Fungicidal compositions

Abstract

A fungicidal composition comprising: (i) a Pantoea agglomerans bacterium, (ii) a bacterium comprising a genomic sequence comprising a first nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 1, a second nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 2, a third nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 3 and a fourth nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 4, or a bacterium comprising a genomic sequence which has at least 96% identity to the to the combined sequence of SEQ ID NOs: 1 to 4; and (iii) at least one yeast-like ascomycetous microorganism selected from the group consisting of Hyphopichia pseudoburtonii and Wickerhamomyces anomalus is provided.

Description

[0001] FUNGICIDAL COMPOSITIONS

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (“P3970PC00 Sequence Listing.xml”; Size: 11 ,642,000 bytes; and created on 25 March 2026) filed with this application is herein incorporated by reference in its entirety.

[0004] FIELD

[0005] This disclosure relates to the field of fungicidal compositions comprising two bacteria species and a yeast. In particular, it relates to fungicidal compositions for inhibiting the growth of pathogenic fungi on berry-producing plants and on post-harvest berries, such as grapes.

[0006] BACKGROUND

[0007] Pathogenic fungi are known to contribute to numerous plant diseases, including diseases of berryproducing plants such as grey rot, powdery mildew and downy mildew, causing widespread damage and crop losses globally, both during cultivation and post-harvest storage of berries.

[0008] Chemical (or synthetic) fungicides are available, but their use in pre- or post-harvest conditions is not considered sustainable because of the frequent appearance of resistant strains and the adverse effects of the chemicals in the fungicides on the environment and human health. There is a growing demand from consumers for agricultural products that are grown or produced without synthetic product application. This shift towards organic food production and away from the use of chemical or synthetic fungicides has highlighted the need for biological methods to inhibit pathogen growth.

[0009] Various strategies have been developed for the biological control of plant pathogens, including the use of ingredients such as plant extracts, minerals, and beneficial microbes. Common options include copper soap, sulphur, neem oil, baking soda mixtures, and potassium bicarbonate, typically applied as foliar sprays. However, not all of these are suitable for edible crops or for crops that will be used in a fermentation process, such as grapes. Organic fungicides comprising single-species microorganisms are commercially available for use in vineyards; however, not all of them work effectively and consistently under varying field conditions.In particular, many organic fungicides have a lower effectiveness against pathogens compared to chemical fungicides and have a particularly poor performance where there is a change in environmental conditions. To overcome these problems, many so-called environmentally friendly fungicides combine organic fungicides with chemical or synthetic fungicides. This alternative control method nevertheless still faces the associated disadvantages of chemical control methods and does not address the legislative and community shift towards environmentally safe solutions which do not pose adverse risks to human and animal health.

[0010] Therefore, there is a need for biological plant pathogen control methods which are well adapted to different and changing environmental conditions without the associated risks of chemical pathogen control methods, and which show enhanced performance over known microbial antagonist biocontrol methods.

[0011] The preceding discussion of the background is intended only to facilitate an understanding of the present disclosure. It should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was part of the common general knowledge in the art as at the priority date of the application.

[0012] SUMMARY

[0013] In accordance with an aspect of the disclosure there is provided a fungicidal composition, the composition comprising:

[0014] (i) a Pantoea agglomerans bacterium;

[0015] (ii) a bacterium having a genomic sequence comprising a first nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 1, a second nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 2, a third nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 3 and a fourth nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 4, or a bacterium which has at least 96% identity to the combined sequence of SEQ ID NOs: 1 to 4; and (iii) a yeast-like ascomycetous microorganism selected from the group consisting of Hyphopichia pseudoburtonii and Wickerhamomyces anomalus.

[0016] The yeast-like ascomycetous microorganism may be Hyphopichia pseudoburtonii.

[0017] The yeast-like ascomycetous microorganism may be Wickerhamomyces anomalus.The bacterium of (ii) may have a genomic sequence comprising a first nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 1 , a second nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2, a third nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 and a fourth nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 4, or may have a genomic sequence comprising at least 96% identity to the entire length of SEQ ID NOs: 1 to 4 when combined. The bacterium of (ii) may have a genomic sequence comprising a first nucleic acid sequence of SEQ ID NO: 1 , a second nucleic acid sequence of SEQ ID NO: 2, a third nucleic acid sequence of SEQ ID NO: 3 and a fourth nucleic acid sequence of SEQ ID NO: 4.

[0018] The bacterium of (ii) may have a genomic sequence comprising a first nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 5, a second nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and a third nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 7, or may have a genomic sequence comprising at least 96% identity to the entire length of SEQ ID NOs: 5 to 7 when combined. The bacterium of (ii) may have a genomic sequence comprising a first nucleic acid sequence of SEQ ID NO: 5, a second nucleic acid sequence of SEQ ID NO: 6, and a third nucleic acid of SEQ ID NO: 7.

[0019] The Pantoea agglomerans may be the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4020.

[0020] The Hyphopichia pseudoburtonii may be the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number Y963.

[0021] The Wickerhamomyces anomalus may be the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number Y934.

[0022] The bacterium of (ii) may be a Bacillus species.

[0023] The bacterium of (ii) may be the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4001 .

[0024] The bacterium of (ii) may be the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4022.

[0025] The fungicidal composition may comprise:

[0026] (i) Pantoea agglomerans strain B4020;

[0027] (ii) Bacillus strain B4022; and(iii) Wickerhamomyces anomalus strain Y934.

[0028] The fungicidal composition may comprise:

[0029] (i) Pantoea agglomerans strain B4020;

[0030] (ii) Bacillus strain B4001 ; and

[0031] (iii) Hyphopichia pseudoburtonii strain Y963.

[0032] The fungicidal composition may be capable of inhibiting the growth of Botrytis cinerea, Plasmopara viticola and Erysiphe necator, and optionally also Alternaria alternata and Aspergillus niger.

[0033] The bacteria and yeast of (i), (ii) and (iii) may be present in the composition in approximately a 1 :1 :1 ratio.

[0034] The fungicidal composition may not include any chemical or synthetic fungicidal agents.

[0035] In accordance with a second aspect of the disclosure there is provided a use of a fungicidal composition as described above for treating, preventing or reducing bunch rot, grey rot, noble rot, downy mildew, powdery mildew, black mold or gray mold on plants and fruit.

[0036] The plants may be grapevines.

[0037] The fruit may be berry fruits, such as grapes, strawberries, raspberries, blueberries or blackberries.

[0038] In accordance with another aspect of the disclosure there is provided a method of treating, preventing or reducing bunch rot, grey rot, noble rot, downy mildew, powdery mildew, black mold or gray mold on plants and fruit, the method comprising applying a fungicidal composition as described above to the plants or fruit.

[0039] BRIEF DESCRIPTION OF THE FIGURES

[0040] Figure 1 is a schematic graph illustrating the mycelial growth inhibition ability of yeast isolates against three Botrytis cinerea strains (B05.10; IWBT-FF1 ; and IWBT-FF2);

[0041] Figure 2 is a distribution scheme illustrating the antifungal activity of 25 selected bacterial strains against Botrytis cinerea strains B05.10, IWBT-FF1 and IWBT-FF2;Figure 3 is a schematic graph illustrating the mycelial growth inhibition ability of bacterial isolates against three Botrytis cinerea strains (B05.10; IWBT-FF1 ; and IWBT-FF2);

[0042] Figure 4 is a genome-based phylogenetic tree illustrating the genomic similarity of strain B4001 and strain B4022 to Bacillus strains;

[0043] Figure 5 is a heatmap showing Average Nucleotide Identity (ANI) values based on wholegenome comparisons between strain B4001 and strain B4022and their conspecific close relatives;

[0044] Figure 6 is a schematic heatmap comparing relative concentrations of volatile organic compounds produced by selected yeasts, namely Lodderomyces elongisporus, Wickerhamomyces anomalus and Hyphopichia pseudoburtonii, in the presence and absence of Botrytis cinerea;

[0045] Figure 7 is a schematic heatmap illustrating the spore germination inhibition activity of cell-free extracts collected at different time intervals from nine selected bacterial strains against three Botrytis cinerea strains, with a commercially available biocontrol product Bacillus amyloliquefaciens D747) used as a positive control;

