WO2010118463A1

WO2010118463A1 - Biocontrol fungi

Info

Publication number: WO2010118463A1
Application number: PCT/AU2010/000411
Authority: WO
Inventors: Robin Bruce Coles
Original assignee: BIOCENTRAL LABORATORIES Ltd
Current assignee: BIOCENTRAL LABORATORIES Ltd
Priority date: 2009-04-17
Filing date: 2010-04-15
Publication date: 2010-10-21
Anticipated expiration: 2011-10-17
Also published as: AU2010237601A1

Abstract

The present invention relates to fungi which have been identified as having the ability to control one or more plant pathogens. Tn specific forms, the fungi of the present invention belong to the species Aspergillus ustus.

Description

BIOCONTROL FUNGI

PRIORITY CLAIM

This patent application claims priority to Australian provisional patent application 2009901848, filed 17 April 2009, the content of which is hereby incorporated by reference.

FIELD

The present invention relates to fungi which have been identified as having the ability to control one or more plant pathogens. In specific forms, the fungi of the present invention belong to the species Aspergillus ustus.

BACKGROUND

Nearly all cultivated plants are susceptible to attack by a range of plant pathogens. In Australia, and other parts of the world, plant diseases are serious pests and cause major horticultural and agricultural losses. Within the range of plant pathogens the ones most damaging to agricultural yields are the fungal and oomycete plant pathogens.

At present, the most common methods for the control of plant pathogens control is with chemical pesticides such as fungicides.

Fungicidal control includes the use of various chemicals in different activity groups.

For example: Group A, activity the Benzimidazole group has the active constituent benomyl and goes under the trade name Benlate®, whilst Group B, activity and chemical grouping Dicarboximide with active constituent iprodione, trade name Rovral® are widely used as a vegetable seed coating and is regularly used to spray crops during early growth to prevent leaf disease. Alternative fungicides are in Group C, e.g. Score®. The Group D fungicides with activity group Phenylamide, active constituent metalaxyl and trade name Ridomil® are frequently used to prevent damping-off of seedlings in horticultural crops. The Group K fungicides belonging to the Strobilurin chemical grouping with trade names such as Amistar®, Stroby® and Flint®.

A problem with the use of the above-mentioned fungicides is that they all require withholding periods after application. Furthermore, a rapid build up of fungicide resistance occurs if the same group of fungicides is constantly used. Thus, the use of different fungicide groups must be implemented to reduce the probability of a plant pathogen becoming fungicide resistant.

Other factors which reduce the effectiveness of the use of fungicides, are the random distribution of fungal diseases in the soil and on host weeds. This results in some fungal diseases surviving because they have not received an adequate fungicide treatment. Other undesirable effects are that some fungicides destroy "non-target" organisms such as beneficial soil microbes and earthworms. There is also the problem that fungal diseases rapidly re-establish into an environment devoid of antagonistic microbial activity. This occurs when contaminated soil is blown onto the site or diseased seed and leaf material are re-introduced into a microbial reduced environment.

In light of the above, it would be desirable to provide an alternative to chemical treatment for the control of plant pathogens, particularly fungal or oomycete plant pathogens.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

SUMMARY

In a first aspect the present invention provides an isolated fungus of the species Aspergillus ustus, wherein the fungus is capable of controlling one or more plant pathogens.

In some embodiments, fungi may be classified as Aspergillus ustus in terms of their morphology.

In some embodiments, the fungus according to the present invention comprises an Internal Transcribed Spacer rDNA sequence which is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 1.

An exemplary fungus according to the present invention has been deposited in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The fungus has been deposited at the National Measurement Institute of 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia on 25 February 2009 and has been accorded accession number V09/005065.

In some embodiments, the A. ustus fungus of the present invention controls a fungal plant pathogen.

In a second aspect, the present invention also provides a method for controlling a plant pathogen, the method comprising administering to a plant or its growing environment a fungus according to the first aspect of the invention.

The method of the second aspect of the invention also contemplates co-administration of the fungus with one or more additional treatments. For example, the fungus may be sequentially applied or co-applied with one or more other biological control agents, one or more chemical pesticides, fungicides or plant growth enhancers; one or more fertilisers or plant nutrients and the like.

In some embodiments, the fungus of the present invention may be co-administered with another biological control agent for a pathogen. In some specific embodiments, the co-administered biological control agent is a fungus of the genus Gliocladium, including species such as Gliocladium vixens or Gliocladium roseum.

