon the involvement of hydrogen peroxide in plant defense responses in incompatible plant-pathogen interactions, wherein it may activate the production of phytoalexins and other cellular protectants such as, for example, salicylic acid and glutathione-S-transferases; induces cross-linking of hydroxyproline-rich glycoproteins (HPGP); and is involved in triggering hypersensitive cell death in response to pathogen invasion. Additionally, hydrogen peroxide is a product of the enzymic action of glucose oxidase, which catalyzes the oxidation of glucose to 8-gluconolactone and hydrogen peroxide. As a consequence, the expression of glucose oxidase in plants was considered a suitable means for producing hydrogen peroxide as an anti-fungal agent. The

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O attractiveness of this approach arose, in part, from the knowledge that the glucose oxidase gene of Penicillium dangearii is involved in the effective biocontrol of V Sdahliae by this organism (Kim et al., 1988, 1990; US Patent No. 5, 516, 671).

Plants expressing a range of leucine-rich repeat proteins that comprise nucleotide binding sites have also been described as providing improved protection against various 00 diseases in plants, including rust fungi (see, for example, International Patent Application No. PCT/AU95/00240).

OO 10 SUMMARY OF THE INVENTION In work leading up to the present invention, the present inventors sought to identify an

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environmentally-acceptable means for the treatment of a wide range of fungal pathogens in or on agricultural, horticultural or silvicultural post-harvest products. The inventors identified the anti-fungal component produced by Pseudomonas strain as a sugar acid, and have determined the biochemical and genetic pathway for its production. The identification of this particular anti-fungal agent, and the cloning of the genetic sequences encoding proteins involved in the biosynthesis of the antifungal agent enables the development of strategies for the control of a wide range of fungal pathogens in or on agricultural, horticultural or silvicultural post-harvest products.

Accordingly, a related embodiment of the present invention is directed to a method of prophylactic or therapeutic treatment of infection by a fungal pathogen in an animal or plant comprising administering an effective amount of a sugar acid thereto for a time and under conditions sufficient to inhibit or prevent fungal growth or reproduction. In an alternative related embodiment, a biocontrol agent which is capable of producing an anti-fungal active amount of a sugar acid in the presence of an aldose substrate is administered to the plant of animal to inhibit or prevent fungal growth or reproduction.

Another related embodiment provides a biocontrol agent which is capable of producing an anti-fungal effective amount of a sugar acid in the presence of an aldose substrate is applied to the plant product to inhibit or prevent fungal growth or reproduction. This related embodiment applies to any plant product, in particular those edible products intended for human or animal consumption, which are susceptible to rapid degradation due to fungal infection, such as, for example, fruit and vegetables, including tomatoes, apples, pears, citrus fruits, grapes, and berries, amongst others.

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0 Another related embodiment of the invention provides a biocontrol agent for the treatment of a fungal infection in a plant or animal, wherein said agent comprises an Sisolated bacterial cell which is capable of producing a sugar acid when cultured in the n presence of a carbon source comprising an aldose, and wherein said biocontrol agent is further capable of colonising the infection site. In a particularly preferred related embodiment, the biocontrol agent consists of an isolate of Pseudomonas sp. having the 00 characteristics of Pseudomonas strain AN5 rif(AGAL Accession No. NM 00/09624).

SA further related embodiment provides a phytoprotective composition for the treatment 00 10 of a fungal infection of a plant comprising an effective amount of a sugar acid in Scombination with a phytopathologically-acceptable diluent or wetting agent. The

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r phytoprotective composition may also include a biocontrol agent which produces the sugar acid in the presence of an appropriate aldose substrate by virtue of the expression of a PQQ-dependent sugar oxidase enzyme therein.

A further related embodiment provides a composition for the: treatment of a fungal infection in a human or other mammal comprising an effective amount of a sugar acid in combination with one or more pharmaceutically-acceptable carriers or diluents. As with other compositions described herein, those compositions for animal use may comprise a biocontrol agent which expresses a PQQ-dependent sugar oxidase enzyme.

The active sugar acid and/or biocontrol agent, and compositions comprising same, as described herein, can be produced by any means known to those skilled in the art.

Additionally, certain sugar acids, including gluconic acid, are readily available to the public from any one of a number of sources. One related embodiment, however, clearly extends to a method of producing a sugar acid comprising introducing an isolated nucleic acid molecule which encodes a PQQ-dependent sugar acid biosynthetic enzyme to an organism and culturing said organism in the presence of an aldose substrate for a time and under conditions sufficient to produce a sugar acid. Preferably, the sugar acid is subsequently extracted from the organism. More preferably, the sugar acid is partially or substantially purified from the organism or an extract thereof.

A preferred embodiment provides an isolated nucleic acid molecule comprising a nucleotide sequence which encodes one or more proteins in the biosynthesis of a sugar acid or is complementary to said nucleotide sequence. Preferably, the nucleotide sequence of the invention encodes a sugar oxidase enzyme.

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It will be evident from the foregoing discussion that not all cells produce PQQ as a Scofactor, and, as a consequence, to produce a sugar acid in accordance with the inventive concept of the related embodiment, it are preferred in certain circumstances to express the enzymes involved in PQQ biosynthesis, in addition to those gene(s) encoding the sugar oxidase enzyme. The present inventors have identified one or more 0 0 genes of the PQQ operon of Pseudomonas strain AN5, in particular genes designated PQQA, PQQB, PQQC, PQQD, PQQE, and PQQF. Accordingly, a preferred 0 embodiment of the present invention further provides an isolated nucleic acid molecule 00 10 which comprises a nucleotide sequence encoding one or more polypeptides involved in the biosynthesis of PQQ, preferably selected from the group consisting of PQQA, c PQQB, PQQC, PQQD, PQQE, and PQQF.

A still further related embodiment of the invention provides a method of enhancing the tolerance of a plant to infection by a fungal pathogen comprising expressing therein a first isolated nucleic acid molecule encoding a sugar oxidase, and optionally a second isolated nucleic acid molecule encoding one or more PQQ-biosynthesis enzymes for a time and under conditions sufficient for a sugar acid to be produced by said plant, or by a cell, tissue or organ of said plant.

According to one embodiment of the invention there is provided an anti-fungal composition comprising an effective amount of a sugar acid selected from the group consisting of mannonic acid, gluconic acid and galactonic acid when used to prevent or inhibit the growth or reproduction of a fungal pathogen in or on an agricultural, horticultural or silvicultural post-harvest product. Preferably, the sugar acid is gluconic acid.

In a preferred embodiment, the anti-fungal composition further comprises an additional chemical or biological pesticide.

In a further preferred embodiment, the anti-fungal composition further comprises a diluent; a wetting agent; a humectant; a wax or setting agent; or any combination thereof.

00 0 O Preferably, the anti-fungal composition according to the invention is formulated as a wettable powder, a dry flowable powder, a granule, an aqueous suspension, an Semulsion, or as a microencapsulated particle.

According to another embodiment of the invention there is provided an agricultural, horticultural or silvicultural post-harvest product having applied thereto a composition 00 00according to the invention. Preferably, the post-harvest product is a post-harvest product of an ornamental plant, a field crop, a vegetable plant; a fruit tree, a nut tree, a Swood tree or a herb plant. Also preferably, the post-harvest product is a fruit, a 00 10 vegetable, a cereal, a grain, a nut, a seed, a floral bulb, wood, timber, silage, bark, natural latex, a tuber, a bloom, a leaf, a stem, a branch, or a root.

In a preferred embodiment, the cereal is wheat, maize, sorghum, rice, rye, oats, millet, or barley.

In a further preferred embodiment, the seed is a legume seed. Preferably, the legume seed is a soybean.

In a further preferred embodiment, the nut is a peanut, almond, Brazil nut, or pecan.

In a further preferred embodiment, the fruit is a pome fruit, stone fruit, citrus fruit, grape, tomato, potato, persimmon, strawberry,, papaya, banana, or tomato. Preferably, the pome fruit is an apple or a pear. Also preferably, the apple is a Granny Smith, Red or Golden Delicious, Jonathan, Gala, Fuji, Newton, or Macintosh strain. Also preferably, the pear is a d'Anjou, Packham's Triumph, William's Bon Chretian, or Beurre Bose strain.

In a further preferred embodiment, the citrus fruit is grapefruit, orange, lemon, kumquat, lime, mandarin or pomelo.

In a further preferred embodiment, the stone fruit is a peach, nectarine, apricot, plum, or cherry.

In a further preferred embodiment, the vegetable is an asparagus, carrot, beet, sugar beet, cabbage, cauliflower, brussel sprout, artichoke, Jerusalem artichoke, lettuce, spinach or potato.

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In a further preferred embodiment, the post-harvest product is a processed post-harvest Sproduct. Preferably, the processed post-harvest product is selected from the group consisting of raisins, prunes, figs, dried apricots, and dates.

According to yet another embodiment of the invention there is provided a method of 0 0 preventing or inhibiting the growth or reproduction of a fungal pathogen in or on a post-harvest agricultural, horticultural or silvicultural product comprising applying to Sthe post-harvest product an effective amount of the composition according to the 00 10 invention for a time and under conditions sufficient to prevent or inhibit the growth or reproduction of the fungal pathogen in or on the post-harvest product.

According to yet another embodiment of the invention there is provided a method for enhancing storage of an agricultural, horticultural or silvicultural post-harvest product comprising applying to the post-harvest product an effective amount of the composition according to the invention for a time and under conditions sufficient to prevent or inhibit the growth or reproduction of a fungal pathogen in or on the post-harvest product.

In preferred embodiments of the invention, the fungal pathogen is a Phycomycetes, an Ascomycetes or a Basidiomycetes. Preferably, the fungal pathogen is selected from the group consisting of Alternaria spp.; Armillaria spp.; Arthrobotrys spp.; Aspergillus spp.; Boletus spp.; Botrytis spp.; Candida spp.; Claviceps spp.; Cronartium spp.; Epicoccum spp.; Epidermophyton spp.; Eurotium spp.; Fomes spp.; Fusarium spp.; Gaeumannomyces spp.; Geotrichum spp.; Glomerella spp.; Gymnosporangium spp.; Leptosphaeria spp.; Microsporum spp.; Monilinia spp.; Mucor spp.; Penicillium spp.; Pezicula spp.; Phialophora spp.; Physoderma spp.; Phytopthera spp.; Pityrosporum spp.; Polyporus spp.; Puccinia spp.; Rhizoctonia spp.; Rhizopus spp.; Saccharomyces spp.; Scedosporium spp.; Sclerotinia spp.; Septoria spp.; Trichoderma spp.; Trichophyton spp.; Ustilago spp.; Venturia spp.; and Verticillium spp. More preferably, the fungal pathogen is selected from the group consisting of Armillaria mellae; Arthrobotrys oligosporus; Aspergillus flavus; Aspergillus fumigatus; Aspergillus ochraceous; Boletus granulatus; Botrytis cinerea; Botrytis fabae; Candida albicans; Claviceps purpurea; Cronartium ribicola; Epicoccum purpurescens; Epidermophyton floccosum; Eurotium rubrum; Fomes annosus; Fusarium graminearum; Fusarium oxysporum; Fusarium oxysporum f. apii Snyder Hansen; Fusarium oxysporum f.

i 00 0 cubense; Gaeumannomyces gramminis; Gaeumannomyces gramminis var. tritici; Geotrichum candidum; Glomerella cingulata; Gymnosporangium juniperi-virginianae; SLeptosphaeria maculans; Microsporum canis; Monilinia fructicola; Mucor piriformis; Penicillium digitatum; Penicillium expansum; Physoderma alfalfae; Phytopthera infestans; Pityrosporum orbiculare (Malassezia furfur); Polyporus sulphureus; Puccinia graminis.; Rhizoctonia solani; Rhizoctonia solani 9760; Rhizoctonia solani 00 9834; Rhizopus stolonifer; Saccharomyces cerevisiae; Scedosporium prolificans; Sclerotinia sclerotiorum, Sclerotinia minor; Sclerotinia trifoliorum; Septoria apiicola; 0Trichoderma harzianum; Trichophyton mentagrophytes; Trichophyton mentagrophytes 00 10 var interdigitale; Trichophyton rubrum; Trichophyton tonsurans; Ustilago nuda Rostr Venturia inaequalis; and Verticillium dahliae.

In a preferred embodiment, the post-harvest product is immersed in the composition according to the invention.

In a further preferred embodiment, the composition according to the invention is sprayed onto the post-harvest product.

In a further preferred embodiment, the composition according to the invention is brushed onto the post-harvest product.

In a further preferred embodiment, the composition according to the invention is applied to the post-harvest product in admixture with an oil and/or a wax.

In a further preferred embodiment, the composition according the invention is applied to the post-harvest product under pressure.

In a further preferred embodiment, the post-harvest product is a post-harvest product of an ornamental plant, a field crop, a vegetable plant; a fruit tree, a nut tree, a wood tree or a herb plant. Preferably, the post-harvest product is a fruit, a vegetable, a cereal, a grain, a nut, a seed, a floral bulb, wood, timber, silage, bark, natural latex, a tuber, a bloom, a leaf, a stem, a branch, or a root. Also preferably, the cereal is wheat, maize, sorghum, rice, rye, oats, millet, or barley. Also preferably, the seed is a legume seed.

More preferably, the legume seed is a soybean. Also preferably, the nut is a peanut, almond, Brazil nut, or pecan. Also preferably, the fruit is a pome fruit, stone fruit, citrus fruit, grape, tomato, potato, persimmon, strawberry,, papaya, banana, or tomato. More 00

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Spreferably, the pome fruit is an apple or a pear. Particularly preferably, the apple is a SGranny Smith, Red or Golden Delicious, Jonathan, Gala, Fuji, Newton, or Macintosh strain. Also preferably, wherein the pear is a d'Anjou, Packharn's Triumph, William's Bon Chretian, or Beurre Bosc strain. Also preferably, the citrus fruit is grapefruit, orange, lemon, kumquat, lime, mandarin or pomelo. Also preferably, the stone fruit is a 0 0 peach, nectarine, apricot, plum, or cherry.

SIn a further preferred embodiment, the vegetable is an asparagus, carrot, beet, sugar 00 beet, cabbage, cauliflower, brussel sprout, artichoke, Jerusalem artichoke, lettuce,, spinach or potato.

Also preferably, the post-harvest product is a processed post-harvest product.

Particularly preferably, the processed post-harvest product is selected from the group consisting of raisins, prunes, figs, dried apricots, and dates.

According to yet another embodiment of the invention there is provided an isolated nucleic acid molecule which comprises a nucleotide sequence capable of encoding one or more proteins involved in the biosynthesis of a sugar acid, wherein said nucleotide sequence is selected from the group consisting of: a nucleotide sequence which is at least about 50% identical to at least about 30 contiguous nucleotides of SEQ ID NO: 7 preferably other than the sequence set forth in SEQ ID NO: 1; (ii) a nucleotide sequence which is capable of hybridising under at least low stringency conditions to at least about 30 contiguous nucleotides of the complement of SEQ ID NO: 7 preferably other than the sequence set forth in SEQ ID NO: 1; (iii) a nucleotide sequence which encodes the amino acid sequence set forth in SEQ ID NO: 8; and (iv) a sequence complementary to any one of to (iii).

In a preferred embodiment, an encoded protein involved in the biosynthesis of a sugar acid is a sugar oxidase, more preferably a PQQ-dependent sugar oxidase.

In a particularly preferred embodiment, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 7 or a sequence that encodes the 00 21 amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof comprising one or Smore conservative amino acid substitutions relative to SEQ ID NO: 8.

In another embodiment of the present invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding a protein involved 00 in the synthesis of PQQ, wherein said nucleotide sequence is selected from the group Sconsisting of: a sequence that comprises at least about 50 contiguous nucleotides of SEQ 00 ID NO: 9 preferably other than a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6; (ii) a nucleotide sequence which is capable of hybridising under at least low stringency conditions to at least about 50 contiguous nucleotides of the complement of SEQ ID NO: 9 preferably other than the complement of a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, and 6; (iii) a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID Nos: 10, 12, 14, 16, 18 and 20; and (iv) a sequence that is complementary to any one of to (iii).

Preferably, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 9 or 11 or 13 or 15 or 17 or 19, or a sequence that encodes an amino acid sequence set forth in SEQ ID NO: 10 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 12 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 14 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 16 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 18 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: In another embodiment, the present invention provides for the use of the isolated nucleic acid encoding a sugar oxidase to produce a sugar acid in a cell. Preferably, the sugar acid production in such a use involves a PQQ-dependent sugar oxidase encoded by the isolated nucleic acid in combination with one or more isolated nucleic acid molecules of the PQQ operon as described herein to produce PQQ for said PQQdependent sugar oxidase.

In an alternative embodiment, the present invention provides for the use of the isolated nucleic acid of the PQQ operon to produce PQQ in a cell. The ]present invention clearly 00 22 contemplates the use of one or more of said nucleic acid molecules to produce PQQ in Sa cell.

In yet a further embodiment, the present invention provides an i:solated or recombinant polypeptide capable of catalyzing the biosynthesis of a sugar acid and comprising an 00 Samino acid sequence selected from the group consisting of: an amino acid sequence encoded by the nucleic acid comprising a sequence selected from the group consisting of: 00 a nucleotide sequence which is at least about 50% identical to at least about 30 contiguous nucleotides of SEQ ID NO: 7 preferably other than the sequence set forth in SEQ ID NO: 1; a nucleotide sequence which is capable of hybridising under at least low stringency conditions to at least about 30 contiguous nucleotides of the complement of SEQ ID NO: 7 preferably other than the sequence set forth in SEQ ID NO: 1; a nucleotide sequence which encodes the amino acid sequence set forth in SEQ ID NO: 8; and a sequence complementary to any one of to and (ii) an amino acid sequence which is at least about 50% identical to the sequence set forth in SEQ ID NO: 8.

In a particularly preferred embopdiment, the isolated or recombinant polypeptide capable of catalyzing the biosynthesis of a sugar acid comprises the amino acid sequence set forth in SEQ ID NO: 8.

Yet another embodiment of the present invention provides for the use of the isolated or recombinant polypeptide to produce a sugar acid.

In yet another embodiment, the present invention provides an isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ and comprising an amino acid sequence selected from the group consisting of: an amino acid sequence encoded by one or more nucleic acid acids each comprising a nucleotide sequence selected from the group consisting of: a sequence that comprises at least about 50 contiguous nucleotides of SEQ ID NO: 9 preferably other than a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6; 00 23 S(b) a nucleotide sequence which is capable of hybridising under at least Slow stringency conditions to at least about 50 contiguous nucleotides of Ithe complement of SEQ ID NO: 9 preferably other than the complement of a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6; 0 a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID Nos: 10, 12, 14, 16, 18 and 20; and S(d) a sequence that is complementary to any one of(a) to and 00 (ii) an amino acid sequence which is at least about 50% identical to a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18 and Preferably, an isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ comprises an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 18 or SEQ ID NO: Yet another embodiment of the present invention provides for the use of the isolated or recombinant polypeptide to produce PQQ.

A further embodiment of the present invention provides an isolated or recombinant protein complex or mixture capable of catalyzing the PQQ-dependent biosynthesis of a sugar acid and comprising: a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 7 or comprising the amino acid sequence set forth in SEQ ID NO: 8; and (ii) one or more polypeptides selected from the group consisting of: a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 9; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18; and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 00 Preferably, the polypeptide at of the isolated or recombinant protein complex or Smixture has PQQ-dependent sugar oxidase activity and a polypeptide at (ii) of the Sisolated or recombinant protein complex or mixture is involved in the biosynthesis of In A further embodiment of the present invention provides a method of producing a sugar acid comprising expressing the isolated nucleic acid molecule capable of encoding a sugar oxidase according to any embodiment described herein or the recombinant 00 10 polypeptide encoded thereby in a cell, tissue or organism and culturing said cell, tissue or organism in the presence of an aldose substrate for a time and under conditions N sufficient to produce a sugar acid. Preferably, the subject method further comprises introducing the nucleic acid molecule to the cell, tissue or organ in a expressible format. Preferably, the method further comprises extracting or purifying the sugar acid produced. In one embodiment. the cell is a bacterial cell such as a Pseudomonas sp. In an alternative embodiment, the cell, tissue or organ is a plant cell, tissue, or organ. In a preferred embodiment, the method further comprises expressing a nucleic acid molecule of the PQQ operon according to any embodiment described hererin or a polypeptide encoded thereby for a time and under conditions sufficient to produce PQQ in the cell, tissue or organ.

A further embodiment of the present invention provides a method of producing PQQ comprising expressing one or more nucleic acid molecules comprising the PQQ operon or one or more genes or open reading frames of the PQQ operon or one or more recombinant polypeptides encoded thereby in a cell, tissue or organism and culturing said cell, tissue or organism in the presence of a suitable substrate for a time and under conditions sufficient to produce PQQ. Preferably, the subject method further comprises introducing said one or more nucleic acid molecules to the cell, tissue or organ in a expressible format. Preferably,the method further comprises extracting or purifying the PQQ produced. Preferably, the cell is a bacterial cell such as a Pseudomonas sp. In an alternative embodiment, the cell, tissue or organ is a plant cell, tissue, or organ.

