WO2000071735A1

WO2000071735A1 - Composition of plant antimicrobial proteins

Info

Publication number: WO2000071735A1
Application number: PCT/GB2000/001905
Authority: WO
Inventors: Andrew James Greenland; Angel Manuel Fuentes Mateos
Original assignee: Syngenta Limited
Priority date: 1999-05-21
Filing date: 2000-05-18
Publication date: 2000-11-30
Also published as: GB9911933D0; AU4599400A

Abstract

The invention relates to certain combinations of antimicrobial proteins which result in an enhanced improvement of antifungal activity against a range of fungi wherein the antifungal activity of the combination has a larger effect than the combination of the antifungal properties of the individual components. The combination of antifungal protein from Radish and a second antimicrobial protein selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens is claimed.

Description

COMPOSITION OF PLANT ANTIMICROBIAL PROTEINS

FIELD OF THE INVENTION

The present invention relates to an antimicrobial composition comprising a first and second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second antimicrobial agents being such as to produce an enhanced antimicrobial activity. The invention further relates to a DNA construct comprising a first DNA sequence encoding an antifungal agent from Radish or a homologue, variant or active derivative thereof and a second DNA sequence encoding a second antimicrobial agent selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof and wherein expression of said first and second DNA sequences may be under the control of the same or different promoter region; to a biological system transformed with the DNA construct of the invention; to a plant cell stably transformed with a DNA construct of the invention; to a method of combating microbial diseases comprising exposure of the microorganism to an antimicrobial composition according to the invention; a plant having improved resistance to a microbial pathogen and containing recombinant DNA which encodes an antifungal agent from Radish or a homologue, variant or active derivative thereof and a second antimicrobial agent selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof .

BACKGROUND TO THE INVENTION

Plants produce a wide array of antifungal compounds to combat potential invaders and it has become increasingly clear that proteins with antifungal activity are a very important part of these defences. Several classes of such proteins have been described including thionins, osmotins, glucose oxidases, beta-l,3,-glucanases, ribosome inactivating proteins, zeamatins, chitin-binding lectins and chitinases and antifungal proteins derived from certain plant species. We have previously described the structural and antifungal properties of several such proteins including Rs-AFPl and Rs-AFP2 from Raphanus, Dm- AMP 1 and Dm-AMP2 from Dahlia merckii (Published International Patent Application No. WO 93/05153), Hs-AFPl from Heuchera sanguinea (Published International Patent Application No. WO 95/18229), Ace- AMP 1 from Allium (Published International Patent Application No. WO 95/04754), osmotin from tobacco (Published International Patent Application WO 91/18984), glucose oxidase (Published International Patent Application WO 90/13470) and chitinase/glucanase mixture where both proteins are isolated from tobacco (Published European Patent Application No. EP 440304 ). We have now most unexpectedly found that certain combinations of antimicrobial proteins result in an enhanced improvement of antifungal activity against a range of fungi wherein the antifungal activity of the combination has a larger effect than the combination of the antifungal properties of the individual components. The enhancement of antifungal activity may be regarded as being a synergistic effect. For example we have found that the combination of an antifungal protein from Radish and a second antimicrobial protein selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens results in an enhanced improvement of antifungal activity against a range of fungi wherein the antifungal activity of the combination is larger than the combination of the antifungal properties of the individual components. Furthermore the spectrum of fungi against which the combination of the first and second antifungal agents is active may be broader than that observed with either antifungal agent used separately. SUMMARY OF THE INVENTION Accordingly in a first aspect the invention provides an antimicrobial composition comprising a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity. As used herein the term enhanced is used to denote an improvement in antimicrobial activity on a given organism which is greater than the additive antimicrobial effect of the individual antimicrobial agents acting independently.

We have found that the antimicrobial composition according to the invention is particularly effective against fungi which are food spoilage fungi such as for example members of Alternaria sp and Penicillium roquefortii.

As used herein the term antimicrobial denotes activity against fungi and/or bacteria.

It is particularly preferred that the composition of the invention is an antifungal composition i.e. where the first and second agents in the composition have antifungal activity and where the combination of the two agents leads to enhanced antifungal activity.