The fungus of the first aspect of the invention may be formulated into a composition together with a carrier for administration to a plant or its growing environment. Thus, in a third aspect, the present invention provides a composition comprising a fungus according to the first aspect of the invention together with an appropriate carrier. In some embodiments, the composition comprises spores of the fungus.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that the following description is for the purpose of describing particular embodiments only, and is not intended to be limiting with respect to the above description.

The term "isolated" refers to material removed from its original environment (e.g. the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, while an isolated fungus may be a pure culture removed from a natural source, the isolated fungus need not be pure and may include a mixture of the fungus with other organisms including, for example, other fungi or bacteria or other matter, provided that it is removed from its original environment.

In some embodiments, fungi may be classified as Aspergillus ustus in terms of their morphology. For example, A. ustus may be characterized by drab olive to dull brown colonies, sometimes with dark purple exudate. Microscopically, A. ustus displays conidiophore stipes smooth walled, coloured in brown shades. Conidial heads radiate to loosely columnar with age, commonly splitting into more or less well defined columns. Vesicles are hemispherical to subspherical, 7-15 micrometres in diameter. Conidiogenous cells are biserate. Metulae cover the upper half to three-fourths of the vesicle. Conidia have very rough walls, are spherical, 3-4.5 micrometre in diameter and dark yellow-brown in colour. Irregular to elongate Hulle cells are also sometimes present.

In some embodiments, fungi may also be characterised as A. ustus genetically, for example on the basis of a ribosomal RNA gene sequence.

Ribosomal RNAs (rRNA) are highly conserved in all cells. For this reason, genes that encode the rRNA (rDNA) may be sequenced in order to identify an organism's taxonomic group, calculate related groups, and estimate rates of species divergence. For this reason many thousands of rRNA sequences are known and stored in specialized databases such as RDP-II and the European SSU database.

In fungi, and most eukaryotes, the 18S rRNA is in the small ribosomal subunit, and the large subunit contains three rRNA species, the 5S, 5.8S and 28S rRNAs.

The nuclear-encoded ribosomal RNA genes of fungi exist as a multiple-copy gene family comprised of highly similar DNA sequences (typically from 8-12 kb each) arranged in a head-to-toe manner. Each repeat unit has coding regions for one major transcript (containing the primary rRNAs for a single ribosome), punctuated by one or more intergenic spacer (IGS) regions. In some groups (mostly basidiomcyetes and some ascomycetous yeasts), each repeat also has a separately transcribed coding region for 5S RNA whose position and direction of transcription may vary among groups. A detailed review of rDNA and fungal systematics is provided by Hibbett (Trans. Mycol. Soc. Jm. 33: 533-556, 1992)

In some embodiments, A. ustus may be characterised by the sequence of one or more rDNAs including, for example, the sequence of the Internal Transcribed Spacer region.

The Internal Transcribed Spacer (ITS) region of the rDNA is now perhaps the most widely sequenced DNA region in fungi. It has typically been most useful for molecular systematics at the species level, and even within species. Because of its higher degree of variation than other genie regions of rDNA (SSU or 18S rDNA and LSU), variation among individual rDNA repeats can sometimes be observed within both the ITS and IGS regions. In addition to the commonly used ITS1+ITS4 primers, several taxon- specific primers have also been described that allow selective amplification of fungal sequences (for example see Gardes and Bruns, MoI. Ecol. 2: 113-118, 1993)

Reference herein to the ITS region may include any one or more of the ITS-I region, the ITS-2 region and/or all or part of the 5.8S rDNA, which may be interspersed with the ITS regions.

Accordingly, in some embodiments, the fungus according to the present invention comprises an Internal Transcribed Spacer rDNA sequence which is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 1.

As previously mentioned, reference herein to "at least 90%" should be understood to also include thresholds higher than this including, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100%.

When comparing nucleic acid sequences to calculate a percentage identity to SEQ ID NO: 1, the compared nucleotide sequences should be compared over a comparison window of at least 100 nucleotide residues, at least 200 nucleotide residues, at least 300 nucleotide residues, at least 400 nucleotide residues or over the full length of SEQ ID NO: 1. The comparison window may comprise additions or deletions (ie. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. {Nucleic Acids Res. 25(17): 3389-402, 1997).

In some embodiments, the fungus comprises an Internal Transcribed Spacer rDNA sequence comprising one or more poly-cytosine repeat motifs. In some embodiments, the "one or more poly-cytosine repeat motifs" comprise at least 3, at least 4, at least 5, at least 6 or at least 7 contiguous cytosine residues. In some embodiments, the one or more poly-cytosine repeat motifs comprise 7 contiguous cytosine residues. In some embodiments, the fungus comprises an Internal Transcribed Spacer rDNA sequence comprising at least two poly-cytosine repeat motifs

In some embodiments, the fungus comprises an Internal Transcribed Spacer rDNA sequence as set forth in SEQ ID NO: 1.