[0046] Figure 8 is a schematic heatmap illustrating the mycelial growth inhibition activity of cell-free extracts collected at different time intervals from the selected nine bacterial strains against the three Botrytis cinerea strains, with Bacillus amyloliquefaciens D747 used as a positive control;

[0047] Figure 9 is a schematic graph illustrating cyclic lipopeptide profiles in cell-free extracts obtained from selected bacterial supernatants with higher, moderate and low to no antifungal activity. The lowercase letters (a-c) identify significant differences between similar lipopeptides produced by selected bacterial strains. The data was based on ANOVA analysis using Tukey at p<0.05.;

[0048] Figure 10 is a schematic graph illustrating in vitro anti-oomycete activity of selected yeast strains against Phytophthora cinnamomi Steu 6825 and Phytophthora capsici DCS 585 on Malt Extract Agar (MEA). The mycelia inhibition (%) was calculated by measuring mycelial growth towards bacteria or yeast cells after 8 and 11 days with respective to pathogens. The lowercase letters (a-e) identify significant differences between yeast or bacterial strains against their respective pathogens. The data was based on ANOVA analysis using Tukey at p<0.05;Figure 11 is a schematic graph illustrating in vitro anti-oomycete activity of selected bacterial strains against Phytophthora cinnamomi Steu 6825 and Phytophthora capsici DCS 585 on MEA;

[0049] Figure 12 is a schematic representation of a dual culture assay used for screening of antifungal activity in single and mixed cultures;

[0050] Figure 13 is a schematic graph of growth curves illustrating Hyphopichia pseudoburtonii (Hp) Y963 viability in monoculture (Mo) compared with co-cultures (Co) of the bacterial strains Pantoea agglomerans (Pa), bacterial strain B4001, bacterial strain B4022, and Bacillus wiedmannii (Bw) B4016;

[0051] Figure 14 is a schematic graph of growth curves illustrating Wickerhamomyces anomalus (Wa) Y934 viability in monoculture (Mo) compared with co-cultures (Co) of the bacterial strains Pantoea agglomerans (Pa), strain B4001, strain B4022, and Bacillus wiedmannii (Bw) B4016;

[0052] Figure 15 is a schematic graph of growth curves illustrating Hyphopichia burtonii (Hb) Y951 viability in monoculture (Mo) compared with co-cultures (Co) of the bacterial strains Pantoea agglomerans (Pa), strain B4001 , strain B4022, and Bacillus wiedmannii (Bw) B4016;

[0053] Figure 16 is a schematic graph of growth curves illustrating Wickerhamomyces anomalus (Wa) Y541 viability in monoculture (Mo) compared with co-cultures (Co) of the bacterial strains Pantoea agglomerans (Pa), strain B4001, strain B4022, and Bacillus wiedmannii Bw) B4016;

[0054] Figure 17 is a schematic graph of growth curves illustrating Wickerhamomyces anomalus (Wa) Y517 viability in monoculture (Mo) compared with co-cultures (Co) of the bacterial strains Pantoea agglomerans (Pa), strain B4001, strain B4022, and Bacillus wiedmannii Bw) B4016;

[0055] Figure 18 is a schematic graph of growth curves illustrating the viability of Pantoea agglomerans (Pa) B4020 in monoculture (Mo) and in co-culture (Co) with Hyphopichia pseudoburtonii (Hp) Y963, Hyphopichia burtonii (Hb) Y951, and Wickerhamomyces anomalus (Wa) Y934, Y541 and Y517;

[0056] Figure 19 is a schematic graph of growth curves illustrating the viability of the bacterial strain B4022 in monoculture (Mo) and in co-culture (Co) with Hyphopichia pseudoburtonii (Hp) Y963, Hyphopichia burtonii (Hb) Y951 , and Wickerhamomyces anomalus (Wa) Y934, Y541 and Y517;

[0057] Figure 20 is a schematic graph of growth curves illustrating the viability of the bacterial strain B4001 in monoculture (Mo) and in co-culture (Co) with Hyphopichia pseudoburtonii (Hp) Y963,Hyphopichia burtonii (Hb) Y951 , and Wickerhamomyces anomalus (Wa) Y934, Y541 and Y517;

[0058] Figure 21 is a schematic graph of growth curves illustrating the viability of Bacillus wiedmannii (Bw) B4016 in monoculture (Mo) and in co-culture (Co) with Hyphopichia pseudoburtonii (Hp) Y963, Hyphopichia burtonii (Hb) Y951, and Wickerhamomyces anomalus (Wa) Y934, Y541 and Y517;

[0059] Figure 22 is a schematic graph illustrating mycelial growth inhibition of bacterial strains against Botrytis cinerea B05.10 and the two oomycetes, Phytophthora cinnamomi and Phytophthora capsici;

[0060] Figure 23 is a schematic graph illustrating mycelial growth inhibition of yeast strains against Botrytis cinerea B05.10 and the two oomycetes, Phytophthora cinnamomi and Phytophthora capsici;

[0061] Figure 24 is a schematic graph illustrating yeast-bacterial mixed culture mycelial inhibition activity against Botrytis cinerea B05.10 and the two oomycetes, Phytophthora cinnamomi and Phytophthora capsici;

[0062] Figure 25 is a schematic graph illustrating mycelial inhibition activity of a further set of yeast-bacterial mixed cultures against Botrytis cinerea B05.10 and the two oomycetes, Phytophthora cinnamomi and Phytophthora capsici;

[0063] Figure 26 is a schematic graph illustrating mycelial inhibition activity of a further set of yeast-bacterial mixed cultures against Botrytis cinerea B05.10 and the two oomycetes, Phytophthora cinnamomi and Phytophthora capsici;

[0064] Figure 27 is a schematic graph illustrating mycelial inhibition activity of a further set of yeast-bacterial mixed cultures against Botrytis cinerea B05.10 and the two oomycetes, Phytophthora cinnamomi and Phytophthora capsici;

[0065] Figure 28A is a schematic graph illustrating growth dynamics of Wickerhamomyces anomalus strain (Y934) in a co-culture with various Bacillus species and Pantoea agglomerans, in which the yeast and bacterial growth in monocultures (indicated by asterisks) was compared to those in mixed cultures;

[0066] Figure 28B is a schematic graph illustrating growth dynamics of Wickerhamomyces anomalus strain (Y934) in a co-culture with various Bacillus species and Pantoea agglomerans, in which theyeast and bacterial growth in monocultures (indicated by asterisks) was compared to those in mixed cultures;

[0067] Figure 28C is a schematic graph illustrating growth dynamics of Wickerhamomyces anomalus strain (Y934) in a co-culture with various Bacillus species and Pantoea agglomerans, in which the yeast and bacterial growth in monocultures (indicated by asterisks) was compared to those in mixed cultures;

[0068] Figure 28D is a schematic graph illustrating growth dynamics of Wickerhamomyces anomalus strain (Y541) in a co-culture with various Bacillus species and Pantoea agglomerans, in which the yeast and bacterial growth in monocultures (indicated by asterisks) was compared to those in mixed cultures;

[0069] Figure 28E is a schematic graph illustrating growth dynamics of Wickerhamomyces anomalus strain (Y541) in a co-culture with various Bacillus species and Pantoea agglomerans, in which the yeast and bacterial growth in monocultures (indicated by asterisks) was compared to those in mixed cultures; and

[0070] Figure 28F is a schematic graph illustrating growth dynamics of Wickerhamomyces anomalus strain (Y934) in a co-culture with various Bacillus species and Pantoea agglomerans, in which the yeast and bacterial growth in monocultures (indicated by asterisks) were compared to those in mixed cultures;

[0071] Figure 29A is a visual representation of the effect of selected multispecies consortia on the growth of Erysiphe necator(the causative agent of powdery mildew) and Plasmopara viticola (the causative agent of downy mildew) using leaf-disc assays;

[0072] Figure 29B is a visual representation of the effect of selected multispecies consortia on the growth of Erysiphe necator(the causative agent of powdery mildew) and Plasmopara viticola (the causative agent of downy mildew) using leaf-disc assays;

[0073] Figure 29C is a visual representation of the effect of selected multispecies consortia on the growth of Botrytis cinerea (the causative agent of grey rot) using grape bioassays;

[0074] Figure 30 is a photograph of powdery mildew disease load on grapevine leaves to illustrate the ranking used to score disease level; and

[0075] Figure 31 is a schematic stacked bar graph showing the effect of the consortia on thepowdery mildew disease load on potted grapevine leaves at harvest.

