Control of fungal infections in plant, soil or growing media

Abstract

The present application relates to a bacterial composition, bacterial strains and culture, which can be used to control of fungal infections in plant, soil or growing media, such as compost. According to the present invention, there is provided an anti-fungal bacterial composition comprising at least a Bacillus velezensis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology.

Description

[0001] Control Of Fungal Infections In Plant, Soil Or Growing Media

[0002] The present application relates to a bacterial composition, bacterial strains and culture, which can be used to control of fungal infections in plant, soil or growing media, such as compost.

[0003] Background Of The Invention

[0004] Plant pathogens cause severe losses and damage to crops worldwide. Different strategies have been used to reduce the occurrence of plant diseases including pesticides, less susceptible cultivars, crop rotation, and other control measures, but their efficacy is usually insufficient due to the survival and resistance of soil-borne pathogens. During the previous decades there has been a clear sustainability objective to reduce the use of chemically synthesized pesticides, and biocontrol methods, strategies and approaches are now being used in plant disease management. Various bacterial species are known to play a significant role in controlling plant pathogens and diseases which can be used as biocontrol agents (BCAs).

[0005] For example, Agaricus bisporus (the white button mushroom) is one of the most cultivated and consumed mushrooms in the world. Fungal diseases are prevalent on all mushroom farms and render mushrooms unfit for sale. This causes significant (20-25%) yield losses for growers, equating to €40 million in turnover. There are four main diseases that affect mushroom production: dry bubble (caused by Lecanicillium fungicola), wet bubble (caused by Mycogene perniciosa), green mould (caused by Trichoderma aggressivum) and cobweb (caused by Cladobotryum spp.).

[0006] Dry bubble, caused by L. fungicola, is currently one of the most prevalent fungal disease in mushroom farms and, therefore, the most detrimental to commercial mushroom cultivation. It was originally assumed that mushroom compost is an unlikely source of L. fungicola as spores decompose at 40°C and could not survive the composting process, where temperatures reach at least 70- 80°C. Therefore, infection was thought to occur when the peat casing, which is placed over the compost to induce mushroom formation, containing L. fungicola comes into contact with the fruiting mushroom bodies (Berendsen et al., 2010). However, recent data suggests that L. fungicola DNA and RNA is present in mushroom compost throughout the cropping process even in crops not exhibiting the physical manifestation of the disease (McGee et al., 2017, Carrasco et al., 2019). L. fungicola produces large numbers of conidia that can attach to numerous objects around the farm including harvesting equipment, machinery, workers gloves and shoes, doors, floors, dust and flies (Berendsen, 2010). Mycogone produces two types of spores, conidia and chlamydospores (Pyck et al., 2015). If the disease is left uncontrolled, the spores will be spread by water splashes onto neighbouring crops, floor and surrounding surfaces (Kouser et al., 2015). The pathogen can also be spread via gloves of pickers and use of equipment. The tough outer wall of the chlamydospores allows the spores to survive in organic debris such as casing and compost. The mixing of the spores and casing / compost debris could be spread and blown around the farm in the dust fraction. New infections can develop if infected debris and dust settle on casing and / or casing equipment (Pyck et al., 2015).

[0007] Several species of Cladobotryum cause cobweb disease in A. bisporus including Cladobotryum mycophilum and Cladobotryum mycophilum. Infection spreads due to conidia that become air borne with physical disturbance, such as watering. Cobweb disease has become an issue in mushroom farming due to the increasing resistance of Cladobotryum spp. to chemical fungicides.

[0008] T. aggressivum is a filamentous fungus that colonises the mushroom mycelium substrate causing a green mould to appear on the surface of the mushroom beds. Upon colonisation of A. bisporus, T. aggressivum causes severe reductions in mushroom yields, possibly through a combination of mycoparasitic and saprotrophic activities (O’Brien et al., 2014).

[0009] A. bisporus has little effective natural resistance to the aforementioned pathogens and control of these diseases has traditionally been through a combination of the use of stringent hygiene practises, strategic cultural practices and with the use of chemicals. Chemical control is limited due to regulations permitting the use of certain chemicals and the fact that both the host and the pathogen are fungi. Therefore, highly selective fungicides are necessary to prevent the establishment of the pathogens without damaging the host, A. bisporus.

[0010] Sporgon™ WP 50 (prochloraz manganese) is currently approved in Europe to control dry and wet bubble disease in mushrooms. However, it does not give 100% disease control (Arney et al., 2002) and is being phased out due to concerns over pathogen resistance and high residue levels in mushroom farm waste (Dragt et al., 1996). Typically Sporgon™ is applied onto the surface of the casing layer during irrigation and it is possible that this can lead to a concentration gradient within the casing layer (Grogan and Jukes, 2003), rendering it less effective. Vivando® (metrafenone) is approved for the control of mushroom cobweb disease, caused by Cladobotryum spp. However, it has been reported recently that some Cladobotryum spp have recently developed resistance to Vivando®. Biological control through the use of natural antagonistic microorganisms has been suggested as an alternative method to chemical pesticides for safer crop management. Bacillus species are one of the most predominant bacterial species that reside within mushroom compost (Ntougias et al., 2004). Members of these species have been well documented as effective antagonists of numerous plant pathogens. In addition, many Bacillus species are already regarded as non-pathogenic and some have been granted qualified presumption of safety (QPS) status by EFSA.

[0011] The present invention is directed to new bacterial strains, cultures and compositions for protecting against and inhibiting the growth of fungal pathogens in plant, soil or growing media, such as compost.

[0012] Summary of the Invention

[0013] According to a first aspect of the invention, there is provided a plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus velezensis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology.

[0014] Optionally, there may also be provided a plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus subtilis or Bacillus amyloliquefaciens strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white / cream, rough / wrinkled and circular colony morphology.

[0015] Still optionally, there is provided a plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus velezensis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology; and a Bacillus subtilis or Bacillus amyloliquefaciens strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white / cream, rough / wrinkled and circular colony morphology.

[0016] According to a preferred embodiment of this first aspect, the Bacillus velezensis strain is Bacillus velezensis MM223 having accession number NCIMB 42692 or a mutant or variant derived therefrom having anti-fungal activity. Additionally, the Bacillus subtilis strain is Bacillus subtilis MM232 having accession number NCIMB 43953 or a mutant or variant derived therefrom having anti-fungal activity. The anti-fungal activity may be against fungal pathogens, preferably plant or mushroom fungal pathogens. Optionally, the anti-fungal activity provides protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb, green mould and / or Fusarium head blight (FHB).

[0017] Optionally, the Bacillus velezensis strain has a 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1. Still optionally, the Bacillus velezensis strain has a 16S rRNA at least 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.5, 99.6, 99.7, 99.8, 99.9% identical to SEQ ID NO:1. In this manner the Bacillus velezensis strain comprises a 16S rRNA-encoding gene having >95% identity with SEQ ID NO: 1; preferably > 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identity with SEQ ID NO: 1.

[0018] Additionally, the Bacillus subtilis strain has a 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2. Optionally, the Bacillus subtilis strain has a 16S rRNA at least 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.5, 99.6, 99.7, 99.8, 99.9% identical to SEQ ID NO:2. In this manner the Bacillus subtilis strain comprises a 16S rRNA-encoding gene having >95% identity with SEQ ID NO: 1; preferably > 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identity with SEQ ID NO: 2.

[0019] Optionally, the Bacillus velezensis strain has a genome sequence defined in SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO:9. Still optionally, the Bacillus velezensis strain has a genome sequence at least 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identical to SEQ ID NO:9. In this manner the Bacillus velezensis strain comprises a genome having >95% identity with SEQ ID NO: 9; preferably > 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identity with SEQ ID NO: 9.

[0020] Advantageously, the anti-fungal bacterial compositions described herein may also have insect or fly pest control activity.

[0021] According to a second aspect of the invention, there is provided an isolated bacterial strain comprising Bacillus velezensis MM223 strain with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1; preferably comprising Bacillus velezensis MM223 strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO:9, more preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692 or a mutant or variant derived therefrom which inhibits the growth of plant fungal pathogens.

[0022] Advantageously, the strains described herein may also have insect or fly pest control activity. According to a third aspect of the invention, there is provided an isolated bacterial strain comprising Bacillus subtilis MM232 having accession number NCIMB 43953 or a mutant or variant derived therefrom which inhibits the growth of plant fungal pathogens.

[0023] Preferably, there is provided an isolated bacterial strain culture which inhibits the growth of plant fungal pathogens comprising Bacillus velezensis MM223 strain with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1, and Bacillus subtilis MM232 with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2. More preferably there is provided an isolated bacterial strain culture which inhibits the growth of plant fungal pathogens comprising Bacillus velezensis MM223 strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO:9, and Bacillus subtilis MM232 with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2.

[0024] According to a fourth aspect of the invention, there is provided an isolated bacterial strain culture which inhibits the growth of plant fungal pathogens comprising

[0025] Bacillus velezensis MM223 having accession number NCIMB 42692, and

[0026] Bacillus subtilis MM232 having accession number NCIMB 43953; or mutants or variants derived therefrom having anti-fungal activity.

[0027] Advantageously, the strain cultures described herein may also have insect or fly pest control activity. According to a fifth aspect of the invention, there is provided the use of the antifungal bacterial composition or the isolated bacterial strain or culture to confer improved fungal pathogen resistance to plant, soil or growing media comprising applying said composition to said plants, soil or growing media. Additionally, the use of the anti-fungal bacterial composition or the isolated bacterial strain or culture confers improved control of insect or fly pests, preferably sciarid fly pests, to the plant, soil or growing media. We have shown that the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention has larvicidal activity. In this manner, the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention may be used as a larvicide. Additionally, the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention may also be used as an adulticide.

[0028] According to a sixth aspect of the invention, there is provided a method for preparing augmented soil or growing media, preferably wherein the growing media is compost, the method comprising the steps of: providing the anti-fungal bacterial composition or the isolated bacterial strain or culture; providing soil or growing media; and inoculating the soil or growing media with the composition or isolated bacterial strain or culture.

[0029] According to a seventh aspect of the invention, there is provided an augmented soil or growing media, preferably wherein the growing media is compost, comprising the anti-fungal bacterial composition or the isolated bacterial strain or culture.

[0030] According to an eighth aspect of the invention, there is provided the use of augmented soil or growing media, preferably wherein the growing media is compost, as a nutrient source for farming, the use comprising the steps of: providing the augmented soil or growing media; providing at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed; cultivating the at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed in an environment comprising the augmented soil or growing media.