An exemplary fungus according to the present invention has been deposited in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The fungus has been deposited at the National Measurement Institute of 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia on 25 February 2009 and has been accorded accession number V09/005065.

Accordingly, in one specific embodiment, the fungus comprises the fungus deposited at the National Measurement Institute under accession number V09/005065.

As set out above, the present invention contemplates an isolated fungus of the species Aspergillus ustus, wherein the fungus is capable of controlling one or more plant pathogens.

Reference herein to the "control" of one or more plant pathogens should be understood to include, for example: the treatment or prevention of disease in a plant caused by a particular pathogen; antagonism of growth, reproduction or spreading of a pathogen either on or within a plant or in soil; antagonism of the ability of a pathogen to infect a plant and/or direct; or indirect killing of a pathogen on or within a plant or in soil.

Control of a particular pathogen may occur via one or several mechanisms including, for example, production of a compound which inhibits the growth of, or kills, the pathogen; parasitism of the pathogen; competitive inhibition for nutrients and/or infection or colonisation sites on a plant; and the like.

In some embodiments, the A. ustus fungus of the present invention controls a fungal plant pathogen at least via the production of one or more antifungal compounds.

In light of the above, control of a plant pathogen by A. ustus may occur in planta, on the surface of a plant tissue such as the leaf or root surface, in the rhizosphere, in soil, on harvested plant material (for example for the control of post-harvest pathogens), on harvested timber or wood from plants (for the control of post-harvest rots and pest damage) and the like.

The present invention may be used to control a wide variety of plant pathogens including fungal pathogens as described below; oomycete pathogens such as Phytophthora spp. and Pythium spp.; plant-pathogenic nematodes such as root knot nematodes and cyst nematodes; bacterial pathogens; phytoplasmas; and the like.

In some embodiments, the pathogen is a fungal plant pathogen. The majority of phytopathogenic fungi belong to the Ascomycetes and the Basidiomycetes. Spores or other propagules of fungal pathogens may be spread by air or water, or they may be soil borne. Many soil borne spores, may be capable of living saprotrophically and/or carrying out the first part of their lifecycle in the soil.

Examples of significant fungal plant pathogens which might be controlled using the A. ustus fungi of the present invention include: Fusaήum spp.; Thielaviopsis spp. (Causal agents of: canker rot, black root rot, Thielaviopsis root rot); Verticillium spp.; Rhizoctonia spp.; Puccinia spp.; Colletotrichum spp.; Botrytis spp.; Alternaria spp.; Cladosporium spp.; and Sclerotinia spp.

In light of the above, the A. ustus fungi of the present invention might be used to control pathogens on a range of plant species depending on the host range of the pathogen to be controlled.

In some embodiments, the pathogen is Colletotrichum coccodes. C. coccodes is the causative agent of black spot on potatoes (Solatium tuberosum) and anthracnose on a number of other plant species such as hemp and tomato.

In some embodiments, the pathogen is Botrytis cinerea. B. cinerea is the causative agent of botrytis bunch rot on grapes (Vitis vinifera) and grey mould on a number of other horticultural crops such as strawberries and bulb crops.

In some embodiments, the pathogen is Rhizoctonia solani. R. solani has a wide host range and can cause root rots and damping off in a number of plant species depending on the anastomosis group of the particular R. solani isolate. In some embodiments, the pathogen is R. solani anastomosis group 8 (AG8), which can infect wheat and other cereals.

Notwithstanding the examples disclosed above, the fungi of the present invention are thought to have the ability to control a range of fungal or other pathogens, which infect a range of other plant species.

In some embodiments, the method of the second aspect of the invention is for controlling a fungal plant pathogen as hereinbefore described in connection with the first aspect of the invention.

As set out above, the method of the second aspect of the invention contemplates administering the fungus of the first aspect of the invention to a plant or a plant's growing environment. Such administration should be understood to include, for example, application of the fungus (or a composition comprising the fungus) as: a seed coating applied before or at sowing; a soil amendment which may be incorporated into a soil during cultivation, seed sowing or after seed sowing; a seedling treatment or drench applied to seedlings or plantlets to be sown; a foliar spray or dust applied to germinated plants; or any other suitable application means.