[0076] Figure 32 is a schematic graph illustrating as fermentation curves the fermentation kinetics of Saccharomyces cerevisiae, Torulaspora delbrueckii, Lachancea thermotolerans, Consortium 1 and Consortium 2 in a synthetic grape juice medium. The following abbreviations were used: Sc, S. cerevisiae; Td, T. delbrueckii; Lc, L. thermotolerans; CO1 , Consortium 1 ; CO2, Consortium 2. The data points represent the average of three replicates ± standard deviation;

[0077] Figure 33A is a schematic graph illustrating the fermentation kinetics and population dynamics in a synthetic grape juice medium co-inoculated with a combination of Saccharomyces cerevisiae and Consortium 1. The inoculation conditions are: Sc-CO1 (A), Sc-CO2 (B), Sc-CO1-Td-Lc (C), Sc-CO2-Td-Lc (D), CO1-Td-Lc (E), CO2-Td-Lc (F). The following abbreviations were used: Sc, S. cerevisiae; Wa, W. anomalus; Hp, H. pseudoburtonii; Td, T. delbrueckii; Lc, L. thermotolerans; Bn, strain B4001 (CO2) or strain B4022 (CO1); Pa, P. agglomerans; CO1, Consortium 1; CO2, Consortium 2. The population is expressed as colony forming units per mL (CFU / mL). The data points represent the average of three replicates ± standard deviation.;

[0078] Figure 33B is a schematic graph illustrating the fermentation kinetics and population dynamics in a synthetic grape juice medium co-inoculated with a combination of Saccharomyces cerevisiae and Consortium 2. Abbreviations, population and data points are as for Figure 33A;

[0079] Figure 33C is a schematic graph illustrating the fermentation kinetics and population dynamics in a synthetic grape juice medium co-inoculated with a combination of Saccharomyces cerevisiae, Consortium 1 , Torulaspora delbrueckii, and Lachancea thermotolerans. Abbreviations, population and data points are as for Figure 33A;

[0080] Figure 33D is a schematic graph illustrating the fermentation kinetics and population dynamics in a synthetic grape juice medium co-inoculated with a combination of Saccharomyces cerevisiae, Consortium 2, Torulaspora delbrueckii, and Lachancea thermotolerans. Abbreviations, population and data points are as for Figure 33A;

[0081] Figure 33E is a schematic graph illustrating the fermentation kinetics and population dynamics in a synthetic grape juice medium co-inoculated with a combination of Consortium 1 , Torulaspora delbrueckii, and Lachancea thermotolerans. Abbreviations, population and data points are as for Figure 33A;

[0082] Figure 33F is a schematic graph illustrating the fermentation kinetics and population dynamics in a synthetic grape juice medium co-inoculated with a combination of Consortium 2, Torulasporadelbrueckii, and Lachancea thermotolerans. Abbreviations, population and data points are as for Figure 33A;

[0083] Figure 34 is a schematic graph illustrating the relative abundance of yeast species during alcoholic fermentation in cv. Merlot. The x-axis indicates the fermentation period, starting from day zero at the beginning of fermentation and extending to day ten at the end of fermentation; and

[0084] Figure 35 is a schematic graph illustrating the relative abundance of bacterial species during alcoholic fermentation in cv. Merlot. The x-axis represents the fermentation period, starting from day zero at the beginning of fermentation and extending to day four, after which the bacterial species became undetectable.

[0085] DETAILED DESCRIPTION

[0086] Multi-species microbial compositions having fungicidal activity are described herein. The fungicidal compositions include microorganisms from both the Fungi and Monera kingdoms, and more particularly include:

[0087] (i) a bacterium belonging to the species Pantoea agglomerans,

[0088] (ii) a fungus belonging to the species Hyphopichia pseudoburtonii or Wickerhamomyces anomalus (both yeast-like ascomycetous microorganisms), and

[0089] (iii) a bacterium having a genomic sequence including a first nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 1 , a second nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 2, a third nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 3 and a fourth nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 4, or a bacterium which has at least 96% identity to the entire length of SEQ ID NOs: 1 to 4 when combined, typically in the order of SEQ ID NOs: 1 to 4. Bacteria with these sequences in their genome are members of the Bacillus genus and were initially thought to belong to Bacillus nakamurai. However, genomic analysis of these bacteria (described below) suggests that they are more likely a distinct, as yet unclassified species.

[0090] As used herein, a “fungicidal composition” is a composition that destroys fungi (such as yeasts and molds) or inhibits their growth, in particular pathogenic fungi which cause disease on plants and fruit. A fungicidal composition can be natural or chemical (synthetic), or a combination thereof depending on its composition.

[0091] SEQ ID NOs: 1 to 10 are contigs, which are long, continuous DNA sequences reconstructed byaligning and merging shorter, overlapping DNA fragments called reads which are generated during sequencing. In this specification, SEQ ID NOs: 1 to 4 form part of the genome of Bacillus strain B4001 ; SEQ ID NOs: 5 to 7 form part of the genome of Bacillus strain B4022; and SEQ ID NOs: 1 to 4 form part of the genome of Bacillus strain B4023. The total length of the sequences of SEQ ID NOs: 1 to 4 represents at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total genomic sequence of strain B4001. The total length of the sequences of SEQ ID NOs: 5 to 7 represents at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total genomic sequence of strain B4022. The total length of the sequences of SEQ ID NOs: 8 to 10 represents at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total genomic sequence of strain B4023.

[0092] "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website e.g., at blast. ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_L OC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1 ; and (vi) conditional compositional score matrix adjustment.Percentage identity can be obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0093] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence, and in particular when the sequences of the contigs of SEQ ID Nos: 1 to 4 are combined, the contigs of SEQ ID Nos: 5 to 7 are combined, or the contigs of SEQ ID Nos: 8 to 10 are combined.

[0094] In one embodiment, the bacterium (iii) can include a first, second, third and fourth nucleic acid sequence with at least 96.5% identity, at least 97.0% identity, at least 97.5% identity, at least 98.0% identity, at least 98.5% identity, at least 99.0% identity, at least 99.1% identity, at least 99.2% identity, at least 99.3% identity, at least 99.4% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity or 100% identity to SEQ ID NOs: 1, 2, 3 and 4, respectively. The percentage identity may be calculated over all 4 contigs when read together, or separately for each individual contig. The bacterium having the genomic sequence comprising the contigs of SEQ ID NOs: 1 to 4 has been deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4001.

[0095] In another embodiment, the bacterium (iii) can include first, second and third nucleic acid sequences with at least 96.5% identity, at least 97.0% identity, at least 97.5% identity, at least 98.0% identity, at least 98.5% identity, at least 99.0% identity, at least 99.1% identity, at least 99.2% identity, at least 99.3% identity, at least 99.4% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity or 100% identity to SEQ ID NOs: 5, 6 or 7. The percentage identity may be calculated over all 3 contigs when read together, or separately for each individual contig. The bacterium having the genomic sequence comprising the contigs of SEQ ID NOs: 5 to 7 has been deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4022.

[0096] In another embodiment, the bacterium (iii) can have a first, second, and third nucleic acidsequence each having at least 96.5% identity, at least 97.0% identity, at least 97.5% identity, at least 98.0% identity, at least 98.5% identity, at least 99.0% identity, at least 99.1% identity, at least 99.2% identity, at least 99.3% identity, at least 99.4% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity or 100% identity to SEQ ID NOs: 8, 9 and 10, respectively. The percentage identity may be calculated over all 4 contigs when read together, or separately for each individual contig. The bacterium having the genomic sequence comprising the contigs of SEQ ID NOs: 8 to 10 has been deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4023.

[0097] In one embodiment, the composition includes the Pantoea agglomerans strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4020. The species of Pantoea agglomerans has been given the NCBI taxonomy ID no.

[0098] 549, and other examples of Pantoea agglomerans strains which can be used in the compositions described herein can be found on the NCBI database (https : / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=549).

[0099] In one embodiment, the composition includes the Wickerhamomyces anomalus strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number Y934. The species of Wickerhamomyces anomalus has been given the NCBI taxonomy ID no. 4927, and other examples of Wickerhamomyces anomalus strains which can be used in the compositions described herein can be found on the NCBI database (https : / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=4927).