[0031] According to a ninth aspect of the invention, there is provided a method of biocontrol conferring improved pathogen resistance comprising topically applying the anti-fungal composition, isolated bacterial strain or culture to plants, soil or growing media. Additionally, the method of biocontrol also confers improved control of insect or fly pests.

[0032] According to a tenth aspect of the invention, there is provided a kit of parts for use in preparing augmented compost and / or farming, the kit of parts comprising the anti-fungal composition or the isolated bacterial strain or culture, preferably comprising instructions for use thereof.

[0033] According to an eleventh aspect of the invention, the anti-fungal compositions, strains, or strain cultures of the invention discussed herein comprising Bacillus velezensis MM223 may be used in insect pest control in the production of mushrooms. We have shown that the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention has larvicidal activity. In this manner, the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention may be used as a larvicide. Additionally, the antifungal bacterial composition or the isolated bacterial strain or culture of the invention may also be used as an adulticide. Advantageous aspects of the above embodiments are provided in the dependent claims. Brief Description Of The Figures

[0034] The present application will now be described with reference to the accompanying Figures and Examples in which:

[0035] Figure 1 provides tables showing the percentage inhibition results from in vitro screening of bacterial isolates against the four main pathogens of mushrooms (A) Lecanicillium fungicola', (B) Mycogone perniciosa', (C) Cladobotryum mycophilum', (D) Trichoderma aggressivum as well as to (E) Agaricus bisporus.

[0036] Figure 2 provides an image and description of the colonies formed by A) MM223 and B) MM232 on TSA media, accompanied by a morphological and biochemical assessment.

[0037] Figure 3 provides BLAST® results of MM223 Contig 11 (107,775 bases) when queried against the NCBI nucleotide database. Results revealed a high identity match (>99 %) to Bacillus velezensis, accession CP033054.1.

[0038] Figure 4 is a phylogenetic tree corresponding to Supplementary Figure 1 from Samaras et al., 2021 which confirms that B. velezensis (i.e. MM223) is distinct from both B. amyloliquefaciens and B. subtilis.

[0039] Figure 5 provides BLAST® results of the 16S sequencing results from MM232 when queried against the NCBI nucleotide database. Results revealed a high identity match (>99 %) to Bacillus subtilis.

[0040] Figure 6 provides a process overview of composting and cropping for A. bisporus, highlighting possible application points for MM223 and MM232 (dashed arrows).

[0041] Figure 7 In vitro assay images illustrating the efficacy of MM223 and MM232 against the common mushroom pathogens L. fungicola, M. perniciosa, C. mycophilum and T. aggressivum. Efficacy against the pathogens is displayed by zones of inhibition which are not apparent on the control plates.

[0042] Figure 8 provides results from in vitro bioassay, confirming that MM223 and MM232 were very efficient at controlling the growth of M. perniciosa (>60% inhibition); L. fungicola (>70% inhibition) and C. mycophilum (>60% inhibition) mycelium. Both MM223 and MM232 also inhibited the growth of T. aggressivum though at a lower level (>30% inhibition).

[0043] Figure 9 shows plate trials to test biocontrol agent MM223 efficacy against pathogen T. aggressivum. (A) Phase II compost with Agaricus bisporus spawn; Biocontrol MM223 added before pathogen; (B) Phase II compost with Agaricus bisporus spawn; Pathogen is added before biocontrol MM223; (C) Pathogen only added to plates of Phase II compost with Agaricus bisporus spawn. Figure 10 provides results from amplification of green mould caused by T. aggressivum detected in an environmental sample collected from the plate trial (2 wells per sample): (A) Phase II compost with Agaricus bisporus spawn; Biocontrol MM223 added before pathogen; (B) Phase II compost with Agaricus bisporus spawn; Pathogen added before Biocontrol MM223; (C) Phase II compost with Agaricus bisporus spawn; Only Pathogen added, (D) Phase II compost only with Agaricus bisporus spawn.

[0044] Figure 11 shows the percentage reduction of dry bubble disease by MM223 when L. fungicola was added to casing at concentration of 5.0 x 102cells / 1.5kg of casing / 0.09m2on day 5 of the cropping cycle. MM223 was applied at 109cells; 108cells; 107cells or 106cells / 1.5kg of casing / 0.09m2. PC: Sporgon™ was applied on day 5 at the recommended commercial concentration (1.2g / m2). Regime 1: MM223 applied on day -1 (compost), day 0, day 21 and day 29. Regime 2: MM223 applied on day -1 (compost), day 0, day 1, day 2, day

[0045] 20, 21 and day 30. Regime 3: MM223 added on day 0, day 1 , day 2, day 3, day 5 and day

[0046] 21. Regime 4: MM223 applied on day 6, day 11, day 15 and day 21.

[0047] Figure 12 shows the incidence of dry bubble disease per plot. Data presented is the averages and standard deviations of three replicates per treatment. L. f only: L. fungicola was applied at 102cells / 1.5kg of casing / 0.09m2on Day 4. Microcapsules containing MM223 and L. f: 10g of sodium alginate microcapsules containing MM223 were added to the compost and 5g to the casing at filling. L. fungicola was applied at 102cells / 1.5kg of casing / 0.09m2on Day 4.

[0048] Figure 13 shows the incidence of dry bubble disease per plot after application of MM223 formulated as both fresh endospores (diamonds) and spray dried endospores (squares). Endospores were applied at concentrations of 1.5 x1011spores / msq and 3.0 x 1011spores / msq. Spray dried endospores showed superior percentage inhibition of dry bubble disease with percentage inhibitions greater than 60% observed.

[0049] Figure 14 shows in vitro assay images illustrating the efficacy of MM223 and MM232 against the Fusarium graminearum, the causative pathogen of Fusarium head blight (FHB) in both wheat and barley crops.

[0050] Figure 15 A) the average number of dry bubbles per square meter of mushroom growing houses that had either MM223 or Sporgon™ applied to control disease. B) average yield of mushrooms per square meter.

[0051] Figure 16 The average number of dry bubbles per square meter over 12 months when Sporgon™ or MM223 were applied in 4 Irish commercial mushroom farms. Figure 17 Percentage reduction in viable sciarid fly (Lycoriella species) larvae after 16 h and 24 hr incubation in the presence of phosphate buffered saline (Control), fresh MM223 endospores and cell free MM223 fermentation supernatant.

[0052] Figure 18 compares average weekly sciarid fly numbers at multiple sites / commercial farms in the presence and in the absence of MM223 spray-dried endospores.

[0053] Detailed Description Of The Invention

[0054] In this specification, it will be understood that the terms ‘comprise’ ‘comprises’ or ‘comprising’ may be replaced by the terms ‘consists of’, ‘consisting of’, ‘consists essentially of’ or consisting essentially of’. Furthermore, the words comprises / comprising when used in this specification are to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0055] In this specification, it will be understood that ‘anti-fungal activity’ means the protection against fungal pathogens as described herein, involving the inhibition and reduction of fungal pathogen growth. In this manner, the anti-fungal bacteria or bacterial composition of the invention confers improved pathogen resistance in that it protects against and inhibits the growth of fungal pathogens. In this context, it acts as a biocontrol agent (BCA). The antifungal bacteria or bacterial composition of the invention may be applied to the compost and / or casing at any stage during mushroom production, for example as a pre-treatment step after spawn run, during the cropping cycle or between mushroom flushes (typically flushes 1-3) as shown in Figure 6.

[0056] In this specification, a ‘soil improver’ is defined as an Ell fertilising product the function of which is to maintain, improve or protect the physical or chemical properties, the structure or the biological activity of the soil to which it is added. Soil improvers can be organic (consisting “material 95% of which is of solely biological origin”) or inorganic (“a soil improver other than an organic soil improver”).

[0057] In this specification, reference to mushrooms, will be understood to cover all mushrooms, including brown and white mushrooms and specifically Agaricus bisporus. In this specification, the term anti-fungal activity embraces activity against plant pathogens and will be understood to provide protection against mushroom fungal pathogens, including Agaricus bisporus fungal pathogens.

[0058] In this specification, the term ‘insect pest control’ or ‘insect pest control agent’ will be understood to be any agent, including a strain, strain culture or bacterial composition, with insect pest control activity, preferably fly pest control activity, it will be understood that ‘pest control activity’ means the protection against pests, such as insect or flies, as described herein, involving the inhibition and reduction of insect or fly pest growth. Ideally, the insect pest control activity will provide protection against the main insect or fly pests for mushrooms (such as Agaricus bisporus). The main fly pests associated with commercial mushrooms production are dipteran (two-winged flies), such as the sciarid fly, including Lycoriella ingenua, Lycoriella castanescens, and / or Bradysia ocellaris. The pest control activity may be larvicidal and / or adulticidal activity. Larvicidal activity is essential in preventing damage during mushrooms development and growth, and advantageously this also prevents the development of adult flies which can be a significant factor in the spread of the various fungal pathogens discussed herein. This bacteria or bacterial composition of the invention with insect pest control activity (and thereby an insect pest control agent) may be applied to the compost and / or casing at any stage during mushroom production, for example as a pretreatment step after spawn run, during the cropping cycle or between mushroom flushes (typically flushes 1-3) as shown in Figure 6.

[0059] Furthermore, it will be understood that any mention of bacterial strains means viable strains, cells, spores, live spores and / or endospores. Endospores may be fresh endospores or spray dried endospores or freeze-dried endospores.

[0060] Growing media is a material other than soil in the ground in which plants and mushrooms are grown. In this specification, it will be understood that soil or growing media includes potting soils and other growing media. Growing media components are either organic or inorganic. Organic components include, but are not limited to: peat moss, bark, coconut coir, rice hulls, wood fibre, etc. Inorganic components include, but are not limited to: perlite, pumice, vermiculite, sand, hydrogel, etc. In the context of mushroom growth, growing media can include the compost and / or casing. In this specification, the term “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Percentage identity can be measured using software programs which are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI).

[0061] Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

[0062] For the avoidance of doubt, ‘MM232’, ’MM 232’, or ‘Bacillus subtilis MM232’ refers to the strain deposited at NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, under accession number NCIMB 43953 on 6 April 2022. Likewise, ‘MM223’, ’MM 223’, or ‘Bacillus velezensis MM223’ refers to the strain deposited at NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9Y, under accession number NCIMB 42692 on 21 November 2016.