The method of the second aspect of the invention also contemplates co-administration of the fungus with one or more additional treatments. For example, the fungus may be sequentially applied or co-applied with one or more other biological control agents, one or more chemical pesticides, fungicides or plant growth enhancers, one or more fertilisers or plant nutrients and the like. The fungus may also be co-administered with one or more agents that assist with the growth of the fungus and/or enhance the ability of the fungus to control a plant pathogen. For example, the fungus may be administered in conjunction with one or more nutrients. Nutrients that may be supplied may include, for example, any one or more of: a source of energy, a source of carbon, a source of nitrogen, a source of phosphorus, a source of sulphur, a source of growth factors, a source of vitamins, a source of amino acids, a source of purine, a source of pyrimidine, a source of minerals, and a source of micronutrients. In some embodiments, the nutrients include any one or more of ammonium, various nitrates, amides, amino acids, peptides, mineral (i.e. Iron) or carbon sources (i.e. carbohydrates) to provide energy to promote fungal growth. Nutrients may be applied separately in solid or liquid from and/or may be included in carrier materials or a composition of the fungus as described below.

In some embodiments, the fungus of the present invention may be co-administered with another biological control agent for a pathogen. In some specific embodiments, the co-administered biological control agent is a fungus of the genus Gliocladium, including species such as Gliocladium virens or Gliocladium roseum.

G. virens is used as a commercial biological pesticide to protect against soil-borne pathogens such as Rhizoctonia solani and Pythium ultimum (Kock, Crop Protection 18: 119-125, 1999). Furthermore, G. roseum has been demonstrated to be a mycoparasite of the plant pathogen Botrytis cinerea (Li et ah, Botanical Bulletin of Academia Sinica 43, 2002).

A range of Gliocladium spp. which may be co-administered with the A. ustus fungus of the present invention may be sourced from culture collections including for example, the ATCC. ATCC accession numbers for G. virens isolates include, for example, ATCC numbers 13213, 13362, 20903, 20904, 20906, 24290, 42955, 44327, 44734, 48179, 52045, 52199 and 58676. ATCC accession numbers for G. roseum isolates include, for example, ATCC numbers 10521, 16389, 20010, 201998, 204433, 26441, 28863, 36137, 46475, 48395, 52629, 58993, 62196, 64054, 66680, 66681, 8684.

As will be appreciated by those of skill in the art, suitable Gliocladium spp. may also be sourced from other culture collections such as the Agricultural Research Service Culture Collection (NRRL Collection), the German Resource Centre for Biological Material (DSMZ Collection), the NCIMB and the like. Alternatively, new Gliocladium spp. isolates may be obtained using standard isolation techniques.

As set out above, the fungus of the first aspect of the invention may be formulated into a composition together with a carrier for administration to a plant or its growing environment.

Thus, in a third aspect, the present invention provides a composition comprising a fungus according to the first aspect of the invention together with an appropriate carrier.

The composition may be any suitable composition for a suitable application method. For example the composition may be solid, liquid, semisolid or a gel. Compositions may include, seed dressings, soil additives, foliar sprays or dusts, seedling or plantlet drenches and the like.

A broad range of materials may be used as a carrier. Suitable carriers may include, for example, silica, peat, cereal grains or grain products, an oil, culture media, water- insoluble gels, alginate salts such as calcium alginate, various clays or clay-like materials. The carrier may also comprise mixtures of two or more of these or other carrier materials.

The fungus may be combined with carriers by mixing an inoculum of the fungus with the carrier material in various ratios. Such a mixture may take the form of a suspension, a paste or the like, depending on the particular carrier, the state of the fungus and the ratios used. The composition may also be an aqueous or oil suspension of the fungus, or a dry mix of the fungus and appropriate carrier. In embodiments wherein the composition is a dry mix, the composition may take the form of a powder, granule, dust, pellet or any other acceptable form.

Alternatively, the combination of the fungus with a carrier material may be such that the fungus is encapsulated by, or enclosed within, the carrier material. In the latter case, encapsulating material may be in a variety of shapes, including more or less flat layers or sheets and spheroidal pellets or beads. In addition, encapsulating materials in combination with the fungus may be formed in specific ranges of size. The particular shape and size of the encapsulating material will depend on factors such as the state of the fungus, the composition of the carrier material, and the intended applications.

Carrier materials contemplated herein may also comprise a gel. Such gel materials may be polymeric or copolymeric, and may be derived from either natural sources or from synthetic chemicals. Examples of gel materials which may be used as carrier materials include various salts of alginic acid and polyacrylamide. Two or more materials, including powdered materials and gel materials, may be combined in various proportions to provide a suitable carrier for the fungus.