Yet another embodiment of the present invention provides a method of enhancing the tolerance of a plant to infection by a fungal pathogen comprising expressing therein an isolated nucleic acid molecule capable of encoding a sugar oxidase or a recombinant sugar oxidase polypeptide, and optionally expressing therein one or more second 00

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0 isolated nucleic acid molecules each encoding a PQQ-biosynthesis enzyme or recombinant polypeptides encoded thereby for a time and under conditions sufficient Sfor a sugar acid to be produced by said plant, or by a cell, tissue or organ of said plant.

A further embodiment of the present invneiton provides a transformed plant comprising the isolated nucleic acid molecule according to any embodiment described herein.

00 SA further embodiment clearly extends to a progeny plant, cell, tissue or organ of the Stransformed plant, wherein said progeny, cell, tissue or organ comprises the isolated 00 10 nucleic acid molecule according to any embodiment described herein C BRIEF DESCRIPTION OF DRAWINGS Figure 1 is a copy of a photograph of untreated strawberries which have been inoculated with a fungal pathogen.

Figure 2 is a copy of a photograph of strawberries which have been inoculated with a fungal pathogen and treated with a 5% solution of gluconic acid.

Figure 3 is a copy of a photograph of cauliflower heads without gluconic acid being applied (left) or treated with a 5% solution of gluconic acid (right).

Figure 4 is a copy of a photograph of PDA plates inoculated with Eurotium rubrum.

The plates clockwise from top left contain 1.0% and 4.5% gluconic acid.

Figure 5 is a copy of a photograph of PDA plates inoculated with Aspergillus ochraceous. The plates clockwise from top left contain 1.0% and gluconic acid.

Figure 6 is a copy of a photographic representation of a thin layer chromatogram (TLC) of an agar overlay assay showing the biological activity of the anti-take-all compound from crude extract of Pseudomonas strain AN5. Panel A shows a TLC performed using a solvent comprising 10% methanol in Chloroform. Panel B shows a TLC performed using a solvent comprising 30% methanol in chloroform. Panel C shows a TLC performed using a solvent comprising 50% methanol in chloroform. The take-all fungus was inoculated onto the top of the TLC plate in a potato dextrose overlay agar and has the ability to grow well. An inhibition zone, 00 0 0indicated by the arrow, corresponds to the location of an anti-fungal agent of Pseudomonas strain AN5 against take-all. This anti-fungal agent is at the origin and Sdoes not migrate in the presence of any of the solvents tested.

Figure 7 is a copy of a photographic representation of silica gel 60 F 254 TLC plates using a solvent system comprising n-propanol:ethyl acetate:water [5:2:3 00 showing the separation of compounds in crude extracts of Pseudomonas strain (panel a) and the mutant strain AN5-MN1 (panel The data indicate differences in the simple sugars produced by the mutant strain AN5-MN1, compared to Pseudomonas 00 10 strain 00 CI Figure 8 is a copy of a photographic representation of the TLC plates of Figure 7 in an agar overlay assay, showing the biological activity of a compound produced by Pseudomonas strain AN5 (panel but not the mutant strain. AN5-MN1 (panel b), against the take-all fungus, as evidenced by the arrow (inhibition zone). The solvent conditions are the same as shown in Figure 7.

Figure 9 is a copy of a photographic representation of a silica gel 60 F 2.4 TLC plate using a solvent system comprising n-propanol:ethyl acetate:water [5:2:3 showing silica column fractions of Pseudomonas strain AN5. Numbering at the bottom of each lane indicates the fraction number collected from the silica column; and C indicates a crude extract of Pseudomonas strain AN5, which was used as starting material for the silica column.

Figure 10 is a copy of a photographic representation of a PDA plate inoculated with take-all fungus, showing the biological activity of silica column fractions 17 to shown in Figure 9 against the take-all fungus. Numbering corresponds to the silica column fraction number. The zone of clearing indicates inhibition of fungal growth, compared to the white patches where the fungus grows. Data indicate that fractions 17 to 20 are active against take-all fungus, and that fractions 19 and 20 contain the highest activity.

Figure 11 is a copy of a photographic representation of a PDA plate in an agar overlay bioassay, showing the activity against take-all fungus of silica column fractions 17 to 20 of Pseudomonas strain AN5, following their re-chromatography on TLC as described in the legend to Figure 9. The active fractions were eluted in four samples 00 (based on Rf value), numbered 1-4. Only fraction 3 (Rf value 0.75) was active against take-all fungus, as evidenced by the inhibition zone.

Figure 12 is a copy of a graphical representation of a 'H NMR spectrum of the active fraction purified by silica column and TLC as indicated in the legends to Figures 9 and 11. Numbering on the x-axis indicates ppm.

00 In 3 Figure 13 is a copy of a graphical representation of a 3 C NMR spectrum of the active fraction purified by silica column and TLC as indicated in the legends to Figures 9 and 00 10 11. Numbering on the x-axis indicates ppm.

0 C1, Figure 14 is a copy of a graphical representation of mass spectrum data of the active fraction of Pseudomonas strain AN5 against take-all fungus which was purified by silica column as indicated in the legends to Figure 9 and Figure 11. Pseudomonas strain AN5 was cultured with glucose as the sole carbon source. Top panel: data show abundance of each fragment as a function of mass/charge. Lower panel: data show abundance of each fragment as a function of elution time (min). The arrow indicates the position of gluconolactone.

Figure 15 is is a copy of a graphical representation of mass spectrum data of the active fraction of Pseudomonas strain AN5 against take-all fungus which was purified by silica column as indicated in the legend to Figure 11. Pseudornonas strain AN5 was cultured with glucose as the sole carbon source. Top panel: data show abundance of each fragment as a function of mass/charge. Lower panel: data show abundance of each fragment as a function of elution time (min). The arrow indicates the position of gluconic acid.

Figure 16 is a copy of a photographic representation of a PDA plate inoculated with Pseudomonas strain AN5, showing the biological activity of different amounts of pure gluconic acid (Sigma Chemical Company Pty. Ltd.) against take-all using an agar overlay bioassay. Numbering indicates the amount of sugar acid used, as follows: (1 15mg; 12.5mg; and 7.5mg. Data indicate that there is a positive correlation between the amount of gluconic acid used and the size of the inhibition zone (cleared region), which inhibition zone is indicative of activity against take-all fungus.

y 00

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O Figure 17 is a copy of a photographic representation of a PDA plate inoculated with Pseudomonas strain AN5, showing the biological activities of different purified acids S(malic acid; ascorbic acid), and sugar acids (glutaric acid; gluconic acid) (Sigma Chemical Company Pty. Ltd.) against the take-all fungus, using an agar overlay bioassay. The amount of acid used was 12.5mg in each case. Numbering indicates the acid used, as follows: malic acid; ascorbic acid; glutaric acid; and (4) 00 gluconic acid. All four acids produce strong inhibition zones (cleared regions), indicative of activity against the take-all fungus.

00 10 Figure 18 is a black and white copy of a colour photographic representation of bacterial Splates containing King's B media and inoculated with Pseudomonas strain AN5 (panel or Pseudomonas fluorescens Pf-5 (panel Data indicate that Pseudomonas fluorescens Pf-5 produces 2,4-diacetylphloroglucinol, by virtue of the darker appearance of this plate (which, in the original colour plate, is a red colour), compared to the whitened appearance of the plate in panel a. In contrast, Pseudomonas strain does not produce detectable levels of 2,4-diacetylphloroglucinol in this assay. The original colour photographic representation is available on request.

Figure 19 is a copy of a graphical representation of the 'H NMR spectrum of phenazine- -carboxylic acid. Numbering on the x-axis indicates ppm.

Figure 20 is a copy of a graphical representation of 'H NMR of a crude extract of Pseudomonasfluorescens Pf-5 from malt agar. Numbering on the x-axis indicates ppm.

Figure 21 is a copy of a graphical representation of 'H NMR of a crude extract of Pseudomonas strain AN5 from malt agar. Numbering on the x-axis indicates ppm.

Figure 22 is a black and white copy of a colour photographic representation of three PDA plates inoculated with Pseudomonas strain sp. strain AN5 (panels a and b) or the mutant strain AN5-MNI(panel In panels b and c, the indicator dye bromocresol purple was added to the growth medium, and the yellow colour in panel b of the original photographic representation (darker grey colour over alight grey background in the black and white copy) is indicative of an acidification of the medium by Pseudomonas strain AN5. In panel c, the purple colour of the original photographic representation (very dark grey colour over a lighter grey background in the black and 00 white copy) is indicative of an alkalisation of the medium by Pseudomonas strain MN 1. The original colour photographic representation is available on request.

Figure 23 is a black and white copy of a colour photographic representation of a PDA plate containing the indicator dye bromocresol purple and inoculated with the take-all fungus. In the original photograph, the take-all fungus is shown as a black central zone, 00 surrounded by a yellow zone of growth, and there is a purple zone around the take-all fungus, suggesting it is releasing compounds which increase the pH of the media. In the black and white copy, the take-all fungus is shown as a black central zone, surrounded 00 10 by alight grey zone, and the purple zone around the take-all fungus is represented by a Sdarker grey zone towards the edges of the plate. The original colour photographic ,I representation is available on request.

Figure 24 is a copy of a graphical representation of mass spectrum data of the active fraction of Pseudomonas strain AN5 against take-all fungus which was purified by silica column as indicated in the legend to Figure 9. Pseudomonas strain AN5 was cultured with galactose as the sole carbon source. Top panel: data show abundance of each fragment as a function of mass/charge. Lower panel: data show abundance of each fragment as a function of elution time (min). The arrow indicates the position of galactonolactone.

Figure 25 is a copy of a graphical representation of mass spectrum data of the active fraction of Pseudomonas strain AN5 against take-all fungus which was purified by silica column as indicated in the legend to Figure 9. Pseudomonas strain AN5 was cultured with galactose as the sole carbon source. Top panel: data show abundance of each fragment as a function of mass/charge. Lower panel: data show abundance of each fragment as a function of elution time (min). The arrow indicates the position of galactonic acid.

Figure 26 is a copy of a graphical representation of mass spectrum data of the active fraction of Pseudomonas strain AN5 against take-all fungus which was purified by silica column as indicated in the legend to Figure 9. Pseudomonas strain AN5 was cultured with mannose as the sole carbon source. Top panel: data show abundance of each fragment as a function of mass/charge. Lower panel: data show abundance of each fragment as a function of elution time (min). The arrow indicates the position of mannono- 1,4-lactone.

00 Figure 27 is a copy of a graphical representation of mass spectrum data of the active Dfraction of Pseudomonas strain AN5 against take-all fungus which was purified by silica column as indicated in the legend to Figure 9. Pseudomonas strain AN5 was cultured with mannose as the sole carbon source. Top panel: data show abundance of each fragment as a function of mass/charge. Lower panel: data show abundance of each 0 O fragment as a function of elution time (min). The arrow indicates the position of mannonic acid.

00 10 Figure 28 is a genetic and physical map of the Pseudomonas strain AN5 genome Sshowing the location of the sugar oxidase gene and purA genes, with direction of CI transcription indicated by arrow Figure 29 shows the open reading frame of the sugar oxidase gene in Pseudomonas strain AN5 and encoded polypeptide therefor. The open reading frames of a selectable minimum size in this sequence using the standard or alternative genetic codes are represented graphically. The order of the reading frames from the top is -1, The NCBI ORF Finder (Open Reading Frame Finder) was used to determine the ORF's in this region (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi). The program used was from the National Centre for Biotechnology Information (NCBI) maintained by the National Library of Medicine in the U.S. 277 486; 44 2455; 1332 1433; 1803 2039; 888- 1001; 396- 722; 401 586; 821 2401; -2: 172 525; 754 861; 883 1077; 1351 1503; 1.594 1743; 1938 2186; 219 368 and 2223 2456] Figure 30 shows the Open Reading Frames in the PQQ operon of Pseudomonas strain All the open reading frames of a selectable minimum size in this sequence using the standard or alternative genetic codes are represented graphically. The order of the reading frames from the top is The NCBI ORF Finder (Open Reading Frame Finder) was used to determine the ORF's in this region (http://www.ncbi.nlm.nih.gov/gorf/orf/orfig.cgi). The program used was from the National Centre for Biotechnology Information (NCBI) maintained by the National Library of Medicine in the U.S. [See Table 1 for the location of the ORFs].

Figure 31 is a physical and genetic map of a segment of the Pseudomonas strain genome showing the location of PQQ operon.

00 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS SThe present invention is related to a method of prophylactic or therapeutic treatment of infection by a fungal pathogen in an animal or plant or a cell, tissue or organ thereof, or a product derived therefrom comprising administering an effective amount of a sugar acid thereto for a time and under conditions sufficient to inhibit or prevent fungal 00 growth or reproduction.

By 'fungalpathogen" is meant any fungus or yeast which is capable of infecting a plant 00 10 or animal or part thereof, including a human, in a manner so as to reduce productivity or health of said plant or animal or part thereof, or to cause disease, morbidity or c, mortality therein. In the present context, the term "fungal pathogen" further includes any of the true fungi or yeasts which infect plants or animals or infest post-harvest agricultural, horticultural or silvicultural products.

A "post-harvest product" is herein understood to mean a product derived from an agricultural, horticultural or silvicultural plant or tree but not to include the plant or tree per se.

The present invention is further useful in the delay or prevention of food spoilage due to infection of fungal pathogens, particularly in the case of plant products fruits, vegetables, wood or ornamental products), in which case, the sugar acid or composition comprising same are sprayed directly onto the produce to partially or completely inhibit fungal growth under standard storage conditions, thereby increasing the post-harvest life of produce.

The present invention is directed to an anti-fungal composition comprising an effective amount of a sugar acid selected from the group consisting of rnannonic acid, gluconic acid and galactonic acid when used to prevent or inhibit the growth or reproduction of a fungal pathogen in or on a post-harvest agricultural, horticultural or silvicultural product. The invention is also directed to a post-harvest agricultural, horticultural or silvicultural product having applied thereto a composition according to the invention and method of applying the composition of the invention to a post-harvest agricultural, horticultural or silvicultural product.

00

O

As used herein, the term "sugar acid" shall be taken to refer to a monocarboxylic acid, dicarboxylic acid or tricarboxylic acid selected from the group consisting of aldonic Sacid, uronic acid, and aldaric acid.

Those skilled in the art are aware that aldonic acids are monocarboxylic acids that are produced naturally or synthetically by the oxidation of an aldose at the aldehydic 00 carbon atom, either using weak oxidizing agents, such as, for example, sodium hypoiodate, alternatively, specific enzymes.

0 00 10 Aldaric acids are dicarboxylic acids that are produced using stronger oxidising agents Swhich act at both the aldehydic carbon atom and the carbon atom bearing the primary N hydroxyl group.

Uronic acids are carboxylic acids wherein only the carbon atom bearing the primary hydroxyl group is oxidised.

Preferably, the sugar acid used in the performance of the present invention is a derivative of D-glucose, in particular the aldonic acid gluconic acid; and/or the aldaric acid, glucaric acid; and/or the uronic acid glucuronic acid. The use of sugar acids derived from D-galactose, in particular galactonic acid and/or galactaric acid, and/or galacturonic acid, is also contemplated by the present invention. The use of sugar acids derived from D-mannose, in particular mannonic acid and/or mannonaric acid, and/or mannonuronic acid, is also contemplated by the present invention.

In a particularly preferred embodiment, the present invention is directed to the use of an aldonic sugar acid selected from the group consisting of gluconic acid, mannonic acid and galactonic acid, and, more particularly, to the use of gluconic acid.

The present invention does not extend to the use of a derivative of a sugar acid such as, for example, the use of ascorbic acid, malic acid, citric acid, glutamic acid, aconitic acid, fumaric acid, crotonic acid or maleic acid, in isolated form.

By "effective amount" of a sugar acid is meant an amount which is effective in preventing the growth and/or reproduction of the fungal pathogen, alternatively, killing the fungal pathogen, without significant adverse effects on the health and/or viability 00 of the plant or animal to which the sugar acid is administered and such that the post- Sharvest product subsequently derived therefrom is subjected to unwarranted detriment.

SThe effective amount of sugar acid administered will vary depending upon the nature of the fungal pathogen, the sugar acid being administered, and the mode in which the sugar acid is provided in the form of a biocontrol agent or purified sugar acid, amongst others). Persons skilled in the art will be capable of determining an effective 00 amount of any sugar acid to be administered, by standard empirical means. Such methods will be well-known to those skilled in the art, or are described herein. In general, it is not essential to the performance of a bioassay which tests the growth 10 and/or viability of a fungal pathogen to include host cells which the fungus normally 00 Sinfects, provided that appropriate in vitro culture conditions have been established N which permit the growth of the fungal pathogen. If no such assay is available, then bioassays are conducted using appropriate animal or plant hosts, or isolated cells or tissues, or parts of an appropriate animal or plant as a source of nutrient for the fungal pathogen. In the particular case of human subjects, bioassays, other than those used in human trials, are conducted using appropriate animal models, in particular a mammalian host of a fungal pathogen of humans, such as, for example, mice, rats, or rabbits, or isolated mammalian cells, in particular cultured fibroblast cells.

In this regard, the present inventors have found the agar plug assay described by Poplawsky et al. (1988) to be particularly useful for determining the growth and/or viability of a wide range of fungal pathogens of plants, including Alternaria spp.; A.

mellae; A. oligosporus; B. granulatus; B. cinerea; E. purpurescens; F. annosus; G.

graminis var tritici; M. fructicola; P. sulphureus; and V dahliae.

To determine the efficacy of any particular sugar acid in controlling a fungal pathogen, various dosages of the sugar acid are tested for their ability to slow or inhibit fungal growth, or alternatively, to kill the fungal pathogen, compared to the effectiveness of one or more negative and/or positive control compounds. Suitable negative controls include the use of a suitable growth medium comprising, as a substitute for the sugar acid being tested, the corresponding aldose sugar, or corresponding lactone, or alternatively, the absence of the test compounds from the growth medium. A suitable positive control includes the use of a suitable growth medium comprising gluconic acid at a concentration which is known to inhibit the growth of the fungal pathogen being tested, or which is known to kill the fungal pathogen being tested.

00 Once the parameters have been established within which a particular sugar acid will inhibit fungal growth and/or reproduction, it is preferred for the sugar acid to be tested Sfurther to ensure that it does not produce adverse effects or severe contraindications on the host when used within those parameters.

The purified sugar or biocontrol agent is generally administered, in the case of 00 therapeutic treatments, to the site of infection. Alternatively, in the case prophylactic treatments, the purified sugar acid or biocontrol agent will generally be administered to a probable infection site, or at least in a form suitable for transport to a probable 00 10 infection site.

C Various modes of administration of the sugar acid to a plant, animal or product derived therefrom will be known to those skilled in the art, and the present invention is not to be limited by the nature of the formulation used. Since non-modified sugar acids are soluble hydrophilic molecules, sprays, solutions, lotions and topical ointments for administration are readily formulated without the need for chemical solvent-based solubilising agents, which are detrimental to the plant or animal cells.

Conveniently, the sugar acid is administered to a plant, plant cell or post-harvest agricultural, horticultural or silvicultural product in the form of an aqueous solution, in particular a spray, applied to the infection site and/or the soil or other growth medium.

The application of sugar acids in solid form, in the form of an emulsion, a dry flowable powder, a wettable powder, a granule, or as a microencapsulated particle is not to be excluded.

The compositions of the present invention are provided in any of the standard forms known in the art. The compositions are in a solid or liquid form. Solid compositions are in the form of dusts, granules, or wettable powders. Lyophilized cultures are readily resuspended in aqueous solutions for application to agricultural commodities. Liquid compositions are in the form of aqueous or non-aqueous media, in solutions, suspensions, dispersions, or concentrated form, a slurry or paste.

The compositions of the present invention may comprise one or more agriculturally, horticulturally and/or silviculturally acceptable carriers, excipients, or other materials as are known in the art, and which are agriculturally, horticulturally and/or silviculturally compatible.

00 The term "carriers" includes, for example, a gel or gum based carrier xanthan Sgum) or a water based carrier water, buffer solutions, carbohydrate containing solutions, and saline solutions); an oil based carrier; a wax based carrier; a powdered carrier ingredient to provide the composition in powdered form, and in which the composition is dispersed and thus diluted to a desired concentration starch and 00 talc); and any mixture of afore-mentioned.

The term "excipient" refers to conventional additives, such as surfactants and wetting 00 10 agents Tween 20 and Triton X-100), antioxidants, nutrients, emulsifiers, 00 Sspreading agents, suspending agents, sticking agents, anti-scald agents diphenylamine or ethoxyquin), preservatives, pesticides and the like. Preservatives may include, for example: a gum, a natural gum, such as guar gum, locust bean gum, karaya gum, tragacanth gum or preferably xanthan gum; methyl cellulose; (c) silica gel; and any mixture of the afore-mentioned.

The compositions of the present invention are applied by any method known in the art, including but not limited to immersion, spraying, or brushing. In addition, the compositions of the invention are incorporated into waxes, wraps or other protective coatings used in processing the agricultural or horticultural post-harvest products. The composition of the present invention may also be applied to a post-harvest product under pressure.

Optionally, a wetting agent, such as, for example, a non-ionic detergent, are included to facilitate access of the sugar acid to the fungal pathogen. Wetting agents comprising ionic detergents which are alkaline in nature are less desirable, because they may have a pH-neutralising effect, or even an alkylating effect, on the sugar acid. As will be known to those skilled in the art, the hyphae of fungal pathogens may penetrate the epidermal layers of the plant tissues, and, as a consequence, the wetting agent may further assist in the penetration of the sugar acid to underlying cells of the root, stem, or leaf, alternatively underlying tissues of the post-harvest agricultural or horticultural product. Additionally, the epidermal cells of many plant tissues are covered with a waxy layer, which are penetrated using various wetting agents. Accordingly, the wetting agent may assist the sugar acid in reaching those cells of the plant which are to be treated.