The fungi mentioned above are all important food spoilage organisms giving rise to contamination of foodstuffs which is of commercial significance and so the enhanced activity which we have unexpectedly observed may be of great benefit The antifungal protein from radish is preferably Rs-AFPl (SEQ ID 1), Rs- AFP2 (SEQ ID 2), Rs-AFP3 (SEQ ID 9) or Rs-AFP4 (SEQ ID 10) or a homologue, variant or active derivative thereof, is more preferably Rs-AFPl or Rs-AFP2 or a homologue, variant or active derivative thereof and is most preferably Rs-AFPl or a homologue, variant or active derivative thereof; the antimicrobial protein from Mirabilis is preferably Mj-AMPl (SEQ ID 3) or MJ-AMP2 (SEQ ID 4) or a homologue, variant or active derivative thereof and is most preferably MJ-AMP2; the antimicrobial protein from Impatiens is preferably lb- AMP 1 (SEQ ID 5), Ib- AMP2 (SEQ ID 6), Ib-AMP3 (SEQ ID 7), or Ib-AMP4 (SEQ ID 8) and is most preferably Ib-AMPl. The sequences of Rs-AFPl, Rs-AFP2 are provided in

Published International Patent Application WO 93/05153, the sequence of Rs-AFP3 and Rs-AFP4 in Terras et al (Plant Cell 7 573 1995)), the sequence of M-AMP1 and MJ-AMP2 are provided in Published International Patent Application WO 92/15691 and the sequence of Ib-AMPl, Ib-AMP2, Ib-AMP3 and Ib-AMP4 are provided in Published International Patent Application WO 95/24486 the teachings of which are incorporated herein by reference. The expression "homologues" as used herein refers to any peptide which has some amino acids in common with the given sequence. Suitably at least 60% of the amino acids will be similar, more suitably at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% of amino acids will be similar to the corresponding amino acid in the given sequence.

As used herein the term "similar" is used to denote sequences which when aligned have similar (identical or conservatively replaced) amino acids in like positions or regions, where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein. For example, two amino acid sequences with at least 85% similarity to each other have at least 85% similar (identical or conservatively replaced) amino acid residues in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected. The degree of similarity may be determined using methods well known in the art (see, for example, Wilbur, W . and Lipman, D.J. "Rapid Similarity Searches of Nucleic Acid and Protein Data Banks." Proceedings of the National Academy of Sciences USA 80, 726-730 (1983) and Myers E.and Miller W. "Optimal Alignments in Linear Space". Comput. Appl. Biosci. 4:11-17(1988)). One programme which may be used in determi-αing the degree of similarity is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1228, Selfpark Street, Madison, Wisconsin, 53715, USA as part of the Lasergene system.

Amino acids which differ from the basic sequence may be conservatively or non-conservatively substituted. A conservative substitution is to be understood to mean that the amino acid is replaced with an amino acid with broadly similar chemical properties. In particular conservative substitutions may be made between amino acids with the following groups: (i) Alanine, Serine, Glycine and Threonine; (ϋ) Glutamic acid and Aspartic acid; (ϋi) Arginine and Lysine;

(iv) Asparagine and Glutamine;

(v) Isoleucine, Leucine, Valine and Methionine; (vi) Phenylalanine, Tyrosine and Tryptophan.

In general, more conservative than non-conservative substitutions will be possible without destroying the antimicrobial properties of the compounds. Suitable homologues may be determined by testing antimicrobial properties of the peptide using routine methods, for example as illustrated hereinafter.