Compositions comprising two or more different carrier materials may be homogeneous or heterogeneous with respect to the carrier component. In the latter case compositions may comprise carriers comprising two or more different layers. Compositions comprising one or more carrier materials may be constituted with a suitable liquid, in various ratios, as a pellet, paste, or suspension, and the like. Liquid compositions may be essentially pure compounds, used for suspending the fungus in the composition. Alternatively, a liquid composition may itself comprise one or more additional compounds dissolved or suspended in the liquid. The composition may further comprise one or more nutrients. Such nutrients may constitute the sole source of nutrients for the fungus during a particular period of growth or they may represent nutrients which are supplementary to another source of nutrients. Nutrients to be supplied to a fungus may be organic or inorganic, and may represent, for example: a source of energy, a source of carbon, a source of nitrogen, a source of phosphorus, a source of sulphur, a source of growth factors, a source of vitamins, a source of amino acids, a source of purine, a source of pyrimidine, a source of minerals, and a source of micronutrients.

The composition may be admixed with a carrier material to form initially an amorphous, more or less homogeneous mixture, which subsequently may be cut, rolled, extruded, etc. to produce various forms of the composition. The fungus may be encapsulated within the carrier material to form a capsule. It will be clear to the skilled artisan that the specific shape and size of such capsules can be varied, depending on factors such as the nature of the encapsulated fungus, the intended application of the encapsulated composition, ease of storage and handling, and the like.

In some embodiments, the composition of the third aspect of the invention may also comprise another biological control agent for a pathogen. In some specific embodiments, the co-administered biological control agent is a fungus of the genus Gliocladium as hereinbefore described.

As set out above, preparation of the composition will typically involve the production of an inoculum of the fungus for incorporation into the composition.

An inoculum of the fungus may be prepared by growing the fungus on any liquid or solid culture medium which will support its growth. However, preferably the culture medium will be one which supports vigorous growth and, optionally, sporulation of the fungus. The composition of the culture medium, aeration rate, incubation temperature, etc. will depend on the particular fungus to be grown for production of inoculum. Those skilled in the art will readily appreciate how these parameters may be successfully manipulated to obtain a suitable source of fungal inoculum. After a suitable period of incubation, the fungus may be harvested by suspending it in either spent liquid medium in which the fungus has been grown or in a sterile liquid, to provide a suspension of fungal inoculum. The fungal inoculum may be in the form of a suspension of mycelial hyphae, spores, or other propagules; or in the form of a suspension comprising a combination of mycelium, spores, and other propagules. In the latter case, the mycelium, spores, and other propagules of the suspension may be separated from each other to provide different types of inoculum suspension prior to adjustment of the concentration of the inoculum. If mycelium is used for preparing the inoculum it may be blended or comminuted to provide a suspension of hyphal fragments. The concentration of the hyphal fragments, spores or other propagules in the suspension may be adjusted to the desired concentration range, normally expressed as colony forming units/ml (cfu/ml). In some embodiments, the concentration of inoculum may be in the range of 10³ -10⁹ cfu/ml, 10⁵ -10⁷ cfu/ml or about 10⁶ cfu/ml. In further embodiments, inocula may be prepared on the basis of mass. For example, in some embodiments, an inoculum may comprise a particular wet weight of biomass.

In some embodiments, the inoculum used to prepare the composition comprises spores of the fungus.

The sterile liquid used to harvest the fungal culture for preparation of fungal inoculum may be water (tap, deionized, or distilled water) or fresh culture medium. When the sterile liquid used to prepare fungal inoculum is water, it subsequently may be supplemented with any number of organic or inorganic compounds which may serve as a source of at least one nutrient for the fungus. Such compounds which may be added to the preparation of fungal inoculum may serve as a source of at least one nutrient for the fungus in one or more of the following capacities: a source of energy, a source of carbon, a source of nitrogen, a source of phosphorus, a source of sulfur, a source of growth factors, a source of vitamins, a source of amino acids, a source of purine, a source of pyrimidine, a source of minerals, and a source of micronutrients. When fresh culture medium is used to resuspend a fungal culture for preparation of a suspension of fungal inoculum, it may have the same composition, qualitatively and quantitatively, as the culture medium on which the fungus has been grown. Alternatively, a second, different medium may be used to resuspend the fungal growth. The composition of the second medium will depend on a number of factors which may include, for example, the microorganism, the pathogen to be controlled, and the intended site of application. In general, a second, different medium used to prepare fungal inoculum may comprise at least one of the following types of components: a source of energy, a source of carbon, a source of nitrogen, a source of phosphorus, a source of sulfur, a source of growth factors, a source of vitamins, a source of amino acids, a source of purine, a source of pyrimidine, a source of minerals, and a source of micronutrients.