00 In the treatment of infections by fungal pathogens of animals and humans, the related embodiment of the present invention is primarily related to the treatment of those dinfections of epidermal cells and keratinised tissues, including the skin, genitalia, and nails. Accordingly, the administration of topical lotions, ointments, and powders is to be preferred for such infections. Those skilled in the art will readily be in a position to produce such formulations, which may include various additional pharmaceutical 00 carriers and/or excipients which are of use in the formulation of skin products.

O In addition to being provided in the form of a purified sugar acid, the active ingredient OO 10 the sugar acid) are administered to the plant or animal, alternatively applied to a 0 post-harvest agricultural or horticultural product in the form of a biocontrol agent CN which produces the sugar acid when cultured in the presence of a carbon source comprising an aldose. Such biocontrol agents are isolated from natural sources, or consist of any one or more mutants or derivatives of a naturally occurring organism, or a genetically-engineered organism. Preferably, the biocontrol agent is capable of colonising the tissues or cells which are normally infected by the fungal pathogen, and is capable of producing an anti-fungal effective amount of a sugar acid. Preferably, the biocontrol agent is further limited in respect of the cell types which it is capable of colonising in the host, such as, for example, a non-pathogenic form of the pathogen in question.

In a related embodiment, the biocontrol agent consists of a non-pathogenic bacterium.

Wherein the sugar acid is for treatment of an infection of animals and/or humans, it is particularly preferred that the biocontrol agent consists of a non-pathogenic strain of bacterium selected from the group consisting of Streptococcus sp., Staphylococcus sp., Escherichia coli, Acidophilius sp. and Lactobacillus sp. For the treatment of urethral, intra-vaginal, rectal and anal infections, non-pathogenic strains of Escherichia coli, Acidophilius sp. and Lactobacillus sp. are preferred.

For the treatment of plants, it is preferred that the biocontrol agent consists of a nonpathogenic strain of bacterium selected from the group consisting of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Frankia sp., and Pseudomonas sp. In the case of Agrobacterium sp., the strain should optimally be one which does not cause adverse crown gall disease, or produce phytohormones, such as, for example, auxins, gibberellins, or cytokinins, at levels which are capable of inducing 00 atypical developmental patterns in the plant. Strains suitable for use in the performance c of the present invention include, for example, derivatives of A. tumefaciens strain SLBA4404; derivatives of A. tumefaciens strain AGL1; and Pseudomonas strain Sand derivatives thereof.

In a particularly preferred related embodiment, the biocontrol agent consists of an 00 isolate of Pseudomonas, exemplified by Pseudomonas strain AN5 (Nayudu et. al., 1994b) or a mutant or derivative thereof. These strains have been shown by the present inventors to have particular utility in the protection of plants against the take-all fungus, 10 G. graminis var. tritici, by virtue of their ability to colonize the root rhizosphere of a 00 Swheat plant or other host of G. graminis var. tritici, and to produce a sugar acid when C cultured in the presence of an aldose substrate.

As applied to a biocontrol agent which produces a sugar acid in its native state, such as, for example, a mutant or derivative of Pseudomonas strain AN5, reference herein to "mutants" and "derivatives" shall be taken to mean those mutants with increased colonizing ability and/or increased sugar acid biosynthetic capacity and/or increased sugar acid secretion, compared to the naturally-occurring isolate, including any auxotrophic mutants, replication mutants and recombinant strains that have been produced by the insertion of additional genetic material, such as, for example, extrachromosomal plasmids or integrated DNA, or transposable genetic elements. As used herein, the term "secretion" shall be taken to include both active transport and diffusion processes which result in the extracellular localisation of a sugar acid which is produced by a cell.

As applied to bacteria which do not have the capacity to produce and/or secrete sugar acids in their native state, such as, for example A. tumefaciens, the terms "mutant" and "derivative" shall be taken to mean that the natural isolate has been subjected to mutagenesis, and/or genetically-engineered, to produce and/or secrete the sugar acid when grown on an appropriate aldose substrate. This are achieved, for example, by genetically-engineering or mutating an organism to express a pyrroloquinolinequinone-dependent (PQQ-dependent) sugar oxidase enzyme.

As applied to bacteria which do not have the capacity to colonise the same cells as the fungal pathogen, the terms "mutant" and "derivative" shall be taken to mean that the natural isolate has been subjected to mutagenesis, and/or genetically-engineered, to 00 O have the capacity to colonize the same cells or tissues as those which the fungal Spathogen infects.

t Particularly useful mutants and derivatives further' include those organisms having altered cell membrane or cell wall components. According to this embodiment, the present invention provides a rifampicin-resistant derivative of Pseudomonas strain 00 AN5, designated hereinafter as "Pseudomonas strain AN5 rif (AGAL Accession No.

NM 00/09624), which exhibits higher anti-fungal activity against G. graminis var.

O tritici than the naturally-occurring isolate Pseudomonas strain 00 SPseudomonas strain AN5 rif has been deposited with the Australian Government Analytical Laboratories (AGAL), at 1 Suakin Street Pymble 2073, New South Wales, Australia, on 28 January, 2000, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and assigned AGAL Accession No. NM 00/09624.

In a further preferred related embodiment of the invention, Pseudomonas strain AN5 or Pseudomonas strain AN5 rif are further improved for use as a biocontrol agent, by the introduction of additional copies of the genetic sequences which, when expressed, enable the organism to produce sugar acids. Such improvements are produced by means of standard transformation/transfection procedures for bacteria, or transposon mutagenesis, amongst other procedures known to those skilled in the art.

Means for producing mutants will be well-known to those skilled in the art, and include the use of chemical and physical mutagens, and transposon mutagenesis, amongst others. Particularly preferred chemical mutagens include EMS and methanesulfonic acid ethyl ester. As will be known to those skilled in the art, EMS generally introduces point mutations into the genome of a cell in a random non-targeted manner, such that the number of point mutations introduced into any one genorne is proportional to the concentration of the mutagen used. Accordingly, in order to identify a particular mutation, large populations of cells are generally treated with EMS and the effect of the mutation is then screened in the mutated cells or, more usually, the progeny cells thereof. Methods for the application and use of chemical mutagens such as EMS are well-known to those skilled in the art.

00 0 O Preferred physical mutagens include the irradiation of cells, in particular ultraviolet and N gamma irradiation of cells, to introduce point mutations into one or more genes present Sin the genome of the cell, or alternatively, to create chromosomal deletions in the t genome. Methods for the application and use of such mutagens are well-known to those skilled in the art.

00 In Insertional mutagenesis are achieved by introducing a DNA molecule which encodes genes for the biosynthesis of sugar acids, into one or more genes present in the genome Sof the cell in an expressible format, such that the ability of the cell to convert aldose to 00 10 sugar acid is enhanced. Alternatively, a nucleic acid molecule which is capable of 0inactivating a negative regulator of a gene involved in the biosynthesis of a sugar acid are introduced ifito the cell.

Preferred DNA molecules for introducing genes into cells, in particular bacterial or plant cells, include transposon molecules, and T-DNA molecules. The use of transposons carries the advantage of providing a marker for the cloning of genetic material capable of encoding molecules that catalyse or otherwise facilitate the conversion of a carbon source to a corresponding sugar acid. Many transposons are known for use in bacterial and plant systems, including TnlO (bacterial), TnS (bacterial), Spm (plant), Ac/Ds (plant), and DSG (plant:), and their use is well-documented.

The biocontrol agents, including mutant and derivatives, described herein are of utility in the preparation of compositions for the treatment of fungal infections in plants and animals or products derived therefrom, and the present invention clearly extends to any and all such uses. For example, the organisms are used in fermentation or other culture conditions, to produce the sugar acid, which are then be extracted from the cells using conventional extraction and/or purification procedures, or alternatively, secreted into the growth medium.

The present invention further relates to a phytoprotective composition for the treatment of a fungal infection of a plant comprising an effective amount of a sugar acid in combination with a phytopathologically-acceptable diluent or wetting agent.

00 0 The wetting agent are any compound which is capable of permeating the epidermal N layer and/or the waxy layer of a plant tissue, such as, but not limited to, a non-ionic Sdetergent.

The concentration of the sugar acid in such compositions will vary depending upon the fungal pathogen, the progression of the disease, the tolerance of the plant to mild 0 acidification, and the soil type. For plants which are less tolerant to acidification, a weaker acid, such as, for example, gluconic acid, are preferred, in which case, a higher 0 concentration of the sugar acid may alleviate symptoms on the plant. Stronger acids, 00 10 such as glucuronic acid and glutaric acid, are used at lower concentrations than 0gluconic acid, however the suitability of any particular sugar acid for a particular fungal infection should be determined empirically in trials prior to large scale applications. Such trials are well within the knowledge of those skilled in the art.

Preferably, the concentration of sugar acid in the compositions of the invention is in the range of about 0.001 to about 10 sugar acid, more preferably in the range of about 0.01 to about 8 sugar acid, and more preferably in the range of about 0.1% to about 5% sugar acid.

In an alternative related embodiment, the phytoprotective composition comprises a biocontrol agent which produces the sugar acid in the presence of an appropriate aldose substrate by virtue of the expression of a PQQ-dependent sugar oxidase enzyme therein. Additionally, the phytoprotective composition are in the form of a spray, emulsion, or dry powder.

The related embodiments of the present invention further provides a composition for the treatment of a fungal infection in a human or other mammal comprising an effective amount of a sugar acid in combination with one or more pharmaceutically-acceptable carriers or diluents. As with other compositions described herein, those compositions for animal use may comprise a biocontrol agent which expresses a PQQ-dependent sugar oxidase enzyme. The composition of the related embodiments of the present invention is particularly directed to the treatment of conditions associated with epidermal and sub-cutaneous infections, such as, for example, those conditions caused by the dermatophytes T. rubrum, T. mentagrophytes, E. floccosum, M. canis, C.

albicans, and P. orbiculare furfur). Accordingly, it is preferred that the composition of the related embodiments of the present invention be formulated for 00

O

O topical application, including intra vaginal, intra-anal, intra-nasal, and intra-oral uses.

Emulsions, lacquers, spray powders, and dry powders are also contemplated. Except Sinsofar as any conventional media or agent is incompatible with an active ingredient, t use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

00 0t In light of the nature of the active ingredient and the intended application, it is preferred Sthat compositions for topical application are formulated so as to have a pH in the range O of about 2.0 to about 7.0, more preferably in the range of about 2.0 to about 5.0, and 00 10 more preferably in the range of about 2.5 to about Creams, lotions, ointments, and tinctures comprising the sugar acid of the related embodiments of the invention, which are intended for topical or intra-vaginal or intra-anal or intra-aural use, can be prepared in a base comprising any standard lotion or ointment for dermatological use, such as, for example, vanishing cream, sorbolene, glycerol and fatty acid esters thereof, liquid polyethylene glycols including polyoxyethylglycol, ethylene glycol, butylated hydroxanisole, liquid paraffin, dimethicone, sorbitan, cetyl palmitate or other fatty acid, polysorbate, and mixtures thereof.

In such topical compositions, alcohols and other organic solvents are not generally required to maintain solubility of the sugar acid, which is hydrophilic. As the related embodiments of the present invention includes modified sugar acids which have altered hydrophilicity compared to their non-modified counterparts, and because such compositions may include less-soluble components, the invention also encompasses the use of non-aqueous based compositions.

Powders are generally prepared using a base of zinc oxide, talc, or other homogeneous powder base.

Lacquers for nail applications are generally prepared in a lacquer base comprising, for example, methacrylic acid copolymer, glycerol triacetate, butyl acetate, ethyl acetate, and ethanol.

00 Spray powders are prepared using a powdered or aqueous base, such as, for example, talc, sorbitan, or a fatty acid; and will also generally comprise a propellant, such as, for Sexample, ethanol, propane, or butane.

Under ordinary conditions of storage and use, these preparations will further contain a preservative to prevent the growth of microorganisms. In any event, it must be stable under the conditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, at least until point-of-sale. This are achieved, for example, by using various known anti-bacterial and anti-fungal agents, including 00 10 parabens, chlorobutanol, phenol, sorbic acid, thimerosal, benzoic acid, phenethyl alcohol, and benzyl alcohol, amongst others. The use of antibiotics, such as, for example, tetracycline, rifampicin, ampicillin, penicillin, and streptomycin, or a derivative thereof, is also encompassed by the present invention.

The compositions of the related embodiments of the invention may further include another anti-fungal compound to provide broad cross-protection against several different pathogens. Such other antifungal compounds include., for example, bifonazole, clotrimazole, econazole nitrate, miconazole nitrate, terbafine hydrochloride, and amorolfine hydrochloride, amongst others.

Preferably, the formulations are prepared by incorporating the active sugar acid compounds, or a biocontrol agent producing same, in an effective amount, in an appropriate base with various of the other ingredients enumerated above, preferably followed by filter-sterilization.

2 The active sugar acid and/or biocontrol agent, and compositions comprising same, as described herein, can be produced by any means known to those skilled in the art.

Additionally, certain sugar acids, including gluconic acid, are readily available to the public from any one of a number of sources. Alternatively, the sugar acid are purified partially or completely using any one of a number of procedures known to those skilled in the art, including chromatographic procedures, such as, for example, separations based on charge, size, hydrophobicity, or affinity. Phase separation techniques, thin layer chromatography, and separations using silica gel are particularly contemplated by the invention. Column chromatographic procedures are carried out conveniently under conditions of high pressure

HPLC).

00 0 O In a related embodiment, the sugar acid is produced by introducing an isolated nucleic acid molecule which encodes a PQQ-dependent sugar acid biosynthetic enzyme to an Sorganism and culturing said organism in the presence of an aldose substrate for a time In and under conditions sufficient to produce a sugar acid. Preferably, the sugar acid is subsequently extracted from the organism or the culture medium. More preferably, the sugar acid is partially or substantially purified from the culture medium, the organism 00 or an extract thereof.

O The nucleotide sequence which encodes one or more proteins involved in the oO 10 biosynthesis of a sugar acid or is complementary to said nucleotide sequence is 0preferably derived from Pseudomonas sp.

Preferably, the nucleotide sequence of the invention encodes a sugar oxidase enzyme.

As used herein, the term "sugar oxidase" shall be taken to refer to a PQQ-dependent enzyme, or a PQQ-dependent enzyme complex, or a number of different enzymes which act in the same biochemical pathway wherein at least one of said enzymes is PQQ-dependent, to convert an aldose to a sugar acid. For example, the term "sugar oxidase" includes the action of an oxidase enzyme, with or without a lactonase enzyme, to convert glucose or galactose to any one or more of their corresponding aldonic, aldaric, or uronic acids gluconic acid; glucaric acid; glucaronic acid; and galactonic acid). As will be known to those skilled in the an:, the glucose oxidase enzyme is capable of converting glucose to S-gluconolactone, which may subsequently be oxidised non-enzymatically to a sugar acid. Notwithstanding that the velocity of such a non-enzymatic oxidation reaction is slow compared to the enzyme-catalysed reaction, the activity of glucose oxidase combined with such non-enzymatic oxidation of a lactone to a sugar acid clearly falls within the scope of the term "sugar acid", subject to the requirement that a sugar acid is the active anti-fungal compound. The term "sugar oxidase" further encompasses the action of a dehydrogenase enzyme with or without a lactonase enzyme, to convert D-glucose to any one or more of the corresponding sugar acids, gluconic acid and/or glucaric acid and/or glucaronic acid.

By "PQQ-dependent" is meant that the sugar oxidase uses PQQ as a cofactor for optimum enzyme activity. As will be known to those skilled in the art, PQQ is a metaldependent enzyme cofactor produced primarily by bacteria, wherein the metal is generally calcium, or copper. The advantage of using a PQQ-dependent sugar oxidase 00 O in the performance of the invention is that the production of PQQ is not linked to glycolysis, and, as a consequence, there is less glycolytic load on a transgenic cell expressing such a sugar oxidase, compared to the expression of an FAD- or NAD- I dependent enzyme.

Preferably, the isolated nucleic acid molecule of the preferred embodiments of the OO invention comprises sequence of nucleotides set forth in SEQ ID NOs: 1 or 7 or the nucleotide sequence of the sugar oxidase gene present in cosmid pMN M53 (AGAL O Accession No. NM 00/09622).

00 SFor the purposes of nomenclature, cosmid pMN M53 comprises the nucleotide (N sequence of a sugar oxidase-encoding gene derived from Pseudomonas strain AN5. To produce cosmid pMN M53, a mutant strain of Pseudomonas strain AN5 was produced by Tn5: uidA insertional mutagenesis of Pseudomonas strain AN5, and screening for bacterial cells which had lost activity against G. graminis var. tritici and did not produce anti-fungal effective amounts of sugar acid when cultured in the presence of aldose substrate. A sub-genomic fragment of the mutant strain was obtained and subcloned into the cosmid pLAF R3, which includes a gene conferring tetracycline-resistance to cells containing the cosmid. The resulting cosmid clone, designated pMN M53, contains sufficient structural gene information of Pseudomonas sp. to confer on any prokaryotic cell harbouring this cosmid, or a derivative nucleic acid molecule thereof having the sugar oxidase-encoding region thereof, the ability to synthesise sugar acids from aldose. Cosmid clone pMN M53 has been deposited with the Australian Government Analytical Laboratories (AGAL), at 1 Suakin street Pymble 2073, New South Wales, Australia, on 27 January, 2000, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and assigned AGAL Accession No. NM 00/09622.

The nucleotide sequence set forth in SEQ ID NO: 1 relates to the terminal 757 bp of a sugar oxidase-encoding gene contained in cosmid clone pMN M53. To obtain the nucleotide sequence set forth in SEQ ID NO: 1, a sequencing primer was used which annealed to the end of the Tn5: uidA insertion in cosmid clone pMN M53. The nucleotide sequence set forth in SEQ ID NO: 1 has less than 50% nucleotide sequence identity overall to any known genes, as determined by BLAST search analysis. BLAST search analysis, however, revealed that a fragment of 23 nucleotides in length derived from SEQ ID NO: 1, comprising nucleotides 553 to 575 of this sequence, has absolute 00

O

O identity to the glucose dehydrogenase-encoding genes of E. coli, or Pantoea citrea.

These limited homologies indicate that the nucleotide sequence present in cosmid clone SpMN M53 is unlikely to comprise a glucose dehydrogenase encoding gene, however is tt most likely involved in glucose metabolism. Moreover, functional expression data indicate that there is sufficient structural gene sequence in cosmid clone pMN M53 to confer on any prokaryotic cell harbouring this cosmid, or a derivative nucleic acid 00 t molecule thereof having the sugar oxidase-encoding region thereof, the ability to convert glucose to gluconic acid. Clearly, the bacterial sugar oxidase gene is not highly conserved with any other known sugar oxidase, such as, for example, the glucose 00 10 oxidase genes of Talaromyces flavus or Aspergillus niger. The nucleotide sequence O SEQ ID NO: 7, on the other hand, relates to the Pseudomonas strain AN5 sugar oxidase-encoding gene.

Accordingly, the preferred embodiments of the present invention clearly extends to homologues, analogues and derivatives of the Pseudomonas sp. nucleotide sequence present in the deposited cosmid designated pMN M53, or the nucleotide sequence present in SEQ ID NOs: 1 or 7. The full-length nucleotide sequence of the Pseudomonas sp. nucleotide sequence present in cosmid pMN M53 is particularly contemplated by the present invention.

For the present purpose, "homologues" of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the preferred embodiments of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence identity to said 00

O

O sequence or a part thereof. Generally, the nucleotide sequence of the present invention are subjected to mutagenesis to produce single or multiple nucleotide substitutions, Sdeletions and/or insertions. Nucleotide insertional derivatives of the nucleotide ttr sequence of the preferred embodiments of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one 00 I or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible 0 with suitable screening of the resulting product being performed. Deletional variants 00 10 are characterised by the removal of one or more nucleotides from the nucleotide 0sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

Particularly preferred homologues, analogues or derivatives include any one or more isolated nucleic acid molecules selected from the following: an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 50% identical to at least about 30 contiguous nucleol:ides of SEQ ID NOs: 1 or 7, or a complementary sequence thereto; (ii) an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 50% identical to at least about 30 contiguous nucleotides of the Pseudomonas gene sequence contained in the cosmid clone pMN M53 (AGAL Accession No. NM 00/09622); (iii) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least about 30 contiguous nucleotides of SEQ ID NO: 1 or 7 or a complementary sequence thereto; (vi) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least about 30 contiguous nucleotides of the Pseudomonas gene sequence contained in the cosmid clone pMN M53 (AGAL Accession No. NM 00/09622); and (vii) an isolated nucleic acid molecule which comprises a nucleotide sequence which is degenerate to SEQ ID NOs: 1 or 7, or the Pseudomonas gene sequence contained in the cosmid clone pMN M53 (AGAL Accession No. NM 00/09622).

Preferably, such homologues will be of a sufficient length and sequence identity to the exemplified sequence and deposited clone to encode a polypeptide having sugar 00 0 O oxidase activity. Preferably, because the nucleic acid molecule of the invention encodes Sa PQQ-dependent enzyme, and this cofactor is prevalent in bacterial cells, the Shomologue, analogue or derivative thereof is obtained from a bacterial source, more t preferably a Pseudomonas sp.