[0100] In one embodiment, the composition includes the Hyphopichia pseudoburtonii strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number Y963. The species of Hyphopichia pseudoburtonii has been given the NCBI taxonomy ID no. 717741 , and other examples of Hyphopichia pseudoburtonii strains which can be used in the compositions described herein can be found on the NCBI database (https : / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=717741).

[0101] In one embodiment, designated herein as “Consortium 1”, the composition includes:

[0102] (i) Pantoea agglomerans strain B4020;

[0103] (ii) Wickerhamomyces anomalus strain Y934; and

[0104] (iii) Bacillus strain B4022.

[0105] In another embodiment, designated herein as “Consortium 2”, the composition includes:

[0106] (i) Pantoea agglomerans strain B4020;(ii) Hyphopichia pseudoburtonii strain Y963; and

[0107] (iii) Bacillus strain B4001 .

[0108] The microorganism species may be present in the compositions described herein in approximately equal proportions, that is, the relative ratios of the three species may be approximately 1 :1 :1 based on cell counts or colony forming units (CFU). For example, each of the three species may be inoculated at approximately 106cells / mL or 106CFU / mL. It will be appreciated, however, that other proportions and relative ratios of the component species may be used depending upon requirements, applications, plant or fruit type, target pathogens and conditions in the field.

[0109] In some embodiments, the microorganisms (cells, spores, mycelia or cultures) are frozen or freeze-dried (lyophilised) (usually in the presence of a cryoprotectant such as glycerol, skim milk, lactose or sucrose) or are dried dessicated.

[0110] In one embodiment, the fungicidal compositions described herein do not include any chemical or synthetic fungicidal agents and are suitable for organic farming uses or organic production processes.

[0111] In another embodiment, the fungicidal compositions described herein can be used in conjunction with existing chemical fungicide spray programmes, thereby reducing the amount of chemical fungicide used.

[0112] The composition can be formulated in various types of formulations, such as solutions, wettable powders, soluble powders, tablets and water-soluble or dispersible granules. The composition can also be formulated as a concentrated stock (which is diluted in an aqueous solution prior to conventional spray application) or as a ready to use product.

[0113] A surfactant can be used as a wetting, solubilizing and penetrating agent. Suitable surfactants include peptide derived surfactants (i.e. surfactin and iturin), non-ionic surfactants, anionic surfactants and amphoteric surfactants, such as cholic acids, alkyl sulfate salts, alkylsulfonic acid salts, alkylarylsulfonic acid salts, alkyl aryl ethers and their polyoxyethylene derivatives, polyethylene glycol ethers, polyol esters and sugar alcohol derivatives.

[0114] Other components of the formulation can include additional surface active agents, solvents, cosolvents, dyes, U.V. (ultra-violet) protectants, antioxidants, antifoams, stickers, spreaders, antifoaming agents, preservatives, humectants, buffers, carriers, emulsifiers, wetting agents, dispersants, fixing agents, disintegrators, acid solubilisers or other components which facilitateproduct handling and application. These carriers, diluents, auxiliary agents and so forth are preferably selected to optimize the antifungal action on plants or fruit.

[0115] Solid carriers can include, for example, the following materials in fine powder or granular form: agarose / agar containing cell culture media or dried cell culture media; organic-type fertilisers; clays (e.g. kaolinite, diatomaceous earth, synthetic hydrated silicon oxide, Fubasami clay, bentonite, acid clay); talc and other inorganic minerals (e.g. sericite, quartz powder, sulfur powder, activated carbon, calcium carbonate); and chemical fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium chloride, urea). Liquid carriers can include, for example, cell culture media, water; alcohols (e.g. methanol, ethanol, isopropanol); ketones (e.g. acetone, methyl ethyl ketone, cyclohexanone); esters (e.g. ethyl acetate, butyl acetate); nitriles (e.g. acetonitrile, isobutyronitrile); and acid amides (e.g. dimethylformamide, dimethylacetamide), as well as dilute bases (e.g. sodium hydroxide, potassium hydroxide and amines).

[0116] Other auxiliary agents can include, for example, adhesive agents and dispersing agents, such as casein, gelatin, polysaccharides (e.g. powdered starch, gum arabic, cellulose derivatives, alginic acid, chitin), lignin derivatives and synthetic water-soluble polymers (e.g. polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid); salts (eg. citrate, chloride, sulphate, acetate, ammonium, bicarbonate, phosphate salts and like) and stabilizers such as PAP (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (2- / 3-tert-butyl-4-methyoxyphenol), vegetable oils, mineral oils, phospholipids, waxes, fatty acids and fatty acid esters.

[0117] Conventional plant growth regulators, herbicides, fungicides, bactericides, insecticides, nematicides, acaricides, biochemical pesticides, plant produced pesticides (botanicals), cell culture media components or plant nutrients and so forth can also be incorporated into the composition of the present invention.

[0118] The composition can also include one or more other antimicrobial or antifungal compounds, including natural peptides, lipopeptides or antibiotics from animal, microbial or plant origin or chemically produced fungicides or antibiotics.

[0119] The composition may be diluted in water, organic mixtures or with a liquid carrier and sprayed or applied on the plants or fruit to be treated.

[0120] The fungicidal compositions described herein are capable of inhibiting the growth of at least one of Botrytis cinerea, Plasmopara viticola and Erysiphe necator, and are preferably capable of inhibiting the growth of all three of these pathogens. They may also be capable of inhibiting thegrowth of Altemaria altemata and Aspergillus niger. Thus, these fungicidal compositions can be used to treat, prevent or reduce bunch rot, grey rot, noble rot, downy mildew, powdery mildew, black mold or gray mold on plants and fruit on which the above fungi grow. The fungicide compositions can be used as a pre-harvest treatment or post-harvest treatment to prevent fruit spoilage. Exemplary plants include grapevine plants, strawberry plants, raspberry plants, blueberry plants, blackberry plants and the like. Exemplary fruits include berry fruits such as grapes, strawberries, raspberries, blueberries, blackberries and the like.

[0121] The bacteria and yeast species in the fungicide compositions described herein were selected not only on their ability to inhibit the growth of Botrytis cinerea, Plasmopara viticola and Erysiphe necator but also because no mutually antagonistic interactions were observed between microorganisms in these species. Other selection criteria were that the microorganisms should thrive in the presence of oxygen so that they can compete with targeted pathogens on plant surfaces; they should have poor fermentation capacity so that they do not interfere with the wine fermentation process; that the microorganisms should be well-adapted to conditions in a vineyard; that the microorganisms should have different modes of action, so that if one of the microorganisms were to be ineffective under certain conditions, at least one of the others should be effective under those same conditions.

[0122] The fungicidal compositions disclosed herein are not only environmentally friendly, but they are also more effective than existing bioproducts for the control of pathogenic fungi. As mentioned earlier, biofungicides comprising single microbe species have been shown to perform poorly in the field due to the inability of the microbes to establish themselves on plants and to withstand different or changing environmental conditions (e.g. due to cold, hot, dry, humid or wet conditions).

[0123] Compositions described herein which comprise compatible yeast and bacteria with different modes of action, different optimal growth conditions and nutritional requirements offer an opportunity to promote biocontrol efficiency. For instance, in Consortium 1 described above, W. anomalus is a strong producer of fungal cell wall degrading enzymes, while the unknown bacterial species comprising strains B4001 and B4022 produces cyclic lipopeptides which are heat labile and form biofilms which can facilitate adherence to plant surfaces and protect the consortium from desiccation and UV irradiation. In Consortium 2, H. pseudoburtonii forms pseudohyphae and can rapidly occupy more space. Studies by the inventors have confirmed that these consortia are more inhibitory than the yeast alone and maintain a similar level of inhibition as the bacteria alone.

[0124] These compositions are also less likely to develop resistance against them than compositions containing a single agent with fungicidal activity.The yeast and bacteria used in the compositions can be grown on affordable substrates, and they grow rapidly and do not require many processing steps to be used in the composition. This makes the production of these compositions and their ultimate use as fungicides less expensive by lowering manufacturing costs compared to conventional methods.

[0125] The invention will now be described in more detail by way of the following non-limiting examples.