[0063] Mushroom dry bubble disease, caused by L. fungicola, and wet bubble disease, caused by M. perniciosa, can cause significant yield losses for growers of the white button mushroom, A. bisporus. A. bisporus has little effective natural resistance to L. fungicola and M. perniciosa and control of these diseases have historically been through a combination of stringent hygiene and cultural practices, and though the use of chemicals, such as Sporgon WP 50 ™(prochloraz manganese). In order to reduce reliance on chemical pesticides, we have isolated a Bacillus velezensis strain and a Bacillus subtilis strain from mushroom compost that both demonstrate antagonistic activity against the four main diseases that affect mushroom production: dry bubble (caused by Lecanicillium fungicola), wet bubble (caused by Mycogene perniciosa), green mould (caused by Trichoderma aggressivum) and cobweb (caused by Cladobotryum spp.). These bacteria do not exhibit antagonistic activity against A. bisporus mycelium or fruiting bodies. We observed a reduction in disease severity in compost (and resultant mushrooms grown in said compost) treated with the strains of the invention.

[0064] In a general context, the present invention is directed to new bacterial strains, cultures and compositions for protecting against and inhibiting the growth of fungal pathogens in plant, soil or growing media, such as compost, preferably mushroom compost.

[0065] According to a first aspect of the invention, there is provided a plant, soil or growing media anti-fungal bacterial composition comprising, consisting of or consisting essentially of a Bacillus velezensis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology.

[0066] There may also be provided a plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus subtilis or Bacillus amyloliquefaciens strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white / cream, rough / wrinkled and circular colony morphology.

[0067] Optionally, there is provided a plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus velezensis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology; and a Bacillus subtilis or Bacillus amyloliquefaciens strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white / cream, rough / wrinkled and circular colony morphology.

[0068] Preferably, the Bacillus velezensis strain is Bacillus velezensis MM223 having accession number NCIMB 42692 or a mutant or variant derived therefrom having anti-fungal activity. Preferably, the Bacillus subtilis strain is Bacillus subtilis MM232 having accession number NCIMB 43953 or a mutant or variant derived therefrom having anti-fungal activity.

[0069] Accordingly, it will be understood that the anti-fungal composition may comprise Bacillus velezensis MM223 alone, or Bacillus subtilis MM232 alone, or both Bacillus velezensis MM223 and Bacillus subtilis MM232. Any such composition, comprising at least one of Bacillus velezensis MM223 and / or Bacillus subtilis MM232 or mutants or variants derived therefrom, has anti-fungal activity and is able to inhibit the growth of fungal pathogens in plant or growing media, such as compost.

[0070] Optionally, the anti-fungal bacterial composition comprises Bacillus velezensis MM223 having accession number NCIMB 42692 or mutants or variants derived therefrom having anti-fungal activity. Still optionally, the anti-fungal bacterial composition comprises Bacillus subtilis MM232 having accession number NCIMB 43953 or mutants or variants derived therefrom having anti-fungal activity.

[0071] Optionally, the anti-fungal bacterial composition comprises Bacillus velezensis MM223 having accession number NCIMB 42692 or mutants or variants derived therefrom having anti-fungal activity; and Bacillus subtilis MM232 having accession number NCIMB 43953 or mutants or variants derived therefrom having anti-fungal activity, preferably against plant or mushroom pathogens.

[0072] Optionally, Bacillus velezensis MM223 has accession number NCIMB 42692 with 16S rRNA sequence defined in SEQ ID NO: 1 :

[0073] GGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTT GCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGAC TGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGSTTGTTTGAACCGCATGGTTC AGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAG TTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATC GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAAT CTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG GATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTT GACGGTACCTAACCAGAAAGCCACGGCTAACTACGT or a 16S rRNA sequence with at least 99, 98, 97, 96, 95% identity to SEQ ID NO: 1 or a variant or mutant having all the identifying characteristics thereof.

[0074] Optionally, the Bacillus velezensis strain has a 16S rRNA at least 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.5, 99.6, 99.7, 99.8, 99.9% identical to SEQ ID NO:1. In this manner the Bacillus velezensis strain comprises a 16S rRNA-encoding gene having >95% identity with SEQ ID NO: 1 ; preferably > 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identity with SEQ ID NO: 1.

[0075] Optionally, the Bacillus velezensis strain has a genome sequence defined in SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO:9;

[0076] Still optionally, the Bacillus velezensis strain has a genome sequence at least 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identical to SEQ ID NO:9. In this manner the Bacillus velezensis strain comprises a genome having >95% identity with SEQ ID NO: 9; preferably > 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identity with SEQ ID NO: 9.

[0077] Optionally, Bacillus subtilis MM232 has accession number NCI MB 43953 with 16S rRNA sequence defined in SEQ ID NO: 2:

[0078] ATCTGAGAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGG GAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCA CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAAT TATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCT CAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAA TTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCG ACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG ATACCCCAGTAGTCAA or a 16S rRNA sequence with at least 99, 98, 97, 96, 95% identity to SEQ ID NO: 2 or a variant or mutant having all the identifying characteristics thereof.

[0079] Optionally, the Bacillus subtilis has a 16S rRNA at least 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.5, 99.6, 99.7, 99.8, 99.9% identical to SEQ ID NO:2. In this manner the Bacillus subtilis strain comprises a 16S rRNA-encoding gene having >95% identity with SEQ ID NO: 1 ; preferably > 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% identity with SEQ ID NO: 2.

[0080] Preferably, the anti-fungal bacterial composition comprising said strains provides protection against fungal pathogens, optionally mushroom fungal pathogens, causing at least one of wet bubble disease, dry bubble disease, cobweb, green mould and / or Fusarium head blight (FHB),

[0081] Preferably, the anti-fungal bacterial composition comprising said strains protects against fungal pathogens, optionally mushroom fungal pathogens, selected from Lecanicillium fungicola, Mycogene perniciosa, Trichoderma aggressivum, Cladobotryum spp. and / or Fusarium graminearum.

[0082] Optionally, the anti-fungal bacterial composition is used with growing media in the form of compost, preferably mushroom compost. In this manner, the composition provides protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb and / or green mould. These are typically fungal pathogens which are detrimental to mushroom growth and present in mushroom compost.

[0083] The control of insect or fly pests is an important component of producing high yielding, quality crops of such mushrooms. Unfortunately, the reliable control of such pests has been complicated by the development of resistance to pesticides, toxicity of pesticides to mushroom mycelium, persistent pesticide residues in the compost / casing material and concerns about exposure of workers to toxic pesticides. Current methods of controlling insect or fly pests include the use of larvicides and adulticides. Commonly used larvicides include diflubenzuron, cyromazine and methoprene while permethrin and dichlorvos are popular for use as adulticides. However, various problems can be associated with the use of larvicides and adulticides. Consequently, new types of pesticide preparations and methods of application are necessary for the continued production of commercial mushrooms.

[0084] The most significant insect pests of mushroom cultivation belong to the Order Diptera (twowinged flies) comprising three families:

[0085] Sciaridae (sciarids),

[0086] Phoridae (phorids); and

[0087] Cecidomyiidae (cecids).

[0088] In Ireland and the UK, the sciarid fly (including Lycoriella ingenua, Lycoriella castanescens, and Bradysia ocel laris) is particularly prevalent. These insect pests often affect the yield and quality of production in the mushroom growing industry, with heavy infestations leading to serious economic consequences. Mushroom yield losses associated with these insect pests can either be direct, through the action of the larvae feeding on the mycelia or fruiting bodies or indirect, as the adults are vectors of several fungal pathogens of mushrooms (including Trichoderma aggressivum, Lecanicillum fungicola and mushroom blotch disease). Control of both fly vector and fungal pathogen is important to reduce mushroom disease incidence.

[0089] Unexpectedly, we have established that Bacillus velezensis MM223, in addition to its antifungal activity, may be used as an insect / fly pest control agent. We have shown that the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention has larvicidal activity. Its use as an insect control agent directly impacts insect / fly larval growth, and indirectly positively impacts the anti-fungal activity by reducing adult vectors need for fungal pathogen transmission.

[0090] Advantageously, the bacterial compositions, strains and strain cultures of the invention discussed herein comprising Bacillus velezensis MM223 have both anti-fungal activity and insect pest control activity, specifically an insect or fly pest control agent. According to one embodiment, the bacterial compositions, strains and strain cultures of the invention discussed herein comprising Bacillus velezensis MM223 may be used as a sciarid fly, including Lycoriella ingenua, Lycoriella castanescens, and Bradysia ocellaris, control agent. We have shown that the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention has larvicidal activity. In this manner, the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention may be used as a larvicide or adulticide.

[0091] The bacterial strains and bacterial composition of the invention may involve delivery means such as cells, spores, live spores and / or endospores. In this manner, the bacterial strains and bacterial composition of the invention may be provided in spore form, as fresh or live spores. Alternatively, the bacterial strains and bacterial composition of the invention may be provided as endospores, including fresh endospores or spray dried endospores or freeze- dried endospores. Still, alternatively, the bacterial strains and bacterial composition may also be encapsulated, for example in calcium alginate beads or other similar medium.

[0092] According to a preferred embodiment, the anti-fungal bacterial composition comprises bacterial strains provided in endospore form, preferably as a dried endospore powder. Optionally, the anti-fungal bacterial composition is in the form of a freeze-dried or spray-dried endospore powder. The powder may be a wettable powder.

[0093] In contrast to Sporgon™ (conventional chemical fungicide in wettable powder form), the antifungal bacterial compositions or bacterial strains of the invention are organic, non-toxic, and non-pathogenic with no toxic effects on mushrooms. Advantageously, the anti-fungal bacterial compositions or bacterial strains of the invention aim to reduce reliance on conventional crop protection fungicide products.

[0094] It will be understood that the anti-fungal bacterial compositions or bacterial strains of the invention may be used alone or together with conventional crop protection products, such as insecticide and fungal spore product in wettable form Botanigard®.

[0095] Optionally, the endospores or endospore powder may be re-suspended in water or embedded in alginate beads prior to application to the plant, soil or growing media.

[0096] Optionally, the endospores or endospore powder may be encapsulated in alginate beads, preferably sodium or calcium alginate beads, optionally 1 %-10% sodium or calcium alginate beads. Most commercially available biocontrol agents or anti-fungal agents will comprise additional carriers / excipients. Advantageously, the anti-fungal composition of the invention does not comprise any additional excipients, such as carriers, dispersants etc. In this manner, the anti-fungal composition comprises approximately 100% spray dried endospores or endospore powder.

[0097] The anti-fungal composition or culture comprising either Bacillus velezensis MM223 alone or Bacillus subtilis MM232 alone has been shown to be effective, while a composition comprising both Bacillus velezensis MM223 or Bacillus subtilis MM232 is similarly effective. Furthermore, the two strains Bacillus velezensis MM223 or Bacillus subtilis MM232 do not inhibit each other. In particular, neither Bacillus velezensis MM223 nor Bacillus subtilis MM232 significantly inhibited the growth of the other strain.