The related embodiments, however, clearly contemplate shorter molecules than those 00 oO which are full-length, which are at least useful in identifying further sugar oxidaseencoding nucleotide sequences falling within the scope of the invention described herein. Preferably, the homologue, analogue or derivative is at least about 100 00oO 10 nucleotides in length, more preferably at feast about 500 nucleotides in length, and even more preferably, comprises at least about 1-10 kb ofnucleotides in length.

Preferably, the percentage identity to the nucleotide sequence of SEQ ID NOs: 1 or 7, or the deposited clone, is at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, and more preferably at least about 90%, or 95%, or 99%. In determining whether or not two nucleotide sequences fall within a particular percentage identity limitation recited herein, those skilled in the art will be aware that it is preferred to conduct a side-by-side comparison or multiple alignment of sequences.

In such comparisons or alignments, differences may arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage identity between two or more nucleotide sequences shall be taken to refer to the number of identical residues between said sequences as determined using any standard algorithm known to those skilled in the art.

For example, nucleotide sequences are aligned and their identity calculated using the BESTFIT programme or other appropriate programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984).

The homologues, analogues and derivatives are obtained by any standard procedure known to those skilled in the art, such as by nucleic acid hybridization (Ausubel et al, 1987), polymerase chain reaction (McPherson et al, 1991) s;creening of expression libraries using antibody probes (Huynh et al, 1985) or by functional assays.

In nucleic acid hybridizations, genomic DNA, mRNA or cDNA or a part of a fragment thereof, in isolated form or contained within a suitable cloning vector such as a plasmid or bacteriophage or cosmid molecule, is contacted with a hybridization-effective 00

O

O amount of a nucleic acid probe derived from SEQ ID NO: 1 or SEQ ID NO: 7 or a Scomplementary sequence thereto, or the deposited clone, for a time and under rconditions sufficient for hybridization to occur and the hybridized nucleic acid is then tt detected using a detecting means. Detection is performed preferably by labelling the probe with a reporter molecule capable of producing an identifiable signal, prior to hybridization. Preferred reporter molecules include radioactively-labelled nucleotide Itriphosphates and biotinylated molecules.

Preferably, variants of the genes exemplified herein, including genomic equivalents, are 00 10 isolated by hybridisation under medium or more preferably, under high stringency Sconditions, to the probe.

For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6 x SSC buffer, 0.1% SDS at 28 0 C or alternatively, as exemplified herein. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. A medium stringency comprises a hybridisation and/or a wash carried out in 0.2 x SSC 2 x SSC buffer, 0.1% SDS at 42 0 C to 65 0 C, while a high stringency comprises a hybridisation and/or a wash carried out in 0.1 x SSC 0.2 x SSC buffer, 0.1 (w/v) SDS at a temperature of at least 55 0 C. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of further clarification only, reference to the parameters affecting hybridisation between nucleic acid molecules is found in Ausubel et al. (1992), which is herein incorporated by reference.

In the polymerase chain reaction (PCR), a nucleic acid primer molecule comprising at least about 20 nucleotides in length, and more preferably at least 30 nucleotides in length, derived from SEQ ID NOs: 1 or 7 or a complementary sequence thereto, or the deposited clone, is hybridized to a nucleic acid template molecule and specific nucleic acid molecule copies of the template are amplified enzymatically as described in McPherson et al., (1991), which is incorporated herein by reference.

In expression screening of cDNA libraries or genomic libraries, protein- or peptideencoding regions are placed operably under the control of a suitable promoter sequence in the sense orientation, expressed in a prokaryotic cell or eukaryotic cell in which said 00 0 O promoter is operable to produce a peptide or polypeptide, screened with a monoclonal or polyclonal antibody molecule or a derivative thereof against one or more epitopes of Sa sugar oxidise polypeptide and the bound antibody is then detected using a detecting t means, essentially as described by Huynh et al. (1985) which is incorporated herein by reference. Suitable detecting means according to this embodiment include 125I-labelled antibodies or enzyme-labelled antibodies capable of binding to the first-mentioned 0 0 antibody, amongst others.

SIt will be evident from the foregoing discussion that the bacterial sugar oxidase of the 00 10 related embodiments of the invention is a PQQ-dependent enzymae. Since not all cells produce PQQ as a cofactor, in order to produce a sugar acid in accordance with the N inventive concept, it are preferred in certain circumstances to also express the enzymes involved in PQQ biosynthesis, in addition to those genes) encoding the sugar oxidise enzyme. The inventors of the related embodiments have identified the PQQ operon of Pseudomonas strain AN5, comprising open reading frames of the genes PQQA, PQQB, PQQC, PQQD, PQQE, and PQQF, which are involved in the production of PQQ.

To produce a functional PQQ cofactor in a cell, it is preferred to ensure that a sufficient number of PQQ-biosynthetic genes are expressed therein to convert one or more of the precursors of PQQ, in the PQQ biosynthetic pathway, to PQQ, and to insert an appropriate metal ion therein, such as, for example, Cu(II) or Ca 2 1 The appropriate substrate, and metal ion, must be either endogenous to the cell, alternatively provided exogenously to the cell. In this regard, certain cells are deficient in only one or two or three or four of the PQQ-biosynthetic genes described herein, and, as a consequence, the present invention clearly relates to the use of only a sub-set of the five genes exemplified herein. Additionally, if a precursor of PQQ which is later in the biosynthetic pathway is provided to the cell, then the genes which encode enzymes acting earlier in the pathway may not be required. For example, the immediate precursor of PQQ are provided to a cell which expresses only the PQQE gene, and a functional PQQ cofactor may still be produced therefrom. Such modifications will be readily apparent to those skilled in the art.

Accordingly, the preferred embodiments further relate to an isolated nucleic acid molecule which comprises a nucleotide sequence encoding one or more polypeptides involved in the biosynthesis of PQQ. Preferably, but not necessarily, such sequences are derived from a bacterial cell, such as, for example, Pseudomonas sp. Several 00 O bacterial PQQ-biosynthesis genes have been described and are readily available from public sources.

SIn a particularly preferred embodiment, the embodiment is a novel isolated nucleic acid molecule comprising one or more of the PQQA, PQQB, PQQC, PQQD, PQQE, and PQQF genes of Pseudomonas strain 00 t Preferably, the isolated nucleic acid molecule of the preferred embodiment is the O sequence of nucleotides set forth in any one of SEQ ID NOs: 2 to 6, 9, 11, 13, 15, 17 or 00 10 19, or the nucleotide sequence of the sugar oxidase gene present in cosmid pMN-L2 (AGAL Accession No. NM 00/09621).

For the purposes of nomenclature, cosmid pMN-L2 comprises the nucleotide sequence of the PQQA, PQQB, PQQC, PQQD, PQQE, and PQQF genes derived from Pseudomonas strain AN5. To produce cosmid pMN-L2, a mutant strain of Pseudomonas strain AN5 was produced by TnS: uidA insertional mutagenesis of Pseudomonas strain AN5, and screening for bacterial cells which had lost activity against G. graminis var. tritici. A sub-genomic fragment of the mutant strain was obtained and sub-cloned into the cosmid pLAF R3, which includes a gene conferring tetracycline-resistance to cells containing the cosmid. The resulting cosmid contains sufficient structural gene information of Pseudomonas sp. to produce PQQ from its natural substrate in a bacterial cell, to confer on any prokaryotic cell harbouring this cosmid, or a derivative nucleic acid molecule thereof having these genes therein, the ability to produce PQQ. Cosmid clone pMN-L2 has been deposited with the Australian Government Analytical Laboratories (AGAL), at 1 Suakin Street Pymble 2073, New South Wales, Australia, on 27 January, 2000, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and assigned AGAL Accession No. NM 00/09621.

The nucleotide sequence set forth in any one of SEQ ID NOs: 2, 3, 5, and 6 have less than 90% nucleotide sequence identity overall to any known PQQ-biosynthesis genes, whilst there is no significant nucleotide sequence identity to SEQ ID NO: 4 overall, as determined by BLAST search analysis. BLAST search analysis, however, revealed that smaller fragments of less than 42 contiguous nucleotides in length derived from any one of SEQ ID NOs: 2 to 6, have greater identities to prior PQQ-biosynthesis genes.

00 0 0 Accordingly, the preferred embodiments of the present invention clearly relates to homologues, analogues and derivatives of the Pseudomonas sp. nucleotide sequence Spresent in the deposited cosmid designated pMN-L2 (AGAL Accession No. NM i^ 00/09621), or the nucleotide sequence present in any one of SEQ ID NOs: 2 to 6, 9, 11, 13, 15, 17 or 19. The full-length nucleotide sequence of the Pseudomonas sp. PQQA, PQQB, PQQC, PQQD, PQQE, and PQQF genes derived from cosmid pMN-L2 is 00 particularly contemplated by the preferred embodiments.

0 Particularly preferred homologues, analogues or derivatives include any one or more 00 10 isolated nucleic acid molecules selected from the following: 0(i) an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 90% identical to at least about 50 contiguous nucleotides of any one of SEQ ID NOs: 2 to 6, 9, 11, 13, 15, 17 or 19 or a complementary sequence thereto; (ii) an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 90% identical to at least about 50 contiguous nucleotides of the Pseudomonas gene sequence contained in the cosmid clone pMN-L2 (AGAL Accession No. NM 00/09621); (iii) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least about 50 contiguous nucleotides of any one of SEQ ID NOs: 2 to 6, 9, 11, 13, 15, 17 or 19,or a complementary sequence thereto; (iv) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least about 50 contiguous nucleotides of the Pseudomonas gene sequence contained in the cosmid clone pMN-L2 (AGAL Accession No. NM 00/09621); and an isolated nucleic acid molecule which comprises a nucleotide sequence which is degenerate to any one of SEQ ID NOs: 2 to 6, 9, 11, 13, 15, 17 or 19 or the Pseudomonas gene sequence contained in the cosmid clone pMN-L2 (AGAL Accession No. NM 00/09621).

Preferably, such homologues will be of a sufficient length and sequence identity to the exemplified sequence and deposited clone to encode functional PQQ-biosynthesis enzymes. The present invention clearly contemplates shorter molecules than those which are full-length, which are at least useful in identifying further PQQ biosynthesis genes falling within the scope of the invention described herein. Preferably, the homologue, analogue or derivative is at least about 100 nucleotides in length, more 00 0 preferably at least about 500 nucleotides in length, and even more preferably, comprises at least about 1-10 kb of nucleotides in length.

t Preferably, the percentage identity to the nucleotide sequence of SEQ ID NOs: 1 or 7, or the deposited clone, is at least about 90%, more preferably at least about 95%, even more preferably at least about 97%, and more preferably at least about 99%, as 00 0t determined using standard nucleotide sequence analysis software.

O Preferably, the hybridization stringency is at least moderate stringency, and more 00 10 preferably a high stringency hybridization is employed to identify new sequences.

The nucleotide sequences set forth herein as SEQ ID Nos: 9, 11, 13, 15, 17 and 19 are comprised within the PQQ operon, and show the positions of the open reading frames therein that encode six proteins involved in PQQ biosynthesis. The present invneiton clearly encompasses homologues, analogues and derivatives of an open reading frame of the PQQ operon, including a homologue, analogue or derivative of a PQQ biosynthesis gene. Such homoloues, analogues or derivatives are obtained by any standard procedure known to those skilled in the art, such as by nucleic acid hybridization (Ausubel et al., 1987), polymerase chain reaction (McPherson et al, 1991) screening of expression libraries using antibody probes (Huynh et al, 1985) or by functional assays.

The amino acid sequences set forth in SEQ ID NOs: 8, 10, 12, 14, 16, 18 and 20 relate to the amino acid sequence of the Pseudomonas strain AN5 sugar oxidase-encoding gene (SEQ ID NO: and the Pseudomonas strain AN5 PQQ synthesis proteins A-F (SEQ ID Nos: 10, 12, 14, 16, 18 and 20 In one embodiment the invention provides an isolated protein which participates in the catalysis of the biosynthesis of a sugar acid and which comprises an amino acid sequence selected from the group consisting of: an amino acid sequence encoded by a nucleotide molecule according to the invention; (ii) an amino acid sequence which is at least about 50% identical any one or more set forth in SEQ ID NOs: 8, 10, 12, 14, 16, 18 or In preferred embodiments the invention provides provided an isolated protein which participates in the catalysis of the biosynthesis of a sugar acid and which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID 00

O

0NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: Although not intending to limit the preferred embodiments to any one theory or mode of action, it is proposed that Pseudomonas strain AN5 or a derivative thereof, in particular Pseudomonas strain AN5 rif (AGAL Accession No. NM 00/09624), or 0 0 another organism carrying the sugar oxidase-encoding nucleotide sequences described herein (optionally with the PQQ-biosynthesis gene nucleotide sequences hereinbefore 0described), oxidizes an aldose such as D-glucose at the aldehydic carbon atom to form 00 10 the corresponding sugar acid, wherein the first step in this conversion is catalysed by the glucose oxidase (GOD) activity of the PQQ-dependent sugar oxidase, involving the C N formation of a lactone, and wherein the second step in this conversion is catalysed by the lactonase activity of the PQQ-dependent sugar oxidase.

Accordingly, in those related embodiments which do not employ an organism in which the genes of the related embodiments are known to be expressed, it is preferred to introduce those genes to the organism in an expressible format. Because the genes of the related embodiments are bacterially-derived, the sugar oxidase-encoding genes present in cosmid pMN M53, and the PQQ operon of cosmid pMN-L2 contain the requisite regulatory sequences for expression in most bacterial cells, in particular Pseudomonas sp.

The related embodiments further provide for the configuration of the inventive genes described herein in an expressible format in both prokaryotic and eukaryotic cells. In most cases, this may involve positioning the structural protein-encoding regions of the genes described herein in operable connection with one or more regulatory sequences required for expression in a particular cell type, or under a particular set of environmental conditions.

For expression in eukaryotic cells, the structural gene sequences of the related embodiments are further modified by the inclusion of intron sequences from known eukaryotic genes to improve the stability of mRNA encoded by said structural genes, or otherwise increase expression. For example, to improve expression in plant cells, the intron sequences of the first intron of the rice actin gene, or the maize Adhl gene, or the Arabidopsis thaliana Adhl gene are particularly useful.

00 It is also within the scope of the related embodiments to include modifications of the exemplified nucleotide sequences, or modifications of the genes present in the Smicroorganism deposits, which have been made for the purposes of adapting the codon t usage of the bacterial gene to that which is optimum in the organism into which the gene is to be expressed. Such codon usage modifications are well known in the art and are readily carried out by a skilled person. Accordingly, degenerate sequences to the 00 exemplified sequences are particularly contemplated by the related embodiments.

Accordingly, the related embodiments clearly extends to the use of gene constructs 00 10 designed to facilitate the introduction and/or expression of the nucleic acid molecule of the invention.

To express the nucleic acid molecule in a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as an insect cell, mammalian cell, plant cell or yeast cell, it is preferred that the structural protein-encoding region be placed operably under the control of a strong universal promoter, or a promoter sequence which is capable of regulating expression in response to various external stimuli. Persons skilled in the art will be in a position to select appropriate promoter sequences for expression of the nucleic acid molecule without undue experimentation.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. In the context of the present invention, the term "promoter" also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or a -10 box transcriptional regulatory sequences.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said sense molecule in a cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid molecule to which it is operably connected. For example, copper-responsive regulatory elements are placed 00 O adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible expression thereon.

tPlacing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. A promoter is usually, but not necessarily, positioned upstream or 5' of a 00 00nucleic acid molecule which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the structural protein-encoding nucleotide secluences, or a chimeric 00 10 gene comprising same. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the arl:, some variation in this distance can also occur.

Promoters suitable for use in gene constructs of the related embodiments include those promoters derived from the genes of viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants which are capable of functioning in isolated cells derived from bacteria, yeasts, fungi, animals, or plants, including monocotyledonous and/or dicotyledonous plants, and/or cells, tissues and organs derived from the isolated cells.

The promoter may regulate gene expression constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others.

Examples of promoters useful in performing this embodiment include the CaMV promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, napin seed-specific promoter, P 3 2 promoter:. BK5-T imm promoter, lac promoter, tac promoter, phage lambda XL or XR promoters, CMV promoter (U.S.

Patent No. 5,168,062), lacUV5 promoter, SV40 early promoter Patent No.

5,118,627), SV40 late promoter Patent No. 5,118,627), adenovirus promoter, 00

O

O baculovirus P10 promoter, or polyhedrin promoter Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051 and 5,169,784), bacteriophage T7 promoter, Sbacteriophage T3 promoter, SP6 promoter, SV40 early promoter, RSV-LTR promoter, r SCSV promoter, SCBV promoter and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping genes, including the actin promoters, or promoters ofhistone-encoding genes, are useful.

00 Numerous vectors having suitable promoter sequences for expression in bacteria have O been described, such as for example, pKC30 (XL: Shimatake and Rosenberg, 1981), 00 10 pKK173-3 (tac: Amann and Brosius, 1985), pET-3 (T7: Studier and Moffat, 1986) or the pQE series of expression vectors (Qiagen, CA), amongst others.

The gene construct may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They are isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the gene constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) gene, the zein gene terminator from Zea mays, the Rubisco small subunit (SSU) gene terminator sequences and subclover stunt virus (SCSV) gene sequence terminators, amongst others. Those skilled in the art will be aware of additional promoter sequences and terminator sequences which are suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.

The gene constructs of the related embodiments may further include an origin of replication sequence for replication in a specific cell type, for example a bacterial cell, when said gene construct is to be maintained as an episomal genetic element (eg.

00 plasmid or cosmid molecule) in said cell. Preferred origins of replication include, but Sare not limited to, thefl-ori and co/El origins of replication.

CD

The gene construct may further comprise a selectable marker gene or genes that are functional in a cell into which said gene construct is introduced.

00 0I As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a gene construct of the 00 10 invention or a derivative thereof.

Suitable selectable marker genes contemplated herein include the ampicillin resistance tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene (Kan), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptll), hygromycin resistance gene, (P-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.

Numerous expression vectors suitable for the present purpose have been described and are readily available.

In a preferred related embodiment, the subject method comprises the additional first step of transforming the cell, tissue, organ or organism with a nucleic acid molecule of the related embodiment, in particular the sugar oxidase-encoding sequences with or without additional PQQ-biosynthesis genes, or one or more gene constructs comprising same. As discussed herein, this nucleic acid molecule are contained within a gene construct. The nucleic acid molecule or a gene construct comprising same are introduced into a cell using any known method for the transfection or transformation of said cell. In the case of eukaryotic organisms, a whole organism are regenerated from a single transformed cell, using any method known to those skilled in the art.

By "transfect" is meant that the introduced nucleic acid molecule is introduced into said cell without integration into the cell's genome.

By "transform" is meant that the introduced nucleic acid molecule or gene construct comprising same or a fragment thereof is stably integrated into the genome of the cell.

00 0 Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaC12 and variations thereof, in particular the method Sdescribed by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al., 1982; t Paszkowski et al., 1984), PEG-mediated uptake to protoplasts (Armstrong et al., 1990) microparticle bombardment, electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells 0 (Christou et al., 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant 0 tissue as described essentially by An et al.(1985), Herrera-Estrella et al. (1983a, 1983b, 00 10 1985).

c For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable biolistic cell transformation methodology and apparatus can be used in performing the related embodiments. Exemplary apparatus and procedures are disclosed by Stomp et al. Patent No. 5,122,466) and Sanford and Wolf Patent No. 4,945,050). When using biolistic transformation procedures, the gene construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 Im gold spheres. The DNA construct are deposited on the microparticle by any suitable technique, such as by precipitation.

Alternatively, wherein the cell is derived from a multicellular organism and where relevant technology is available, a whole organism are regenerated from the transformed cell, in accordance with procedures well known in the art. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, are transformed with a gene construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristernatic tissue apical meristem, axillary buds, and root meristems), and induced meristem tissue cotyledon meristem and hypocotyl meristem).

00

O

O The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres.

I The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

00 The regenerated transformed plants are propagated by a variety of means, such as by O clonal propagation or classical breeding techniques. For example, a first generation (or 00 10 T transformed plant are selfed or crossed to another T1 plant and homozygous second generation (or T2) transformants selected. In the case of woody crops, such as citrus c N and grapes and other plants, which are not readily selfed to make homozygous plants, clonal derivatives of primary transformants will need to be crossed to each other to produce homozygous T2 plants. The T2 plant may then be further propagated through classical breeding techniques.

The regenerated transformed organisms contemplated herein may take a variety of forms. For example, they are chimeras of transformed cells and non-transformed cells; clonal transformants all cells transformed to contain the expression cassette); and grafts of transformed and untransformed tissues in plants, a transformed root stock grafted to an untransformed scion).

It will be apparent from the preceding discussion that the transformed organisms have a variety of applications by virtue of their ability to express a PQQ-dependent sugar oxidase. In fact, such transformed organisms have applications in any field where treatment of a fungal infection is indicated, as discussed herein. In the case of transformed plants, these may themselves exhibit enhanced resistance or tolerance to a fungal pathogen.