[0126] Examples

[0127] 1. Screening of grape must-derived yeasts for antifungal activity

[0128] A total of 31 yeasts isolated from grape surfaces and grape must were screened for their ability to inhibit the growth of different target pathogens, viz. three strains of Botrytis cinerea (the causative agent of grey rot) (B05.10; IWBT-FF1 ; IWBT-FF2), Alternaria alternata and Aspergillus niger. The isolates were obtained from the culture collection of the South African Grape and Wine Research Institute and are listed in Table 1.

[0129] The screening was conducted using a dual culture method. The yeast and the target pathogen were inoculated opposite each other on agar plates at equal distance from the edge of a Petri dish and allowed to grow for five days, after which the mycelial formation was observed and the diameter measured and compared to the growth of the target pathogen inoculated on its own.

[0130] Table 1 provides a summary of the antifungal properties of the different yeasts. Of the 31 yeast isolates screened against the Botrytis cinerea strains, Aspergillus niger and Alternaria alternata, 23 strains displayed antifungal activity and 15 of those had broad spectrum antifungal activity (i.e., they inhibited all target pathogens). 15 of the yeast strains produced glucanases; 19 of the yeast strains produced chitinases; W. anomalus strains Y517 and Y541 reduced glucan levels in B. cinerea hyphae; and W. anomalus strains Y541 and Y934 reduced chitin levels in B. cinerea hyphae.

[0131] Table 1: Antifungal activity of grape-must derived yeast species and strains

[0132]

[0133]

[0134] Key: (Inhibition spectrum) +++, ++-, and — denote strains capable of inhibiting (i) all three of the pathogens, (ii) two pathogens and (Hi) no activity, respectively. Chitinase and glucanase activity: Strong, Weak or ND denote inhibition percentages (i) > 50%, (ii) < 50% and (iii) not determined, respectively.

[0135] Figure 1 illustrates the different levels of mycelial growth inhibition activity of the yeast isolates against the three strains of B. cinerea. Three strains of Wickerhamomyces anomalus, two strains of Lodderomyces elongisporus, and a single strain of Hyphopichia pseudoburtonii were selected for further investigation (marked with **). The mode of action of these selected yeasts includes production of cell wall lytic enzymes (glucanases and chitinases) and volatile organic compounds.2. Isolating and screening vineyard-derived bacteria for antifungal activity

[0136] A total of 188 bacterial isolates were screened for activity against the Botrytis cinerea strains B05.10; IWBT-FF1 ; IWBT-FF2, using the dual-culture assays described for the yeast screening. Of the 188 bacterial isolates, 174 had been isolated from vineyard soil and 14 from grapevine leaves.

[0137] The 188 bacterial isolates showed different levels of mycelial growth inhibition activity against the three strains of Botrytis cinerea. 25 isolates displayed antifungal activity and could inhibit mycelial growth of the three strains of Botrytis cinerea (Figure 2). Only nine strains displayed > 60% inhibition (Figure 3). These nine strains were identified as Bacillus velezensis (B4003; B4005), Bacillus subtilis (B4000), Bacillus pseudomycoides (B4014), Bacillus wiedmannii (B4016) and Pantoea agglomerans (B4020), and unknown strains B4001 and B4022. It was further observed that only four of the strains produced glucanases, and chitinases were produced by some of the strains but the activity was inhibited by the acidic environment created by B. cinerea (Table 2).

[0138] Table 2: Antifungal activity of bacterial isolates from soil and grapevine leaves

[0139]

[0140]

[0141] Key: (Inhibition spectrum) +++, ++ denote strains capable of inhibiting (i) all three of the B. cinerea strains and both Phytophthora species or (ii) only B. cinerea strains, respectively. Chitinase and glucanase activity: Strong (+++), Weak (++). ND indicates “not determined”. Isolates designated NID were not identified.

[0142] 3. Genome sequencing of Bacillus strains

[0143] Genomic DNA of bacterial isolates B4001, B4003, B4005, B4022 and B4023 was extracted, fragmented and then subjected to library preparation and sequencing using the PacBio SMRT Technology. Subsequently, the genomes were assembled using the Hi Fi analysis pipeline, quality evaluated using Quast v5.3.0 and checkM v1 .0.18, and annotated with Prokka v1.14.6. The genome sequences were queried against the Genome Taxonomy Database through Kbase. Phylogenomic analysis was achieved through the use of the Up-to-date Bacterial Core Gene pipeline. Based on the UBCG phylogeny, the close relatives and the genomes conspecific to the strains isolated from the vineyard soil and grapevine leaves, based on the Genome Taxonomy Database (GTDB), were used for the Average Nucleotide Identity (ANI) calculations. The ANI was calculated using jspecies (https: / / jspecies.ribohost.eom / jspeciesws / #analyse) employing the ANIb (blast) algorithm. The amino acid files of the individual strains were submitted to GhostKOALA (https: / / www.kegg.jp / ghostkoala / ) for KEGG Orthology (KO) search and assignment. The secondary metabolite analysis shell (antiSMASH - V8.0.4) resource was employed for the automatic genomic identification and analysis of biosynthesis gene clusters (BGCs) and further aligned using NCBI BlastP against different databases.

[0144] The bacterial strains were found to have genomes of ~ 3.9 Mb and 45.3% GC content (Table 3). The genome sequence for strain B4001 is shown in SEQ ID NO: 1, the genome sequence for strain B4022 is shown in SEQ ID NO: 2, and the genome sequence for strain B4023 is shown in SEQ ID NO: 3.

[0145] Table 3: Genome features of vineyard-isolated Bacillus strains.

[0146] Feature B4003 B4005 B4001 B4022 B4023

[0147] Size (Mb) 3,964,064 3,916,708 3,925,055 3,996,852 3,988,914 Contigs 3 1 4 3 3

[0148] G + C content (%) 46.53 46.65 45.32 45.29 45.3Completeness 99.81 99.81 98.62 98.43 98.43 Contamination 0.05 0.05 1.29 1.05 1.05

[0149] N50 3,876,353 3,916,708 3,887,865 3,973,861 3,973,861 L50 1 1 1 1 1

[0150] CDSs 3,774 3,745 3,804 3,932 3,920 Predicted genes 2,758 2,755 2,633 2,698 2,697 Pseudogenes 10 13 10 24 24

[0151] Coding density 90.1 90.2 89.1 89.3 89.3

[0152] RNAs 132 131 137 136 135 Transfer RNAs 86 86 86 87 87

[0153] T ransfer-Messenger

[0154] 1 1 1 1 1

[0155] RNAs

[0156] Ribosomal RNAs 27 27 27 27 27

[0157] Non-coding RNAs 18 17 23 21 20

[0158] Non-coding RNA

[0159] 59 60 60 60 60

[0160] regions

[0161] Hypotheticals 97 118 157 212 205

[0162] Short open reading

[0163] 4 3 4 4 4

[0164] frames

[0165] Replication origins 2 2 2 2 2 Vegetative replication

[0166] 2 2 2

[0167] origins

[0168] Analysis of the biosynthetic gene clusters, revealed more than 10 gene clusters responsible for secondary metabolite biosynthesis in the genomes of both strains, including the non-ribosomal peptide synthetases (NRPSs) and NRPS-Hybrids which produce lipopeptides, peptide antibiotics, siderophores, polyketide synthases (PKs), PKS-hybrids and betalactone clusters, terpene and terpene -precursor clusters, ribosome-synthesized and post-translational modified peptides (RiPPs) and others (Table 4). The data show differences in BGCs (Bacterial Core Genes) between the two unknown strains, which could explain differences in their antimicrobial activities. For instance, the NRPS-like cluster responsible for the biosynthesis of locilomycin A / B / C as well as the Lanthipeptide-class-i responsible for subtilin were detected in strain B4022 but not for strain B4001 , while the Lanthipeptide-class-ii, cyclic lactone-autoinducer responsible for Amyloliquecidin GF610 and the Sactipeptide cluster coding for sporulation killing factor were predicted in strain B4001 and not strain B4022.Table 4: Comparison of biosynthetic gene clusters of vineyard-isolated Bacillus species.