Finally reference is made to standard microbiological textbooks that contain methods for carrying out basic techniques encompassed by the present invention including, for example, microbiological media preparation and microbial isolation, culture and identification. For example, reference is made to Benson's Microbiological Applications: Laboratory Manual in General Microbiology Complete Version (10th Ed., McGraw-Hill Science, 2006). Reference is also made to standard techniques may be used for molecular biology. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

The present invention is further described by the following non-limiting examples: BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 shows a multiple sequence alignment of Aspergillus 5.8s rRNA-ITS DNA sequences. A.pseu6135 is the type culture of Aspergillus pseudodeflectus NRRL 6135; A.ustus352 is the type culture of Aspergillus ustus SRRC 352; and Asp.spp4C is Aspergillus strain 4C (morphological identification as A. ustus). Cytosine nucleotide insertions in Asp.spp4C at positions 57-63 and 331-337 are shaded.

FIGURE 2 shows the results of a pot trial for the control of black dot disease (Colletotrichum coccodes) on potatoes (Solatium tuberosum). Disease-free tubers were planted into C. coccodes inoculated soil. The seed treatments were as follows: control - water; fungicide - fludioxonil (31.25 g/1); and A. ustus - Aspergillus ustus spore suspension (5xlO⁶ spores/ml). Each of the seed treatments was applied to seed tubers at a rate of 21 of seed treatment / tonne seed tubers.

FIGURE 3 shows the results of a pot trial for the control of black dot disease (Colletotrichum coccodes) on potatoes (Solanum tuberosum). C. coccodes inoculated tubers were planted into C. coccodes inoculated soil. The seed treatments were as follows: control - water; fungicide - fludioxonil (31.25 g/1); and A. ustus - Aspergillus ustus spore suspension (5xlO⁶ spores/ml). Each of the seed treatments was applied to seed tubers at a rate of 21 of seed treatment / tonne seed tubers.

EXAMPLE 1 Isolation of A. ustus

A. ustus isolate 4C was isolated using a soil dilution method and Alternaria radicina selective agar (ARSA) as described by Pryor et al. (Plant Disease 78: 452-456, 1994). EXAMPLE 2 rDNA sequencing of A. ustus

Aspergillus strain 4C was putatively identified as A. ustus on morphological characters. For general maintenance and growth for DNA extractions, A. ustus 4C was grown on half-strength Potato Dextrose Agar (1/2 PDA) or in 100 ml flasks containing 25 ml of Czapek-Dox medium without shaking for 7-10 days at 25 ⁰C.

Molecular taxonomic identification of Aspergillus ustus strain 4C DNA-based identifications used nucleotide sequence data from the conserved nuclear 5.8S rRNA gene (i.e. rDNA) and the adjacent internal transcribed spacer (ITS) regions (White et al., in PCR Protocols: A Guide to Methods and Applications, Eds Innis et al., pp 315-322, Academic Press, San Diego, USA, 1990). Isolation of fungal genomic DNA from pure cultures (Harvey et al, Plant Pathology 49: 619-627, 2000) and PCR amplification (Eppendorf) of ITS-5.8S rDNA sequences (Wakelin et al, Biology & Fertility of Soils 40: 36-43, 2004) were as described previously.

PCR products were separated on a 2% TBE agarose gels, stained with ethidium bromide and visualised under UV light. DNA amplicons were purified according to the manufactures instructions (Wizard" SV, Promega) and direct sequenced using the PCR primers ITS-I and ITS-4 (Australian Genomics Research Facility, South Australia). Similarity searches and taxonomic identifications were made by comparing DNA sequences on GenBank using the BlastN search.

Molecular taxonomy of Aspergillus strains

DNA read-lengths of the 5.8S rRNA gene and adjacent ITS regions submitted to determine DNA sequence homologies and identification of strain 4C were 504 base pairs (bp) in length. Of the 504 bp DNA sequence submitted for comparison, 498 bp were used for alignments with Aspergillus sequences held in the data base. Sequence alignments among Aspergillus 4C and the A. ustus and A. pseudodeflectus type cultures are shown in Figure 1. Species identities based on the 5 highest significant sequence alignments are given in Table 1.

TABLE 1: Identities of Aspergillus strains with the 5 highest significant 5.8s rRNA- ITS DNA sequence alignments in comparison with Aspergillus strain 4C.

Aspergillus 4C exhibited a 96% DNA sequence homology (over 482 bp) to the A. ustus and A. pseudodeflectus type cultures, the latter two strains both having identical sequences in this region (see Figure 1). Differentiation of Aspergillus 4C from the two type cultures was based on two 7 bp Cytosine nucleotide insertions (positions 57-63 and 331-337 respectively).