[0098] Optionally, it will be understood where both bacterial strains are present in the anti-fungal composition or culture, they are present in substantially equal amounts, or for example 50:50, 75:25 or any other suitable % wt ratio thereof.

[0099] According to a second aspect of the invention, there is provided an isolated bacterial strain comprising a Bacillus velezensis strain with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1; preferably comprising a Bacillus velezensis strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO:9; more preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692 or a mutant or variant derived therefrom, having anti-fungal activity and which inhibits the growth of plant fungal pathogens.

[0100] According to a third aspect of the invention, there is provided an isolated bacterial strain comprising a Bacillus subtilis strain with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO:2; preferably comprising a Bacillus subtilis MM232 having accession number NCIMB 43953 or a mutant or variant derived therefrom having anti-fungal activity with anti-fungal activity and which inhibits the growth of plant fungal pathogens.

[0101] According to a fourth aspect of the invention, there is provided an isolated bacterial strain culture which inhibits the growth of plant fungal pathogens comprising a Bacillus velezensis strain with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1, and Bacillus subtilis strain with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2; preferably comprising a Bacillus velezensis strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 95% identical to SEQ ID NO:9, and Bacillus subtilis strain with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2; more preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692 or a mutant or variant derived therefrom having anti-fungal activity, andBacillus subtilis MM232 having accession number NCIMB 43953 or a mutant or variant derived therefrom, having anti-fungal activity.

[0102] Preferably, the bacterial strains or culture protects against plant or mushroom fungal pathogens selected from Lecanicillium fungicola, Mycogene perniciosa, Trichoderma aggressivum, and / or Cladobotryum spp. In this manner, the bacterial strains or culture provides protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb and / or green mould. These fungal pathogens are typically detrimental to mushroom growth and present in mushroom compost. We have also found that both MM223 and MM232 are effective against the Fusarium graminearum, the causative pathogen of Fusarium head blight (FHB) in both wheat and barley crops.

[0103] Optionally, the isolated bacterial strain culture comprises

[0104] Bacillus velezensis MM223 having accession number NCIMB 42692 with 16S rRNA sequence defined in SEQ ID NO: 1 or a 16S rRNA sequence at least 95% identical to SEQ ID NO:1 ; and / or

[0105] Bacillus subtilis MM232 having accession number NCIMB 43953 with 16S rRNA sequence defined in SEQ ID NO: 2 or a 16S rRNA sequence at least 95% identical to SEQ ID NO: 2. Optionally the sequences may have least 99, 98, 97, 96, 95, 95, 95.5 96, 96.5 97, 97.5 98, 98.5 99, 99.5, 99.6, 99.7, 99.8, 99.9% % identity to SEQ ID NO: 1 or 2 or be a variant or mutant having all the identifying characteristics thereof.

[0106] According to a fifth aspect of the invention, there is provided the use of the anti-fungal composition or the isolated bacterial strain or culture to confer improved fungal pathogen resistance to plant, soil or growing media comprising applying said composition to said plants, soil or growing media.

[0107] Advantageously and additionally, there is provided the use of the anti-fungal bacterial composition or the isolated bacterial strain or culture to confer improved control of insect or fly pests, preferably sciarid fly pests, to the plant, soil or growing media. In this manner the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention utilises its larvicidal activity and acts as a larvicide.

[0108] In this manner, the anti-fungal bacterial compositions or bacterial strains of the invention may be used as a soil conditioner or as a soil additive to composts, soils and technical materials.

[0109] Optionally, the isolated bacterial strain is in the form of an endospore, preferably a dried endospore powder. Optionally, the isolated bacterial strain is in the form of a freeze-dried or spray-dried endospore powder. The powder may be a wettable powder.

[0110] Optionally, the growing media is compost, preferably mushroom compost. In this aspect, the anti-fungal composition or the isolated bacterial strain or culture is applied to the mushroom compost to form augmented compost. In this manner, the bacterial strains may be resuspended in water and delivered to the compost via a conventional water system, such as a watering tree or powered dosing system (such as Dosatron). Still optionally, the anti-fungal bacterial composition comprises bacterial strains are provided in endospore form and may be applied to compost from approximately 1x109endospores per meter squared of application area to approximately 9x1011endospores per meter squared of application area, preferably from approximately 1.0 x 101° CFU / ml endospores per meter squared of application area to approximately 5.0 x1011CFU / ml endospores per meter squared of application area.

[0111] Preferably, application to mushroom compost, is a pre-treatment step after spawn run or between mushroom flushes as shown in Figure 6. For example, during mushroom compost production the anti-fungal composition or the isolated bacterial strain or culture may be applied at or after Phase III at the end of spawn run. Additionally, the anti-fungal composition or the isolated bacterial strain or culture may be applied once or twice during the mushroom cropping cycle when watering during case run and between mushroom flushes also shown in Figure 6.

[0112] While the present invention has application in mushroom farming, in particular farming of Agaricus bisporus, it is clear that an anti-fungal composition comprising Bacillus velezensis MM223 and / or Bacillus subtilis MM232 could be usefully added to any soil or growing media such as compost, fertilizer or another fungus or plant farming medium or growth medium or even plants themselves; to improve the farming of any of a wide variety of fungi and / or plants, including for example edible mushrooms, button mushrooms (Agaricus bisporus), Portobello mushrooms (Agaricus bisporus), straw mushrooms (Volvariella volvacea), oyster mushrooms (Pleurotus ostreatus), shiitakes (Lentinula edodes), enokitake (Flammulina spp.), fruit, bananas, apples, pears, oranges, melons, strawberries, nuts, vegetables, potato, carrot, legume pulses, cassava, groundnuts, beans, peas, green vegetables, broccoli, salad vegetables, celery, lettuce, sweet potatoes, grasses, rice, wheat, sugarcane, maize, corn, soybean, sunflower rapeseed, mustard. We have found that MM223 and MM232 are also effective against the Fusarium graminearum, the causative pathogen of Fusarium head blight (FHB) in both wheat and barley crops.

[0113] Furthermore, it is clear that the anti-fungal composition comprising Bacillus velezensis MM223 and / or Bacillus subtilis MM232 or a mutant or variant derived therefrom having antifungal activity could be used to augment, or in the preparation of, compost, soil, fertilizer or another fungus or plant farming medium for sale.

[0114] In this manner, the anti-fungal composition or the isolated bacterial strain or culture may be applied to soil or growing media to form augmented soil or growing media for sale. According to a sixth aspect of the invention, there is provided a method for preparing augmented soil or growing media, the method comprising the steps of: providing the anti-fungal composition, isolated bacterial strain or culture; providing soil or growing media; and inoculating the soil or growing media with said anti-fungal composition, isolated bacterial strain or culture.

[0115] Optionally, the growing media is compost, preferably mushroom compost.

[0116] Optionally, the isolated bacterial strain is in the form of an endospore, preferably a dried endospore powder. Optionally, the isolated bacterial strain is in the form of a freeze-dried or spray-dried endospore powder. The powder may be a wettable powder.

[0117] Optionally, the method for preparing augmented soil or growing media, such as compost is carried out under temperature-controlled conditions.

[0118] Optionally, the method for preparing augmented soil or growing media, such as compost is carried out under aerobic conditions.

[0119] Optionally, the method for preparing augmented soil or growing media, such as compost is carried out under temperature-controlled and aerobic conditions.

[0120] Optionally, the step of inoculating the soil or growing media, such as compost with the bacterial composition is carried out under temperature-controlled conditions.

[0121] Optionally, the step of inoculating the soil or growing media, such as compost with the bacterial composition is carried out under aerobic conditions.

[0122] Optionally, the step of inoculating the soil or growing media, such as compost with the bacterial composition is carried out under temperature-controlled and aerobic conditions.

[0123] According to a seventh aspect of the invention, there is provided augmented soil or growing media, comprising the anti-fungal composition, the isolated bacterial strain or culture. Optionally, the growing media is compost, preferably mushroom compost.

[0124] Optionally, the isolated bacterial strain is in the form of an endospore, preferably a preferably a dried endospore powder. Optionally, the isolated bacterial strain is in the form of a freeze- dried or spray-dried endospore powder. The powder may be a wettable powder.

[0125] According to an eighth aspect of the invention, there is provided the use of augmented soil or growing media as a nutrient source for farming, the use comprising the steps of: providing the augmented soil or growing media; providing at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed; cultivating the at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed in an environment comprising the augmented soil or growing media.

[0126] Optionally, the growing media is compost, preferably mushroom compost.

[0127] Optionally, the isolated bacterial strain is in the form of an endospore, preferably a dried endospore powder. Optionally, the isolated bacterial strain is in the form of a freeze-dried or spray-dried endospore powder. The powder may be a wettable powder.

[0128] According to a preferred aspect there is provided the use of augmented soil or growing media as a nutrient source for farming, the use comprising the steps of: providing the augmented soil or growing media; providing at least one Agaricus bisporus mushroom, mycelium, spawn, and / or spore; cultivating the at least one Agaricus bisporus mushroom, mycelium, spawn, and / or spore, in an environment comprising the augmented soil or growing media.

[0129] Optionally, the growing media is compost, preferably mushroom compost.

[0130] Optionally, the isolated bacterial strain is in the form of an endospore, preferably a preferably a dried endospore powder. Optionally, the isolated bacterial strain is in the form of a freeze- dried or spray-dried endospore powder. The powder may be a wettable powder.

[0131] According to a ninth aspect of the invention, there is provided a method of biocontrol conferring improved pathogen resistance comprising topically applying the anti-fungal composition, isolated bacterial strain or culture to plants, soil or growing media. According to a tenth aspect of the invention, there is provided a kit of parts for use in preparing augmented compost and / or farming, the kit of parts comprising the anti-fungal composition or the isolated bacterial strain or culture, preferably comprising instructions for use thereof.

[0132] The inventors have also found that the addition of the anti-fungal composition or culture of the invention comprising Bacillus velezensis MM223 and / or Bacillus subtilis MM232 to compost to form augmented compost, also led to a higher yield of heavier mushrooms. In this manner, the compositions, bacterial strains and cultures of the invention are characterized by having a high antifungal activity, particularly against phytopathogenic fungi given its capacity to produce antagonist substances, as well as a good capacity to stimulate plant growth. Additionally, and advantageously, the composition, bacterial strain or cultures of the invention may also be used to improve yield of the plant or plant grown in the augmented soil or growing media. Ideally, the composition, bacterial strain or culture of the invention may be used as a soil conditioner.

[0133] According to an eleventh aspect of the invention, there is provided a method for enhancing plant growth or yield comprising topically applying the anti-fungal composition, isolated bacterial strain or culture of the invention to plants, soil or growing media.