[0169] Cluster Type Secondary Functions B4003 B4005 B4001 B4022 B4023 metabolite

[0170] 1 NRPS-like locilomycinA / B / C Antifungal, - - - + + antibacterial,

[0171] 2 NRPS Surfactin Colonisation, ISR, + + + + + antiviral,

[0172] antibacterial,

[0173] antifungal,

[0174] 3 NRPS, t1PKS, Zwittermicin A Antibacterial and - - + + + HR-T2PKS antifungal (broadspectrum activity

[0175] against

[0176] prokaryotic and

[0177] eukaryotic

[0178] microorganisms)

[0179] 4 PKS-like Butirosin A / B Antibacterial + + + + + 5 Terpene Unknown - 6 transAT-PKS, Bacillaene Antibacterial + + + + + NRPS

[0180] 7 NRPS- Fengycin ISR, antifungal + + + + + transAT-PKS,

[0181] betalactone

[0182] 8 Terpene Unknown - 9 T3PKS Unknown - 10 Terpene- Unknown - precursor

[0183] 11 Terpene- Bacillibactin Antibacterial, + + + + + precursor, antifungal,

[0184] NRP- siderophore

[0185] metallophore, production

[0186] NRPS

[0187] 12 Lanthipeptide- Subtilin Antibacterial - - - + + class-i (Gram-positive

[0188] bacteria)

[0189] 13 Lanthipeptide- Amyloliquecidin Antibacterial +

[0190] class-ii, cyclic GF610 (Gram-positive

[0191] lactone- bacteria)

[0192] autoinducer

[0193] 14 Sactipeptide Sporulation killing Antibacterial +

[0194] factor activity

[0195] 15 transAT-PKS Macrolactin Antibacterial, + + - - - antifungal,

[0196] antiviral

[0197] 16 transAT-PKS Difficidin Antibacterial + + - - -17 Other Bacilysin Antibacterial, + + + + + antifungal

[0198] Total 8 8 9 9 9 tRNA - transfer RNAs; tmRNAs - transfer-messenger RNAs; rRNAs - ribosomal RNAs; ncRNAs - noncoding RNAs; sORFs - short open reading frames; oriCs - replication origins; oriVs - vegetative replication origins.

[0199] A phylogenetic tree was constructed using the UBCG pipeline, incorporating reference genomes retrieved from the GTDB (Figure 4). Stenotrophomonas maltophilia NCTC10257 was used as an outgroup. The numbers on the nodes represent the Gene Support Index (GSI).

[0200] Multigene sequencing using 16S rRNA gene, rpoB, recA, gyrB had placed two of the strains (B4003 and B4005) as B. velezensis and the other three (B4001, B4022 and B4023) as B. nakamurai. Whole genome analysis revealed that all the strains exhibited genome features typical of the Bacillus genus, including circular chromosomes, %GC contents, a standard complement of RNA genes, and genome size similar to those reported earlier for Bacillus spp.. Moreover, phylogenetic classification, together with the AN I values above the 96% threshold border for bacterial species, further validated the classification of strains B4003 and B4005 as B. velezensis. Conversely, strains (B4022, B4001, and B4023), which were identified as B. nakamurai by 16S rRNA sequencing, only displayed =94% ANI compared to B. nakamurai. Since the ANI values are below the species demarcation threshold, the data suggest that these strains belong to a distinct species within the genus Bacillus. The strains consistently clustered with the formally undescribed lineage with placeholder “sp018613535” within the Bacillus genus and displayed an ANI >97% (Figure 5). This placeholder designation represents genome-based species identified through GTDB’s standardised taxonomy but lacking a formal nomenclatural description. The placement of these strains in this cluster suggests that they are closely related to this provisional species and may belong to an emerging or currently uncharacterised Bacillus clade.

[0201] 4. Determination of modes of action using dual-culture assays

[0202] Biological control agents inhibit target phytopathogens by competing for nutrients and space, or through the production of antifungal compounds. Thus, the ability to produce fungal cell wall degrading enzymes was evaluated using dual-culture assays on media supplemented with either laminarin (a substrate for p-glucanases) or colloidal chitin (a substrate for chitinases). This was tested in the selected yeasts and bacteria (Tables 1 and 2). The production of volatile organic compounds (VOCs), a known mechanism to inhibit sporulation in fungi, was also evaluated in selected yeasts by inoculating the target pathogen and the antagonist yeast on opposite slants in a glass vial. Figure 6 lists various higher alcohols such as phenylethanol, isoamyl alcohol and butanol produced by the selected yeasts, which remained relatively abundant in mixed culturesincluding B. cinerea.

[0203] The production of non-volatile organic compounds was tested in the cell free supernatants of selected yeasts and bacteria. For the yeasts, the supernatants of the monocultures of individual yeasts and B. cinerea were compared with the co-cultures of the yeasts with B. cinerea, using gas chromatography-mass spectrometry (GC-MS). In contrast, for the bacteria the supernatants of monocultures were first screened for their ability to inhibit spore germination and mycelial growth, followed by characterization of the composition of the supernatants from selected bacteria. Both the yeast and bacterial supernatants were analysed using liquid chromatographymass spectrometry (LCMS).

[0204] The nine selected bacterial strains were cultivated in a nutrient broth and the cell-free extracts were collected after 24, 48, 72 and 96 hours and evaluated for antifungal activity against the three B. cinerea strains (B05.10, IWBT-FF1 and IWBT-FF2). The cell-free extracts displayed different levels of spore germination and mycelial growth inhibition capabilities (Figures 7 and 8). Strains B4001 and B4022 consistently displayed better inhibitory activity than the control, which was a commercial product, B. amyloliquefaciens D747.

[0205] To identify the inhibitory compounds, the extracellular metabolites of the two most inhibitory isolates (B4001 and B4022), the moderate inhibitor (8. nakamurai B4023) and low inhibitor (8. pseudomycoides B4014) were compared. The LC-MS results revealed divergence in the composition of cyclic lipopeptides, which broadly included iturins, fengycins, surfactins and bacillomycins in varying combinations depending on the strain (Figure 9). The biocontrol provided by the five strains included inhibition of spore germination and mycelial growth through the production of these antimicrobial compounds. These compounds are known to have antimicrobial activity and several Bacillus species and strains have been shown to produce them. Strain B4001 isolated from the test vineyard was shown to have a different composition of cyclic lipopeptides than the commercial strain D747 used as a positive control.

[0206] Since Plasmopara viticola (the causative agent of downy mildew in grapevine) is an obligate parasite it is not cultivable in laboratory media. Phytophthora cinnamomi and Phytophthora capsiciare known for being good surrogates for P. viticola and were thus used to screen selected yeasts as well as bacteria for their ability to inhibit P. viticola. The activity of the selected yeasts and bacteria against these two oomycetes can give a good indication of whether the test organisms would be able to inhibit P. viticola.

[0207] Apart from H. burtonii 95 , the yeast strains broadly displayed higher inhibitory activity against Ph. capsici DCS 585 than Ph. cinnamomi Steu 6825 (Figure 10). Among the bacteria, 8.wiedmannii B4016 and B. nakamurai B4023 show similar levels of inhibition against the two surrogates while B. pseudomycoides B4014 only inhibited Ph. capsici DCS 585. Similarly to the yeasts, the remaining bacterial strains displayed higher inhibitory activity against Ph. capsici DCS 585 (Figure 11).

[0208] 5. Assessing strain compatibility

[0209] Binary cultures were used to assess the compatibility of selected yeast and bacterial strains. Yeast and bacterial strains were inoculated at a 1 :1 ratio and co-cultured in malt extract broth incubated at 25°C with agitation at 150 rpm on a rotary shaker. Samples were withdrawn at various time intervals (0, 6, 12, 18, 24, 36, 48, and 72 h) to monitor the growth of each inoculant. The strains were considered compatible if they remained viable in the mixed culture for more than 48 hours.

[0210] As illustrated in Figure 12, single strains and co-cultures were screened using dual cultures on agar plates, for their ability to inhibit the growth of Botrytis cinerea, Phytophthora cinnamomi and Phytophthora capsici (once again, Ph. cinnamomi and Ph. capsici were used as surrogates for P. viticola). The bacteria were enumerated on Nutrient Agar supplemented with 50 mg / L natamycin to suppress yeast growth, while the yeasts were enumerated on Wallerstein Nutrient Agar supplemented with 34 mg / L chloramphenicol to suppress the bacteria. The growth was compared with that observed in the monocultures of the individual strains.