The term "variant" as used herein includes experimentally generated variants or members of a family of related naturally-occurring peptides as may be identified by molecular genetic techniques. Such techniques are described for example in US Patent No. 5,605,793, US Patent No. 5,811,238 and US Patent No 5,830,721, the content of which is incorporated herein by reference. In essence this technique involves expression of the parental gene in a microbial expression system such as Escherichia coli. The particular system selected must be validated and calibrated to ensure that biologically active peptides are expressed, which may be readily achieved using an in vivo bioassay. The gene, or preferably a collection of related genes from different species, may be subject to mutagenic polymerase chain reaction (PCR) as is known in the art. Fragmentation of the products and subsequent repair using PCR leads to a series of chimeric genes reconstructed from parental variants. These chimeras are then expressed in the microbial system which can be screened in the usual way to determine active mutants, which may then be isolated and sequeπced. Reiteration of this molecular evolution DNA shuffling cycle may lead to progressive enhancement of the desired gene properties. The advantage of a technique of this nature is that it allows a wide range of different mutations, including multi-mutation block exchanges, to be produced and screened. Other variants may be identified or defined using bioinfoπnatics systems. An example of such a system is the FASTA method of W.R. Pearson and D.J. Lipman PNAS (1988) 85:2444-2488. This method provides a rapid and easy method for comparing protein sequences and detecting levels of similarity and is a standard tool, used by molecular biologists. Such similar sequences may be obtained from natural sources, through molecular evolution or by synthetic methods and comparisons made using this method to arrive at "opt scores" which are indicative of the level of similarity between the proteins. Particular variants of the invention will comprise antimicrobial proteins with an amino acid sequence with a FASTA opt score (as defined in accordance with FASTA version 3.0t82 November 1, 1997) against any one of the sequences of the proteins in the antimicrobial composition of the invention described herein as follows. Variants of the invention will comprise antimicrobial proteins with an amino acid sequence with a FASTA opt score (as defined in accordance with FASTA version 3.0t82 November 1, 1997) corresponding to at least 60% of the amino acids being similar, more suitably at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% of amino acids being similar to the corresponding amino acid in the given sequence.

The term "derivative" relates to antimicrobial proteins which have been modified for example by using known chemical or biological methods.

Peptide derivatives and variants of antifungal proteins derived from radish, are described in Published International Patent Application Nos WO 97/21814 and WO 97/21815 and the invention extends to their use in the antimicrobial compositions of the invention.

Preferred combinations according to the invention are Rs-AFPl and Ib- AMPl and Rs-AFPl and MJ-AMP2.

The antimicrobial composition of the invention may contain the first and second antimicrobial agents in relative amounts ranging from 0.1 : 10 to 10 : 0.1, preferably from 0.5 : 2 to 2 : 0.5 most preferably 1:1.

The antimicrobial proteins may be extracted and purified from plant material, manufactured from its known amino acid sequence by chemical synthesis using a standard peptide synthesiser or produced within a suitable organism (for example, a microorganism or a plant) by expression of recombinant DNA.

The DNA sequence may be predicted from the known amino acid sequence and DNA encoding the protein may be manufactured using a standard nucleic acid synthesiser. Alternatively the DNA sequence may be isolated from plant derived DNA libraries. The DNA sequences encoding the first and the second antimicrobial agent may be incorporated into a DNA construct or vector in combination with suitable regulatory regions such as promoter regions and transcription teπninator regions. The DNA encoding the first and second antimicrobial agents may be placed under the control of the same or different regulatory regions. The DNA sequence encoding the first and second antimicrobial agents may be placed under the control of a constitutive or inducible promoter which may be stimulated by for example environmental conditions, presence of a pathogen and/or presence of a chemical. It is possible that the first and second antimicrobial agents are placed under the control of the same regulatory regions. Such a DNA construct may be cloned or transformed into a biological system which allows the expression of the encoded first and second antimicrobial agents or an active part thereof. Suitable biological systems include microorganisms e.g bacteria such as E.coli, and endophytes such as Clavibacter xyli subsp cynodontis; yeast; viruses; cultured cells such as mammalian cells and insect cells; and plants. In some cases the first and second antimicrobial agent may be extracted and isolated for use.

The invention also extends to constructs comprising DNA sequences which hybridise to the DNA sequences encoding the antimicrobial proteins, homologues, variants or derivatives thereof. Preferably, such hybridisation occurs at, or between, low and high stringency conditions. In general terms, low stringency conditions can be defined as 3 x SCC at about ambient temperature to about 65°C, and high stringency conditions as 0.1 x SSC at about 65°C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3 x SSC is three time as strong as lx SSC and so on.

In a further aspect the invention therefore provides a DNA construct comprising a first DNA sequence encoding an antifungal protein from Radish or a homologue, variant or active derivative thereof and a second DNA sequence encoding an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof wherein expression of said first and second DNA sequences may be under the control of the same or different promoter region.

In a preferred embodiment of this aspect the invention provides a DNA construct comprising a first DNA sequence encoding encoding Rs-AFPl, variant or active derivative thereof and a second DNA sequence MJ-AMP2 or Ib-AMPl or a homologue, variant or active derivative thereof wherein expression of said first and second DNA sequences may be under the control of the same or different promoter region.