Accordingly, the related embodiments clearly provide a method of enhancing the tolerance of a plant to fungal infection comprising expressing in said plant, or a cell, tissue or organ thereof, an isolated nucleic acid molecule which encodes a PQQdependent sugar oxidase according to any embodiment described herein. Preferably, nucleotide sequences comprising one or more PQQ-biosynthesis genes are also expressed in the plant and, optionally, an appropriate substrate for the activity of the 00

O

0 expressed PQQ-biosynthetic protein is provided to the transformed plant to ensure that a functional cofactor is formed.

t Preferably, the method further comprises the first step of introducing nucleic acid encoding the sugar oxidase and PQQ-biosynthesis genes to the plant cell.

00 Preferably, the inventive method further comprises the step of regenerating a whole plant from the transformed cell.

00 10 By "enhanced tolerance" is meant that the plant is less susceptible to a sustained infection by the fungal pathogen, and/or is less likely to develop disease following an

N

I initial infection by the fungus, because the fungal pathogen is unable to grow uninhibited on the transformed plant.

This embodiment of the related embodiments clearly extends to those progeny of the transformed plants which also express the introduced nucleic acid molecules.

According to one embodiment of the present invention there is provided an anti-fungal composition comprising an effective amount of a sugar acid selected from the group consisting ofmannonic acid, gluconic acid and galactonic acid when used to prevent or inhibit the growth or reproduction of a fungal pathogen in or on an agricultural, horticultural or silvicultural post-harvest product. Preferably, the sugar acid is gluconic acid. Preferably, the concentration of gluconic acid in the compositions of the invention is in the range of about 0.001 to about 10 gluconic acid, more preferably in the range of about 0.01 to about 8 gluconic acid, and more preferably in the range of about 0.1% to about 5% gluconic acid.

In a preferred embodiment, the anti-fungal composition further comprises an additional chemical or biological pesticide. In a further preferred embodiment, the anti-fungal composition further comprises a diluent; a wetting agent; a humectant; a wax or setting agent; or any combination thereof. Preferably, the anti-fungal composition according to the invention is formulated as a wettable powder, a dry flowable powder, a granule, an aqueous suspension, an emulsion, or as a microencapsulated particle.

According to another embodiment of the invention there is provided an agricultural, horticultural or silvicultural post-harvest product having applied thereto a composition 00 according to the invention. Preferably, the post-harvest product is a post-harvest product of an ornamental plant, a field crop, a vegetable plant; a fruit tree, a nut tree, a wood tree or a herb plant. Also preferably, the post-harvest product is a fruit, a vegetable, a cereal, a grain, a nut, a seed, a floral bulb, wood, timber, silage, bark, natural latex, a tuber, a bloom, a leaf, a stem, a branch, or a root. In a preferred embodiment, the cereal is wheat, maize, sorghum, rice, rye, oats;, millet, or barley. In a 00 further preferred embodiment, the seed is a legume seed. Preferably, the legume seed is a soybean. In a further preferred embodiment, the nut is a peanut, almond, Brazil nut, or pecan. In a further preferred embodiment, the fruit is a pome fruit, stone fruit, citrus 10 fruit, grape, tomato, potato, persimmon, strawberry, papaya, or banana. Preferably, the 00 Spome fruit is an apple or a pear. Also preferably, the apple is a Granny Smith, Red or C Golden Delicious, Jonathan, Gala, Fuji, Newton, or Macintosh strain. Also preferably, the pear is a d'Anjou, Packham's Triumph, William's Bon Chretian, or Beurre Bosc strain. In a further preferred embodiment, the citrus fruit is grapefruit, orange, lemon, kumquat, lime, mandarin or pomelo. In a further preferred embodiment, the stone fruit is a peach, nectarine, apricot, plum, or cherry.

In a further preferred embodiment, the vegetable is an asparagus, carrot, beet, sugar beet, cabbage, cauliflower, brussel sprout, artichoke, Jerusalem artichoke, lettuce, spinach or potato.

In a further preferred embodiment, the post-harvest product is a processed post-harvest product. Preferably, the processed post-harvest product is selected from the group consisting of raisins, prunes, figs, dried apricots, and dates.

According to yet another embodiment of the invention there is provided a method of preventing or inhibiting the growth or reproduction of a fungal pathogen in or on a post-harvest agricultural, horticultural or silvicultural product comprising applying to the post-harvest product an effective amount of the composition according to the invention for a time and under conditions sufficient to prevent or inhibit the growth or reproduction of the fungal pathogen in or on the post-harvest product.

According to yet another embodiment of the invention there is provided a method for enhancing storage of an agricultural, horticultural or silvicultural post-harvest product comprising applying to the post-harvest product an effective amount of the composition according to the invention for a time and under conditions sufficient to prevent or inhibit the growth or reproduction of a fungal pathogen in or on the post-harvest 00 product. In preferred embodiments of the invention, the fungal pathogen is a Phycomycetes, an Ascomycetes or a Basidiomycetes. Preferably, the fungal pathogen is selected from the group consisting of Alternaria spp.; Armillaria spp.; Arthrobotiys spp.; Aspergillus spp.; Boletus spp.; Botrytis spp.; Candida spp.; Claviceps spp.; Cronartium spp.; Epicoccum spp.; Epidermopzyton spp.; Eurotium spp.; Fomes spp.; Fusarium spp.; Gaeumannomyces spp.; Geotrichum spp.; Glomerella spp.; 00 Gymnosporangium spp.; Leptosphaeria spp.; Microsporum spp.; Monilinia spp.; Mucor spp.; Penicillium spp.; Pezicula spp.; Phialophora spp.; Physoderma spp.; Phytopthera spp.; Pityrosporum spp.; Polyporus spp.; Puccinia spp.; Rhizoctonia spp.; 10 Rhizopus spp.; Saccharomyces spp.; Scedosporium spp.; Scierotinia spp.; Septoria spp.; 00 Trichoderma spp.; Trichophyton spp.; Ustilago spp.; Venturia spp.; and Verticillium Ni spp. More preferably, the fungal pathogen is selected from the group consisting of Armillaria mellae; Arthrobotrys oligosporus; Aspergillus flavus; Aspergillus fumigatus; Aspergillus ochraceo us; Boletus granulatus; Botrytis cinerea; Bo erytis fabae; Candida albicans; Claviceps purpurea; Cronartium ribicola; Epicoccum purpurescens; Epidermophyton floccosum; Eurotium rubrum; Fomes annosus; Fusarium graminearum; Fusarium oxysporum; Fusarium oxysporum f. apii Snyder Hansen; Fusarium oxysporum f. cubense; Gaeumannomyces gramminis; Gaeumannomyces gramminis var. tritici; Geotrichum candidum; Glomerella cingulata; Gymnosporangium juniperi-virginianae; Lepbosphaeria maculans; Microsporum canis; Monilinia fructicola; Mucor piriformis; Penicillium digitatum; Penicillium expansum; Physoderma alfalfae; Phytopthera infestans; Pityrosporum orbiculare (Malassezia furfur); Polyporus suiphureus; Puccinia graminis.; Rhizoctonia solani; Rhizoctonia solani 9760; Rhizoctonia solani 9834; Rhizopus stolonifer; Saccharomyces cerevisiae; Scedosporium prolificans; Scierotinia scierotiorum, Scierotinia minor; Scierotinia trifoliorum; Septoria apiicola; Trichoderma harzianum; Trichop~hyton men tagrophytes; Trichophyton mernagrophytes var interdigitale; Trichophyton rubrum; Trichophyton tonsurans; Ustilago nuda Rostr Venturia inae qua/is; and Verticiflium dahliae.

In a preferred embodiment, the post-harvest product is immersed in the composition according to the invention. In a further preferred embodiment, the composition according the invention is sprayed onto the post-harvest product. In a further preferred embodiment, the composition according the invention is brushed onto the post-harvest product. In a further preferred embodiment, the composition according the invention is applied to the post-harvest product in admixture with an oil and/or a wax. In a further preferred embodiment, the composition according the invention is applied to the post- 00 0 harvest product under pressure. The compositions of the present invention are provided N in any of the standard forms known in the art. The compositions are in a solid or liquid form. Liquid compositions are in the form of aqueous or non-aqueous media, in t solutions, suspensions, dispersions, or concentrated form, a slurry or paste. The compositions of the present invention are applied by any method known in the art, including but not limited to spraying, dipping, drenching, brushing, or misting. In 00 addition, the compositions of the invention are incorporated into waxes, wraps or other protective coatings used in processing the agricultural, horticultural or silvicultural post-harvest products. An aqueous suspension are used to spray the composition 00 10 according to the invention onto a post-harvest agricultural, horticultural or silvicultural product. Alternatively, with regard to post-harvest treatment, the composition according to the invention are combined with prior art wax-containing suspensions for application to the post-harvest agricultural, horticultural or silvicultural product.

Concentrations of the sugar acid useful in the methods of the invention are any concentrations which inhibits the development of fungal pathogens when applied to the agricultural, horticultural or silvicultural post-harvest product. As will be obvious to the person skilled in the art, effective concentrations will vary depending upon such factors as the ripeness of the fruits/vegetables or fragility of the blooms etc and the concentration of the fungal pathogen affecting the agricultural, horticultural or silvicultural post-harvest product. For example, the composition according to the invention are incorporated into admixture with a typical water base non-paraffin wax suspension in a concentration of 0.001 10%, preferably 0.01 more preferably 0.1 5% gluconic acid and thereafter applied to the post-harvest product.

Alternatively, the composition according to the invention are added to a prior art mineral oil base paraffin wax which typically is melted and then brushed onto fruit. Oil base waxes typically are applied in lower volumes than water base waxes whereby the concentration of composition according to the invention in an oil base wax carrier should be greater than in a water base wax carrier, as will be readily apparent to those skilled in the art. Whatever carrier is employed to apply the composition according to the invention to a post-harvest agricultural, horticultural or silvicultural products, the optimum concentration of composition according to the invention in the carrier will depend upon such factors as the flow rate through the spray nozzle (in the case of spray application), and the speed at which the fruit is moving past the application device. In order to achieve good dispersion and adhesion of compositions within the present invention, it are advantageous to formulate the compositions of the present invention 00

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O with components that aid dispersion and adhesion. Accordingly, suitable formulations will be known to those skilled in the art (wettable powders, granules and the like, or can be microencapsulated in a suitable medium and the like, liquids such as aqueous flowables and aqueous suspensions, and emulsifiable concentrates). Other suitable formulations will be known to those skilled in the art. A surface-active agent among the different families accepted as emulsifying food additives such as lecithin, ammonium 00 phosphatides, fatty acid esters, polysorbates, sucrose esters and saccharose esters and fatty acids and polyglycerides are added to the formulations of the compositions of present interest. Preferably sorbitans and polysorbates comprise the surface-active 00 10 agents.

In a further preferred embodiment, the post-harvest product is a post-harvest product of an ornamental plant, a field crop, a vegetable plant; a fruit tree, a nut tree, a wood tree or a herb plant. Preferably, the post-harvest product is a fruit, a vegetable, a cereal, a grain, a nut, a seed, a floral bulb, wood, timber, silage, bark, natural latex, a tuber, a bloom, a leaf, a stem, a branch, or a root. Also preferably, the cereal is wheat, maize, sorghum, rice, rye, oats, millet, or barley. Also preferably, the seed is a legume seed.

More preferably, the legume seed is a soybean. Also preferably, the nut is a peanut, almond, Brazil nut, or pecan. Also preferably, the fruit is a pome fruit, stone fruit, citrus fruit, grape, tomato, persimmon, strawberry, papaya, or banana. More preferably, the pome fruit is an apple or a pear. Particularly preferably, the apple is a Granny Smith, Red or Golden Delicious, Jonathan, Gala, Fuji, Newton, or IMacintosh strain. Also preferably, wherein the pear is a d'Anjou, Packham's Triumph, William's Bon Chretian, or Beurre Bose strain. Also preferably, the citrus fruit is grapefruit, orange, lemon, kumquat, lime, mandarin or pomelo. Also preferably, the stone fruit is a peach, nectarine, apricot, plum, or cherry.

In a further preferred embodiment, the vegetable is an asparalgus, carrot, beet, sugar beet, cabbage, cauliflower, brussel sprouts, artichoke, Jerusalem artichoke, lettuce, spinach or potato.

Also preferably, the post-harvest product is a processed post-harvest product.

Particularly preferably, the processed post-harvest product is selected from the group consisting of raisins, prunes, figs, dried apricots, and dates.

00 Post-harvest agricultural, horticultural or silvicultural product treated in accordance with the methods of this invention are stored at standard comestible, where appropriate, Sstorage temperatures, such as 0°C 4°C or room temperature, free of the effects of n fungal infection or with reduced incidence and/or severity of infection. Post-harvest agricultural, horticultural or silvicultural products treated in accordance with this invention may also be stored in a controlled atmosphere (such as 2.5 oxygen and 00 carbon dioxide).

SThe post-harvest agricultural, horticultural or silvicultural products are treated at any 00 10 time before or after harvest. The preferred time of treatment is after harvest and prior to storage or shipment.

It is within the compass of the invention to treat the agricultural, horticultural or silvicultural post-harvest product with the composition according to the invention alone or in combination with other biological and/or chemical control agents which are effective to control other pathogens inciting post-harvest diseases or infestations in or on agricultural, horticultural or silvicultural post-harvest products. When used, these agents should be used in amount, as readily determined by one skilled in the art, which will not interfere with the effectiveness of the composition of the invention.

Antifungals are also important during the processing of materials. For example, animal skins are susceptible to attack by microorganisms, in particular fungal pathogens, both prior to and after the tanning process. Prior to the tanning process, bactericides and fungicides are used in the brine solutions to prevent bacteria and fungal pathogens from damaging the hide grain. After the tanning process, the so called wet blue hides are subject to fungal attack during storage or transport and fungicides are used to inhibit this fungal growth. Antifungals such as the composition of the present invention can be used in the fat liquors and leather finishing products to prevent the growth of fungal pathogens.

When preparing formulations of the compositions of the present invention for specific applications the composition also will likely be provided with adjuvants conventionally employed in compositions intended for such applications such as organic binding agents, additional fungicides, auxiliary solvents, processing additives, fixatives, plasticizers, UV-stabilizers or stability enhancers, water soluble or water insoluble 00

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0 dyes, colour pigments, siccatives, corrosion inhibitors, anti-settlement agents, antiskinning agents and the like.

It is envisioned that the temperatures at which the compositions of the present invention are effective would range from about 0.5 0 C. to about 30°C. The preferred temperature range is 2 °C 15 and the optimal range is considered to be 3°C 6°C for storage 00 of post-harvest agricultural, horticultural and silvicultural products when the postharvest product is temperature sensitive. Therefore, the compositions of the present 0invention can theoretically be applied at any time during the harvest, grading, or OO 10 shipping process, or during the early stages of storage.

C, The compositions are in the form of dusting powders or granules comprising the active ingredient and a solid diluent or carrier, for example fillers such as kaolin, bentonite, kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, gypsum, Hewitt's earth, diatomaceous earth and China clay. Such granules can be preformed granules suitable for application to the soil without further treatment. These granules can be made either by impregnating pellets of filler with the active ingredient or by pelleting a mixture of the active ingredient and powdered filler. Compositions for dressing seed, for example, may comprise an agent (for example a mineral oil) for assisting the adhesion of the composition to the seed; alternatively the active ingredient can be formulated for seed dressing purposes using an organic solvent (for example Nmethylpyrrolidone or dimethylformamide). The compositions may also be in the form of dispersible powders, granules or grains comprising a wetting agent to facilitate the dispersion in liquids of the powder or grains which may also contain fillers and suspending agents.The aqueous dispersions or emulsions are prepared by dissolving the active ingredient(s) in an organic solvent optionally containing wetting, dispersing or emulsifying agent(s) and then adding the mixture to water which may also contain wetting, dispersing or emulsifying agent(s). Suitable organic solvents are ethylene dichloride, isopropyl alcohol, propylene glycol, diacetone alcohol, toluene, kerosene, methylnaphthalene, the xylenes, trichloroethylene, furfuryl alcohol, tetrahydrofurfuryl alcohol, and glycol ethers 2-ethoxyethanol and 2-butoxyethanol). The compositions to be used as sprays may also be in the form of aerosols wherein the formulation is held in a container under pressure in the presence of a propellant, e.g.

fluorotrichloromethane or dichlorodifluoromethane.The compounds can be mixed in the dry state with a pyrotechnic mixture to form a composition suitable for generating in enclosed spaces a smoke containing the compounds. By including suitable additives, 00 0 for example additives for improving the distribution, adhesive power and resistance to rain on treated surfaces, the different compositions can be better adapted for various D utilities. The compositions may also be in the form of liquid preparations for use as t dips or sprays which are generally aqueous dispersions or emulsions containing the active ingredient in the presence of one or more surfactants e.g. wetting agent(s), dispersing agent(s), emulsifying agent(s) or suspending agent(s). These agents can be 00 cationic, anionic or non-ionic agents. Suitable cationic agents are quaternary ammonium compounds, for example cetyltrimethylammonium bromide. Suitable Sanionic agents are soaps, salts of aliphatic monoesters of sulphuric acid (for example oO 10 sodium lauryl sulphate), and salts of sulphonated aromatic compounds (for example sodium dodecylbenzenesulphonate, sodium, calcium or ammonium lignosulphonate, CN butylnaphthalene sulphonate, and a mixture of sodium diisopropyl and triisopropylnaphthalene sulphonates). Suitable non-ionic agents are the condensation products of ethylene oxide with fatty alcohols such as oleyl or cetyl alcohol, or with alkyl phenols such as octyl- or nonyl-phenol and octylcresol. Other non-ionic agents are the partial esters derived from long chain fatty acids and hexitol anhydrides, the condensation products of the said partial esters with ethylene oxide, and the lecithins. Suitable suspending agents are hydrophilic colloids (for example polyvinylpyrrolidone and sodium carboxymethylcellulose), and the vegetable gums (for example gum acacia and gum tragacanth). The compositions for use as aqueous dispersions or emulsions are generally supplied in the form of a concentrate containing a high proportion of the active ingredient(s), the concentrate to be diluted with water before use. These concentrates often should be able to withstand storage for prolonged periods and after such storage be capable of dilution with water in order to form aqueous preparations which remain homogeneous for a sufficient time to enable them to be applied by conventional spray equipment. The concentrates may conveniently contain up to suitably 10-85%, for example 25-60%, by weight of the active ingredient(s).

The compositions of this invention may also comprise other compound(s) having biological activity, e.g. compounds having similar or complementary fungicidal or or compounds having pesticidal activity. Examples of the other fungicidal compound are imazalil, benomyl, carbendazim, thiophanate-methyl, captafol, captan, sulphur, triforine, dodemorph, tridemorph, pyrazophos, furalaxyl, ethirimol, dimethirimol, bupirimate, chlorothalonil, vinclozolin, procymidone, iprodione, metalaxyl, forsetylaluminium, carboxin, oxycarboxin, fenarimol, nuarimol, fenfuram, methfuroxan, nitrotal-isopropyl, triadimefon, thiabendazole, etridiazole, triadimenol, biloxazol, 00 O dithianon, binapacryl, quinomethionate, guazitine, dodine, fentin acetate, fentin hydroxide, dinocap, folpet, dichlofluanid, ditalimphos, kitazin, cycloheximide, Sdichlobutrazol, a dithiocarbamate, a copper compound, a mercury compound, DPX 3217, RH 2161, Chevron RE 20615, CGA 64250, CGA 64251 and RO 14-3169.

The composition according to the invention can also be used broadly in industrial 0 systems and more particularly with substrates such as aqueous and wet state products, cardboard, carpet backing, caulking, cementaceous surfaces, coatings, concrete, O emulsions, inks, leather, paints, paper, plastic articles, preservatives, pulp, resins, 00 10 sealants, silicone rubbers, starch-based compositions, stone, stucco, textiles, timber, Swood, and the like. Further use of the compositions according to the invention is in C( cooling tower systems brewery pasteurization plants, closed loop cooling systems; in detergents and fabric softeners, soaps, cosmetics, animal bedding, cat litter, ophthalmic solutions, swimming pool additives, and the like.

Materials which need protection against fungal pathogen attack include, for example, materials such as paints and other coating formulations, surfactants, proteins, starchbased compositions, inks, emulsions and resins, stucco, concrete, stone, wood, adhesives, caulks, sealants, leather, and spin finishes. Other important commercial materials such as polymer dispersions or aqueous latex paints containing polyvinyl alcohol, polyacrylates or vinylpolymers, thickener solutions containing cellulose derivatives, clay and mineral suspensions and metal working fluids, also are prone to degradation by the action of fungal pathogens which can spoil and significantly impair the usefulness of such compositions. Such degradation may produce, inter alia, changes in pH values, gas formation, discolouration, the formation of objectionable odours, and/or changes in rheological properties.

The composition according to the invention can thus also be used broadly in industrial systems and more particularly with substrates such as aqueous and wet state products, cardboard, carpet backing, caulking, cementaceous surfaces, coatings, concrete, emulsions, inks, leather, paints, paper, plastic articles, preservatives, pulp, resins, sealants, silicone rubbers, starch-based compositions, stone, stucco, textiles, timber, wood, and the like. Further use of the compositions according to the invention is in cooling tower systems brewery pasteurization plants, closed loop cooling systems; in detergents and fabric softeners, soaps, cosmetics, animal bedding, cat litter, ophthalmic solutions, swimming pool additives, and the like.