[0134] According to a twelfth aspect of the invention, there is provided a method for insect pest control comprising topically applying the anti-fungal compositions, isolated bacterial strains or cultures of the invention Bacillus velezensis MM223 described herein to plants, soil or growing media. This is based on our findings that the anti-fungal bacterial composition or the isolated bacterial strain or culture of the invention has larvicidal activity. Ideally, this method for insect pest control is used in the production of mushrooms. According to one embodiment, the method may be used for fly pest control agent, specifically sciarid fly control, including Lycoriella ingenua, Lycoriella castanescens, and Bradysia ocellaris.

[0135] According to a thirteenth aspect of the invention, there is provided an insect pest control strain, strain culture or bacterial composition comprising a gram-positive strain, catalase positive, oxidase positive Bacillus velezensis strain strain with a white, rough / wrinkled and irregular colony morphology. a Bacillus velezensis strain with a 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95%, preferably 99.9%, identical to SEQ ID NO:1; a Bacillus velezensis strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 97% identical to SEQ ID NO:9; or

[0136] Bacillus velezensis MM223 having accession number NCIMB 42692.

[0137] In this manner, the insect pest control strain, strain culture or bacterial composition may be used as a larvicide or adulticides.

[0138] According to a preferred embodiment, the strain, strain culture or bacterial composition comprises Bacillus velezensis MM223 having accession number NCIMB 42692.

[0139] It will be understood that the insect pest control strain, strain culture or bacterial composition is ideally a fly pest control agent, preferably a sciarid fly control agent for the for the protection against sciarid fly species including Lycoriella ingenua, Lycoriella castanescens, and / or Bradysia ocellaris.

[0140] As described in relation to the anti-fungal composition, the insect pest control strain may be provided in endospore form, preferably as a dried endospore powder, more preferably as a freeze-dried or spray-dried endospore powder.

[0141] Other aspects described in relation to the anti-fungal composition, such as delivery means and method of use etc will be applicable to this aspect.

[0142] Examples

[0143] Summary

[0144] We have isolated a large number (>500) bacterial and fungal strains from mushroom compost at different stages of its production (constituting the MBio Microbial Biobank). A further bacterial strain was isolated from the fruiting body of a mushroom that was symptomatic for dry bubble disease. All of the bacterial strains from the MBio Microbial Biobank have been identified by DNA sequencing. In silico research into biocontrol agents led to the identification of a proportion of the bacterial strains (n=136) within the MM Microbial Biobank as possibly having efficacy against four fungal pathogens of commercial mushrooms, Agaricus bisporus. These strains have been assessed for their antagonistic effect against the four main fungal pathogens of mushrooms in vitro. A summary of the results from the in vitro screening assessment are presented in Figure 1A-1D. Those bacterial isolates that scored highly in terms of percentage inhibition of pathogen growth were selected for further assessment of their effect on the mycelial growth of A. bisporus, results shown in Figure 1E.

[0145] Two bacterial strains have been selected for more detailed analysis to form a biocontrol solution due to their good control of the four main mushroom pathogens, and their negligible effects on the growth of A. bisporus.

[0146] 1. Materials and Methods

[0147] 1.1. Isolation of bacteria from mushroom compost

[0148] Isolation of bacteria from compost was as follows: 10 g of Phase 1 mushroom compost was added to 90 ml of Maximum Recovery Diluent (MRD) and agitated for 30 mins at 150 rpm. Serial dilutions were prepared and spread onto different media agar plates. These were incubated at 25°C, 37°C and 55°C for 24-48 hrs and any colonies that appeared on the plates were sub-cultured. Pure colonies were stored as 50% glycerol stocks at -80°C for further analysis.

[0149] Catalase and oxidase activities of all isolates were assayed using hydrogen peroxide breakdown and tetramethyl- or dimethyl p-phenlyenediamine. Gram characterisation was conducted using a commercially available Gram staining kit.

[0150] 1.2. Purification and amplification of DNA

[0151] Total genomic DNA was extracted from bacterial isolates using the Invitrogen Purelink Genomic DNA kit according to the manufacturer’s instructions. PCR amplification of the 16S rRNA gene was conducted for sequence identification of bacterial isolates. The DNA fragments from compost isolates were amplified using the either the 16S rDNA universal primers 27F and 1492R (Jiang et al., 2006) or the 16S rDNA primers 341 F and 785R (Klindworth et al., 2013). Sequencing was performed in both forward and reverse. The primer set containing 341 F and 785R is a popular choice for bacteria profiling.

[0152] Bacterial forward primer Bac27F (5 -AGAGTTTGGATCMTGGCTCAG-3') (SEQ ID No: 3); and

[0153] Universal reverse primer Univ1492R (5-CGGTTACCTTGTTACGACTT-3') (SEQ ID No: 4) Bacterial forward primer 341 F (5'- CCTACGGGNGGCWGCAG -3') (SEQ ID No: 5); and Bacterial reverse primer 785R (5'- GACTACHVGGGTATCTAATCC -3') (SEQ ID No: 6)

[0154] The PCR was performed using a commercial high-fidelity PCR kit (such as the Q5® Hot Start High Fidelity master mix; New England Biolabs® Inc.), in a final volume of 25pl per reaction. The PCR condition used were the following: initial denaturation at 98°C for 60 seconds; 35 cycles of denaturation (98°C, 10 seconds), annealing (55°C, 20 seconds) and 40 DNA extension (72°C, 35 seconds); final DNA extension at 72°C for 2 minutes. The amplicons obtained were sequenced both in forward and reverse.

[0155] This resulted in a ~1.5 Kbp long consensus sequence being obtained for the primer pair, 27F and 1492R and a -0.5 Kbp long consensus sequence obtained for the primer pair, 341 F and 785R. The sequences were queried on Blast® nucleotide database (for example on the website of the National Center for Biotechnology Information of the US National Library of Medicine available at https: / / blast.ncbi.nlm.nih.aov / Blast.cgi) and identifications were considered reliable for results matching >98% of identity.

[0156] Two strains have been selected for more detailed analysis to form a biocontrol solution due to their good control of the four main mushroom pathogens, and their negligible effects on the growth of A. bisporus. Following the isolation and selection of these 2 strains of interest, MM232 and MM223, DNA was extracted from a pure culture and sent to Source BioScience, Cambridge for 16S ribosomal sequencing. For MM223, DNA was extracted from a pure culture and sent to Eurofins Genomic Services, Germany for full genome sequencing and de novo assembly. The assembled sequences produced 44 contigs ranging in size from 1 ,029,743 to 107 bases with a minimum coverage depth of 0.28x. The largest contigs were queried on BLAST® nucleotide database for sequence homology. Homology results for contig 11 (107,775 bases) are shown in Figure 3 (MM223). The results from the BLAST® nucleotide database search for MM232 are shown in Figure 5. 1.3. In vitro assessment of the efficacy of bacterial biocontrol agents MM223 and MM232 against L. fungicola, M. perniciosa, C. mycophilum and T. aggressivum and Fusarium graminearum

[0157] The agar plug diffusion method was used to highlight the ability of used to highlight the antagonism between microorganisms (Elleuch et al., 2010).

[0158] Mycoparasites were cultured in PDA plates: Cladobotryum mycophilum (4-5 days); Lecanicillium fungicola (7-9 days); Mycogone perniciosa and Fusarium graminearum (5-7 days); Trichoderma aggressivum (4-5 days). The plates were incubated at 25°C.

[0159] TSA media plates of each of the bacterial isolates were prepared by tightly streaking a single pure colony on to the TSA media plate surface. After incubation for 16 hrs at 37°C, the plates were removed from the incubator.

[0160] A sterile inoculating loop was then used to scrape a loopful of mycelia from a pure plate culture (PDA) of the mycoparasite under investigation. The mycelia were then resuspended in 5 mL of sterile water. The samples were then vortexed before spreading onto fresh PDA plates, using a sterile L-shaped spreader.

[0161] In order to perform the assay, a sterile cork borer was used to aseptically remove an 8 mm 0 sterile agar-plot or cylinder from the centre of the PDA plate containing the mycoparasite under investigation. This cylinder was then replaced by an 8 mm 0 sterile agar-plot or cylinder that was aseptically removed from the TSA media plates containing the bacterial isolate that had been incubated for 16 hrs at 37°C.

[0162] For the control plates, an 8 mm 0 sterile punch was used to remove an agar-plot or cylinder from a TSA media plate that had not been inoculated with any microorganism.

[0163] The plates were then incubated at 25°C for 5 days. Any antimicrobial activity of the microbial secreted molecules was detected by the appearance of zones of inhibition around the central agar plug (Figure 7)

[0164] Biocontrol efficiency was quantified by measuring the percentage of inhibition (PI) of mycelia growth when fungal mycoparasites were co-cultured with biocontrol agents according to the formula: PI (%) = (FR - AR) / FR x 100, where FR represents the fungal growth radius (cm) of a control culture and AR the fungal growth radius distance (cm) in the direction of bacterial bio control growth (Yuan and Crawford, 1995). Four replicates per combination of isolate and fungicide concentration were conducted, and the experiments were carried out in duplicate. Results are shown in Figure 8 and Figure 14. 1.4. Microcosm assessment to confirm the efficacy of Bacillus velezensis strain MM 223 against T. aggressivum

[0165] Phase II compost (50g) was placed into a glass petri dish (90mm x 15mm) and 4x A. bisporus spawn was placed in a square formation within the dish. Three different trials were conducted to investigate the efficacy of MM223 endospores against T. aggressivum at spawn run: i) MM223 added to Phase II compost prior to T. aggressivum inoculation; ii) MM223 added to Phase II compost post T. aggressivum inoculation; and iii) T. aggressivum only. Specifically, 250pl of T. aggressivum solution (1 x 108CFU / ml) and / or 5ml of MM223 solution were added to the compost. All trials were run in triplicates with a total of 9 plates for this investigation. The plates were kept in a dark container at room temperature for 21 days. At end of the trial, DNA was extracted from the compost and amplified using primers targeting an arbitrary T. aggressivum sequence:

[0166] Th-F (CGGTGACATCTGAAAAGTCGTG) (SEQ ID No: 7); and

[0167] Th-R (TGTCACCCGTTCGGATCATCCG) (SEQ ID No: 8) in a 50pl reaction mixture (25pl DreamTaq, 1 l forward primer, 1 l reverse primer, 5pl DNA template and 18pl PCR water), using a Biorad CFX 96 Real Time System and the following amplification steps: Denaturation 95°C for 3 minutes, 95°C for 30 seconds, annealing temperature at 59°C for 30 seconds for 35 cycles and extension time at 72°C for 1 min and final extension at 72°C for 10 min. Amplified DNA was then visualised on a 2% agarose gel. Results are shown in Figures 9 and 10.