In a yet further aspect the invention provides a biological system transformed with the DNA constructs of the invention. Plant cells may be transformed with recombinant DNA constructs according to the invention using a variety of methods such as for example, Agrobacterium Ti plasmids, microinjection, microprojectile gun. The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocotyledonous and dicotyledonous plants may be obtained in this way.

The invention further provides a plant cell stably transformed with a DNA construct of the invention as described herein.

In a still further aspect the invention provides a plant having improved resistance to a microbial pathogen and containing recombinant DNA construct which expresses a first antimicrobial agent selected from the group consisting of an antimicrobial protein from Radish or a homologue, variant or active derivative thereof in combination with a second antimicrobial agent selected from the group consisting of an antimicrobial protein from the group Mirabilis or Impatiens or a homologue, variant or active derivative thereof. In a preferred embodiment of this aspect the invention provides a plant having improved resistance to a fungal pathogen and containing a recombinant DNA construct which expresses Rs-AFPl a homologue, variant or active derivative thereof in combination with a MJ-AMP2 or Ib-AMPl or a homologue, variant or active derivative thereof. A plant having improved resistance to a microbial pathogen may be used as a parent in standard plant breeding crosses to develop hybrids and lines having improved microbial resistance.

In this context a plant having improved resistance is defined as having enhanced tolerance to a microbial pathogen when compared to wild type plant, to a plant expressing the first or second antimicrobial agent in isolation and to the predicted tolerance obtained by an additive effect of the first and second antimicrobial agent. Where the first and second antimicrobial agents are expressed within a transgenic plant or its progeny, the microbe is exposed to the antimicrobial agents at the site of pathogen attack on the plant. In particular, by use of appropriate gene regulatory sequences, the protein may be produced in vivo when and where it will be most effective.

Exposure of a plant pathogen to the antimicrobial composition and combination of antimicrobial proteins according to the present invention may be achieved in various ways, for example; a) a composition comprising the first and or second antimicrobial agents in isolated form may be apphed to plant parts or the surrounding soil using standard agricultural techniques (such as spraying); the first and/or second antimicrobial agents may have been extracted from plant tissue or chemically synthesised or extracted from microorganisms genetically modified to express the protein; b) a composition comprising a microorganism genetically modified to express the first and/or second antimicrobial agents may be applied to a plant or the soil in which a plant grows; c) an endophyte genetically modified to express the first and or second antimicrobial agents may be introduced into the plant tissue for example, via seed treatment d) recombinant DNA encoding the first and second antimicrobial agents may be introduced into the plant genome so that the protein is expressed within the plant

(the DNA may be cDNA, genomic DNA or DNA manufactured using a standard nucleic acid synthesiser).

In a further aspect the invention provides a method of treating microbial diseases comprising exposure of the microbe to an antimicrobial composition according to the invention.

In a further aspect the invention provides a method of treating microbial diseases comprising exposure of the microbe to a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an anti-microbial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity.

A method of treating microbial diseases according to the above aspect of the invention wherein the first and second microbial agents are administered separately. A method of treating microbial diseases according to the above aspect of the invention wherein the first and second microbial agents are administered sequentially.

A method of treating microbial diseases according to the above aspect of the invention wherein the first and second microbial agents are administered simultaneously.

Examples of genetically modified plants which may be produced according to the invention include for example field crops, cereals, fruit and vegetables such as: canola, oil seed rape, sunflower, tobacco, sugar beet, cotton, soya, maize, wheat, barley, rice, sorghum, toamatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage and onion.

In a further aspect the invention provides a method of inhibiting microbial growth in foodstuffs comprising applying to a foodstuff or to the locus of a foodstuff a microbiocidally effective amount of an antimicrobial composition according to any of the above aspects of the invention. In a further aspect the invention provides a method of inhibiting microbial growth in foodstuffs comprising applying to a foodstuff or to the locus of a foodstuff a microbiocidally effective amount of an first antimicrobial agent and a second anti-microbial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity.

A method of inhibiting microbial growth according to the above aspect of the invention wherein the first and second microbial agents are administered separately. A method of inhibiting microbial growth according to the above aspect of the invention wherein the first and second microbial agents are administered sequentially.