The previous morphological characterisation of the strain combined with information now provided by 5.8 rRNA-ITS sequence analyses may be sufficient to identify strain 4C as A. ustus. Notably, the DNA insertions revealed by comparative sequence analysis of strain 4C with the type cultures of A. ustus and A. pseudodeflectus (Figure 1) may be useful for strain 4C-specific identification.

EXAMPLE 3 Control of black dot on potatoes

Trials were undertaken to investigate the relationship between the amount of inoculum in soil and the disease on progeny tubers, to assess the efficacy of A. ustus to reduce black dot disease (caused by Colletotrichum coccodes) on potatoes.

Preparation of treated seed tubers

Either surface-sterilised and visually disease free tubers ('clean tubers') or Colletotrichum coccocfes-inoculated tubers ('diseased tubers') of Solatium tuberosum cv.

Coliban were treated with either water, a spore suspension of A. ustus (5xlO⁶ spores/ml) or the fungicide fludioxonil. The water and A. ustus spore suspension were applied to the seed tubers at a rate of 21/tonne of seed tuber. For the fungicide treatment, a 250 g/1 fludioxonil solution was diluted at the rate of 1 part fungicide to 7 parts water, and 21 of the diluted fungicide solution (31.25 g/1) was applied to the seed tubers at a rate of 21 per tonne of seed.

For the preparation of small batches of seed tubers, tubers were placed into plastic bags and the appropriate seed treatment was applied to the bag. The tubers were the agitated in the bag to coat the tuber with the seed treatment. After coating, the tubers were removed from the bag and air-dried for two days.

Pot trial

UC soil inoculated with Colletotrichum coccodes at a rate of 0.2 cfu/g of soil was used in pots of 20 cm diameter and 4.71 capacity.

Clean tubers and diseased tubers, each treated with each of the three seed treatments, were planted into the inoculated soil. 10 replicate pots for each treatment were prepared.

The pots were maintained in a shade house to allow plants to grow. At 28-30 days post sowing, progeny tubers were removed from the pots, stored and washed. These tubers were assessed for disease incidence and disease severity.

Results For the water control seed treatment, 43.8% of progeny tubers developed black dot when clean tubers were planted into inoculated soil (Figure 2). Higher disease incidence (59%) occurred in the control group when C. coccodes inoculated seed tubers were planted into C. coccodes inoculated soil (Figure 3).

As shown in Figures 2 and 3, both the fungicide and the A. ustus spore suspension treated groups exhibited lower levels of disease incidence and disease severity compared to the water control. It was also noted that both the fungicide and A. ustus exhibited the greatest control of Colletotrichum coccodes when applied to clean seed.

The disease severity data required transformation by log (+0.1) and back transformed data is shown in the Figures 2 and 3.

EXAMPLE 4 A. ustus compositions

Solid composition

A. ustus was grown on potato dextrose agar until the plate was covered by spores (conidia). Spores were then harvested using standard techniques. The harvested spores were then aseptically transferred to sterile liquid shake cultures containing 100ml of water, 5% wheat bran and 1% peat moss at a pH of 4. After 7 days incubation at 150 rpm and 27°C, the solid material was filtered from the culture and allowed to air dry.

Liquid composition A. ustus was grown in liquid culture media such as potato dextrose broth, Rawlin- Thom medium and malt extract medium. After 7 days incubation at 150 rpm and 27°C, the fungal biomass was filtered out from the culture medium and suspended in sterile water. Suspension composition

100ml of liquid medium was inoculated with 2 mycelial discs (5mm diameter) from the actively growing edge of a 5 day old A. ustus culture on potato dextrose agar. The inoculated liquid media were grown in flasks on a rotary shaker at 300 rpm in the dark for 14 days at 25°C. After growth, the liquid cultures were blended to a homogenate using a Waring commercial blender on low speed for 30 seconds in three equal bursts, which gave a fine spore/mycelial mixture (<150μm particle size).

One litre quantities of water containing 35g of ground wheat bran (800 μm) and 35g of fine diatomaceous earth (attapulgite) were autoclaved for 30 minutes at 121°C and 15 psi on two successive days. Cooled quantities of this mixture were inoculated by adding 70 ml of the A. ustus homogenate (described above) followed by shaking. Flasks containing the inoculated mixture were incubated in the dark at 25°C for 5 days.

The above composition may be applied to soil at a rate of approximately 50Og per cubic metre of soil.

EXAMPLE 5

Control of grape pathogens using A. ustus

A grape Botrytis cinerea trial tested the effects of Aspergillus ustus, Gliocladium vixens and a combination of the two fungi to control Botrytis cinerea on maturing grapes.

A laboratory trial established that complete control of B. cinerea was achieved after pre- treating individual grape berries (7 reps per treatment) by dipping in a spore suspension of A. ustus and G. virens at a dose approximating 10⁶ spores/ml.