[0211] Figures 13-17 illustrate growth curves showing yeast viability in monoculture (Mo) compared to co-cultures (Co) with different bacterial strains. The yeasts tested were Hyphopichia pseudoburtonii (Hp) Y963, Hyphopichia burtonii (Hb) Y951 and three strains of Wickerhamomyces anomalus (Wa) Y934, Y541 and Y517, co-cultured with the bacteria Pantoea agglomerans (Pa), the unidentified strains B4001 and B4022, and Bacillus wiedmannii (Bw) strain B4016.

[0212] Figures 18-21 illustrate growth curves showing the viability of Pantoea agglomerans (Pa), strains B4001 and B4022, as well as Bacillus wiedmannii (Bw) strain B4016, in monoculture (Mo) and in co-culture (Co) with Hyphopichia pseudoburtonii (Hp) strain Y963, Hyphopichia burtonii (Hb) strain Y951 , and Wickerhamomyces anomalus (Wa) strains Y934, Y541 and Y517.

[0213] H. pseudoburtonii Y963 declined gradually in the presence of B4001 and B4022 (Figure 13), while W. anomalus Y541 and Y517 showed a decline only in the presence of B4001 (Figures 16 and 17). Conversely, P. agglomerans B4020 was able to grow and maintain viability in the presence of all yeast strains (Figure 18), while strain B4022 declined rapidly in the presence of H. burtoniiY951 (Figure 19) and strain B4001 only showed decline in the presence of H. burtoniiY95t (Figure 20). B. wiedmannii B4016 displayed varying rates of decline in the presence of each yeast strain (Figure 21).

[0214] The bacterial strains displayed higher inhibition activity than the yeasts against all three target pathogens (Figures 22 and 23). By contrast, the yeasts displayed higher inhibition against Ph. capsici than Ph. cinnamomi (Figure 23). As shown in Figures 24-27, the overall level of inhibition was maintained at more than 50% for all the combinations tested.

[0215] The strains were co-cultured in liquid media for 72 hours and samples were withdrawn at 12 hour intervals in order to determine viable counts of both the yeast and bacteria. Strains were considered compatible if both inoculants remained viable for the 72 hour duration, as shown in Figures 28B and 28F, and incompatible if the viability of one of the inoculants declined before 72 hours.

[0216] For example, Bacillus subtilis B4016 lost viability within 24 hours in the presence of Wickerhamomyces anomalus Y934 and Y541 (Figures 28A and 28D), while Bacillus nakamurai only lost viability in the presence of W. anomalusY54t (Figure 28E). Similarly, W. anomalusY934 only remained viable for approximately 50 hours in the presence of B. amyloliquefaciens (Figure 28C). Strains were considered compatible if antifungal activity was maintained or enhanced in the mixed culture compared to the monocultures.

[0217] After the binary cultures, compatible strains were used to construct consortia with ternary cultures.

[0218] 6. Selection of microbial consortia as biocontrol agents against grapevine pathogens

[0219] Following the data obtained from compatibility and biocontrol activities, two consortia composed of a mixture of bacterial strains and yeast strains were developed and named CO1 (comprising P. agglomerans B4020, strain B4022, and l / IZ. anomalus Y934) and CO2 (comprising P. agglomerans B4020, strain B4001, and H. pseudoburtonii Y963). P. agglomerans B4020 displayed compatibility with all yeast strains, while combinations of strains B4001 and B4022 with the yeasts resulted in different inhibitory patterns than P. agglomerans.

[0220] 7. Leaf disc assays and grape bioassavs

[0221] Leaf disc assays and grape bioassays were conducted to test the two consortia ex vivo for their ability to prevent the development of powdery mildew, downy mildew and grey rot.For the leaf disc assays, young leaves were collected from potted vines and surface sterilized with a 0.25% calcium hypochlorite (CaCI2O3) solution for 2 minutes and washed three times for 2 minutes each time with sterile distilled water. Subsequently, 15 mm diameter leaf discs were cut out using a sterilized cork borer and placed in 24-well plates pre-filled with 2 mL of water agar. The discs were inoculated with either monocultures or consortia and left to dry, following which pathogen (Erysiphe necator or Plasmopara viticola) spores were inoculated. The 24-well plates were incubated in the dark for 24 hours at 25°C before being transferred to a growth chamber under 16 / 8 hour dark / light for 10 to 14 days, depending on mildew development on control plates. Development of mildew diseases on the leaf discs was visually evaluated. Mildew diseases were scored according to a scale of 0 to 7 based on leaf visible sporulation or necrosis spots using the OIV descriptor 452-1. For scoring the mildew: 1 = none or very weak, 3 = low sporulation / necrotic spot, 5 = medium or moderate necrotic spot, and 7 = high sporulation. Both consortia displayed an ability to prevent powdery mildew formation as no sporulation was observed for CO1 and very weak sporulation was observed for CO2 (Figure 29A). Similarly, for downy mildew, no necrosis was observed for both consortia (Figure 29B).

[0222] For the grape bioassays, the grapes were surface sterilized and then an artificial wound was created with a sterile 6 mm diameter cork borer before inoculation. The grapes were incubated at 25°C for 7 days and inhibitory activity of the consortia and monocultures on the development of grey mold was visually assessed based on the color changes (browning) and mycelial growth on the treated grapes. The disease severity was further evaluated using an empirical visual scale of 1 to 4. For the scoring: 1 = no visible grey mold spot, 2 = development of brown colour and soft mycelia, 3 = distribution of mycelium on the wounds, and 4 = sporulation of mycelium from wounds). For all experiments the consortia and monocultures were inoculated at 106cells / mL while the pathogens were inoculated at 106spores / mL. Overall, the monocultures as well as the two consortia displayed less rot, and all scoring a level 2 (i.e., browning and soft mycelia development) compared to full sporulation observed in the control which was only inoculated with B. cinerea (Figure 29C).

[0223] 8. Grapevine disease prevention in potted vines

[0224] To determine the influence of the biocontrol consortia on grapevine disease incidence, twenty potted vines were used. All the potted vines were kept outdoors at Stellenbosch University’s Welgevallen experimental vineyard. The two biocontrol consortia were prepared by growing the cultures overnight in malt extract broth and then harvesting the cells by centrifugation at 5000 rpm for 5 minutes. The cells were resuspended in saline (0.9% NaCI) and the OD600nm was measured. Two consortia were prepared in spray bottles with each strain mixed at equal concentration (~ 2 x 106cfu / mL). Each consortium was sprayed onto five vines (i.e., consortium1 on 5 vines and consortium 2 on 5 vines). Another 5 vines were sprayed with water as a negative control and another 5 were sprayed with Switch® (a chemical fungicide from Syngenta, South Africa). The treatments were applied as foliar spray at flowering and at berry pea size stage. The vine leaves as well as the grape bunches were then monitored for disease incidence until harvest. Leaves were collected from the vines and disease incidence was scored as either low (limited sporulation and mycelia), medium (mycelial patches 2 - 5 cm diameter) or high (unlimited patches, strong sporulation and abundant mycelia) (Figure 30).

[0225] All vines displayed varying levels of powdery mildew. Overall, the vines treated with the two consortia and with the chemical fungicide had less disease load than the negative control vines (Figure 31). Importantly, the vines treated with the consortia had a higher percentage of leaves with low disease incidence and a lower percentage of leaves with high disease incidence. In contrast, the negative control treated with water had a higher percentage of leaves with the high disease incidence, while the vines treated with the chemical fungicide had more leaves with moderate disease incidence. The data show that even though the application of the chemical fungicide and the consortia was not optimal (i.e. , only at two phonological stages) the consortia show promise in the control of powdery mildew. None of the grape bunches developed Botrytis cinerea which is the target pathogen for Switch®, the chemical fungicide tested. Consequently, it was not possible to draw any conclusions regarding the performance of the consortia versus the Switch® chemical fungicide.

[0226] 9. Influence of the biocontrol consortia on wine fermentation

[0227] The fermentation ability of the two consortia, Consortium 1 (CO1) and Consortium 2 (CO2), and their impact on desirable wine fermentation yeasts were evaluated in a synthetic grape juice medium. The consortia were co-inoculated with Saccharomyces cerevisiae (Sc), Lachancea thermotolerans (Lt) and Torulaspora delbrueckii (Td) monocultures and mixed cultures thereof.