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The present invention is further described with reference to the accompanying SExamples and drawings.

EXAMPLE 1 Use of Pseudomonas strain AN5 as a biocontrol agent against 00 G. graminis var tritici (take-all) In small scale field trials, the inventors were able to consistently increase wheat yield at 0 different dry-land trial sites in New South Wales, Australia, from between 12% to 36% 00 10 by application of Pseudomonas strain AN5 (Table 1).

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TABLE 1 Summary of wheat yields (kg/plot) Trial AN5 Bai Ba 2 Ba 3 Pf5 Control Goolgowi 10.23 11.00* n.t n.t 8.86" 9.07 Forbes n 10.7610.28 11.28* 10.95* 9.70A 9.12 WestWyalong 5.70* 5.88* 5.17 5.41 4.65A 4.32 n.t not tested; A, not significant;*, significant at p<0.01.

Subsequently, the Harden District Rural Services (HRS) group has established large scale trials using Pseudomonas strain AN5 in acre plots at a trial site at Harden, New South Wales, Australia. Prior to the commencement of the trial, there was significant take-all disease at this site. Following biocontrol treatment using Pseudomonas strain AN5, the treated plot monitored through the wheat growing season exhibited 30% to suppression of disease, as determined by the level of white head formation and visual scoring of symptoms on the roots. Additionally, determination of wheat crop yield using a bio-informatic satellite-based global positioning system (GPS) to accurately determine the location of the harvested crop, and an NIH image analyser, indicated that there was increase in wheat yield due to biocontrol protection conferred by Pseudomonas strain In biocontrol protocols, the survival of Pseudomonas strain AN5 bacteria on the roots of plants, and in the soil, is one of the most crucial factors in effective biological control protection. A typical survival pattern of Pseudomonas strain AN5 bacteria on the roots of 00 wheat over a season is that they decrease in numbers. The rate of decrease of SPseudomonas strain AN5 is related to the moisture content of the soil. In different soil )types, and at different field sites, the numbers of Pseudomonas strain AN5 present in the soil decreases over a number of seasons. High numbers of the bacteria build up at the start of the season (approximately 106-107 per gram of wheat root), with the numbers dropping off towards the drier end of the season. In very dry years, a much more dramatic fall in the 00 numbers of bacteria is observed on the wheat root and this correlates well with loss of biological control protection.

010 00 EXAMPLE 2 C- Biocontrol of Botrytisfabae (chocolate spot fungus) using Pseudomonas strain Chocolate spot (causative agent Botrytis fabae) is a significant disease of faba beans.

Currently, fungicides are applied a number of times during the growing season.

We have shown that Pseudomonas strain AN5 is effective against the chocolate spot disease of faba bean, and is able to suppress the growth of B. fabae, in bioassays carried out in vitro.

In the glasshouse, the present inventors were able to induce chocolate spot by spraying B.

fabae fungus onto the plant. Symptoms of the disease were observable within a few days of spraying. A scale was devised to score the severity of disease symptoms, and, using this scale, plants sprayed with biocontrol bacteria comprising Pseudomonas strain AN5, either before or after inoculation with B. fabae, were shown to have reduced symptoms by up to compared to untreated plants. The present inventors have further shown that up to protection against chocolate spot disease can be obtained by inoculation with Pseudomonas strain Furthermore, we have shown that sugar acids, such as gluconic acid, suppress the growth and/or reproduction of B. fabae fungus in bioassays carried out in-vitro. Accordingly, it is within the scope of the present invention to produce transgenic faba bean plants which produce sugar acids, using the methods described herein, to enhance tolerance of faba beans to B. fabae.

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Sgraminis var tritici (take-all) Microbiological and Molecular biological methods Standard methods were employed as described by Nayudu and Holloway, (1981), Nayudu 0 0 and Rolfe (1987), Nayudu et. al., (1994a) and Nayudu et. al., (1994b).

SBacterial strains 00 10 Pseudomonas strains designated AN5, Pf5, and AN5 rif, possess anti-fungal activity against take-all, and were used in all experiments, unless otherwise stated. The mutant c strains AN5-MN1 and AN5-MN2 do not possess significant anti-fungal activity and were used as negative controls unless otherwise stated.

Bioassay procedure A modification of the agar plug assay (Poplawsky et al., 1988) was used to assay for bioactivity of extracts of Pseudomonas strain AN5, or derivative strains thereof, against take-all fungus, wherein an agar overlay assay was used to test activity of specific fractions. This assay involves macerating the take-all fungus grown in Potato Dextrose (PD) broth and seeding it to a Potato Dextrose Agar (PDA) overlay. This PDA overlay was poured onto thick PDA plates. Fractions to be tested for activity were spotted on top of the overlay, and the plates were dried and incubated at 16 0 C for 3-5 days.

Extraction and analysis of the anti-fingal compound Pseudomonas sp, strain AN5 bacteria were cultured in 250ml flasks, at 25 0 C, for two days, using potato dextrose broth as the growth medium. Following this culture period, 150 ml isopropanol was added to 90ml of cell culture, and the solution mixed by shaking for minutes, after which time cells were collected by centrifugation for 10 minutes at 5,000 rpm. The supernatant was removed and evaporated using a rotavapor at 40 0 C. The crude evaporate was dissolved in 100ml of ethanol (30ml three times). To the ethanol insoluble triturate, 50ml water was added, followed by 50ml acetone, to precipitate proteinaceous material.

The resulting suspension was centrifuged and a total of 0.02ml, drawn from the supernatant as well as from ethanolic solution, was applied by a micropipette (Gilson Pipette) to a silica I r 00 O gel G F254 TLC plate (Aszalos et al., 1968). Thin layer chromatography was performed on the crude extract, using several different solvent systems: 1. methanol; 2. methanol: chloroform [1:9 3. chloroform; 4. pyridine: water [1:1 pyridine: water: ethanol [1:1:1 6. pyridine: water: ethanol [1:1:3 S7. 2-propanol: water [17:3 00 10 8. 2-propanol: water [4:1 9. 2-propanol: water [7:3 c 10. acetone: water 11. acetone: n-butanol:acetic acid:water 12. n-propanol: water [7:1 13. n-propanol: water [7:1.5 14. n-propanol: ethyl acetate: water [5:1:4 n-propanol: ethyl acetate: water [5:2:3 16. n-propanol: ethyl acetate: water [5.5:2:2.5 (v/v/v)J; 17. methyl acetate: 2-propanol: water [18:1:1 18. 2-propanol: ethyl acetate: water [1:1:2 and 19. 2-propanol: ethyl acetate: water [6:1:3 This large number of solvent systems was used in attempts to improve the resolution of the active anti-fungal compound of Pseudomonas sp. strain AN5 on silica TLC plates.

To determine whether any particular compound in TLC plates possessed anti-fungal activity, the bioassay described supra was modified such that the PDA overlay, seeded with take-all fungus, was poured onto the TLC plates and incubated for a week at 16 0

C.

Active fractions were identified by the appearance of a clearance zone in the plates, within an opaque background of cultured fungus.

Using this bioassay, the compound having anti-fungal activity in Pseudomonas strain against take-all fungus did not migrate from the origin using the solvent systems comprising methanol; methanol: chloroform [1:9 or chloroform. The biological activity was tested on TLC plates as well as after scratching bands and extracting the compounds from plates on PDA plates. In particular, there was a clearance zone at the 00 origins of these TLC plates, as well as on PDA plates having the same active fraction (Figure 6).

Using TLC in conjunction with this bioassay, the bioactive anti-fungal compound of Pseudomonas strain AN5 was shown to have an Rf value of about 0.7, when a solvent system comprising pyridine: water: ethanol [1:1:1 or pyridine: water: ethanol 0 0 [1:1:3 was used, however resolution of the compound was poor in this system also.

Silica Gel Reverse Phase Thin Layer Chromatography (SGRPTLC) using a solvent system 10 comprising acetonitrile:methanol:water was unable to produce better resolution than these 00 0 TLC solvent systems.

We subsequently tested solvent systems usually applied to the separation of carbohydrates, in particular organic solvents of binary or ternary composition. Water is an indispensable component of such solvent systems, because water-free solvents, or solvents having low water content, produce diffuse spots, as were obtained for crude extracts of AN5 and mutants thereof (Ghebregzabher et al., 1976).

From the large number of solvent systems listed supra, only one system, n-propanol: ethyl acetate: water has been successful in resolving the anti-fungal component of Pseudomonas strain AN5 in TLC procedures, wherein optimum results were obtained using n-propanol: ethyl acetate: water [5:2:3 Data are shown in Figures 7A and 7B. This solvent system is normally used for separating hexoseamines (Ghebregzabher et al., 1976; Gal, 1968). Accordingly, these data suggested that the anti-fungal compound of Pseudomonas strain AN5 might be a carbohydrate-like molecule.

EXAMPLE 4 Purification of an anti fungal compound in Pseudomonas sp. which is active against G.

graminis var tritici (take-all) Adsorption chromatography on columns or on TLC plates is a useful method for separation and purification of simple sugars, sugar derivatives and oligosaccharides. This technique is, however, time consuming and frequently results in poor resolution due to band tailing.

Accordingly, the inventors followed the substantially faster technique of "flash chromatography" to purify the compound (Still et al., 1978). Flash chromatography is

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00 basically an air pressure driven hybrid of medium pressure and short column chromatography.

Biologically-active extracts of Pseudomonas strain AN5, or the mutants AN5-MN1 or AN5-MN2, were separated on silica columns, using SGRPTLC with the n-propanol: ethyl acetate: water [5:2:3 solvent system, essentially as described by Still et al. (1978).

00 Partially-purified extracts column fractions) of Pseudomonas strain AN5, or the mutants AN5-MN1 or AN5-MN2, were separated on TLC plates using the standardised 00 10 solvent system n-propanol: ethyl acetate: water [5:2:3 vv/vv)]) and different bands Swere scratched and extracted. The extracted compounds of Pseua'omonas strain AN5 were CN tested for their anti-fungal activity on PDA plates, using the bioassay described in the preceding Example. The bioassay was also performed using the modified procedure on TLC plates to confirm the activity of the band of interest. The active fractions, which have similar active band on TLC plates, were pooled for further analysis.

As shown in Figure 9, bioactive column fractions of Pseudomonas strain AN5 were also shown to possess bioactivity using TLC, wherein they migrated to a point corresponding to an Rf value of about 0.75 on TLC. In contrast, the identical silica gel fractions, extracted from the biologically-inactive mutants AN5-MNl or AN5-MN2, were inactive in the bioassay conducted on TLC plates with a PDA overlay, suggesting that the active fraction ofPseudomonas strain AN5 extracts was acting specifically.

The silica gel column fractions obtained from extracts of Pseudomonas strain AN5 were, however, not considered to comprise pure compounds, because many bands were observed on TLC plates containing these fractions (Figure 9).

The bioactive compound was further purified by separating pooled active fractions of Pseudomonas strain AN5 on TLC and excising the bands of interest. The compounds in these excised bands were tested for activity against take-al! on PDA plates, and shown to possess bioactivity (Figure 11).

EXAMPLE Chemical characterisation of an anti-fungal compound in Pseudomonas sp.

which is active against G. graminis var tritici (take-all) as a sugar acid 00

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SThe active anti-fungal fractions from Pseudomonas strain AN5 were further analysed by 'H NMR, 1C NMR, and the mass spectra of compounds determined, to determine the structure of the biologically-active compound.

As shown in Figure 12, 'H NMR of the active fraction purified using silica gel and TLC as 00 described in the preceding Example, produced peaks having resonances at 5.09, 3.57, 3.69, 3.39, 3.26, 3.69 and 3.63, which resonances correspond to protons attached to carbon atoms Cl, C2, C3, C4, C5, and C6, suggesting that the active compound was a carbon-containing O 10 compound. Peaks characteristic of C2 to C6 of gluconic acid were also observed at 3.99, 00 O 3.89, 3.62, 3.62, 3.68 and 3.52.

As shown in Figure 13, 13C NMR of the active fraction purified using silica gel and TLC as described in the preceding Example, produced peaks having resonances at 94.67, 75.35, 74.07, 74.02, 72.23 and 63.16, characteristic of carbons Cl to C6 of a-D-glucose; and peaks having resonances at 98.49,78.53, 78.34, 76.71, 72.19, and 63.32, characteristic of Cl to C6 of P-D-glucose; and peaks having resonances at 181.2, 76.67, 73.54, 75.16, 73.76 and 65.2, which are characteristic of C1 to C6 of gluconic acid.

As shown in Figure 14, mass spectral analysis of the active fractions derived from silica gel and TLC as described in the preceding Example, also suggested that D-glucose and gluconic acid were present in the active fraction.

Proceeding on the basis that the active compound was a sugar acid, pure gluconic acid and other sugar acids were tested for bioactivity against take-all using the PDA plate bioassay shown in Figure 16, purified gluconic acid possessed strong biologically activity against take-all. Data presented in Figure 17 also indicated that, purified malic acid, ascorbic acid, glutaric acid, and glucuronic acid possessed activity against take-all fungus at the concentrations tested.

Furthermore, the active compound produced by Pseudomonas strain AN5 is different to the anti-fungal compounds produced by other bacterial strains. The two bacterial strains, designated Pseudomonas strain AN5, and Pseudomonas strain Pf5, which have anti-fungal activity against take-all, were tested for the presence of 2, 4-diacetylphloroglucinol according to the procedure of Keel et al. (1996). Extracts of these two bacterial strains were also analysed for the presence of amines, sugars, and carbonyl compounds, using art- 00 recognised ninhydrin, silver nitrate, and dinitrophenylhydrazine tests, respectively. Some Sof the results indicating these differences are summarized below: 1. Solubility and pigment production Phenazine-1-carboxylic acid (PCA) is a pigmented greenish-yellow antibiotic which accumulates in cultures of P. fluorescens strain 2-79 (Gurusiddaiah et al, 1986). PCA is 0O highly soluble in chloroform and methylene chloride and insoluble in water, methanol and ethyl acetate. In contrast, the anti-fungal compound produced by Pseudomonas strain 0 is highly-soluble in water. Moreover, Pseudomonas strain AN5 does produce coloured O 10 pigments while growing in media. Similarly, 2,4-diacetylphloroglucinol-producing strains O of Pseudomonas sp. produce red pigments when grown on King's B medium (Keel et al, C1 1996). In contrast, no coloured substances were produced by Pseudomonas strain when grown on King's medium (Figure 18).

2. Separation on TLC The TLC profile of the active anti-fungal agent of Pseudomonas strain AN5 did not correspond to that observed for known anti-fungal agents. In particular, the sugar acids identified to have anti-fungal activity could not be separated on TLC using solvents that resolve previously-identified compounds. For example, on TLC plates of silica gel, using chloroform: methanol 2,4-diacetylphloroglucinol has an Rf value of 0.2; and pyoluteorin has an Rf value of 0.5 (Keel et al, 1992). Additionally, using reverse-phase C18 TLC with an acetonitrile: methanol: water solvent system, 2,4diacetylphloroglucinol has an Rf value of 0.88; pyoluteorin has an Rf value of 0.75; and pyronitrine has an Rf value of 0.28 (Rosales et al, 1995 and Pfender et al, 1993). In contrast, the sugar acid compound of the present invention did not migrate from the origin using a chloroform: methanol [19: solvent system (Figure and was only resolved poorly using acetonitrile: methanol: water 3. Proton NMR spectra In 1H NMR of PCA, proton peaks are only observed above 7 ppm (Gurusiddaiah et al., 1986; Figure 19), whereas no peaks were observed in this region in spectra of the new compound. In proton NMR of 2,4-diacetylphloroglucinol, two peaks at about 6.00 and ppm are characteristic of the attachment of hydrogen to the six carbon protons and the six acetyl protons, respectively (Keel et al, 1992) and this did not match with proton NMR spectra obtained for crude extracts of Pseudomonas strain AN5, or Pseudomonas strain (Figures 20 and 21).

00 In summary, all of the chemical data obtained indicate that the anti-fungal compound of Pseudomonas strain AN5 is a carbohydrate, and, in particular, a sugar acid.

EXAMPLE 6 Sugar acids produce by Pseudomonas sp. induce 00 a pH change in culture media The take-all fungus was grown on potato dextrose plates with the indicator dye, 00 10 bromocresol purple (0.015g/l). After 6-7 days of culture, a purple ring developed around Stake-all, indicating that the media was alkaline (Figure 23). Thus, the take-all fungal CI hyphae grow and releases compounds into the media which are alkdline in nature.

To determine whether this alkylating effect of take-all is modified by the sugar acid of the invention, the parental strain, Pseudomonas strain AN5, and the mutant strains, AN5-MN1, AN5-MN2, and AN5-MN3, were streaked onto PDA supplemented with bromocresol purple (0.015g/l). Data presented in Figure 22 indicate that Pseudomonas strain acidifies media supplemented with 2% glucose when incubated therein, as detected by a yellow halo on PDA plates in the presence of with bromocresol purple. In contrast, the mutant strain AN5-MN1 was unable to produce this yellow halo after 3-4 days (Figure 22).

The plates with the mutants on them went purple, which is an indicator of alkalinity. The absence of this colour change was noted for all mutants. These data indicate that, whereas Pseudomonas strain AN5 decreases the pH of the PDA growth medium, the mutants increase the pH of PDA. The correlation of these data with the anti-fungal activity of these bacterial strains further supports the conclusion that the anti-fungal compound produced by Pseudomonas strain AN5 is acidic in nature.

The effects of different concentrations of purified acids on the take-all root disease is shown in Table 2. Data presented in Table 2 suggest that acidification of the medium may play an important role in protecting against take-all. In particular, whilst gluconic acid confers protection against take-all, the sodium salt of gluconic acid did not confer significant protection against the fungal pathogen.

EXAMPLE 7 Pseudomonas strain AN5 produces different sugar acids 00

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Pseudomonas strain AN5 grown on nutrient agar, Kings B medium, or Malt agar, without D aldose substrate, does not produce significant amounts of sugar acids, as indicated by the pH of the media. Extracts from these media have little or no biological activity against take-all fungus. Additionally, Pseudomonas strain AN5 on potato agar (which contains starch as its main carbon source) does not produce sugar acid, and the media is alkaline, 00 and there is no anti-fungal biological control activity of extracts of Pseudomonas strain AN5 from potato agar.

00 10 In contrast, strong biological activity is detected when Pseudomonas strain AN5 is Sextracted from potato dextrose medium which contains glucose as a carbon source.

CI Furthermore, the pH of this medium turns acidic in nature by virtue of the production of gluconic acid.

We have demonstrated that, if a different carbon source is added to potato agar inoculated with Pseudomonas strain AN5, such as galactose or mannose, then sugar acids are produced. The media extracted from such cultures have biological activity against take-all fungus.

Data presented in Figures 24 and 25 indicate that galacturonic acid is produced from cultures of Pseudomonas strain AN5 grown on PD medium supplemented with galactose.

Additionally, data presented in Figures 26 and 27 indicate that mannonic acid is produced from cultures of Pseudomonas strain AN5 grown on PD medium supplemented with mannose.

The biological activities against take-all of mannonic acid or galacturonic acid are not, however, as strong as gluconic acid. From what is known about the efficiency of conversion of different aldose substrates by Aspergillus sp., it is possible that glucose is merely converted more efficiently into sugar acids than mannose or galactose.

EXAMPLE8 Broad anti-fungal activity of gluconic acid produced by Pseudomonas strain 00 SPseudomonas strain AN5 was tested against a range of microorganisms for biological c control as listed in Table 3, using the PDA plate bioassay, to determine the efficacy of gluconic acid in controlling growth of these pathogens. The anti-fungal agent produced by SPseudomonas strain AN5 showed activity against a broad spectrum of fungi which represent plant pathogens, human pathogens and saprophytes. When tested against bacterial species there were some Gram negative and Gram positive species which were inhibited by 00 the agent produced by Pseudomonas strain AN5, however the species-spectrum of protection was not as broad as in the case of fungi. The range of inhibition observed was in the range of total protection to only partial protection.

00 CN EXAMPLE 9 Mutant strains ofPseudomonas sp. which have modified activity against G. graminis var.

tritici (take-all) The inventors have isolated naturally-occurring mutants of Pseudomonas AN5 bacteria, and genetically engineered new strains of Pseudomonas AN5 bacteria, which produce different amounts of sugar acids when grown on aldose substrates. This has been achieved, for example, by culturing the parent strain on different antibiotics, by introducing a multi-copy plasmid with additional anti-fungal genes into Pseudomonas strain AN5, or by transposon mutagenesis of Pseudomonas strain AN5. Strains which produce less EPS, to facilitate the secretion of more sugar acid into the surrounding medium have also been produced. These mutants and derivatives of Pseudomonas strain AN5 produce larger clearance zones in agar plate bioassays. These strains have been tested for biological control protection against take-all in controlled environment cabinet trials.

00 TABLE 2 Acid Amount Media pH Clearance zone radii pKa Malic acid 7.5 3.4 12.5 2.0_ 25.0 1.5 Ascorbic acid 7.5 0.0 4.17 12.5 0.9 25.0 1.9 2.0 Glutaric acid 7.5 -1.5 4.31 12.5 25.0 2.0 Glucuronic acid 7.5 12.5 25.0 1.7 Gluconic acid 7.5 0.7 3.6 12.5 About 5.0 2.0 Sodium gluconate 7.5 0.0 12.5 0.0 25.0 About 7.0 0.0 Galacturonic acid 7.5 0.7 4.76 12.5 2.0 25.0 1.7 2.5 Acetic acid 1.05 2.1 to 8.4 12.4 >3.0 00 oO 00 00 0 0~ Mutants having reduced anti-fungal activity Mutants having reduced anti-fungal activity are particularly useful as negative controls in experiments to identify anti-fungal compounds, or as vectors into which different anti-fungal properties, such as genes encoding sugar oxidases, are introduced.