A method of inhibiting microbial growth according to the above aspect of the invention wherein the first and second microbial agents are administered simultaneously.

In a further aspect the invention provides a method of inhibiting microbial growth in foodstuffs comprising exposing an environment in which said growth is to be inhibited to a microbiocidally effective amount of an antimicrobial composition according to any of the above aspects of the invention.

In a further aspect the invention provides a method of inhibiting microbial growth in foodstuffs comprising exposing an environment in which said growth is to be inhibited to a microbiocidally effective amount of a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity. A method of inhibiting microbial growth according to the above aspect of the invention wherein the first and second microbial agents are administered separately. A method of inhibiting microbial growth according to the above aspect of the invention wherein the first and second microbial agents are administered sequentially. A method of inhibiting microbial growth according to the above aspect of the invention wherein the first and second microbial agents are administered simultaneously.

The method and compositions of the invention are particularly suitable for use with a wide range of foods and beverages including fruits and jams and dairy products such as yoghurts, cheeses, cream desserts, milk shakes. The antimicrobial compositions and agents according to the invention are in a form suitable for use with foodstuffs for human and animal consumption. Other components of the composition may be chosen according to the nature of the foodstuff and to its method of consumption and this will be readily apparent to a man skilled in the art.

The environment in which it is desired to inhibit microbial growth may be exposed to the composition and the antimicrobial agents in a variety of ways which will most usually be determined by the nature of the foodstuff to be protected. The foodstuff and the agents and/or composition of the invention may, for example, be mixed together during the manufacturing process. Alternatively or additionally, the container in which the foodstuff is packaged may be sprayed with the agent and/or composition before the foodstuff is added and/or sprayed with the agent and/or composition after packing and/or filling. The composition and the antimicrobial agent of the invention may also be used in conjunction with coating products e.g. cheese wax.

The antimicrobial agents may be synthesised chemically or produced using recombinant DNA technology using methods well known in the art. Where the antimicrobial agent is produced using recombinant techniques in a microorganism host the host used for the transformation and production of the desired agent will be a GRAS organism. GRAS organisms being those organisms such as, yeast, Pichia, lactic acid bacteria and certain E.coli strains which are regarded by the Regulatory Authorities as being 'safe'. Proteins thus produced using GRAS organisms may then be purified and added to foodstuffs. The antimicrobial agents may also be used in the form of extracts such as seed or plant extracts where the extract contains the antimicrobial agent in partially purified or enriched form. This is described more fully in the examples herein.

RRTFF nRSPRIPTION OF THE DRAWINGS

The invention is further illustrated in the following non-liiniting examples and figures in which

Figure 1 shows : Isobologram of the minimum inhibitory concentrations of the proteins Ib-AMPl and Rs-AFPl tested against the food spoilage fungus Alternaria sp. The test was carried out in 1/2PDB and assessment of fungal growth inhibition was carried out after 48 hours. Figure 2 shows : Isobologram of the minimum inhibitory concentrations of the proteins Ib-AMPl and Rs-AFPl tested against the food spoilage fungus Penicilliwn roquefortii. The test was carried out in 1/2PDB and assessment of fungal growth inhibition was carried out after 9 days. Figure 3 shows : Isobologram of the minimum inhibitory concentrations of the proteins MJ-AMP2 and Rs-AFPl tested against the food spoilage fungus Alternaria sp. The test was carried out in 1/2PDB and assessment of fungal growth inhibition was carried out after 48 hours.

SEQUENCE LISTING The sequence listing provides the amino acid sequence for Rs-AFPl, Rs-AFP2,

MjAMPl, MJAMP2, IbAMPl, IbAMP2, IbAMP3 and IbAMP4, Rs-AFP3 and Rs-

AFP4.

DETAILED DESCRIPTION OF THE INVENTION

Examples Materials and methods

Materials

Seeds of Raphanus sativus, Mirabilis jalap a and Impatiens balsamina were purchased from Chiltern Seeds (Cumbria, UK). Non-sterile flat-bottom 96-well microplates were used throughout the experiments. Purification of the antifungal proteins

Purification of the antifungal proteins Mj-AMPl, Rs-AFPl and Ib-AMPl was performed as described by Cammue et al. (1992, /. Biol. Chem., 267, 4, 2228-

2233), Terras etal. (1992, /. Biol. Chem., 267, 22, 15301-15309) and by Broekart et al. (in Published International Patent No. WO95/24486), respectively.