[0228] On their own, the two consortia display a poor fermentation tempo and could not ferment to dryness (that is, < 2.5 g / L residual sugar). Consortium 1 (CO1) appeared to retard the fermentation with S. cerevisiae, while Consortium 2 (CO2) had little effect on any of the desirable yeasts (Figure 32), since all the fermentations were completed successfully and the viability of all the yeasts was maintained (Figures 33A to 33D). The wines produced from the different combinations resulted in varying amounts of primary and secondary metabolites.

[0229] The two biocontrol consortia were sprayed on Vitis vinifera L. cv. Merlot bunches two weeks prior to harvest using hand-held spray bottles to determine whether the species in the consortia can adhere to berry surfaces and establish themselves amongst the existing communities. Furtherobjectives were to test if the microorganisms in the consortia would be detected in the wine fermentation process and whether, if sprayed closer to harvest, the consortia would affect wine fermentation negatively. The three species of microorganisms in each of the two sprayed consortia were present in a relative ratio of 1 :1 :1 based on cell counts, e.g. with each strain being kept at approximately 106CFU / mL.

[0230] The sprayed grapes were then harvested and processed for wine production. On the control vines, the bunches were sprayed with distilled water. Two sets of fermentation were conducted: (i) spontaneous fermentation of the grape must prepared from the grapes sprayed with CO1 and CO2, as well as the control grapes sprayed distilled water, and (ii) grape must prepared from untreated bunches was inoculated with Saccharomyces cerevisiae, CO1 and CO2, separately. The microbial population dynamics as well as the fermentation kinetics were monitored.

[0231] Overall, all spontaneous and inoculated fermentation was successfully completed and generated different fermentation profiles. It was observed that in the spontaneous fermentations of the grapes where the consortia had been sprayed, the individual yeasts (i.e., Wickherhamomyces anomalus in CO1 and Hyphopichia pseudburtonii in CO2) could be detected.

[0232] Figure 34 shows the relative abundance of yeast species during alcoholic fermentation in cv. Merlot. In spontaneous grape fermentation which occurred after spraying the consortia onto the grapes, the abundance levels of the yeasts were low and declined below detection within the first three days of fermentation, while in the inoculated ferments the yeasts survived until the end of fermentation. Figure 35 shows the relative abundance of bacterial species during alcoholic fermentation in cv. Merlot. Among the bacteria, only Pantoea agglomerans could be detected together with a wide variety of Bacillus species. Nonetheless, in all cases the bacterial population declined within the first four days of fermentation.

[0233] Overall, the data showed that the species in the two consortia are capable of colonizing grape surfaces, establishing themselves among the existing population and persisting into the early stages of wine fermentation. The results indicate that should the disclosed consortia be applied onto vines within two weeks of harvest and thereafter be transferred into the grape must, the microorganisms composing the consortia would be unlikely to be detrimental to wine fermentation as they have little or no negative influence on the desirable microorganisms.

[0234] The biogenic amines in the final wines were measured. These compounds are known to induce allergic reactions in sensitive consumers and some countries, such as Germany, Australia and Finland, have set an upper limit of 10 mg / L for histamine for example. In this study, the total concentration of biogenic amines in all the wines was below 3 mg / L ,with histamine levels rangingfrom 14 to 30 pg / L (Table 5), indicating that the consortia pose a negligible risk with regard to the accumulation of high levels of biogenic amines.

[0235] Table 5: Quantification of biogenic amines (BAs) levels (gg / L) at the end of alcoholic fermentation in Merlot treatments. Values represent the average of three replicates ± standard deviation.

[0236]

[0237] Values with the same letter in the same column are statistically similar when compared with Tu key’s HSD posthoc test at 95 % confidence level.

[0238] The foregoing description has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the technology to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.

[0239] The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the present disclosure be limited not by this detailed description, but rather by any claims that issue on an application basedhereon. Accordingly, the present disclosure is intended to be illustrative, but not limiting, of the scope of any accompanying claims.

[0240] Finally, throughout the specification and any accompanying claims, 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 integer or group of integers but not the exclusion of any other integer or group of integers.

Claims

CLAIMS:

1. A fungicidal composition, the composition comprising:(i) a Pantoea agglomerans bacterium;(ii) a bacterium having a genomic sequence comprising a first nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 1, a second nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 2, a third nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 3 and a fourth nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 4, or a bacterium which has at least 96% identity to the combined sequence of SEQ ID NOs: 1 to 4; and (iii) a yeast-like ascomycetous microorganism selected from the group consisting of Hyphopichia pseudoburtonii and Wickerhamomyces anomalus.

2. The fungicidal composition of claim 1 , wherein the yeast-like ascomycetous microorganism is Hyphopichia pseudoburtonii.

3. The fungicidal composition of claim 1 , wherein the yeast-like ascomycetous microorganism is Wickerhamomyces anomalus.

4. The fungicidal composition of any one of the preceding claims, wherein the genome of the bacterium of (ii) comprises a first nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 1 , a second nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2, a third nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 and a fourth nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 4.

5. The fungicidal composition of any one of the preceding claims, wherein the genome of the bacterium of (ii) comprises a first nucleic acid sequence of SEQ ID NO: 1 , a second nucleic acid sequence of SEQ ID NO: 2, a third nucleic acid sequence of SEQ ID NO: 3 and a fourth nucleic acid sequence of SEQ ID NO: 4.

6. The fungicidal composition of any one of claims 1 to 3, wherein the genome of the bacterium of (ii) comprises a first nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 5, a second nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and a third nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 7.

7. The fungicidal composition of claim 6, wherein the genome of the bacterium of (ii) comprises a first nucleic acid sequence of SEQ ID NO: 5, a second nucleic acid sequence of SEQ ID NO: 6, and a third nucleic acid of SEQ ID NO: 7.

8. The fungicidal composition of any one of the preceding claims, wherein the Pantoea agglomerans is the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4020.

9. The fungicidal composition of claim 1 or 2, wherein the Hyphopichia pseudoburtonii is the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number Y963.

10. The fungicidal composition of claim 1 or 3, wherein the Wickerhamomyces anomalus is the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number Y934.

11. The fungicidal composition of any one of the preceding claims, wherein the bacterium of (ii) is a Bacillus species.

12. The fungicidal composition of any one of the preceding claims, wherein the bacterium of (ii) is the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4001.

13. The fungicidal composition of any one of claims 1 to 11 , wherein the bacterium of (ii) is the strain deposited at the culture collection of the South African Grape and Wine Research Institute under accession number B4022.

14. The fungicidal composition of claim 1 , which comprises:(i) Pantoea agglomerans strain B4020;(ii) Bacillus strain B4001 ; and(iii) Hyphopichia pseudoburtonii strain Y963.

15. The fungicidal composition of claim 1 , which comprises:(i) Pantoea agglomerans strain B4020;(ii) Bacillus strain B4022; and(iii) Wickerhamomyces anomalus strain Y934.

16. The fungicidal composition of any one of the preceding claims, which is capable of inhibiting the growth of Botrytis cinerea, Plasmopara viticola and Erysiphe necator.

17. The fungicidal composition of any one of the preceding claims, which is capable of inhibiting the growth of Botrytis cinerea, Plasmopara viticola, Erysiphe necator, Alternaria alternate and Aspergillus niger.

18. The fungicidal composition of any one of the preceding claims, wherein (i), (ii) and (iii) are present in the composition in about a 1 :1 :1 ratio.

19. The fungicidal composition of any one of the preceding claims, wherein the composition does not include any chemical or synthetic fungicidal agents.

20. Use of a fungicidal composition of any one of the preceding claims for treating, preventing or reducing bunch rot, grey rot, noble rot, downy mildew, powdery mildew, black mold or gray mold on plants and fruit.

21. Use according to claim 20, wherein the plants are grapevines.

22. Use according to claim 20, wherein the fruit are berry fruits.

23. Use according to claim 20, wherein the fruit are grapes, strawberries, raspberries, blueberries or blackberries.

24. A method of treating, preventing or reducing bunch rot, grey rot, noble rot, downy mildew, powdery mildew, black mold or gray mold on plants and fruit, the method comprising applying a fungicidal composition according to any one of claims 1 to 19 to the plants or fruit.

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