[0168] 1.5. Microcosm assessment to confirm the efficacy of Bacillus velezensis strain MM223 against dry bubble disease of button mushrooms

[0169] Small scale microcosms were used to test the efficacy of the top performing bacterial isolate, MM223, against mushroom dry bubble disease at a trial farm in Co. Monaghan. Specifically, 3kg of spawned mushroom compost were filled into containers of dimensions 26cm (L) x 34cm (W) x 13cm (H). This was overlaid with 1.5kg of a peat- based casing substrate. The punnets of compost and casing were incubated in a climate chamber with controlled temperature, CO2 and humidity.

[0170] Spores of a virulent L. fungicola strain (originally isolated from a dry bubble sampled on a Monaghan Mushrooms farm) were inoculated in water onto the casing layer at 102-105 / 0.09m2on day 4 of the cropping cycle. Sporgon™ was applied on day 4 in 0.1 L of water at a farm rate of 1.2g / m2, as recommended by the manufacturer (BASF), as a positive control. Healthy mushrooms and dry bubbles were harvested from all three mushroom flushes, and mushroom yield and quality assessed. Vegetative MM223 cells were added in water to the compost at filling and to the casing at different concentrations (106- 109cells / 1.5kg of casing / 0.09m2per plot in each watering of 0.15L) and at different time points (combination of days from 0 - 15, after flush 1 and after flush 2) that corresponded to normal commercial watering regimens (Table 1).

[0171] Table 1 Formulations, concentrations and application regimen of the Bacillus velezensis strain MM223 trialled at microcosm level.

[0172] A formulation for MM223 was developed consisting of MM223 encapsulated in 2% calcium alginate beads. 10g of dried beads were mixed throughout the compost and 5g were mixed throughout the casing to give 6.9 x 109cells / 1.5kg casing / 0.09m2(Table 1). Results are shown in Figures 11 and 12. Example 1 : Isolation, identification and characterisation of Bacillus strain MM223

[0173] Biocontrol strain MM223 was isolated from phase I compost (composed of fermented wheat straw, horse manure, chicken litter) from a bunker at Carbury Compost, Co. Kildare, Ireland. MM223 was isolated using TSA media plates, which had been incubated at 25°C for 48hrs. Individual colonies were re-streaked onto TSA plates to obtain pure bacterial cultures. The colony and cellular morphology of MM223 were assessed and it was determined to be a gram positive, catalase positive and oxidase positive Bacillus (Figure 2).

[0174] Initial sequencing of the 16S ribosomal RNA indicated that MM223 is a Bacillus amyloliquefaciens strain. On the 16thNovember 2016, MM223 was deposited under the Budapest Treaty in NCIMB (Accession number: NCIMB 42692) as a Bacillus amyloliquefaciens. However, in June 2021 the full genome of MM223 was sequenced in order to fully confirm taxonomy. There are 4 main groups in the Bacillus subtilis group that are closely related: Clade 1 (B. subtilisy Clade 2 (8. amyloliquefaciens ‘operational group’ comprising B. amyloliquefaciens, B. velenzsis and B. siamensisy Clade 3 (8. licheniformusy and Clade 4 (8. pumilus) (Fan et al., 2017). The full genome sequencing of MM223 produced 44 contigs ranging in size from 1 ,029,743 to 107 bases with a minimum coverage depth of 0.28x. The largest contigs were queried on BLAST® nucleotide database for sequence homology, all showing 96-100% identity to 8. velezensis. Homology results for one of the larger contigs (contig 11, 107,775 bases) are shown in Figure 3. This provides evidence that MM223 is from the B. velezensis Clade 2 group. Based on this information on 10 August 2021, NCIMB reclassified MM223 NCIMB 42692 as B. velezensis.

[0175] BASF commercial biofungicide Serifel®, a 8. amyloliquefaciens strain MBI600, was originally registered with the United States Environmental Protection Agency (US EPA) as 8. subtilis strain MBI600. Further taxonomy characterisation was conducted by Samaras et al. in 2021 using whole genome proteome data from MBI600 and the proteomes of 147 selected Bacillus species. MBI600 strain was used as a reference point for best reciprocal Blastp hits against the other proteomes, which resulted in 2,317 core proteins shared between all species. The proteins were then grouped according to function and aligned. The alignments were concatenated to one protein super-alignment and used to compute a Neighbour Joining tree (Figure 4). Interestingly, the resulting tree indicates that MBI600 is a member of the B. subtilis subsp. subtilis Clade 1 evolutionary group (see arrow referencing MBI600 in Figure 4) and not a member of the B. amyloliquefaciens Clade 2 ‘operational group’. These results may be derived from the different analytical techniques used. In any event, these results confirm our findings that MM223, a B. velenzsis strain, is in a different clade from MBI600.

[0176] Example 2: Isolation, identification and characterisation of Bacillus strain MM232

[0177] Biocontrol strain MM232 was isolated from phase I compost (composed of fermented wheat straw, horse manure, chicken litter) from a bunker at Carbury Compost, Co. Kildare, Ireland. MM223 was isolated using TSA media plates, which had been incubated at 25°C for 48hrs. Individual colonies were re-streaked onto TSA plates to obtain pure bacterial cultures. The colony and cellular morphology of MM232 were assessed and it was determined to be a gram positive, catalase positive and oxidase positive Bacillus (Figure 2).

[0178] Initial sequencing of the 16S ribosomal RNA indicated that MM232 is a Bacillus subtilis strain. On the 23rd March 2022, MM232 was deposited under the Budapest Treaty in NCIMB (Accession number: NCIMB 43953) as a Bacillus subtilis.

[0179] Example 3: In vitro assessment of the efficacy of bacterial isolates, including MM223 and MM 232, against Mycogene perniciosa, Lecanicillium fungicola, Trichoderma aggressivum, Cladobotryum mycophilum and fungal pathogen Fusarium graminearum

[0180] In silico research into biocontrol agents led to the identification of a proportion of the bacterial strains (n=136) within the MM Microbial Biobank as possibly having efficacy against fungal pathogens of commercial mushrooms, Agaricus bisporus. These bacterial strains were assessed for the in vitro efficacy against the four most prevalent fungal pathogens of A. bisporus'. M. perniciosa, L. fungicola, T. aggressivum, C. mycophilum.

[0181] Table 2 Percentage inhibition in vitro of two Bacillus strains from the MBio Microbial Biobank (MM223 and MM232) against four fungal mushroom pathogens, Mycogene perniciosa, Lecanicillium fungicola, Trichoderma aggressivum, Cladobotryum mycophilum

[0182]

[0183] In vitro bioassay results confirmed that MM223 and MM232 were very efficient at controlling the growth of M. perniciosa (>50% inhibition); L. fungicola (>60% inhibition) and C. mycophilum (>50% inhibition) mycelium (Figures 8 and 9). Both MM223 and MM232 also inhibited the growth of T. aggressivum though at a lower level (>30% inhibition) (Figures 7 and 8).

[0184] Figure 14 shows in vitro assay images illustrating the efficacy of MM223 and MM232 against the Fusarium graminearum, the causative pathogen of Fusarium head blight (FHB) in both wheat and barley crops.

[0185] Example 4: Microcosm assessment to confirm the efficacy of Bacillus velezensis strain MM223 against green mould caused by T. aggressivum

[0186] Trichoderma spp. parasitise the mycelial form of Agaricus, so microcosm assessment on Phase 3 (mycelial colonisation) was carried out. The other pathogens ( / W. perniciosa, L. fungicola and C. mycophilum) parasitize the fruiting body stage, so microcosm growing trials were carried out in Example 3.

[0187] Strictly in terms of visual symptoms, the application of MM223 freeze-dried endospores to Phase II compost prior to T. aggressivum inoculation resulted in a reduction in disease severity relative to the control (Pathogen only). The amount of green mould present on the compost appeared to be less than that present on the control (Figure 9). The presence of T. aggressivum in the compost was confirmed with the use of PCR (Figure 10). The results indicate that when MM223 is added before the pathogen, T. aggressivum is not only visually absent from the plates, but it cannot be detected by PCR either.

[0188] On the other hand, the application of biological control to compost previously inoculated with T. aggressivum spores seemed to have no effect on the suppression of green mould disease (Figure 10), confirming the importance of pre-inoculation with MM223. Example 5: Microcosm trials to confirm the efficacy of Bacillus velezensis strain MM223 against dry bubble disease of mushrooms

[0189] MM223 (in cell form) was tested at microcosm scale for biocontrol efficacy against an aggressive strain of L. fungicola, which was obtained from a dry bubble from an Irish farm. The efficacy of MM223 against mushroom dry bubble disease was quantified as the percentage reduction in disease compared to no control (either chemical or biological) and was compared to that of Sporgon™ (positive control).

[0190] Different MM223 application rates and points were assessed in 10 microcosm trials. Data indicated that MM223 controlled dry bubble disease when applied to the mushroom substrate between 106to 109cfu / / 1.5kg of casing / 0.09m2. Results showed dry bubble inhibition up to 90% with 106cfu / / 1.5kg of casing / 0.09m2when compared to positive control (Figure 11).

[0191] Example 6 - Delivery means

[0192] Calcium alginate bead delivery

[0193] A new formulation for MM223 has been developed comprising of MM223 encapsulated in calcium alginate beads in which L. fungicola was applied at 102cells / 1.5kg of casing / 0.09m2on Day 4; and 10g of calcium alginate microcapsules were added to the compost and 5g to the casing at filling. L. fungicola was applied at 102cells / 1 ,5kg of casing / 0.09m2on Day 4. The broth from a fermentation of MM223, yielding 1012cells / L, was centrifuged at 3500 rpm for 2 hrs in order to obtain a cell pellet. The supernatant was discarded and the pellet was mixed with a 3% w / v solution of sterile sodium alginate solution at a ratio of 2:5 (v / v). The 3% w / v sodium alginate / MM223 mixture was then injected slowly into a stirred solution of 2% CaCh, using a 20 mL disposable syringe. A cross linking reaction occurred with the results that alginate microcapsules containing MM223 formed in the CaCh solution. The solidified microcapsules were then washed with sterile water, and filtered to obtain a microcapsule containing embedded MM223.

[0194] A disease efficacy trial testing the new MM223 formulation at microcosm level was completed. When L. fungicola was applied on Day 4, MM223 calcium alginate microcapsules inhibited dry bubble disease in the second flush by 43% as shown in Figure 12. Example 7 - Delivery means

[0195] Endospore delivery

[0196] Bacillus species are known to be able to form endospores. This is a commercial formulation of choice for Bacillus based products. Endospores are highly differentiated, dormant cells, which can withstand harsh conditions such as extreme temperatures, chemicals, radiation, dryness, or lack of nutrients. Endospores are a reversible status in the life cycle of Bacillus species with vegetative cells having the ability to convert to an endospore when environmental conditions are unfavourable to it, and, in turn, the endospore reverting back to vegetative growth when conditions become favourable again.