Antifungal Activity Assays

The antifungal activity assays were carried out in flat-bottom 96-well microplates. 20μl of test solution containing appropriate amounts of the antifungal protein(s) in sterile distilled water were mixed with 80μl of a suspension of fungal spores (lx 10⁴ spores/ml) in Medium A (half-strength potato dextrose broth or 1/2

PDB). Control microcultures contained 20μl of sterile distilled water and 80μl of the fungal spore suspension. Growth of fungi, collection and harvest of fungal spores are done as previously described (Broekaert et al., 1990, FEMS Microbiol. Lett., 69, 55- 60).

The following fungal strains are used: Fusarium culmorum, Penicillium chrysogenum, Penicillium roquefortii, Penicillium commune, Penicillium nalgiovense, Mucor plumbeus, Scopulariopsis brevicaulis, Aspergillus versicolor, Alternaria sp., Cladosporium sp. and Trichoderma harzianum.

Unless otherwise stated the microplates were incubated at 24°C for 48 hours. Percentages of fungal growth were estimated by microscopic examination of the microplates after the incubation period. Control microcultures were used as a reference in order to estimate fungal growth inhibition. The minimum concentration of protein required to give strong fungal growth inhibition, i.e. more than 90% fungal growth inhibition, was taken as the minimum inhibitory concentration (MIC) of the protein being tested.

Assessment of antibacterial activity

The purified antifungal proteins to be studied are initially in a freeze-dried form. The amount of each protein is previously determined by weighing out the freeze-dried material. Each of the freeze-dried proteins is suspended in sterile deionised water to provide a concentration of 2500 μg/ml . These stock suspensions are maintained at -20 °C and diluted to a series of halving concentrations in sterile deionised water prior to use.

Gram-negative pathogenic and spoilage bacteria and Gram-positive pathogenic bacteria are used to study the activity of the proteins. The following bacterial strains are used: Listeria monocytogenes NCTC 11994, Staphylococcus aureus NCTC 4136, Bacillus cereus NCTC 11145, Salmonella typhimurium NCTC 10413, Escherichia coli 0157 NCTC 12900, Yersinia enterocolitica NCTC 11175, Pseudomonas euginosa NCTC 12934, Shewanella putrefaciens NCTC 10735 and Hafhia alvei NCTC 6578.

The antibacterial activity assays are performed using an absorbance method based on previously published work (Cammue et al., 1992 J. Biol. Chem. 267, 2228- 33). Preliminary experiments determined the suitability of all media for these experiments. Yeast extract is added to the media used for the Listeria monocytogenes assays to improve the growth of the organism.

The organisms are grown overnight in tryptone water (TW) to provide cultures in the exponential phase of growth. For the Listeria monocytogenes culture 0.5% yeast extract is added to the tryptone water (TWΥE).

A soft agarose (SAM) is inoculated with bacteria to a cell density of ca 10⁵ cfu/g. For the Listeria monocytogenes assay a soft agarose medium containing 0.5 yeast extract (SAMYE) is used. All recipes are detailed herein. Aliquots (80-μl) of the prepared bacterial suspension are added to the diluted proteins (20-μl) in flat-bottom 96-well microplates. Concentrations ranging from 0 to 500 μg/ml of each of the proteins are obtained on the wells for each of the microorganisms. All assays are carried out in duplicate microtitre plates. The absorbance at 620 nm of each culture is measured with the aid of a microtitre plate reader after 30 min and 48 h incubation at 28 °C.

The mean readings of the two replicate sets of plates is determined and the percentage of growth inhibition is calculated using the following method: 100 times the ratio of the corrected absorbance at 620 nm of the control microculture minus the corrected absorbance of the test microculture over the corrected absorbance of the control microculture. The corrected absorbance values equal the absorbance at 620 nm of the culture measured after 48 h incubation minus the absorbance of the same culture after 30 min incubation. The IC₅₀ values of the proteins against the organisms are the concentrations required for 50% growth inhibition after 48 h incubation at 28°C. Results a) Antifungal potency of the antifungal proteins

The antifungal activity of the purified antifungal proteins Ib-AMPl, Mj- AMP2 and Rs-AFPl is assessed on different fungi using the assay described above. A summary of the π-inimum inhibitory concentrations (MIC) required to strongly inhibit growth of a series of fungal strains is shown in Table 1.