One particularly useful mutant, designated Pseudomonas strain AN5-M1, is a single transposon mutant strain of AN5 which has lost anti-fungal activity. A Tn5 transposon has been inserted into a gene which is required for activity against take-all in this mutant.

Three transposon mutants, AN5-MN1, AN5-MN2, and AN5-MN3 were isolated which have lost biocontrol activity, do not produce the anti-fungal metabolite and are unable to control the take-all root disease of wheat. These mutants cannot utilise glucose in a similar pathway to the parent strain, Pseudomonas strain AN5. These mutants are deficient in an enzyme which converts glucose to sugar acid.

TABLE 3 Host range of protection conferred by Pseudomonas strain AN5 cultured in the presence of glucose substrate TYPE OF ORGANISM SPECIES TESTED MODE OF INFECTION Deuteromycetes E. pupurescens Saprophytic Alternaria sp. Saprophytic A. oligosporus Saprophytic M. fructocola Pathogenic cinerea Pathogenic V dahliae Pathogenic /Saprophytic Basidiomycetes F. annosus Pathogenic A. mellea Pathogenic B. granulates Mycorrhizal P. suphureus Saprophytic Yeast S. cerevisiae Saprophytic Gram negative bacterium number ofspecies Varied Gram positive bacterium number ofspecies Varied 00 Mutants having reduced anti-fungal activity Mutants having reduced anti-fungal activity are particularly useful as negative controls in dexperiments to identify anti-fungal compounds, or as vectors into which different anti-fungal properties, such as genes encoding sugar oxidases, are introduced.

One particularly useful mutant, designated Pseudomonas strain AN5-M1, is a single 00 transposon mutant strain of AN5 which has lost anti-fungal activity. A Tn5 transposon has been inserted into a gene which is required for activity against take-all in this mutant.

00 10 Three transposon mutants, AN5-MN1, AN5-MN2, and AN5-MN3 were isolated which Shave lost biocontrol activity, do not produce the anti-fungal metabolite and are unable to C1 control the take-all root disease of wheat. These mutants cannot utilise glucose in a similar pathway to the parent strain, Pseudomonas strain AN5. These mutants are deficient in an enzyme which converts glucose to sugar acid.

Two additional transposon mutants, carrying the Tn5:uidA transposon, were identified by virtue of their inability to confer biocontrol against take-all fungus. These mutants were designated PQQ and SOX As shown in Example 13, the PQQ and SOX mutants contained the Tn5 element in the PQQD gene, and sugar oxidase gene, respectively, of the Pseudomonas sp. genome.

Mutants having increased anti-fungal activity A spectinomycin-resistant strain, designated Pseudomonas strain AN5 spec, was isolated as a naturally-occurring mutant of Pseudomonas strain AN5, by screening for growth on the antibiotic spectinomycin. Pseudomonas strain AN5spec has adequate biocontrol characteristics against take-all fungus.

Pseudomonas strain AN5 rif (AGAL Accession No. NM 00/09624) was isolated as a naturally-occurring mutant of Pseudomonas strain AN5, by screening for growth on the antibiotic rifampicin. Accordingly, Pseudomonas strain AN5rif is rifampicin-resistant.

Bioassays using this strain indicate that is has superior biocontrol characteristics against take-all fungus, on both PDA plates and in pot trials, compared to the parent strain of Pseudomonas strain Pseudomonas strain AN5-M1 was modified by introducing a cosmid comprising the pLAF3 vector with a wild-type region of Pseudomonas strain AN5 genome inserted 00 Stherein. Cosmid pLAF3 is generally multi-copy in Pseudomonas sp., however copy number is relatively low (10-15 copies per chromosome), compared to many bacterial plasmids.

DTransformants were screened for anti-fungal activity against take-all.

One transformant was identified which protected against take-all as efficiently as the parent strain, Pseudomonas strain AN5. This modified bacterial strain, however, only poorly 00 colonises the roots of wheat and there are very few numbers of bacteria present on the roots compared to the colonising ability of the parent strain, suggesting that, for the transformant, each individual cell which colonises the roots may provide superior protection compared to 0 10 an individual cell of the parent strain, presumably by producing greater levels of Santi-fungal agent on a cell basis. These data also suggest that, by increasing the capacity of C a single cell to produce the anti-fungal compound, a more effective biological control strain are created.

The inventors have also created a range of genetically-engineered strains which colonize the roots of wheat in a normal manner and show a greater clearance zone in agar plate bioassays.

The inventors have also genetically-engineered a number of novel biocontrol strains that produce higher amounts of anti-fungal agent, either by introducing multi-copy plasmids (eg. Pseudomonas strain AN5-M1, and Pseudomonas strain AN5-P1) or by obtaining a transposon mutant of this strain (eg. Pseudomonas strain These enhanced biological control strains were shown to be more potent against take-all in agar plate bioassays by their larger clearance zones. Results with strains Pseudomonas strain AN5-P1 and Pseudomonas strain AN5-T5 indicated that an increase in biological control protection can be obtained against take-all fungus by increasing the anti-fungal nature of the strain without compromising colonisation ability. Therefore, an increased in anti-fungal activity increases biological control protection ability.

In glasshouse trials, these modified strains were shown to protect significantly better than the parent strain, Pseudomonas strain AN5, as determined by the scoring of symptoms on the roots of wheat. In particular, inhibition of growth of G. graminis in the presence of these strains was determined following inoculation of pots with the fungus on millet seed.

Control samples contained either no added fungus or biocontrol bacterium, or fungus without biocontrol bacteria. Disease score for take-all was determined, using a scale 00 wherein a numeric indicator is assigned to the severity of the disease, as follows: zero no N disease; 1 disease which is barely detectable; and 5 maximum disease on the crown of dthe plant. At the end of the experiment, the plants were also dried and weighed, and their dry weights expressed as a percentage of the dry weight of control plants. Data presented in Table 4 indicate the mean data obtained over six treatments. Enhanced antibiosis is indicated by the enhanced antifungal activities of the genetically-engineered strains, oO compared to the parental strain.

In such pot trials, where an excess of take-all fungus was artificially added to the potting O 10 medium, these improved biocontrol strains provided significant protection against take-all Sfor much longer periods of time than the parent strain. Pseudomonas strain AN5-P1, and Cl Pseudomonas strain AN5-T5, which produce higher concentrations of sugar acids, have the potential to provide greater protection when they are lower in number on wheat roots.

Therefore, these strains should give better protection against take-all when reduced in number on the roots of wheat during drier winter growing seasons.

EXAMPLE Mass spectrum analysis of sugar acids produced by Pseudomonas strain AN5 rif Pseudomonas strain AN5 rif(AGAL Accession No. NM 00/09624), was cultured for two days at 25 0 C in 100 ml of sterile (autoclaved) pontiac broth (potatoes 400g/l), comprising either 2% glucose, or 4% glucose, or 4% galactose, or 4% mannose as a carbon source. The identities of the sugar acids produced were determined using mass spectrum as described in the preceding Examples.

Mass spectrum analysis of active fractions indicated that, in each sample, there is only one sugar acid produced which corresponds to the aldonic acid product of the aldose sugar substrate provided to the bacterial cultures grown under these conditions. In particular, glucose was converted to gluconic acid the peak for gluconic acid was as in Figure galactose was converted to galactonic acid (the peak for galactonic acid was as in Figure 25), and mannose was converted to mannonic acid (the peak for mannonic acid was as in Figure 27).

c r TABLE 4 Comparison of genetically engineered strains Bacterial Treatment Bacteria Take-all Plant dry weight (cells/gram wheat disease score of control plant) root) 1. Control (no take- 0 0 100% all or bacteria) 2. Take-all treatment 0 5 9% (no bacteria) 3. Pseudomonas 8x10 7 2 78% strain 4. Pseudomonas 1 x 10 3 2 83% strain Pseudomonas 8 x 10 5 1 93% strain AN5-P 1 6. Pseudomonas 5 x 10 7 1 strain EXAMPLE 11 Quantitation of gluconic acid produced by Pseudomonas strain AN5 rif cultures using glucose as the sole carbon source 1. Quantitation ofsugar acids produced by Pseudomonas sp. strain AN5 rif Pseudomonas strain AN5 rif(AGAL Accession No. NM 00/09624) was cultured for two days at 25 0 C in 100 ml of sterile (autoclaved) pontiac broth (potatoes 400g/I), comprising either 2% glucose, or 4% glucose, or 2% galactose, or 4% (w/v) galactose. A negative control solution was also prepared which contained no additional aldose substrate. Sugars were added from stock solutions sterilised by the addition of chloroform.

The total numbers of viable cells were determined after two days of culture, using standard procedures. The viable count for cultures containing 2% glucose in pontiac broth were, on average 7.325 x 10 7 cells/ml, compared to only 6.0 x 104 cell/ml for cultures grown on pontiac broth without added sugar.

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c The pH values of the culture supernatants were determined following collection of the dbacterial cells after two days of culture, by centrifugation at 6,000 rpm to collect bacterial cells, and titration of an aliquot of each supernatant against 0.001 N to 0.01 N NaOH. To confirm the titrable pH value obtained for each supernatant, equal volumes of acetone were added to the remaining supernatants, and samples incubated overnight at 4 0 C to precipitate 00 any proteinaceous components or bacterial cells. The precipitates were removed by centrifugation at 6,000 rpm and the pH values of the culture supernatants determined by titration as before. Results were as follows: 00 Bacteria cultured with 2% glucose: pH 8.0 CI Bacteria cultured with 4% glucose: pH 7.9 Bacteria cultured with 2% galactose: pH 8.2 Bacteria cultured with 4% galactose: pH 7.9 Bacteria cultured without sugar: pH 9.0 and Pontiac broth alone: pH pH values in parentheses indicate the pH of supematants following acetone precipitation.

Pontiac broth comprising 2% galactose produced a magenta (dark pink) colour using the indicator dye phenolphthalein, and, as a consequence, that medium was not titratable using sodium hydroxide solution. Other media include small volumes of 0.001 N sodium hydroxide to produce the same pink colour as that which was produced for pontiac broth inoculated with Pseudomonas strain AN5 rif(AGAL Accession No. NM 00/09624) in the absence of aldose. The average amount of 0.001N NaOH required to neutralize acid sugars produced by each 100 ml culture (n was as follows: Bacteria cultured with 2% glucose: 0.6 ml; Bacteria cultured with 4% glucose: 0.8 ml; Bacteria cultured with 2% galactose: nd Bacteria cultured with 4% mannose: 0.45 ml; and Bacteria cultured without sugar: 0.0 nl.

not determined) For solid media, pontiac agar plates were prepared and adjusted to pH values of 6.5, 10.5, and 12.5. Bromocresol purple (15mg/I) was added to each plate. Pontiac agar plates 00 87

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were inoculated by streaking Pseudomonas strain AN5 rif (AGAL Accession No. NM D00/09624), and incubated for one day at 25'C. After this period, those plates which were at a pH in the range of pH 6.5 to pH 10.5 began to turn yellow. No colour change was apparent for those plates at pH 12.5. Bacterial growth was also affected by the pH of the culture media, and more growth was observed at lower pH values, in the order: 6.5=8.5 00 10.5> 12.5. Growth was very poor at pH 12.5.

In summary, titration of bacterial cultures is not the optimum method for the quantitation of 0O sugar acids, wherein the carbon source is other than glucose, because of the high alkaline pH of the solution which is required for bacterial growth. This method is preferred for use C1 in conjunction with solid media. Alternatively, sugars are quantitated using the procedure described in the following citation: Microbiology 143: 1595-1603, 1997.

2. Assay ofgluconic acid production by Pseudomonas strain AN5 rif Pseudomonas strain AN5 rif (AGAL Accession No. NM 00/09624) was streaked onto nutrient agar containing rifampicin (100 lg/ml) and incubated at 25 0 C for two days. From these plates, nutrient broth and pontiac broth were inoculated with a bacterial loop. These broths were also incubated at 25 0 C on a shaker for two days. 5ml of these broths was used to inoculate 100ml of respective broths. These were grown overnight for about 15-18 hour.

Small aliquot's of these were used with a number of dilutions to obtain viable cell counts.

These cultures were centrifuged at 6000rpm for 15 minutes to pellet cells. The bacterial pellets were washed twice with distilled autoclaved water to remove any broth, and resuspended in 100ml 0.01M glucose solution (pH 7 glucose at 1.8 Bacterial suspensions were incubated on shaker at 25 0 C. After 3.5h, these solutions were observed to produce a yellow colour in the presence of bromocresol purple (15mg/ Supematant's were frozen at -30 0 C and then lyophilised. An aliquot of the lyophilised material was dissolved in water, and 5ml of solution was titrated with 0.01N NaOH. Sugar acids were quantitated as described above.

Data indicate that bacteria obtained from pontiac broth as a starter culture and then grown in pure glucose solution are capable of converting 40% of the glucose substrate present to gluconic acid in 3.5 hour under these conditions. In nutrient broth as a starter culture in a similar experiment, 32% of the glucose substrate was converted to gluconic acid in same time. These results suggest that pontiac broth is better medium for starter culture of bacteria for the production of gluconic acid, when glucose is used as a sole carbon source.

EXAMPLE 12 Summary of sugar acid production by different Pseudomonas strains on different carbon sources The levels of sugar acids produced by Pseudomonas strain AN5 ri'(AGAL Accession No.

NM 00/09624), and the two transposon mutants, PQQ" and SOX-, carrying the transposon in the PQQD and sugar oxidase genes (Example were determined following growth in pontiac media, with or without 2% aldose in the culture medium. Data are presented in Table 0 Bacterial added sugar time (hr) Culture O.D. media pH sugar acid strain _(mg/ml) PQQ- none 17 240 7.0-8.3 0.00 glucose 63 410 5.95 0.117 SOX- none 17 232 7.0-8.3 0.00 glucose 63 380 5.9 0.117 if none 17 212 6.1-8.6 0.00 glucose 47 450 2.95 5.10 glucose 64 305 3.0 5.10 none 23 235 8.6 0.00 galactose 47 435 3.35 1.61 galactose 70 390 3.1 2.24 none 23 235 6.1-8.6 0.00 mannose 70 380 3.25 3.92 Data are consistent with glucose being the preferred carbon source for Pseudomonas strain and derivatives thereof to produce sugar acids from aldose substrate.

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EXAMPLE 13 C Production of anti-fungal effective amounts of sugar acids in situ on wheat roots by Pseudomonas strain Wheat seeds were treated with various biocontrol bacteria and grown in sterile magenta jar 00 assays in vermiculite. We collected the root exudate of wheat roots inoculated with these various biocontrol strains. These included the parent strain Pseudomonas strain Pseudomonas strain AN5 rif(AGAL Accession No. NM 00/09624), and various mutants, 00 including the PQQ- and SOX- mutants. The exudate were concentrated by freeze drying and then resuspended in water. Only Pseudomonas strain AN5. and Pseudomonas strain CI AN5rif showed strong biological activity in bioassays against the take-all pathogen. The extracts from the remaining mutant strains, or untreated wheat, showed no biological activity. These data suggest that the sugar acid is produced by Pseudomonas strain AN5, or Pseudomonas strain AN5rif (AGAL Accession No. NM 00/09624), on wheat roots, in suppression of the take-all pathogen on wheat roots.

EXAMPLE 14 Isolation of nucleotide sequences of Pseudomonas sp. encoding PQQ-dependent sugar oxidases and PQQ-biosynthesis genes To identify the gene region of Pseudomonas sp. which encodes genes for the production of sugar acids, mutant lines were produced by transposon mutagenesis using a transposon. Two mutant lines were identified which failed to confer protection against take-all fungus in bioassays.

Restriction enzyme fragments BamHl, Hind III, EcoR 1) were produced from these mutant lines. Mapping and sequence analyses of the cloned fragments indicated that the transposon was inserted into two non-contiguous regions of the genome, as follows: 1. a first region encoding the proteins involved in catalysing the conversion of aldose to sugar acid, the partial sequence of which is set forth in SEQ ID NO,: 1; and 2. a second region encoding the enzymes involved in synthesis of the cofactor, PQQ, the partial sequence of which is presented in SEQ ID NO: 4.

Limited homology of the nucleotide sequence set forth in SEQ ID NO: 1 to bacterial dehydrogenases suggests that this sequence encodes an enzyme involved in sugar metabolism.

00 SNucleotide sequence analysis also indicated that the transposon was inserted into the SPQQD gene of the second region. In particular, an alignment of the PQQD gene of Pseudomonas strain AN5, with the PQQD genes of other bacteria, indicating limited identity (not shown). These data indicated that this tagged region of the Pseudomonas sp.

00 genome comprises the PQQ operon.

Southern blot hybridisation mapping analyses indicate that the sugar oxidase gene of 00 Pseudomonas strain AN5 is contained on the following DNA fragments: a 3.8 kb BamHI 00 fragment; a 7.6 kb Bgl 11 fragment; a 6.5 kb Pstl fragment; an approximately 18 kb Hindlll Ci fragment; and an approximately 22 kb EcoRl fragment.

Southern blot hybridisation mapping analyses indicate that the PQQ operon oxidase gene of Pseudomonas strain AN5 is contained on the following DNA fragments: an approximately 20 kb BamH fragment; an 8.0 kb Pstl fragment; an approximately 18 kb Hindlll fragment; and an approximately 20 kb EcoRl fragment.

The appropriate genomic regions were isolated from Pseudomonas strain AN5, which possesses anti-fungal activity, and cloned into the cosmid vector pLAF3, to produce the following cosmid clones: 1. Cosmid pMN M53 (AGAL Accession No. NM 00/09622), comprising 30-40 kb of Pseudomonas strain AN5 DNA which comprises nucleotide sequences encoding a sugar oxidase enzyme; and 2. Cosmid pMN-L2 (AGAL Accession No. NM 00/09621), comprising 30-40 kb of Pseudomonas strain AN5 DNA which comprises nucleotide sequences of the PQQ operon.

The nucleotide sequence of a part of the sugar oxidase-encoding gene is set forth in SEQ ID NO: 1. Figure 28 shows a genetic and physical map of the Pseudomonas strain genome showing the location of the sugar oxidase gene therein. The nucleotide sequence of this region was determined. Open reading frames in this region, including open reading frames in both the forward and reverse direction, were determined fromteh nucleotide sequence using the software of the NCBI NCBI ORF Finder). The open reading frames are depicted in Figure 29. The longest open reading frame identified belongs to the only transcription unit detected and encodes a sugar oxidase protein. The nucleotide sequence of the sugar oxidase is set forth in SEQ ID NO: 7 and the encoded amino acid sequence is set forth in SEQ ID NO: 8.

00 91 0 0 SThe nucleotide sequences of the various genes of the PQQ operon were also partially determined (SEQ ID NOs: 2 to Open reading frames in the PQQ operon region of the Pseudomonas strain AN5 genome, including open reading frames in both the forward and reverse direction, were determined from the nucleotide sequence using the software of the 00 NCBI NCBI ORF Finder). The open reading frames are depicted in Figure 30. A physical and genetic map of the PQQ operon of Pseudomonas strain AN5 is also shown in Figure 31. The nucleotide sequence of the PQQ operon region was determined (SEQ ID 0 N NOs: 9, 11, 13, 15, 17 and 19). The open reading frames encoding proteins involved in O 10 PQQ biosythesis were determined by BLAST searching for homologous proteins in other CI bacteria. This approach yielded open reading frames encoding PQQF (SEQ ID NO: PQQA (SEQ ID NO: 12), PQQB (SEQ ID NO: 14), PQQC (SEQ ID NO: 16), PQQD (SEQ ID NO: 18) and PQQE (SEQ ID NO: 20) polypeptides.

EXAMPLE Inhibition of fungal growth by gluconic acid Data presented in Table 2 show a spectrum of fungal pathogens: that are susceptible to treatment with gluconic acid.

TABLE 6. Inhibition of fungal growth by luconic acid Fungal strain Main Area of Growth Rate Level of Conc. of Significance in Lab AN5 gluconic acid Control giving Total Inhibition* Rhizopus stolonifer Fruit Rot, Very Fast 48% postharvest Trichoderma Mushroom Very Fast 52% harzianum cultivation Eurotium rubrum Wheat, Very Fast 67% 0.8% Postharvest Rhizoctonia solani Root Rot wheat Fast 62% 0.4% 9760 Mucorpiriformis Fruit Rot Fast 73% 0.4% Postharvest Fusarium Crown Rot, wheat Medium 10% graminearum Moniliniafructicola Postharvest fruit Medium 70% rot Rhizoctonia solani Root Rot wheat Medium 65% 0.7% 9834 Geotrichum Oranges, Sour Rot Medium 43% 0.8% candidum Penicillium Oranges, Green Medium 15% digitatum Mould Aspergillus Grain, medium 32% ochraceous Mycotoxins Producer Aspergillusflavus Grain, Mycotoxin Medium 35% Producer Penicillium Post Harvest rot, Medium 23% expansum Apples Verticillium dahliae Verticillium wilt Slow 76% 0.2% cotton Leptosphaeria Black leg Canola Slow 82% 0.2% maculans Take All Wheat Pathogen Slow 77% 0.05% Trichophyton Human Slow 80% 0.2% tonsurans Pathogenic Dermatophyte Trichophyton Human Slow 85% 0.4% mentagrophytes var Pathogenic interdigitale dermatophyte Aspergillus Human pathogen Slow 77% fumigatus Scedosporium Human pathogen Slow 66% 0.2% prolificans *Total Inhibition defined in this experiment as no fungal growth, during the time taken for the control plate to be 70% to 85% covered.