[0197] Fermentations of MM223 were set up to support the trial for the application MM223 in the form of endospores, processed as both fresh endospores and spray dried endospores. Endospore formation was achieved through conventional nutrient depletion and percentage sporulation was calculated by heating the samples to 80°C for 10 mins to distinguish vegetative cells from endospores.

[0198] In order to prepare fresh endospores, the broth from a fermentation of MM223, yielding 1012endospores / L, was centrifuged at 3500 rpm for 2 hrs in order to obtain a cell pellet. The supernatant was discarded and the pellet was further resuspended in a minimum amount of remaining supernatant. Further processing of these endospores by spray drying resulted in a free flowing, off-white powder.

[0199] A disease efficacy trial testing the endospore formulation was completed. When spray-dried endospores of MM223 were applied over 2 applications (Day 0 and Day 21), A reduction in the incidence of dry bubble in the second flush by >70% were observed (Figure 13).

[0200] Example 8

[0201] Farm scale assessment to confirm the efficacy of Bacillus velezensis strain MM 223 against dry bubble disease of button mushrooms compared to Sporgon™

[0202] Materials and Methods

[0203] Spray-dried endospores of MM223 in powdered form were tested at farm scale for biocontrol efficacy against naturally occurring L. fungicola.

[0204] The first trial was conducted on one farm in Ireland over a 4 month summer period (May to August) when dry bubble disease is prevalent. MM223 was applied in spore form at 1x 1011cfu / msq during watering after case run and after flush 1 of the cropping cycle. The efficacy of MM223 against mushroom dry bubble disease was quantified as the percentage reduction in disease compared to that of Sporgon™ (positive control).

[0205] The second trial was conducted across number of farms in Ireland during a full calendar year (12 months). MM223 was applied in spore form at 1x 1011cfu / msq to compost before filling into a house and during the cropping cycle after flush 1. The efficacy of MM223 against mushroom dry bubble disease was quantified as the percentage reduction in disease compared to that of Sporgon™ (positive control), which was applied to all of the farms the previous year.

[0206] Results

[0207] Results of the first trial showed both MM223 and Sporgon™ had good efficacy against endogenous dry bubble disease in a farm environment (Figure 1). There was no significant difference in the average number of dry bubbles / msq (P = 0.923). Moreover, the addition of MM223 had a positive and significant (P <0.05) impact on mushroom yield, increasing it by an average of 1kg / msq.

[0208] Results of the second trial indicated that the application of MM223 to the compost before filling and after the first flush of mushrooms reduced the number of dry bubbles compared to Sporgon™ in a farm setting, though this difference was not statistically significant (P = 0.46) (Figure 2)

[0209] Conclusions

[0210] MM223 is as effective against mushroom dry bubble disease in a commercial mushroom growing environment as Sporgon WP 50™ (prochloraz manganese). In addition, MM223 can have a beneficial effect on mushroom yield compared to Sporgon™.

[0211] Example 9

[0212] In vitro assessment of MM223 to kill sciarid fly larvae

[0213] Materials and Methods

[0214] Sciarid fly (Lycoriella species) eggs were collected. Briefly, from the fly cage, gravid females were collected with a vacuum aspirator and transferred into egg-laying chamber (made from a 1000 mL Nalgene filter unit). The lid of the Nalgene unit was replaced with a dark coloured velvet cloth and secured with the aid of elastic bands. The unit was placed onto a punnet containing mushroom compost with actively growing Agaricus bisporus mycelium and left in a dark humid location for 2 days at room temperature. During this time the female flies laid eggs onto the wet velvet cloth. The eggs were collected from the velvet cloth using a fine paint brush under a stereo microscope and placed into 1.5 ml Eppendorf tubes containing phosphate buffered saline (PBS) After 1 hr, the PBS was removed and replaced with 1 ml of 0.1% bleach / 0.05% sodium hypochlorite solution for 2 min. The eggs were washed three times with sterile distilled water and carefully transferred onto agar plates with actively growing Agaricus bisporus mycelium. After 2-3 days incubation at 22°C in the dark the eggs developed into larvae.

[0215] To evaluate the larvicidal activity of the biocides, a rapid potato based acute toxicity test was adapted from a previous study (Taylor et al., 2007). Briefly, 30 grams of potato flakes were rehydrated with 40 mL of sterile water and mixed thoroughly. A portion of 4.85 grams was taken and mixed with 5 mL of known concentration of test substance:

[0216] • Phosphate buffered saline (negative control)

[0217] • MM223 endospores at 109cfu

[0218] • MM223 fermentation supernatant

[0219] From the prepared potato-test substance mixtures ~1 gram was transferred and placed as a potato wedge in the centre of the petri plate. Duplicate plates were prepared for each treatment. A total 10 larvae (first and second instar) were placed on the potato wedge and the plates were incubated at ~22°C for 16- 24 hours. At 16 h and 24 h, the number of live and dead larvae in each plate was determined under a stereomicroscope. The percentage reduction in live larvae was determined.

[0220] Results

[0221] In vitro assessment of MM223 to kill sciarid fly larvae

[0222] Two larvicidal assays were conducted two weeks apart. Results both trials showed phosphate buffered saline had no effect on the viability of sciarid fly larvae at 16 h or 24 h. In contrast, both fresh MM223 endospores and the undiluted fermentation media (cell-free) led to significant reductions in viable sciarid larvae (Figure 17). Example 10

[0223] On site average fly level assessment

[0224] Average weekly fly levels without and with MM223 (as a spray dried endospore powder) application were assessed at multiple mushroom growing sites (commercial farms) over a 2 year period.

[0225] We found that in Year 1 (MM223 not applied) compared to Year 2 (MM223 applied to compost) there was a reduction of average fly levels on all sites.

[0226] The invention will now be described by the following non-limiting sequentially numbered statements:

[0227] 1. A plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus velezensis strain which is a gram positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology.

[0228] 2. The anti-fungal bacterial composition according to statement 1 wherein the Bacillus velezensis strain is Bacillus velezensis MM223 having accession number NCIMB 42692.

[0229] 3. The anti-fungal bacterial composition according to statement 2 in which Bacillus velezensis MM223 with accession number NCIMB 42692 has a 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1.

[0230] 4. The anti-fungal bacterial composition according to statement 1, 2 or 3 further comprising a Bacillus subtilis strain which is a gram positive strain, catalase positive, oxidase positive strain with a white / cream, rough / wrinkled and circular colony morphology.

[0231] 5. The anti-fungal bacterial composition according to statement 4 wherein the Bacillus subtilis strain is Bacillus subtilis MM232 having accession number NCIMB 43953. 6. The anti-fungal bacterial composition according to statement 5 in which Bacillus subtilis MM232 with accession number NCIMB 43953 has a 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2.

[0232] 7. The anti-fungal bacterial composition according to any of the preceding statements which protects against one or more of fungal pathogens selected from Lecanicillium fungicola, Mycogene perniciosa, Trichoderma aggressivum, Cladobotryum spp. and / or Fusarium graminearum.

[0233] 8. The anti-fungal bacterial composition according to any of the preceding statements wherein the growing media is compost, preferably mushroom compost.

[0234] 9. The anti-fungal bacterial composition according to statement 7 or statement 8 for the protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb, green mould and / or Fusarium head blight (FHB).

[0235] 10. The anti-fungal bacterial composition according to any of the preceding statements wherein said bacterial strains are provided in endospore form, preferably as spray dried endospores.

[0236] 11. An isolated bacterial strain comprising Bacillus velezensis MM223 having accession number NCIMB 42692 which inhibits the growth of plant fungal pathogens; preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692 with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1.

[0237] 12. An isolated bacterial strain culture which inhibits the growth of plant fungal pathogens comprising

[0238] Bacillus velezensis MM223 having accession number NCIMB 42692, and

[0239] Bacillus subtilis MM232 having accession number NCIMB 43953; preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692 with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95% identical to SEQ ID NO:1; and

[0240] Bacillus subtilis MM232 having accession number NCIMB 43953with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2.

[0241] 13. Use of the anti-fungal bacterial composition or the isolated bacterial strain or culture according to any of the preceding statements to confer improved fungal pathogen resistance to plant, soil or growing media comprising applying said anti-fungal composition to said plants, soil or growing media; optionally wherein said bacterial strains are provided as endospores; further optionally applied from approximately 1x109endospores per meter squared of application area to approximately 9x1011endospores per meter squared of application area.

[0242] 14. A method for preparing augmented soil or growing media, preferably wherein the growing media is compost, the method comprising the steps of: providing the anti-fungal bacterial composition according to any of statements 1 to 10 or the isolated bacterial strain or culture according to statement 11 or 12; providing soil or growing media; and inoculating the soil or growing media with the composition or isolated bacterial strain or culture.

[0243] 15. Augmented soil or growing media, preferably wherein the growing media is compost, comprising the anti-fungal bacterial composition of statements 1 to 10 or the isolated bacterial strain or culture according to statement 11 or 12.

[0244] 16. Use of augmented soil or growing media, preferably wherein the growing media is compost, as a nutrient source for farming, the use comprising the steps of: providing the augmented soil or growing media prepared according to statement 14 or 15; providing at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed; cultivating the at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed in an environment comprising the augmented soil or growing media; preferably wherein the use comprising the steps of: providing the augmented soil or growing media prepared according to statement 14 or 15; providing at least one Agaricus bisporus mushroom, mycelium, spawn, and / or spore; cultivating the at least one Agaricus bisporus mushroom, mycelium, spawn, and / or spore, in an environment comprising the augmented soil or growing media.

[0245] References

[0246] Amey, R.C., Athey Pollard, A. I., Burns, C., Mills, P. R., Bailey, A. and Foster, G. D. (2002). PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola. Mycol Res, 106,4-11.

[0247] Berendsen, R. L, Baars, J. J., Kalkhove, S. I., Lugones, L. G., Wdsten, H. A., & Bakker, P. A. (2010). Lecanicillium fungicola: causal agent of dry bubble disease in white-button mushroom. Molecular plant pathology, 11(5), 585-595.

[0248] Carrasco, J., Tello, M., de Toro, M., Tkacz, A., Poole, P., Perez-Clavijo, M. and Preston, G. (2019). Casing microbiome dynamics during button mushroom cultivation: implications for dry and wet bubble diseases. Microbiology, 165(6), pp.611-624.