In addition, a combination of both A. ustus and G. virens spore suspension dip achieved control of B. cinerea 12 days after pre-application of spore suspensions. In comparison, the control of B. cinerea pre treated berries developed infection after 12 days, A. ustus and G. virens spore suspension treatments had no infections. Therefore A. ustus and G. virens treated grapes did not infect the grapes but provided protection from B. cinerea when applied two days before inoculation with the disease B. cinerea.

Experiment

Individual grapes of the Chardonnay variety were harvested. Individual grapes were dipped (seven per treatment) into individual spore suspensions of A. ustus, G. virens and Botrytis cinerea of 10⁶ spores/ml for 10 seconds, then a combination of both fungi, A. ustus, G. virens were used to dip grapes and placed in sterile ice cube trays with moist paper to maintain humidity. The trays were incubated at 23°C for 2 days on a laboratory bench in 12 hrs daylight and 12 hrs dark for 2 days then removed and dipped in a B. cinerea suspension for 10 seconds and incubated for a further 10 days.

B. cinerea dipped grapes all developed the disease after 12 days, while the A. ustus dipped grapes followed by B. cinerea dipping had a low level of B. cinerea while G. virens dipped grapes followed by B. cinerea dipping had no B. cinerea after 12 days. The combination of A. ustus and G. virens dipping followed by B. cinerea had no infection after 12 days.

EXAMPLE 6

In field control of grape pathogens using A. ustus

A field trial may also be performed to assess control of B. cinerea on grapes by A. ustus. Individual bunches of grapes on grapevines are treated with A. ustus, G. virens and two days later B. cinerea in a preventative and protective application using the same spore suspension doses as above using 6 reps/treatment. Whole bunches are to be sprayed until run off using the same dose of fungal spore suspensions used in the individual grape berry experiment described above. Replicate bunches will be enclosed in sealed moist plastic bag for 24 hrs to induce high humidity and encourage fungal germination. The bunches will be harvested after 14 days and incubated in a moist environment to induce sporulation.

EXAMPLE 7

Control of Rhizoctonia solani using Aspergillus ustus and Gliocladium virens

Rhizoctonia solani strain AG8 was co-inoculated onto potato dextrose agar with Aspergillus ustus and a combination of Aspergillus ustus and Gliocladium virens.

Inhibition of the growth of Rhizoctonia solani by Aspergillus ustus as well as a combination of Aspergillus ustus and Gliocladium virens was observed after 10 days co- incubation at 24°C with a 12hr/12hr day/night photoperiod.

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 element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

It must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

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

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An isolated fungus of the species Aspergillus ustus, wherein the fungus is capable of controlling one or more plant pathogens.

2. The fungus of claim 1, wherein the fungus comprises an Internal Transcribed Spacer rDNA sequence which is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 1.

3. The fungus of claim 1 or 2 wherein the fungus comprises an Internal

Transcribed Spacer rDNA sequence including one or more poly-cytosine motifs.

4. The fungus of any one of claims 1 to 3, wherein the fungus comprises the fungus deposited under National Measurement Institute accession number V09/005065.

5. The fungus according to any one of claims 1 to 4, wherein the fungus is capable of controlling one or more fungal plant pathogens.

6. The fungus of any one claims 1 to 5, wherein the pathogen is Colletotήchum coccodes.

7. The fungus of any one of claims 1 to 6, wherein the plant is Solanum tuberosum.

8. The fungus of any one of claims 1 to 5 wherein the pathogen is Botrytis cinerea.

9. The fungus of any one of claims 1 to 5 or 8, wherein the plant is Vitis vinifera.

10. The fungus of any one of claims 1 to 5 wherein the pathogen is Rhizoctonia solani.

11. A method for controlling a plant pathogen, the method comprising administering to a plant or its growing environment a fungus according to any one of claims 1 to 10.

12. The method of claim 11, wherein the pathogen is a fungal plant pathogen.

13. The method of claim 11 wherein the pathogen is Colletotrichum coccodes.

14. The method of any one of claims 11 to 13, wherein the plant is Solanum tuberosum.

15. The method of claim 11 wherein the pathogen is Botrytis cinerea.

16. The method of any one of claims 11, 12 or 15, wherein the plant is Vitis vinifera.

17. The method of claim 11 wherein the pathogen is Rhizoctonia solani.

18. A composition comprising the fungus of any one of claims 1 to 10 together with an appropriate carrier.

19. The composition of claim 18, wherein the fungus comprises spores.

20. The composition of claim 18 or 19 wherein the composition comprises a plant seed dressing.