FUNGUS Ib-AMPl (ppm) MJ-AMP2 (ppm) Rs-AFPl (ppm)

Altemaria sp. 12.5 25 100 Cladosporium sp. 100 50 >200

Penicillium sp. 50 1.5 12.5

Table 1. Antifungal potency of the antifungal proteins Ib-AMPl, MJ-AMP2 and Rs- AFPl in medium 1/2 PDB.

b) Antifungal potency of the combination of antifungal proteins The combination of subinhibitory concentrations of the antifungal proteins enhances synergistically the activity of the mixture. The potentiation of the activities of the antifungal proteins by other antifungal proteins are able to provide strong inhibition of fungal growth in vitro with concentrations of both antifungal proteins that are much lower than those required if they are used individually. In a similar way, the application of combinations of other antifungal proteins allows a reduction in the amount of such proteins needed to strongly inhibit growth of food spoilage fungal strains and/or of plant or human pathogenic fungi.

In the experiments described, where antifungal activity is measured using dilution tests, the presence of a synergistic effect can be determined using isobolograms (Parish M.E. and Davidson P.M. (1993) Methods for evaluation. In: Antimicrobials in foods. Ed. By P.M. Davidson and A.L. Branen. Marcel Dekker, Inc. New York). Isobolograms are graphical representations of the minimum inhibitory concentrations (MIC) of two different antifungal agents required to strongly inhibit growth of a fungus. The concentrations of the two antifungal agents are arranged in each axis from lowest to highest concentration. If the concentrations that strongly inhibit growth fall on an approximately straight line that connects the individual MIC values on each axis the combined effect is additive. Deviation of the linearity to the left of the additive line will mean synergism. Isobolograms for some of the combinations are included in figures 1 to 3 shown below.

Claims

1. An antimicrobial composition comprising a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity.

2. A DNA construct comprising a first DNA sequence encoding an antifungal protein from Radish or a homologue, variant or active derivative thereof and a second DNA sequence encoding an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof wherein expression of said first and second DNA sequences may be under the control of the same or different promoter region.

3. A biological system transformed with a DNA construct according to claim 2.

4. A plant cell stably transformed with a DNA construct according to claim 2.

5. A plant having improved resistance to a microbial pathogen and containing recombinant DNA construct which expresses a first antimicrobial agent selected from the group consisting of an antimicrobial protein from Radish or a homologue, variant or active derivative thereof in combination with a second antimicrobial agent selected from the group consisting of an antimicrobial protein from the group Mirabilis or Impatiens or a homologue, variant or active derivative thereof.

A method of treating microbial diseases comprising exposure of the microbe to an antimicrobial composition according to claim 1.

A method of treating microbial diseases comprising exposure of the microbe to a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity.

A method of inhibiting microbial growth in foodstuffs comprising applying to a foodstuff or to the locus of a foodstuff a microbiocidally effective amount of an antimicrobial composition according to claim 1.

9. A method of inhibiting microbial growth in foodstuffs comprising applying to a foodstuff or to the locus of a foodstuff a microbiocidally effective amount of a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from

Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity.

10. A method of inhibiting microbial growth in foodstuffs comprising exposing an environment in which said growth is to be inhibited to a microbiocidally effective amount of an antimicrobial composition according to claim 1.

11. A method of inhibiting microbial growth in foodstuffs comprising exposing an environment in which said growth is to be inhibited to a microbiocidally effective amount of a first antimicrobial agent and a second antimicrobial agent in which said first antimicrobial agent is an antifungal protein from Radish or a homologue, variant or active derivative thereof and said second antimicrobial agent is selected from the group consisting of an antimicrobial protein from Mirabilis or Impatiens or a homologue, variant or active derivative thereof the relative amounts of the first and second agents being such as to produce an enhanced antimicrobial activity.

12. A method according to claim 7, 9 or 11 wherein the first and second microbial agents are administered separately.

13. A method according to claim 7, 9 or 11 wherein the first and second microbial agents are administered sequentially.

14. A method according to claim 7, 9 or 11 wherein the first and second microbial agents are administered simultaneously.