[0249] Dragt, J. W., Geels, F. P., Debruijn, W. C. and van Griensven, I. J. I. D. (1996). Intracellular infection of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola var. fungicola. Mycol Res, 100,1082-1086.

[0250] Elleuch L., Shaaban M., Smaoui S., et al., (2010). Bioactive secondary metabolites from a new terrestrial Streptomyces sp. TN262, Applied Biochemistry and Biotechnology, V162 (2),P: 5779-593.

[0251] Fan Ben, Blom Jochen, Klenk Hans-Peter, Borriss Rainer (2017). Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an “Operational Group B. amyloliquefaciens” within the B. subtilis Species Complex. Frontiers in Microbiology, Volume 8.

[0252] Grogan, H.M. and Jukes, A.A., (2003). Persistence of the fungicides thiabendazole, carbendazim and prochloraz-Mn in mushroom casing soil Pest Manag Sci. 59:1225-1231 Jiang, H., Dong, H., Zhang, G., Yu, B., Chapman, L.R. and Fields, M.W. 2006. Microbial diversity in water and sediment of Lake Chaka, an athalassohaline lake in northwestern China. Applied and Environmental Microbiology, Vol. 72 (6), pp. 3832- 3845.Klindworth, A., Pruesse, E., Schweer, T., Replies, J., Quast, C., Horn, M., et al. (2013). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and nextgeneration sequencing-based diversity studies. Nucleic Acids Res. 41 :e1.

[0253] Kouser, S, Shah, S, Ahmed, M, Shah, M.D., Sheikh. (2015). Morphological characterisitics of wet bubble disease (Mycogone pernicosa) isolated from button mushroom (Agaricus bisporus) and assessment of factors affecting disease development and spread. African Journal of Microbiology Research. Vol. 9(3), pp.185-193, 21 January 2015.

[0254] McGee, C. F., Byrne, H., Irvine, A. and Wilson, J. (2017). Diversity and dynamics of the DNA- and cDNA-derived compost fungal communities throughout the commercial cultivation process for Agaricus bisporus. Mycologia 109 (3) 475-484.

[0255] Ntougias,S., Zervakis,G.I., Kavroulakis, N., Ehaliotis, C., Papadopoulou, K.K. (2004). Bacterial Diversity in Spent Mushroom Compost Assessed by Amplified rDNA Restriction Analysis and Sequencing of Cultivated Isolates. Systematic and Applied Microbiology, 27 (6) 746-754.

[0256] O'Brien, M., Grogan, H., Kavanagh, K. (2014). Proteomic response of Trichoderma aggressivum f. europaeum to Agaricus bisporus tissue and mushroom compost, Fungal Biology, Volume 118, Issues 9-10, Pages 785-791.

[0257] Pyck et al. 2015. Fungal diseases of mushroom and their control. Factsheet. Teagasc

[0258] Samaras A, Nikolaidis M, Antequera-Gomez ML, Camara-Almiron J, Romero D, Moschakis T, Amoutzias GD, Karaoglanidis GS. (2021). Whole Genome Sequencing and Root Colonization Studies Reveal Novel Insights in the Biocontrol Potential and Growth Promotion by Bacillus subtilis MBI 600 on Cucumber. Front Microbiol. 2021 Jan 12; 11.

[0259] W.M. Yuan, D.L. Crawford (1995) Characterization of streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots, Applied and Environmental Microbiology, Vol. 61, No. 8, pages 3119-3128.

Claims

Claims1. A plant, soil or growing media anti-fungal bacterial composition comprising a Bacillus velezensis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white, rough / wrinkled and irregular colony morphology.

2. The anti-fungal bacterial composition according to claim 1 in which the Bacillus velezensis strain has a 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95%, preferably 99.9%, identical to SEQ ID NO:1 ; wherein said strain provides anti-fungal activity, including protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb, green mould and / or Fusarium head blight (FHB), preferably wherein said strain provides protection against mushroom fungal pathogens, including Agaricus bisporus fungal pathogens.

3. The anti-fungal bacterial composition according to claim 1 or claim 2 in which the Bacillus velezensis strain has a genome sequence defined in SEQ ID NO: 9 or a sequence at least 97% identical to SEQ ID NO:9; wherein said strain provides anti-fungal activity, including protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb, green mould and / or Fusarium head blight (FHB), preferably wherein said strain provides protection against mushroom fungal pathogens, including Agaricus bisporus fungal pathogens.

4. The anti-fungal bacterial composition according to any of claims 1 to 3 wherein the Bacillus velezensis strain is Bacillus velezensis MM223 having accession number NCIMB 42692.

5. The anti-fungal bacterial composition according to any of claims 1 to 4, wherein said Bacillus velezensis strain has insect pest control activity; preferably fly pest control activity; more preferably sciarid fly pest control activity, including Lycoriella ingenua, Lycoriella castanescens, and / or Bradysia ocellaris control activity.

6. The anti-fungal bacterial composition according to any of the preceding claims further comprising a Bacillus subtilis strain which is a gram-positive strain, catalase positive, oxidase positive strain with a white / cream, rough / wrinkled and circular colony morphology.

7. The anti-fungal bacterial composition according to claim 6 in which the Bacillus subtilis strain has a 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2.

8. The anti-fungal bacterial composition according to claim 6 or claim 7 wherein the Bacillus subtilis strain is Bacillus subtilis MM232 having accession number NCIMB 43953.

9. The anti-fungal bacterial composition according to any of the preceding claims which protects against one or more of fungal pathogens selected from Lecanicillium fungicola, Mycogene perniciosa, Trichoderma aggressivum, Cladobotryum spp. and / or Fusarium graminearum.

10. The anti-fungal bacterial composition according to any of the preceding claims wherein the growing media is compost, preferably wherein the growing media is mushroom compost.

11. The anti-fungal bacterial composition according to claim 9 or claim 10 for the protection against fungal pathogens causing at least one of wet bubble disease, dry bubble disease, cobweb, green mould and / or Fusarium head blight (FHB).

12. The anti-fungal bacterial composition according to any of the preceding claims wherein said bacterial strains are provided in endospore form, preferably as a dried endospore powder, more preferably as a freeze-dried or spray-dried endospore powder.

13. An isolated bacterial strain which inhibits the growth of plant fungal pathogens comprising a Bacillus velezensis strain with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95%, preferably 99.9%, identical to SEQ ID NO:1 ;preferably comprising a Bacillus velezensis strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 97% identical to SEQ ID NO:9; more preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692.

14. An isolated bacterial strain culture which inhibits the growth of plant fungal pathogens comprising a Bacillus velezensis strain with 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95%, preferably 99.9%, identical to SEQ ID NO:1, and a Bacillus subtilis strain with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2; preferably comprising a Bacillus velezensis strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 97% identical to SEQ ID NO:9, and a Bacillus subtilis strain with 16S rRNA sequence defined in SEQ ID NO: 2 or a sequence at least 95% identical to SEQ ID NO: 2; more preferably comprising Bacillus velezensis MM223 having accession number NCIMB 42692, and Bacillus subtilis MM232 having accession number NCIMB 43953.

15. An isolated bacterial strain or strain culture according to claim 13 or 14, wherein said Bacillus velezensis strain has insect pest control activity; preferably fly pest control activity; more preferably sciarid fly pest control activity, including Lycoriella ingenua, Lycoriella castanescens, and / or Bradysia ocellaris control activity.

16. Use of the anti-fungal bacterial composition or the isolated bacterial strain or culture according to any of the preceding claims to confer improved fungal pathogen resistance to plant, soil or growing media comprising applying said anti-fungal composition or the isolated bacterial strain or culture to said plants, soil or growing media; optionally wherein said bacterial strains are provided as endospores; further optionally wherein the endospores are applied from approximately 1x109endospores per meter squared of application area to approximately 9x1011endospores per meter squared of application area.

17. Use of the anti-fungal bacterial composition or the isolated bacterial strain or culture according to claim 16 to confer improved control of insect or fly pests, preferably sciarid fly pests, to the plant, soil or growing media.

18. A method for preparing augmented soil or growing media, preferably wherein the growing media is compost, the method comprising the steps of: providing the anti-fungal bacterial composition according to any of claims 1 to 12 or the isolated bacterial strain or culture according to any of claim 13 to 15; providing soil or growing media; and inoculating the soil or growing media with the composition or isolated bacterial strain or culture.

19. Augmented soil or growing media, preferably wherein the growing media is compost, comprising the anti-fungal bacterial composition of claims 1 to 12 or the isolated bacterial strain or culture according to any of claims 13 to 15.

20. Use of augmented soil or growing media, preferably wherein the growing media is compost, as a nutrient source for farming, the use comprising the steps of: providing the augmented soil or growing media prepared according to claim 18 or 19; providing at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed; cultivating the at least one fungus, mycelium, spawn, spore, plant, cutting, turf, and / or seed in an environment comprising the augmented soil or growing media; preferably wherein the use comprising the steps of: providing the augmented soil or growing media prepared according to claim 18 or 19; providing at least one Agaricus bisporus mushroom, mycelium, spawn, and / or spore; cultivating the at least one Agaricus bisporus mushroom, mycelium, spawn, and / or spore, in an environment comprising the augmented soil or growing media.

21. An insect pest control strain, strain culture or bacterial composition comprising a gram-positive strain, catalase positive, oxidase positive Bacillus velezensis strain strain with a white, rough / wrinkled and irregular colony morphology.a Bacillus velezensis strain with a 16S rRNA sequence defined in SEQ ID NO: 1 or a sequence at least 95%, preferably 99.9%, identical to SEQ ID NO:1; a Bacillus velezensis strain with a genome sequence defined in SEQ ID NO: 9 or a sequence at least 97% identical to SEQ ID NO:9; orBacillus velezensis MM223 having accession number NCIMB 42692.

22. The insect pest control strain, strain culture or bacterial composition according to claim 21 comprising Bacillus velezensis MM223 having accession number NCIMB 42692.

23. The insect pest control strain, strain culture or bacterial composition according to claim 21 or claim 22 which has fly pest control activity.

24. The insect pest control strain, strain culture or bacterial composition according to any of claims 21 to 23 which has sciarid fly control activity.

25. The insect pest control strain, strain culture or bacterial composition according to any of claims 21 to 24 for the protection against sciarid fly species including Lycoriella ingenua, Lycoriella castanescens, and / or Bradysia ocellaris.

26. The insect pest control strain, strain culture or bacterial composition according to any of claims 21 to 25 wherein said bacterial strains are provided in endospore form, preferably as a dried endospore powder, more preferably as a freeze-dried or spray-dried endospore powder.

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