00 SEXAMPLE 16 Testing of gluconic acid as anti-fungal agent on post-harvest prcoducts: strawberries and cauliflower 00 t3Several experiments were done to determine the effectiveness of gluconic acid in O controlling fungal deterioration of strawberries. These experiments consisted of visual measurement of the degree of fungal infestation on the fruit between strawberries that 00 had been treated with gluconic acid, those that had been treated with water only and those that had not had any treatment. The strawberries were sprayed with the test solution or relevant control solution, separated into sealed containers and stored at The fruits were examined each day to observe any signs of fungal decay.

Generally after a period of a couple of days the first signs of decay would appear. The progression was scored using a system of strict definition of fungal deterioration. Each level of decay was allocated a number, with a higher number indicative of higher infestation. The results of these experiments indicate that gluconic acid provides protection from fungal deterioration are shown in Figures 1 3.

The process of strawberry fungal decay was found to be complicated by several factors identified during the work leading to the present invention. These factors include the fact that the level of latent fungal infestation proved to be quite variable, a fact that necessitates the use of very large sample sizes to obtain valid results.

In a similar manner, we determined whether we could improve shelf life of cauliflowers by protection against the common brown rot obsened during storage. The results of these tests showed that the length of time before the appearance of brown development was significantly extended on that part of the cauliflower sprayed with gluconic acid (Figure Understandably, the magnitude of the protection was again variable depending on the initial inoculum present on the cauliflower.

00 CN REFERENCES 1. Amann and Brosius (1985).Gene 40.183.

2. An, et al. (1985) EMBOJ. 4:277-284.

3. Armstrong, et al.(1990) Plant Cell Rep. 9: 335-339.

4. Aszalos, et al. (1968) J. Chromatography 37:. 487-498.

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Claims (73)

1. An anti-fungal composition comprising an effective amount of a sugar acid T selected from the group consisting of mannonic acid, gluconic acid and ~galactonic acid when used to prevent or inhibit the growth or reproduction of a fungal pathogen in or on an agricultural, horticultural or silvicultural 00 post-harvest product.

2. The anti-fungal composition according to claim 1, wherein the sugar acid is CN gluconic acid. 00

3. The anti-fungal composition according to claim 2, wherein the concentration of gluconic acid is in the range of about 0.001 to about 10

4. The anti-fungal composition according to claim 2 or claim 3, wherein the concentration of gluconic acid is in the range of about 0.01 to about 8 The anti-fungal composition according to any one of claims 2 to 4, wherein the concentration of gluconic acid is in the range of about 0.1% to about 5%

6. The anti-fungal composition according to any one of claims 1 to 5, further comprising an additional chemical or biological pesticide.

7. The anti-fungal composition according to any one of claims 1 to 6, further comprising a diluent; a wetting agent; a humectant; a wax or setting agent; or any combination thereof.

8. The anti-fungal composition according to any one of claims 1 to 7, wherein the composition is formulated as a wettable powder, a dry flowable powder, a granule, an aqueous suspension, an emulsion, or as a microencapsulated particle.

9. An agricultural, horticultural or silvicultural post-harvest product having applied thereto a composition according to any one of claims 1 to 8. 00 The agricultural, horticultural or silvicultural post-harvest product Saccording to claim 9, wherein the post-harvest product is a post-harvest product of an ornamental plant, a field crop, a vegetable plant; a fruit tree, a nut tree, a wood tree or a herb plant. 00 S11. The post-harvest product according to either claim 9 or claim 10, wherein Sthe post-harvest product is a fruit, a vegetable, a cereal, a grain, a nut, a I seed, a floral bulb, wood, timber, silage, bark, natural latex, a tuber, a 00 bloom, a leaf, a stem, a branch, or a root.

12. The post-harvest product according to claim 11, wherein the cereal is wheat, maize, sorghum, rice, rye, oats, millet, or barIey.

13. The post-harvest product according to claim 11, wherein the seed is a legume seed.

14. The post-harvest product according to claim 12, wherein the legume seed is a soybean. The post-harvest product according to claim 11, wherein the nut is a peanut, almond, Brazil nut, or pecan.

16. The post-harvest product according to claim 11, wherein the fruit is a pome fruit, stone fruit, citrus fruit, grape, tomato, persimmon, strawberry, papaya or banana.

17. The post-harvest product according to claim 16, wherein the pome fruit is an apple or a pear.

18. The post-harvest product according to claim 17, wherein the apple is a Granny Smith, Red or Golden Delicious, Jonathan, Gala, Fuji, Newton, or Macintosh strain. 00 I 19. The post-harvest product according to claim 17, wherein the pear is a d'Anjou, Packham's Triumph, William's Bon Chretian, or Beurre Bosc Sstrain. The post-harvest product according to claim 16, wherein the citrus fruit is 00 grapefruit, orange, lemon, kumquat, lime, mandarin or pomelo.

21. The post-harvest product according to claim 16, wherein the stone fruit is a I peach, nectarine, apricot, plum, or cherry. 00

22. The post-harvest agricultural, horticultural or silvicultural product according to claim 11, wherein the vegetable is an asparagus, carrot, beet, sugar beet, cabbage, cauliflower, brussel sprout, artichoke, Jerusalem artichoke, lettuce, potato, or spinach.

23. The post-harvest agricultural, horticultural or silvicultural product according to any one of claims 9 to 22, wherein the post-harvest product is a processed post-harvest product.

24. The post-harvest product according to claim 23, wherein the processed post-harvest product is selected from the group consisting of raisins, prunes, figs, dried apricots, and dates. A method of preventing or inhibiting the growth or reproduction of a fungal pathogen in or on a post-harvest agricultural, horticultural or silvicultural product comprising applying to the post-harvest product an effective amount of the composition according to any one of claims 1 to 8 for a time and under conditions sufficient to prevent or inhibit the growth or reproduction of the fungal pathogen in or on the post-harvest product.

26. A method for enhancing storage of an agricultural, horticultural or silvicultural post-harvest product comprising applying to the post-harvest product an effective amount of the composition according to any one of claims 1 to 8 for a time and under conditions sufficient to prevent or inhibit the growth or reproduction of a fungal pathogen in or on the post-harvest product. 00

27. The method according to either claim 25 or claim 26, wherein the fuangal pathogen is a Phycomycetes, an Ascomycetes or a Basidiomycetes.

28. The method according to any one of claims 25 to 27, wherein the fungal 00 pathogen is selected from the group consisting of Ahternaria spp.; Armillaria spp.; Arthrobotrys spp.; Aspergillus spp.; Boletus spp.; Botrytis spp.; Candida spp.; Cia viceps spp.; Cronartium spp.; Epicoccum spp.; Epidermophyton spp.; Eurotium spp.; Fomes spp.; Fusarium spp.; 00 Gemnoye etihmGoeel p. Gaumnnmyes spp.; eoicum spp.; Goeel p. Gymnosporangium spp.; Leptosphaeria spp.; Microsporum spp.; Monilinia spp.; Mucor spp.; Pen icillium spp.; Pezicula spp.; Phialophora spp.; Physoderma spp.; Phytopth era spp.; Pityrosporum spp.; Polyporus spp.; Puccinia spp.; Rhizoctonia spp.; Rhizopus spp.; Saccharomyces spp.; Scedosporium spp.; Scierotinia spp.; Septoria spp.; Trichoderma spp.; Trichophyton spp.; Ustilago spp.; Venturia spp.; and Verlicillium spp.

29. The method according to any one of claims 25 to 28, wherein the fungal pathogen is selected from the group consisting of Armillaria mellae; Arthrobotrys oligosporus; Aspergillus flavus; Aspergillus fumigatus; Aspergillus ochraceous; Boletus granulatus; Botrytis cinerea; Botiytis fabae; Candida albicans; Claviceps purpurea; Cronartium ribicola; Epicoccum purpurescens; Epidermophyton floccosum; Eurotium rubrum; Fomes annosus; Fusarium graminearum; Fusarium oxysporum; Fusarium oxysporum f. apii Snyder Hansen; Fusarium oxysporum f. cubense; Gaeumannomyces gramminis; Gaeumannomyces gramminis var. tritici; Geotrichum candidum; Glomerella cingulata; Gymnosporangium juniperi-virginianae; Leptosphaeria maculans; Microsporum canis; Monilinia fructicola; Mucor pirformis; Penicillium digitatum; Penicillium expansum; Physoderma alfajfae; Phytopthera infestans; Pityrosporum orbiculare (Malassezia furfur); Polyporus suiphureus; Puccinia graminis.; Rhizoctonia solani; Rhizoctonia solani 9760; Rhizoctonia solani 9834; Rhizopus stolonifer; Saccharomyces cerevisiae; Scedosporium prolificans; Scierotinia scierotiorum, Scierotinia minor; Scierotinia trifoliorum; Septoria apiicola; Trichoderma harzianum; Trichophyton mentagrophytes; Trichophyton mentagrophytes var interdigitale; Trichophyton rubrum; 00 IVVu Trichophyton tonsurans; Ustilago nuda RostrVenturia inaequalis; and d Verticillium dahliae. The method according to any one of claims 25 to 29, wherein the post- harvest product is immersed in the composition according to any one of 00 claims 1 to 8. S31. The method according to any one of claims 25 to 29, wherein the N composition according to any one of claims 1 to 5 is sprayed onto the post- 00 harvest product.

32. The method according to any one of claims 25; to 29, wherein the composition according to any one of claims lIto 8 is brushed onto the post- harvest product.

33. The method according to any one of claims 25 to 29, wherein the composition according to any one of claim 1 to 8 :is applied to the post- harvest product in admixture with an oil and/or a wax.

34. The method according to any one of claims 25 to 29, wherein the composition according to any one of claims 1 to 8 is applied to the post- harvest product under pressure. The method according to any one of claims 25 to 34, wherein the post- harvest product is a post-harvest product of an ornamental plant, a field crop, a vegetable plant; a fruit tree, a nut tree, a wood tree or a herb plant.

36. The method according to any one of claims 25 to 35, wherein the post- harvest product is a fruit, a vegetable, a cereal, a grain, a nut, a seed, a floral bulb, wood, timber, silage, bark, natural latex, a tuber, a bloom, a leaf, a stem, a branch, or a root.

37. The method according to claim 36, wherein the cereal is wheat, maize, sorghum, rice, rye, oats, millet, or barley. The method according to claim 36, wherein the seed is a legume seed. 00 101

39. The post-harvest product according to claim 38, wherein the legume seed is Sa soybean. The method according to claim 36, wherein the nut is a peanut, almond, OC Brazil nut, or pecan. O 41. The method according to claim 36, wherein the fruit is a pome fruit, stone I fruit, citrus fruit, grape, tomato, potato, persimmon, strawberry, or papaya or 00 banana.

42. The method according to claim 41, wherein the pome fruit is an apple or a pear.

43. The method according to claim 42, wherein the apple is a Granny Smith, Red or Golden Delicious, Jonathan, Gala, Fuji, Newton, or Macintosh strain.

44. The method according to claim 42, wherein the pear is a d'Anjou, Packham's Triumph, William's Bon Chretian, or Beurre Bosc strain. The method according to claim 41, wherein the citrus fruit is grapefruit, orange, lemon, kumquat, lime, mandarin or pomelo.

46. The method according to claim 41, wherein the stone fruit is a peach, nectarine, apricot, plum, or cherry.

47. The method according to claim 36, wherein the vegetable is an asparagus, carrot, beet, sugar beet, cabbage, cauliflower, brussel sprout, artichoke, Jerusalem artichoke, lettuce, potato or spinach.

48. The method according to any one of claims 25 to 47, wherein the post- harvest product is a processed post-harvest product.

49. The method according to claim 48, wherein the processed post-harvest product is selected from the group consisting of raisins, prunes, figs, dried apricots, and dates. 0 102 O An isolated nucleic acid molecule which comprises a nucleotide sequence Scapable of encoding one or more proteins involved in the biosynthesis of a Ssugar acid, wherein said nucleotide sequence is selected from the group consisting of: a nucleotide sequence which is at least about 50% identical to at least o0 about 30 contiguous nucleotides of SEQ ID NO: 7 other than the sequence t set forth in SEQ ID NO: 1; (ii) a nucleotide sequence which is capable of hybridising under at least low C stringency conditions to at least about 30 contiguous nucleotides of the oO Scomplement of SEQ ID NO: 7 other than the sequence set forth in SEQ ID SNO: 1; (iii) a nucleotide sequence which encodes the amino acid sequence set forth in SEQ ID NO: 8; and (iv) a sequence complementary to any one of to (iii).

51. The isolated nucleic acid molecule of claim 50 wherein an encoded protein involved in the biosynthesis of a sugar acid is a sugar oxidase.

52. The isolated nucleic acid molecule of claim 51 wherein the sugar oxidase is a PQQ-dependent sugar oxidase.

53. The isolated nucleic acid molecule according to any one of claims 50 to 52 comprising the nucleotide sequence set forth in SEQ ID NO: 7 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 8.

54. An isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding a protein involved in the synthesis of PQQ, wherein said nucleotide sequence is selected from the group consisting of: a sequence that comprises at least about 50 contiguous nucleotides of SEQ ID NO: 9 other than a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6; (ii) a nucleotide sequence which is capable of hybridising under at least low stringency conditions to at least about 50 contiguous nucleotides of the complement of SEQ ID NO: 9 other than the complement of a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6; 00 1 VJ ri (iii) a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID Nos: 10, 12, 14, 16, 18 and 20; and (iv) a sequence that is complementary to any one of(i) to (iii). The isolated nucleic acid molecule according to claim 54 comprising the 00 nucleotide sequence set forth in SEQ ID NO: 9 or a sequence that encodes the t amino acid sequence set forth in SEQ ID NO: N 56. The isolated nucleic acid molecule according to claim 54 comprising the 00 nucleotide sequence set forth in SEQ ID NO: 11 or a sequence that encodes the Samino acid sequence set forth in SEQ ID NO: 12.

57. The isolated nucleic acid molecule according to claim 54 comprising the nucleotide sequence set forth in SEQ ID NO: 13 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 14.

58. The isolated nucleic acid molecule according to claim 54 comprising the nucleotide sequence set forth in SEQ ID NO: 15 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 16.

59. The isolated nucleic acid molecule according to claim 54 comprising the nucleotide sequence set forth in SEQ ID NO: 17 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO: 18. The isolated nucleic acid molecule according to claim 54 comprising the nucleotide sequence set forth in SEQ ID NO: 19 or a sequence that encodes the amino acid sequence set forth in SEQ ID NO:

61. Use of the isolated nucleic acid according to any one of claims 50 to 53 to produce a sugar acid in a cell.

62. Use according to claim 61 wherein sugar acid production utilizes a PQQ- dependent sugar oxidase encoded by the isolated nucleic, acid according to any one of claims 50 to 53 and the isolated nucleic acid molecule according to any one of claims 54 to 60 is used to produce PQQ for saidC PQQ-dependent sugar oxidase. 00 104

63. Use of the isolated nucleic acid according to any one of claims 54 to 60 to T produce PQQ in a cell.

64. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis oO of a sugar acid and comprising an amino acid sequence selected from the group consisting of: (iii) an amino acid sequence encoded by the nucleic acid according to any one of C claims 50 to 53; and oO 00 (iv) an amino acid sequence which is at least about 50% identical to the sequence set forth in SEQ ID NO: 8. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis of a sugar acid and comprising the amino acid sequence set forth in SEQ ID NO: 8.

66. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ and comprising an amino acid sequence selected from the group consisting of: an amino acid sequence encoded by the nucleic acid according to any one of claims 54 to 60; and (vi) an amino acid sequence which is at least about 50% identical to a sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, 16, 18 and

67. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ and comprising the amino acid sequence set forth in SEQ ID NO:

68. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ and comprising the amino acid sequence set forth in SEQ ID NO: 12.

69. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ and comprising the amino acid sequence set forth in SEQ ID NO: 14. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis of PQQ and comprising the amino acid sequence set forth in SEQ ID NO: 16. 00

71. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis Sof PQQ and comprising the amino acid sequence set forth in SEQ ID NO: 18.

72. An isolated or recombinant polypeptide capable of catalyzing the biosynthesis 00 of PQQ and comprising the amino acid sequence set forth in SEQ ID NO: O 73. Use of the isolated or recombinant polypeptide according to any one of claims C 64 or 65 to produce a sugar acid. 00

74. Use of the isolated or recombinant polypeptide according to any one of claims 66 to 72 to produce PQQ. An isolated or recombinant protein complex or mixture capable of catalyzing the PQQ-dependent biosynthesis of a sugar acid and comprising: a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 7 or comprising the amino acid sequence set forth in SEQ ID NO: 8; and (ii) one or more polypeptides selected from the group consisting of: a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 9; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18; and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

76. The isolated or recombinant protein complex or mixture of claim 75 wherein the polypeptide at has PQQ-dependent sugar oxidase activity and a polypeptide at (ii) is involved in the biosynthesis of PQQ.

77. A method of producing a sugar acid comprising expressing the isolated nucleic acid molecule according to any one of claims 50 to 53 or the recombinant polypeptide according to claim 64 or claim 65 in a cell, tissue or organism and 00 Sculturing said cell, tissue or organism in the presence of an aldose substrate for a time and under conditions sufficient to produce a sugar acid.

78. The method according to claim 77 further comprising introducing the nucleic acid molecule to the cell, tissue or organ in a expressible format. 00 S 79. The method according to claim 77 or 78 further comprising extracting or purifying the sugar acid produced. 00

80. The method according to any one of claims 77 to 71) wherein the cell is a Sbacterial cell.

81. The method according to claim 80 wherein the bacterial cell is a Pseudomonas sp.

82. The method according to any one of claims 77 to 79 wherein the cell, tissue or organ is a plant cell, tissue, or organ.

83. The method according to any one of claims 77 to 82 further comprising expressing the nucleic acid molecule according to any one of claims 54 to 60 or the recombinant polypeptide according to any one of claims 64 to 72 for a time and under conditions sufficient to produce PQQ in the cell, tissue or organ.

84. A method of producing PQQ comprising expressing one or more isolated nucleic acid molecules according to any one of claims 54 to 60 or one or more recombinant polypeptides according to any one of claims 64 to 72 in a cell, tissue or organism and culturing said cell, tissue or organism in the presence of a suitable substrate for a time and under conditions sufficient to produce PQQ. The method according to claim 84 further comprising introducing one or more nucleic acid molecules according to any one of claims 54 to 60 to the cell, tissue or organ in a expressible format.

86. The method according to claim 84 or 85 further comprising extracting or purifying the PQQ produced. 00 IV/

87. The method according to any one of claims 84 to 86 wherein the cell is a Sbacterial cell.

88. The method according to claim 87 wherein the bacterial cell is a Pseudomonas sp. 00

89. The method according to any one of claims 84 to 88 wherein the cell, tissue or 8 organ is a plant cell, tissue, or organ. 00 A method of enhancing the tolerance of a plant to infection by a fungal pathogen Scomprising expressing therein the isolated nucleic acid molecule according to any one of claims 50 to 53 or the recombinant polypeptide according to claim 64 or 65, and optionally expressing therein one or more second isolated nucleic acid molecules each encoding a PQQ-biosynthesis enzyme, for a time and under conditions sufficient for a sugar acid to be produced by said plant, or by a cell, tissue or organ of said plant.

91. The method according to claim 90 comprising expressing one or more isolated nucleic acid molecules each according to any one of claims 54 to 60 or one or more recombinant polypeptides each according to any one of claims 66 to 72.

92. A transformed plant comprising the isolated nucleic acid molecule according to any one of claims 50 to

93. A progeny plant, cell, tissue or organ of the plant according to claim 92, wherein said progeny, cell, tissue or organ comprises the isolated nucleic acid molecule according to any one of claims 50 to

94. An anti-fungal composition according to any one of claims 1 to 8, substantially as hereinbefore described with reference to the accompanying Figures and Examples. An agricultural, horticultural or silvicultural post-harvest product according to any one of claims 9 to 24, substantially as hereinbefore described with reference to the accompanying Figures and Examples. I 00 108

96. A method of preventing or inhibiting the growth or reproduction of a fungal Spathogen in or on a post-harvest agricultural, horticultural or silvicultural product according to any one of claims 25, and 27 to 49, substantially as hereinbefore described with reference to the accompanying Figures and Examples. 00

97. A method for enhancing storage of an agricultural, horticultural or silvicultural post-harvest product according to any one of claims 26 to 49, substantially as Q0 hereinbefore described with reference to the accompanying Figures and SExamples.

98. An isolated nucleic acid molecule according to any one of claims 50 to substantially as hereinbefore described with reference to the accompanying Figures and Examples.

99. An isolated or recombinant polypeptide according to any one of claims 64 to 72, substantially as hereinbefore described with reference to the accompanying Figures and Examples. Dated 15 February, 2008 The Australian National University Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON