WO2021255181A1 - Bacteria - Google Patents

Bacteria Download PDF

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Publication number
WO2021255181A1
WO2021255181A1 PCT/EP2021/066441 EP2021066441W WO2021255181A1 WO 2021255181 A1 WO2021255181 A1 WO 2021255181A1 EP 2021066441 W EP2021066441 W EP 2021066441W WO 2021255181 A1 WO2021255181 A1 WO 2021255181A1
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Prior art keywords
crop
bacteria strain
bacteria
seq
growth
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PCT/EP2021/066441
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French (fr)
Inventor
Jaime CARRASCO
Gail PRESTON
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Oxford University Innovation Limited
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Priority to EP21734105.6A priority Critical patent/EP4200400A1/en
Publication of WO2021255181A1 publication Critical patent/WO2021255181A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the invention relates to bacteria strains for controlling the growth of crop pathogenic microorganisms and improving crop growth.
  • Pathogens and pests affect crops, causing substantial economic losses and threatening food security.
  • mycoparasites are responsible for the largest crop losses in commercial mushroom production (Fletcher & Gaze, 2008), causing diseases such as dry bubble ( Lecanicillium fungicola), wet bubble ( Mycogone perniciosa) and green mould ( Trichoderma spp.), which are major challenges for mushroom cultivation, directly affecting growers’ profit margins.
  • Dry bubble disease accounts for estimated 20% of button mushroom crop losses globally (Tasmanian Institute of Agriculture, 2018). Losses of production up to 40%, with 70% of profit shortcuts due to quality depreciation have been described in Spain.
  • biocontrol agents development of biocontrol agents is an active field. For example, bacteria isolated from mushroom compost were tested for activity against Trichoderma spp., the causal agents of green mould in A. bisporus and one isolate was identified to be a Bacillus subtilis strain (Milijasevic-Marcic et al., 2017).
  • Bacillus subtilis strain QST713 Another example of a biocontrol agent is Bacillus velezensis strain QST713, the active ingredient in a commercially available biofungicide, Serenade®, which has been registered for use against many crop pathogens.
  • Serenade® the active ingredient in a commercially available biofungicide
  • B. velezensis QST713 has been used industrially in France for the protection of button mushrooms, Agaricus bisporus, against green mould disease. Reports on the biocontrol mechanism of this bacteria are underway (Pandin et ah, 2018 and 2019).
  • biocontrol agents such as bacteria strains
  • the inventors have surprisingly identified Bacillus velezensis strains in the casing material employed in mushroom cultivation that are capable of exerting antagonism against pathogenic microorganisms responsible for many of the major soil-borne diseases, such as mycoparasites. These strains may be useful as biocontrol agents for controlling the growth of crop pathogenic microorganisms and improving crop growth.
  • the casing is the material applied to cover the fully colonized compost where the following takes place: the transition from vegetative growth (mycelium) to reproductive growth (mushrooms), also known as mushroom fructification.
  • the inhabitants in raw casing material are thought to provide natural suppressive effects against mycoparasites such as L.fungicola, M.
  • perniciosa and Cladobotryum mycophilum although these suppressive effects are weakened during the colonization of the casing material by the mycelium of A. bisporus, resulting in the contribution of disease outbreaks such as cobweb (caused by Cladobotryum spp.), bubble diseases (caused by L. fungicola or M. perniciosa) or green mould (caused by Trichoderma spp.).
  • the inventors identified Bacillus velezensis strains CM5, CM19 and CM35, each of which is capable of exerting antagonism against the mycoparasites Lecanicillium fungicola L1 and Mycogone perniciosa M1, to prevent conidial germination and mycelium growth. Furthermore, the strains do not show detectable toxicity against the host mycelium ( Agaricus bisporus) which suggests a non-detrimental effect on mushroom production when applied as an inoculant.
  • CM5, CM19 and CM35 were effective in exerting antagonism against mycoparasites when the strains were introduced into casing material during mushroom fruitbody formation, because as mentioned above A. bisporus tends to release the mycoparasites from fungistasis in the casing layer.
  • the data from crop trials show that no deleterious effect on mushroom production has been detected due to the application of the bacterial treatments.
  • the inventors have formulated the bacteria strains into powder formulations that would be useful for applying the bacteria to crops.
  • the bacteria strains when introduced in the water-soluble powder form, exhibited effective antagonism against the agents responsible for many of the major soil-borne diseases (e.g. L. fungicola, M. perniciosa, Trichoderma atroviride (mushroom mycoparasites and competitors), Fusarium commune, F. oxysporum, Botrytis cinerea, Alternaria spp. (plant mycoparasites)).
  • the genome sequencing and the metabolism studies of the bacteria strains suggested the production of metabolites with an active antifungal profile. The results therefore suggest that the bacteria strains discovered can be used to control the growth of crop pathogens and improve crop growth, not only for button mushrooms, Agaricus bisporus, but also for other plant crops.
  • the invention provides a bacteria strain which: (a) comprises a 16S rRNA- encoding gene having ⁇ 95% sequence identity with SEQ ID NO: 1, 2 or 3, (b) comprises a genome having ⁇ 90% identity to the genome sequence of B.
  • velezensis CM5, CM 19 or CM35 (c) is Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301, or a variant thereof, (d) is Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302, or a variant thereof, or (e) is Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303, or a variant thereof, wherein said strain is capable of exerting antagonism against a crop pathogenic microorganism.
  • the invention also provides a bioinoculant composition comprising the bacteria strain of the invention.
  • the invention also provides a crop growth medium comprising the bacteria strain of the invention.
  • the invention also provides a method for controlling the growth of a crop pathogenic microorganism, improving crop growth and/or treating or preventing a disease caused by a crop pathogenic microorganism, comprising administering to a crop and/or the environment surrounding the crop, the bacteria strain of the invention, the bioinoculant composition of the invention, or the growth medium composition of the invention.
  • the invention also provides a method of treating and/or preventing a disease, such as dry bubble disease, wet bubble disease or cobweb, comprising administering to a crop and/or the environment surrounding the crop, the bacteria strain of the invention, the bioinoculant composition of the invention, or the growth medium composition of the invention.
  • a disease such as dry bubble disease, wet bubble disease or cobweb
  • the method involves applying to a crop the B. velezensis strain, or a variant thereof, of the invention, or the composition of the invention.
  • the invention also provides the use of the bacteria strain of the invention for controlling the growth of a crop pathogenic microorganism and/or improving crop growth, such as mushroom growth.
  • the invention also provides a growth medium for crops obtainable by treating with the bacteria strain of the invention.
  • the invention also provides a method of isolating a microorganism with antagonistic properties against one or more crop pathogenic microorganisms, comprising the steps of obtaining a sample from casing material, testing the sample for antagonistic properties and isolating microorganism with said antagonistic properties.
  • Figure 1 shows healthy and diseased mushrooms a, b) healthy crop (control); c, d) symptoms of mushrooms infected with L. fungicola (dry bubble); e, f) undifferentiated mass of A. bisporus tissue infected by M. perniciosa (wet bubble).
  • Figure 2 shows mycotoxicity of the strains against the mycoparasites evaluated. Circles indicate strains with antifungal activity towards L. fungicola and M. perniciosa.
  • Figure 3 shows the effect of CM5 co-cultured with mycoparasites in LBA: a) inhibition of L. fungicola, ⁇ b) Inhibition of T. aggressivum; c) inhibition of C. mycophilum; d) inhibition of conidial germination (L. fungicola) compared to non-active strains (circles).
  • a)-c) the mycoparasite is inoculated in the centre of the plate, and candidate biocontrol agents (CBAs) are inoculated adjacent to the growing mycelium.
  • CBAs candidate biocontrol agents
  • Figure 4 shows two time points of mycotoxicity of the selected strains against bisporus Amycel XXX® in agar compost.
  • Figure 5 shows radial inhibition generated by strains CM5, CM 19 and CM35 respectively on the mycelial growth of a) L. fungicola and b) M. perniciosa.
  • Figure 6 shows mycoparasites, L. fungicola L1 and M. perniciosa M1, co-cultured with strains CM5, CM19 and CM35. Negative control with four non-fungitoxic strains was included.
  • Figure 7 shows the Inhibition Zone Diameter (IZD) generated by the strains CM5, CM 19 and CM35, respectively, in the conidial germination of L. fungicola L1 (10 9 conidia/plate).
  • IZD Inhibition Zone Diameter
  • Figure 8 shows L. fungicola L1 co-cultured with the strains CM5, CM19 and CM35 introduced as a water-soluble bacterial powder (50 ⁇ l of 10 9 ufc/ml) against 10 2 , 10 3 , 10 4 and 10 5 conidia per plate (PDA).
  • Top row shows growth of L. fungicola in the absence of the bacterial strains.
  • the second, third, and fourth rows correspond to strains CM5, CM19, and CM35, respectively.
  • Figure 9 shows Trichoderma atroviride TA1 co-cultured with the strains CM5, CM19 and CM35 introduced as a water-soluble bacterial powder (50 ⁇ l of 10 9 ufc/ml) against 10 2 , 10 3 , 10 4 and 10 5 conidia per plate (PDA).
  • Top row shows growth of L. fungicola in the absence of the bacterial strains.
  • the second, third, and fourth rows correspond to strains CM5, CM19, and CM35, respectively.
  • Figure 10 shows Fusarium oxysporum CEBAS-CSIC co-cultured with the strains CM5, CM 19 and CM35 introduced as a water-soluble bacterial powder (50 ⁇ l of 10 9 ufc/ml) against 10 2 , 10 3 and 10 4 conidia per plate (PDA).
  • the first, second, and third rows correspond to strains CM5, CM19, and CM35, respectively.
  • Figure 11 shows Fusarium ses CEBAS-CSIC co-cultured with the strains CM5, CM 19 and CM35 introduced as a water-soluble bacterial powder (50 ⁇ l of 10 9 ufc/ml) against 10 2 , 10 3 , 10 4 and 10 5 conidia per plate (PDA).
  • the first, second, and third columns correspond to strains CM5, CM19, and CM35, respectively.
  • Figure 12 shows Botrytis cinerea CEBAS-CSIC co-cultured with the strains CM5, CM19 and CM35 introduced as a water-soluble bacterial powder (50 ⁇ l of 10 9 ufc/ml) against 10 2 , 10 3 , 10 4 and 10 5 conidia per plate (PDA).
  • the first, second, and third columns correspond to strains CM5, CM19, and CM35, respectively.
  • Figure 13 shows the production (kg m -2 ) of healthy mushrooms in the first trial in crop: a) control room, non-inoculated; b) room infected with Lecanicillium fungicola L1; c) room infected with Mycogone perniciosa M1.
  • Figure 14 shows the production (kg m -2 ) of healthy mushrooms in the second trial in crop: a), control room, non-inoculated; b) room infected with Lecanicillium fungicola L1; c) room infected with Mycogone perniciosa M1.
  • a bacteria strain of the invention may comprise a 16S rRNA-encoding gene having ⁇ 95% (i.e. equal or greater than 95%), ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity with the 16S rRNA-encoding gene of Bacillus velezensis CM5 (SEQ ID NO: 1).
  • the 16S rRNA-encoding gene may have ⁇ 99.6%, ⁇ 99.7%, ⁇ 99.8%, ⁇ 99.9% or 100% sequence identity with SEQ ID NO: 1.
  • the 16S rRNA-encoding gene of the bacteria strain of the invention may differ from SEQ ID NO: 1 by 1, ⁇ 2 (i.e. equal to or less than 2), ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6 or ⁇ 7 bases.
  • the bacteria strain may be a Bacillus velezensis strain.
  • a bacteria strain of the invention may comprise a genome having ⁇ 90% (i.e. equal or greater than 90%), ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, ⁇ 99.1%, ⁇ 99.2%, ⁇ 99.3%, ⁇ 99.4%, ⁇ 99.5%, ⁇ 99.6%, ⁇ 99.7%, ⁇ 99.8%, ⁇ 99.9% or 100% sequence identity with the genome sequence of Bacillus velezensis CM5.
  • the draft genome of Bacillus velezensis CM5 is provided in SEQ ID NOs: 57-106, and it has 4,205,756 bp with 45.85% GC.
  • the genome sequence of Bacillus velezensis CM5 may be obtainable by the assembly of the contigs provided in SEQ ID NOs: 57-106.
  • the bacteria strain may be a Bacillus velezensis strain.
  • a bacteria strain of the invention may be Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301.
  • a bacteria strain of the invention may comprise a 16S rRNA-encoding gene having ⁇ 95% (i.e. equal or greater than 95%), ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity with the 16S rRNA-encoding gene of Bacillus velezensis CM19 (SEQ ID NO: 2).
  • the 16S rRNA-encoding gene may have ⁇ 98.4%, ⁇ 98.5, ⁇ 98.6%, ⁇ 98.7%, ⁇ 98.8%, ⁇ 99%, ⁇ 99.1%, ⁇ 99.2%, ⁇ 99.3%, ⁇ 99.4%, ⁇ 99.5%, ⁇ 99.6%, ⁇ 99.7%, ⁇ 99.8%, ⁇ 99.9% or 100% sequence identity with SEQ ID NO: 2.
  • the 16S rRNA-encoding gene of the bacteria strain of the invention may differ from SEQ ID NO: 2 by 1, ⁇ 2 (i.e.
  • the bacteria strain may be a Bacillus velezensis strain.
  • a bacteria strain of the invention may comprise a genome having ⁇ 90% (i.e. equal or greater than 90%), ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, ⁇ 99.1%, ⁇ 99.2%, ⁇ 99.3%, ⁇ 99.4%, ⁇ 99.5%, ⁇ 99.6%, ⁇ 99.7%, ⁇ 99.8%, ⁇ 99.9% or 100% sequence identity with the genome sequence of Bacillus velezensis CM 19.
  • the complete genome of Bacillus velezensis CM19 is provided in SEQ ID NO: 145, and it has 4,203,670 bp with 45.84% GC.
  • the draft genome is provided in SEQ ID NOs: 107-144.
  • the genome sequence of Bacillus velezensis CM 19 may be obtainable by the assembly of the contigs provided in SEQ ID NOs: 107-144.
  • the bacteria strain may be a Bacillus velezensis strain.
  • a bacteria strain of the invention may be Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302.
  • a bacteria strain of the invention may comprise a 16S rRNA-encoding gene having ⁇ 95% (i.e. equal or greater than 95%), ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99% or 100% sequence identity with the 16S rRNA-encoding gene of Bacillus velezensis CM35 (SEQ ID NO: 3).
  • the 16S rRNA-encoding gene may have ⁇ 98.7%, ⁇ 98.8%, ⁇ 98.9%, ⁇ 99%, ⁇ 99.1%, ⁇ 99.2%, ⁇ 99.3%, ⁇ 99.4%, ⁇ 99.5%, ⁇ 99.6%, ⁇ 99.7%, ⁇ 99.8%, ⁇ 99.9% or 100% sequence identity with SEQ ID NO: 3.
  • the 16S rRNA-encoding gene of the bacteria strain of the invention may differ from SEQ ID NO: 3 by 1, ⁇ 2 (i.e. equal to or less than 2), ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, ⁇ 15, ⁇ 20 or ⁇ 23 bases.
  • the bacteria strain may be a Bacillus velezensis strain.
  • a bacteria strain of the invention may comprise a genome having ⁇ 90% (i.e. equal or greater than 90%), ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98%, ⁇ 99%, ⁇ 99.1%, ⁇ 99.2%, ⁇ 99.3%, ⁇ 99.4%, ⁇ 99.5%, ⁇ 99.6%, ⁇ 99.7%, ⁇ 99.8%, ⁇ 99.9% or 100% sequence identity with the genome sequence of Bacillus velezensis CM35.
  • the draft genome of Bacillus velezensis CM35 is provided in SEQ ID NOs: 6-56 and it has 4,204,667 bp with 45.84% GC.
  • the genome sequence of Bacillus velezensis CM35 may be obtainable by the assembly of the contigs provided in SEQ ID NOs: 6-56.
  • the bacteria strain may be a Bacillus velezensis strain
  • a bacteria strain of the invention may be Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303.
  • a bacteria strain of the invention includes Bacillus velezensis CM5, CM 19 or CM35, and variants thereof. Bacillus velezensis CM5, CM19 and CM35 were initially isolated from casing materials for growing A. bisporus. Hence, a bacteria strain of the invention may be a natural inhabitant in a casing material, e.g. for growing A. bisporus.
  • a bacteria strain of the invention may be wild type, which is the unaltered form of the bacterial strain Bacillus velezensis CM5, CM19 or CM35.
  • a bacteria strain of the invention may be a strain selected from:
  • Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303.
  • a bacteria strain of the invention may be a variant of Bacillus velezensis CM5, CM19 or CM35.
  • a bacteria strain of the invention may be a variant of a strain selected from:
  • the variant typically retains the properties of its corresponding wild type strain, such as being capable of exerting antagonism against a crop pathogenic microorganism, e.g. inhibiting the growth of Lecanicillium fungicola and/or Mycogone perniciosa.
  • a bacteria strain of the invention may have ⁇ 1 ( i. e. one or more), ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8 or ⁇ 9 housekeeping gene having ⁇ 98% ( i.e . equal or greater than 98%), ⁇ 99% or 100% sequence identity with the corresponding housekeeping gene in B. velezensis CM5, CM 19 or CM35, wherein the housekeeping gene is selected from glycerol uptake facilitator ( glpF) (e.g. SEQ ID NO: 146 or 163), dihydroxy-acid dehydratase ⁇ ilvD) (e.g. SEQ ID NO: 147), phosphotransacetylase (pta ) (e.g.
  • SEQ ID NO: 148 or 149 phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH) (e.g. SEQ ID NO: 150), triose phosphate isomerase (tpiA) (e.g. SEQ ID NO: 151), protein translocase subunit SecA ( secA ) (e.g. SEQ ID NO: 152), carbamoyl-phosphate synthase arginine- specific ( carA_l ) (e.g. SEQ ID NO: 153), carbamoyl-phosphate synthase small chain 0 carAJ ) (e.g.
  • purH e.g. SEQ ID NO: 150
  • tpiA phosphoribosylamino-imidazole carboxy formyl formyltransferase
  • secA protein translocase subunit SecA
  • carA_l carbamoyl-phosphate synthase argin
  • SEQ ID NO: 154 for CM19 or SEQ ID NO: 162 for CM5 or CM35 protein RecA ( recA ) (e.g. SEQ ID NO: 155), chaperone protein DnaK ( dnaK) (e.g. SEQ ID NO: 156), ATP synthase subunit beta ( atpD ) (e.g. SEQ ID NO: 157), DNA gyrase subunit B igyrB) (e.g. SEQ ID NO: 158) and/or translation initiation factor IF-2 ( infB ) (e.g. SEQ ID NO: 159).
  • the names of these genes are in accordance with the pubMLST scheme for Bacillus spp. (https://pubmlst.org/).
  • a bacteria strain of the invention may have ⁇ 1 ⁇ i.e. one or more), ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8 or ⁇ 9 housekeeping gene having ⁇ 98% ⁇ i.e. equal or greater than 98%), ⁇ 99% or 100% sequence identity with the corresponding housekeeping gene in B. velezensis CM5, CM 19 or CM35, wherein the housekeeping gene is selected from glycerol uptake facilitator ( glpF) (e.g. SEQ ID NO: 146 or 163), dihydroxy-acid dehydratase ⁇ ilvD) (e.g. SEQ ID NO: 147), phosphotransacetylase ⁇ pta ) (e.g.
  • SEQ ID NO: 148 or 149 phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH) (e.g. SEQ ID NO: 150), triose phosphate isomerase ⁇ tpiA) (e.g. SEQ ID NO: 151), protein translocase subunit SecA ⁇ secA) (e.g. SEQ ID NO: 152), carbamoyl-phosphate synthase arginine- specific ⁇ carA_1) (e.g. SEQ ID NO: 153), carbamoyl-phosphate synthase small chain ⁇ carA_2) (e.g.
  • purH e.g. SEQ ID NO: 150
  • triose phosphate isomerase ⁇ tpiA e.g. SEQ ID NO: 151
  • protein translocase subunit SecA ⁇ secA e.g. SEQ ID NO: 152
  • SEQ ID NO: 154 for CM19 or SEQ ID NO: 162 for CM5 or CM35 protein RecA ⁇ recA) (e.g. SEQ ID NO: 155), chaperone protein DnaK ( dnaK) (e.g. SEQ ID NO: 156), ATP synthase subunit beta ⁇ atpD) (e.g. SEQ ID NO: 157), DNA gyrase subunit B igyrB) (e.g. SEQ ID NO: 158), translation initiation factor IF-2 ( infB ) (e.g. SEQ ID NO: 159) and/or purine nucleotide synthesis repressor (purR ) (e.g. SEQ ID NO: 160 for CM19 or SEQ ID NO: 161 for CM5 or CM35).
  • purR purine nucleotide synthesis repressor
  • a bacteria strain of the invention may have ⁇ 1 ( /. e. one or more), ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8 or ⁇ 9 housekeeping gene, such as the ones listed above, wherein the nucleic acid sequence encoding the housekeeping gene may have ⁇ 90% ( i.e . equal or greater than 90%), ⁇ 95% or 100% sequence identity with the nucleic acid encoding the corresponding housekeeping gene in B. velezensis CM5, CM19 or CM35 (e.g. any of the SEQ ID NOs: 164 to 179).
  • a bacteria strain of the invention may contain gene modifications relative to the wild type strain (e.g. Bacillus velezensis CM5, CM19 or CM35), such as gene disruptions or gene additions. Using known methods in the art, variants may be generated to improve or alter the characteristics of the bacteria of the present invention.
  • a variant typically retains the crop pathogenic microorganism antagonistic properties of its corresponding wild type strain, e.g. inhibiting the growth of Lecanicillium fungicola and/or Mycogone perniciosa.
  • variants include deletions, insertions, inversions, repeats and substitutions in the genome of the bacteria selected according to general rules known in the art.
  • the variants may contain modified genes with codon optimisation.
  • the gene modifications may increase the efficiency of pathogen antagonistic effects, expand antagonistic spectrum, expand the host range, for pest resistance, increase their production or to allow them to be grown outside their original habitat.
  • Methods of codon optimisation are well known in the art.
  • a bacteria strain of the invention may express additional genes relative to the wild type strain (CM5, CM19 or CM35).
  • the additional genes may increase the efficiency of pathogen antagonistic effects, expand antagonistic spectrum, expand the host range, for pest resistance, increase their production or to allow them to be grown outside their original habitat.
  • Methods to express additional proteins in bacteria are known in the art.
  • a bacteria strain of the invention is particularly effective in growing in growth medium for crops, such as casing material for growing mushrooms.
  • a bacteria strain of the invention may be in a culture in the form of a stock (e.g. frozen at -80°C or freeze-dried), in a liquid broth or in a formulation. Methods for reviving bacteria from frozen or freeze-dried cultures are known in the art.
  • the invention also provides a method of isolating a microorganism with antagonistic properties against one or more crop pathogenic microorganisms, comprising the steps of obtaining a sample from casing material for growing mushrooms, testing the sample for antagonistic properties and isolating microorganism with said antagonistic properties.
  • the isolated microorganism may be a bacteria of the invention.
  • a sample may be suspended in a liquid, such as a bacterial growth broth. The suspension may then be centrifuged in order to pellet debris. The supernatant may be serially diluted and grown on nutrient agar plates. Individual colonies can then be isolated and they can then be, for example, transferred directly to storage or further cultured, such as in a fresh nutrient agar plates or in a growth broth, such as LB. Preliminary characterisation can be carried out by microscopic appearance, Gram reaction and catalase test, all of which are well known in the art. Bacterial strains can be identified based on the analyses of the 16S rDNA sequence, e.g. PCR assay. The individual colonies can be screened for desirable properties, such as antagonistic effects against pathogenic microorganisms in crops, e.g. fungal pathogens.
  • a bacteria strain of the invention is selected on the basis of it having particular advantageous properties for controlling the growth of crop pathogenic microorganism and/or improving crop growth.
  • the bacteria strains of the invention are surprisingly effective in exerting antagonism against pathogenic microorganisms in crops, such as fungal crop pathogens, and in particular exhibiting selective suppressive effects against fungal crop pathogens.
  • a bacteria strain of the invention may be superior in exerting antagonism against pathogenic microorganisms in crops compared to other bacteria strains isolated from the casing material for growing mushrooms.
  • a bacteria strain of the invention is capable of exerting antagonism against pathogenic microorganisms in crops.
  • a bacteria strain of the invention may kill, deter, suppress growth or inhibit reproduction of the pathogenic microorganism.
  • a bacteria strain of the invention may exert antagonism by producing specific substances that affect pathogen cell membranes such as those described below, by inducing resistance, and may act as competitors to pathogenic microorganism for nutrient sources and space. Examples of pathogenic microorganisms are provided below.
  • antagonism may be determined by assessing the degree of inhibition on nutrient agar plates, e.g. in a dual culture assay as described by Sakthivel and Gnanamanickam (1986).
  • a bacteria strain may be inoculated at a set distance from a pathogen, and the difference in the growth of the pathogen in the absence and presence of the bacterial strain after a certain amount of time can be measured in order to indicate antagonistic activity.
  • bacterial strains to be tested can be placed on a plate containing a uniform spread of pathogen. The presence of a halo surrounding the bacteria is indicative of anti-pathogen activity.
  • a bacteria of the invention may inhibit growth of a pathogenic microorganism by ⁇ 50% (i.e. equal or greater than 50%), ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95% or 100%.
  • a bacteria strain of the invention may inhibit the growth of Lecanicillium fungicola L1 and/or Mycogone perniciosa M1 by ⁇ 50% ⁇ i.e. equal or greater than 50%), ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95% or 100% in an in vitro dual culture test.
  • An in vitro dual culture test for mycelial growth inhibition typically involves culturing a microorganism (e.g. Lecanicillium fungicola L1 or Mycogone perniciosa M1) on an appropriate medium (e.g. PDA or MEA) at 22°C for 12 days; adding the bacteria colonies of interest (e.g. B.
  • An in vitro dual culture test for spore germination inhibition typically involves plating 50 ⁇ l of a conidial suspension (10 2 - 10 4 conidia ml -1 ) of the selected mycoparasite thoroughly distributed on an appropriate medium (e.g. PDA or MEA); in the middle of the plate a bacterial plug is disposed and the plates are incubated for 5-7 days, to subsequently measure the inhibition halo.
  • an appropriate medium e.g. PDA or MEA
  • the bacteria of the invention may exert antagonism by preventing conidial germination and/or by inhibiting mycelium growth.
  • a bacteria strain of the invention may exert antagonism against a pathogenic microorganism through chemicals produced by them.
  • a bacteria strain of the invention may express secondary metabolites, such as non-ribosomal peptides and/or polyketides, which may have antifungal activity.
  • the bacteria strain may contain gene clusters encoding the enzymatic machinery for synthesis of secondary metabolites. Ways to identify and detect secondary metabolites produced by bacteria are known in the art. Furthermore, ways to identify gene clusters encoding the enzymatic machinery for synthesis of peptides are also known in the art and described in the examples.
  • a bacteria strain of the invention may express antibiotics (e.g. bacilysin, bacillomycin, iturin A, surfactin or fengycin), siderophones or lytic enzymes (e.g. ⁇ -1,3- glucanase, chitinase, protease, lipase or amylase) or polyketides (e.g. bacillaene, diffidin or macrolactin).
  • antibiotics e.g. bacilysin, bacillomycin, iturin A, surfactin or fengycin
  • siderophones or lytic enzymes e.g. ⁇ -1,3- glucanase, chitinase, protease, lipase or amylase
  • polyketides e.g. bacillaene, diffidin or macrolactin.
  • Macrolactin has been described to exhibit antagonistic effects against the soil-borne plant pathogen R
  • Bacillaene has been shown to suppress the growth of fungi such as Pseudoxylaria and Trichoderma (Um et al., 2013).
  • Fengycin was the first discovered antifungal from Bacillus strains (Vanittanakom et al., 1986), and has been shown to exhibit antifungal activity for instance against Fusarium graminearum or Sclerotinia sclerotiorum (Ramarathnam et al., 2007).
  • a bacteria strain of the invention may be capable of expressing one or more metabolites selected from: macrolactin, bacillaene, fengycin, difficidin, bacilysin, teichuronic acid, butirosin, subtilin, bacillibactin, rhizocticin and/or surfactin.
  • the bacteria strain may be capable of expressing subtilin.
  • a bacteria strain of the invention may be capable of expressing macrolactin, bacillaene, fengycin, difficidin, bacilysin, teichuronic acid, butirosin, subtilin, bacillibactin, rhizocticin and surfactin.
  • each of Bacillus velezensis CM5, CM 19 and CM35 is capable of expressing all of these metabolites (see Table 1 at Example 7).
  • a bacteria strain of the invention may have plant growth promoting traits, including the ability to synthesise or degrade plant and fungal hormones and intermediates in plant and fungal hormone biosynthesis, to produce chemicals that modulate plant and fungal hormone biosynthesis, to secrete compounds that promote mobilisation of soil minerals, and to produce volatile or diffusible chemicals that induce plant and fungal immune responses.
  • the invention refers to any pathogenic microorganisms that cause diseases or disease symptoms in crops.
  • pathogenic microorganisms include bacterial, viral, fungal, nematode, phytomyxean, protozoan, algal and parasite crop pathogens.
  • the microorganism may be a fungal pathogen, such as a mycoparasite.
  • a bacteria strain of the invention is capable of specifically inhibiting a mycoparasite without affecting crop growth (e.g . A. bisporus).
  • a pathogenic microorganism that a bacteria strain of the invention is capable of exerting antagonism against may be one or more microorganisms selected from: Lecanicillium fungicola, Mycogone perniciosa, Trichoderma aggresivum., Cladobotryum mycophilum, Trichoderma atroviride, Fusarium ses, Alternaria solani, Alternaria brassiciola, Botrytis cinerea, Fusarium oxysporum, Phytophthora infestans, Sclerotinia sclerotorium and Cladosporium fulvum.
  • a bacteria strain of the invention may have a broad spectrum antifungal effect.
  • the bacteria strain may have antifungal effect against a number of mycoparasites.
  • a bacteria strain of the invention is particularly capable of exerting antagonism against Lecanicillium fungicola, Mycogone perniciosa, Trichoderma aggresivum, Cladobotryum mycophilum, Trichoderma atroviride, Fusarium ses, and/or Botrytis cinerea, as demonstrated in the examples herein.
  • a bacteria strain of the invention may exert antagonism against the mycoparasite, Lecanicillium fungicola, which causes dry bubble disease in crops, such as mushrooms.
  • the bacteria of the invention may prevent conidial germination and mycelium growth of the Lecanicillium fungicola, as shown e.g. in Figures 2, 3, 5-8.
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Lecanicillium fungicola, such as dry bubble disease in crops, for example, mushrooms, e.g. Agaricus bisporus.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • a bacteria strain of the invention may exert antagonism against the mycoparasite, Mycogone perniciosa which causes wet bubble disease in crops, such as mushrooms.
  • the bacteria of the invention may prevent conidial germination and mycelium growth of the Mycogone perniciosa, e.g. see Figures 2 and 5-6.
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Mycogone perniciosa, such as wet bubble disease in crops, for example, mushrooms, e.g. Agaricus bisporus.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • a bacteria strain of the invention may exert antagonism against mycoparasites, Trichoderma spp, such as Trichoderma aggresivum, Trichoderma atroviride, which cause green mould in crops, such as mushrooms (e.g. see Figures 3 and 9).
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Trichoderma spp, such as green mould in crops, for example, mushrooms, e.g. Agaricus bisporus.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • a bacteria strain of the invention may exert antagonism against the fungus, Rhizoctonbia solani.
  • R. solani causes a wide range of commercially significant crop diseases. It is one of the fungi responsible for brown patch (a turfgrass disease), damping off (e.g. in soybean seedlings), black scurf of potatoes, bare patch of cereals, root rot of sugar beet, belly rot of cucumber, sheath blight of rice, and many other pathogenic conditions.
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by R. solani.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • a bacteria strain of the invention may exert antagonism against Cladobotryum spp (e.g. see Figure 3).
  • Cladobotryum including C. dendroides, C. mycophilum, C. varium, C. multiseptatum, and C. verticillatum, are known to be the causal agents of cobweb disease in the mushroom A. bisporus and are found in mushroom- growing countries worldwide.
  • Cladobotryum varium was reported to be a causal agent of fungal disease in mushrooms P. eryngii and F. velutipes.
  • C. mycophilum has also been reported as a causal agent of cobweb disease in mushroom P. eryngii.
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Cladobotryum spp, such as cobweb disease in crops, for example mushrooms, e.g. Agaricus bisporus.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • a bacteria strain of the invention may exert antagonism against Botrytis cinerea (e.g. see Figure 12), which causes grey mould.
  • Botrytis cinerea is a necro trophic fungus that affects a wide variety of hosts including protein crops, fiber crops, oil crops, and horticultural crops.
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Botrytis cinerea, such as grey mould.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • the bacteria of the invention may exert antagonism against Fusarium spp, such as F. ses and F. oxysporum (e.g. see Figures 10 and 11). Fusarium species can cause damping-off and root rot of young conifer seedlings, resulting in severe crop and economic losses in forest nurseries. It is one of the most important soil-borne pathogens within nurseries throughout western North America.
  • a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Fusarium spp.
  • the bacteria of the invention is particularly effective in preventing such diseases.
  • crops are contemplated for use with the invention.
  • crops include food crops, such as fungal crops and horticultural crops.
  • Crops that are useful with the invention include fungal crops, e.g. mushroom.
  • mushrooms include: Agaricus bisporus (commonly known as button mushroom), Pleurotus spp. and in particular Pleurotus ostreatus (commonly known as oyster mushroom), Lentinula edodes (commonly known as shiitake mushroom), Auricularia auricula-judae (commonly known as Jew's ear mushroom), Volvariella volvacea (commonly known as paddy straw mushroom), Flammulina velutipes (commonly known as enoki mushroom), Tremella fuciformis (commonly known as snow fungus mushroom), Hypsizygus tessellatus (aka Hypsizygus marmoreus, commonly known as the beech mushroom), Ganoderma lucidum (commonly known as reishi), Stropharia rugosoannulata (commonly known as wine cap mushroom),
  • Horticultural crops may be useful with the invention and these include vegetables (examples are chickpeas, lettuce, broccoli, and beans) and small fruit crops (examples are grape, strawberry, and raspberry).
  • crops may also be useful with the invention: rice, wheat, sugarcane and other sugar crops, maize (com), soybean oil, potatoes, palm oil, cassava, legume pulses, sunflowerseed oil, rape and mustard oil, sorghum, millet, groundnuts, beans, sweet potatoes, bananas, soybeans, cottonseed oil, groundnut oil and/or yams.
  • the bacterial strains of the invention can be applied to crops to improve crop growth and control pathogenic microorganism growth in crops. Therefore, the bacterial strains can be used to benefit any crop, including healthy crops and crops affected by a crop disease or crop disease symptoms (e.g . the detectable presence of a known crop pathogen, or the presence of rot, mottling, galls, discoloration such as yellowing or browning, fruit greening, stunted growth, crop death, cellular death, cell wall breakdown, and/or the presence of spots, lesions, dieback, wilting, dwarfing, Witch's broom and/or knots).
  • a crop disease or crop disease symptoms e.g . the detectable presence of a known crop pathogen, or the presence of rot, mottling, galls, discoloration such as yellowing or browning, fruit greening, stunted growth, crop death, cellular death, cell wall breakdown, and/or the presence of spots, lesions, dieback, wilting, dwarfing, Witch's
  • the invention involves administering the bacteria strains and bacterial compositions of the invention to a crop and/or the environment surrounding the crop, e.g. the soil, casing material, and/or compost surrounding the crop, the water applied to the crop, or the hydroponic system in which the crop is grown.
  • Methods for administering the bacteria strains and compositions of the invention include application to the surface of the growth medium (e.g. casing material, substrate or compost), to the crop or parts of the crop, such as fruiting body, to the substrate, to the compost and/or to the environment of the crop.
  • Administration methods may include spraying, such as by foliar spraying, injection or soaking.
  • the bacteria strains and compositions of the invention are not applied onto the mushroom.
  • the strains and compositions of the invention are not applied onto the mushroom by foliar application.
  • strains and compositions of the invention may be applied to the surface of the casing material, not the compost.
  • the bacteria strains and compositions of the invention may also be administered by mixing with the irrigation water of the crop and/or with the growth medium (e.g . casing material).
  • the growth medium e.g . casing material
  • the administration method ideally provides an effective amount of the bacteria to the crop for controlling the growth of a crop pathogenic microorganism and improving crop growth.
  • an effective amount of bacteria may be an amount of bacteria which is sufficient to improve health, growth or productivity of the crop, and/or which reduces the effects, titre or symptoms of the crop disease, or prevents worsening of the crop disease, symptoms or infection of the crop.
  • an effective amount may be ⁇ 10, ⁇ 50, or ⁇ 100 cfu of the bacterial strain per gram of crop, e.g. at one week after administration.
  • the bacteria strains and compositions of the invention may be applied to the crop one or more times throughout the growth of the crop.
  • a bacteria strain or a composition of the invention may be applied: (1) after applying the casing layer, e.g. four days after applying the casing layer, (2) after the first flush and/or (3) after the second flush.
  • the invention relates to improving crop growth and controlling the growth of crop pathogenic microorganism.
  • the improvement in crop growth may be due to improved resistance to disease.
  • the invention may also provide a method of improving resistance to disease. This may be an increase of defence in a healthy crop or a decrease in disease severity in a crop or in a population of crops, or in the number of diseased crops in a crop population.
  • the improvement in crop growth may be due to improved ability to defend against disease.
  • the invention may also provide a method of improving the ability of a crop to defend against disease. This may be a measurable increase in crop defence against a disease, e.g. measured in terms of a measurable decrease in disease symptoms, pathogen titre, or loss of crop yield and/or quality, or a measurable increase in growth, crop quantity or quality.
  • the improvement in crop growth may be due to a reduction of disease symptoms.
  • the invention may also provide a method of reducing disease symptoms. This may be a measurable decrease in the number or severity of disease symptoms.
  • the improvement in crop growth may be due to faster growth.
  • the invention may also provide a method of inducing faster growth of a crop. This may be a measurable increase in the rate of growth of a crop, including seedlings, stems, roots, seeds, flowers, fruits, leaves and shoots thereof.
  • the improvement in crop growth may be due to improved crop productivity and/or quality.
  • the invention may also provide a method of improving crop productivity and/or quality. This may be a measurable increase in the quantity or quality of a crop in terms of numbers, size, or weight of crop seeds, fruits, vegetable matter, fibre, grain, and the like, or in terms of sugar content, juice content, unblemished appearance, colour, and/or taste.
  • the improvement in crop growth may be due to improved germination.
  • the invention may also provide a method of improving germination. This may be a measurable increase of the chance of successful germination of an individual seed (or mycelium in the case of mushrooms), a measurable increase in the percentage of seeds/active mycelium successfully germinating, and/or a measurable increase in the speed of germination.
  • the improvement in crop growth may be due to improved seedling emergence, or improved pinning in the case of mushrooms.
  • the invention may also provide a method of improving seedling emergence.
  • the invention may also provide a method of improving pinning in mushrooms. For example, a measurable increase in the speed of growth and/or development of successfully germinated individual seeds or spores can be observed.
  • the invention provides a composition comprising a bacteria strain of the invention.
  • the composition may be a bioinoculant.
  • the bioinoculant may comprise an excipient.
  • the excipient may be water or an organic solvent (. e.g . xilene, methanol, ethylene-glycol or mineral oil), a dispersion stabilizator, a surfactant (. e.g . calcium-dodecyl-benzene-sulphonate, polyglycol-ether, etoxylated alkyl-phenol or alkyl-aryl-sulphonates), optionally waxes.
  • an organic solvent e.g xilene, methanol, ethylene-glycol or mineral oil
  • a dispersion stabilizator e.g. calcium-dodecyl-benzene-sulphonate, polyglycol-ether, etoxylated alkyl-phenol or alkyl-aryl-sulphonates
  • a surfactant e.g . calcium-dodecy
  • the excipient may be montmorillonite, bentonite, wood flour, starch, cellulose and a binder, such as e.g. a mineral oil, polyvinyl-alcohol or saccharose.
  • the excipient may be selected by the skilled person without undue experimentation.
  • a bioinoculant composition of the invention may comprise cryoprotector, such as milk and/or sacarose solution.
  • a bioinoculant composition of the invention may be in the form of a water-soluble powder.
  • the inoculant composition of the invention may be in the form of an aqueous suspension, suspension concentrate, capsulated concentrate, emulsion forming liquid spray, granule, granule dispersible in water, or microgranule.
  • the formulation which may be used according to the invention may be selected by the skilled person without undue experimentation.
  • the concentration of each of the bacteria strain of the invention in the formulation maybe between 10 10 to 10 12 cfu gr -1 (colony forming units per gram of bacteria), e.g. 10 10 , 10 11 or 10 12 cfu gr -1 .
  • the invention also provides a growth medium comprising a bacteria strain of the invention.
  • the invention also provides a growth medium obtainable by treating with a bacteria strain of the invention.
  • a growth medium may be any medium that provides nutrients for a crop to grow, e.g. casing material, substrate, compost or soil. Casing materials and substrates are typically used for growing mushrooms. Casing materials useful with the invention may be black peat with sugar beet lime, blonde peat from Spaghnum peat moss, or a mixture of the two.
  • a bacteria of the invention When a bacteria of the invention is applied to a growth medium, it may influence the microbial ecology of the growth medium, and so a growth medium that has been treated with a bacteria strain of the invention may have a distinctive environment that would be capable of supporting crop growth and controlling the growth of pathogenic microorganisms.
  • the distinctive environment may be the result of having treated the growth medium, e.g. having treated the growth medium with 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5 or ⁇ 6 days, or 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7 or ⁇ 8 weeks.
  • a growth medium of the invention typically has an enriched population of a bacteria strain of the invention compared to the amount that is naturally present in the medium.
  • the growth medium of the invention may have a population of a bacteria of the invention that is ⁇ 10%, ⁇ 20%, ⁇ 30%, ⁇ 40%, >50% compared to the amount that is naturally present in the medium.
  • the amount of bacteria that is naturally present in the medium can be determined using routine methods, such as using PCR or labelling techniques for DNA that is specific to the bacteria of interest.
  • a bacteria strain of the invention may be combined with further agents in an integrated strategy for controlling the growth of crop pathogenic microorganism and/or improving crop growth.
  • the further agent may be further bacteria strains of the invention.
  • the further agent may be a biocontrol agent, such as Bacillus velezensis strain QST713.
  • the further agent may be a chemical fungicide, such as prochloraz -manganese, chlorothalonil or metrafenone, or a plant or fungal growth promoting chemical, and/or an elicitor of plant or fungal immune responses.
  • a bacteria strain of the invention as used herein is a collective term referring to any bacteria strain described herein, i.e. including CM5, CM19, CM35, and variants thereof.
  • ⁇ x herein, this means equal to or greater than x.
  • ⁇ y herein, this means equal to or less than y.
  • a first nucleic acid sequence having at least 70% nucleic acid sequence identity with a second sequence requires that, following alignment of the first nucleic acid sequence with the second sequence, at least 70% of the nucleotides in the first nucleic acid sequence are identical to the corresponding nucleotides in the second sequence.
  • sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 3 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 3, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, the gaps or missing positions should be considered to be non-identical positions.
  • Sequences are typically aligned for identity calculations using a mathematical algorithm, such as the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87 (1990): 22642268), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90 (1993): 5873 5877).
  • a mathematical algorithm such as the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87 (1990): 22642268), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90 (1993): 5873 5877).
  • Such an algorithm is incorporated into the XBLAST programs of Altschul et al. (J. Mol. Biol. 215 (1990): 403 410).
  • Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25 (1997): 3389 3402).
  • the BLAST algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • HSPs high scoring sequence pair
  • Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395).
  • Bacteria strains were isolated from casing materials for growing mushroom crops in the following way.
  • Contamination of the casing material for growing mushroom is considered the primary source of infection for many diseases.
  • the casing is the material applied to cover the fully colonized compost where the following takes place: mushroom fructification, and transition from vegetative growth (mycelium) to reproductive growth (mushrooms).
  • mushroom fructification for example, dry and wet bubble diseases are caused by Lecanicillium fungicola and Mycogone perniciosa, respectively, in the casing layer ( Figure 1).
  • natural suppressive effects against mycoparasites such as L. fungicola, M. perniciosa and Cladobotryum mycophilum can be observed in the absence of the host, and the native microbiota are thought to be responsible for these effects.
  • Mycoparasites are responsible for the largest crop losses in commercial mushroom production (Fletcher & Gaze, 2008), causing such diseases as dry bubble (Lecanicillium fungicola), wet bubble (Mycogone perniciosa) and green mould (Trichoderma spp.), which are major challenges for mushroom cultivation, directly affecting growers' profit margins.
  • dry bubble disease accounts for estimated 20% of button mushroom crop losses globally (Tasmanian Institute of Agriculture, 2018). Losses of production up to 40%, with 70% of profit shortcuts due to quality depreciation have been described in Spain.
  • the growing media used in the examples are the following:
  • Agar-casing 150 gr of dried and milled fresh casing are autoclaved with approx. 800 mL of distilled water at 121°C and 1 at. The supernatant is filtered through a double layer of muslin to remove the bulk of the substrate, and 10 g of tryptone, 5 g of yeast extract and 15 g of agar are added to the supernatant liquid up to 1 L with distilled water.
  • Agar-compost 50 gr of dried and ground phase II compost are autoclaved with approx. 800 mL of distilled water at 121°C and 1 at. The supernatant is filtered through a double layer of muslin to remove the bulk of the substrate, and 15 g of agar is added up to 1 L with distilled water.
  • CFC Pseudomonas supplement content of a vial for 500 mL of medium: cetrimide (5 mg); Fusionic acid (5 mg); Cephalosporin (25 mg).
  • - LBA 10 g Tryptone (Cultimed) + 5 g Yeast extract (Cultimed) + 10 g NaCl + 15 g Agar for 1L.
  • TSA Tryptone soya agar, prepared according to the manufacturer's instructions.
  • the plates were incubated at 25 and 28 ° C for 72h, then the colony forming units (c.f.u.) per plate in each of the media were counted, to estimate the differences between isolated microbial populations of each growing medium.
  • An analysis of variance (ANOVA) was performed using the Statgraphics Centurium XVI software, analyzing the significant differences by Fisher's LSD test (P ⁇ 0.05).
  • the colony multiplication ratio was determined by spectrophotometer reading (Thermo Fisher Scientific - Genesys 10-S) of the optical density of the culture broth at a wavelength of 600 nm (OD600). The fermentation was stopped when the absorbance at OD600 reached a value of 1. The culture broth fermented with the bacteria was resuspended in a 20% glycerol solution. Glycerol is a low molecular weight cryoprotective agent that penetrates cells. Finally, the tubes were stored in ultra-freezers at -80°C.
  • the pathogenic fungi used in the current work were isolated from a commercial crop showing symptoms of disease.
  • a conidial suspension of L. fungicola L1 and M. perniciosa M1 was prepared from a 9-days-old sporulating culture plated on PDA according to Carrasco et al. (2016). 50 ⁇ l of a 10 6 conidia/ml suspension was applied to PDA plates. 10 6 cfu of the bacterial strains previously cultured in Luria-Broth overnight (28°C, 200 rpm) were plated at intervals on the plates and the plates were incubated at 25 °C for 5-8 days.
  • Figure 2 shows an example of the screening plates from which the strains CM5, CM19 and CM35 were identified.
  • Figure 2 shows mycotoxicity of the strains CM5 and CM 19 against L. fungicola and M. perniciosa.
  • the strain was co-cultured in LBA with L. fungicola, T. aggressivum, C. mycophilum by plating a 5 mm agar plug of a 7-days-old PDA culture of mycoparasites.
  • the cultures were incubated at 22°C for 4-12 days (depending of the targeted mycoparasite) and subsequently four plugs of strains CM5 (incubated in LBA overnight) were disposed at 10 mm from the margin of the fungal colony in a cross shape. The plates were incubated for a further 5 days.
  • a 5 mm agar plug of a 7-days-old PDA culture of each of L. fungicola L1 and M. perniciosa M1 was plated. The cultures were incubated at 22°C for 12 days and subsequently four plugs of the bacterial strains (incubated in LB A overnight) were deposited 10 mm from the margin of the fungal colony in a cross shape. The plates were incubated for a further 5 days and the radial growth of the colony was measured and compared to the non-inoculated with bacteria as control.
  • Figure 5 shows that all three candidate strains, CM5, CM19 and CM35, generated radial inhibition on the mycelial growth of L. fungicola and M. perniciosa.
  • the mycelium plug co-cultured with the strains showed a radial inhibition rate of 56.5% (with CM5), 53.6 (with CM19) and 56.7 (with CM35) for L. fungicola L1 and of 54.5% (with CM5), 55.5% (with CM19) and 55.6% (with CM35) for M. perniciosa ML
  • a conidial suspension of L. fungicola L1 and M. perniciosa M1 was prepared from a 9-days-old sporulating culture plated on PDA according to Carrasco et al. (2016). 50 ⁇ l of a 10 6 conidia/ml suspension was applied to PDA plates. 10 6 cfu of the bacterial strains previously cultured in Luria-Broth overnight (28°C, 200 rpm) was plated either at intervals or at the center of the plate and the plates were incubated at 25 °C for 5-8 days.
  • Figure 6 shows growth of L. fungicola L1 and M. perniciosa M1 on plates without a bacterial strain (control) and with CM5, CM 19 and CM35 strains, and a non-fungitoxic bacterial strain. It can be seen that the growth of L. fungicola L1 and M. perniciosa M1 was unaffected in the plate with the non-fungitoxic bacteria. In contrast, CM5, CM 19 and CM35 all inhibited growth of L. fungicola L1 and M. perniciosa ML It can be seen that strains CM19 and CM35 exhibit greater antagonism of M. perniciosa M1 than CM5. The strains show clear inhibition of mycoparasites when co-cultured in vitro.
  • Figure 7 shows the plates from which the radial inhibition rate of the strains against L. fungicola L1 and M. perniciosa M1 was determined.
  • An inhibition halo equal to 25.8 mm (with CM5), 17.1 (with CM19) and 23.4 (with CM35) for L. fungicola L1 and
  • Example 5 Design of a stable water-soluble bacterial powder as a commercial formulation
  • a water-soluble powder has been designed as prototype of commercial formulation to apply computed cfu (colony forming units of bacteria) in crop while watering through spraying systems.
  • LB Luria-Bertani broth
  • Each strain recovered from the biobank stock was fermented overnight in LB broth and the broth, with OD600> 0.7 was used to inoculate a bioreactor (a 5 L Biostat A fermenter (Sartorius)).
  • a 10-fold serial dilution of the water-soluble powder in sterile water is prepared and plated on LB A plates to estimate the concentration of bacteria (cfu) in the powder and to adjust treatments.
  • Example 6 Antagonism against causal agents for major soil-borne diseases
  • Example 5 The water-soluble bacterial powder prepared in Example 5 was used to test the potential of the candidate strains to fight the causal agents for major soil-borne diseases.
  • a sterile 5 mm paper inoculated with 10 6 cfu/ml of each strains have been confronted to increasing doses of mycoparasites (10 2 , 10 3 , 10 4 , 10 5 conidia per plate) and incubated at room temperature.
  • the mycoparasites tested were: L. fungicola L1, M. perniciosa M1, Fusarium commune CEBAS-CSIC, Fusarium oxysporum CEBAS- CSIC, Botrytis cinerea 447, Alternaria spp. CEBAS-CSIC, Trichoderma atroviride Tal.
  • Figures 8-12 show the inhibition halos produced by strains CM5, CM19 and CM35 against L. fungicola L1, Trichoderma atroviride TA1, Fusarium oxysporum CEBAS-CSIC, Fusarium ses CEBAS-CSIC and Botrytis cinerea 447, respectively.
  • each of the candidate strains exhibited inhibition halos even at high concentrations of the different mycoparasites in the plates.
  • the candidate strains appear to have differing activity against different pathogenic fungi.
  • the candidate bacteria strains were identified based on the analyses of the 16S rDNA sequence.
  • gDNA from the candidate bacterial strains was obtained by centrifuging an overnight fermented LB broth at 28°C was extracted using an NucleoSpin® Microbial DNA (Macherey-Nigel). The concentration and purity of the extracted DNA was assessed using a NanoDrop ND1000 spectrophotometer.
  • Amplification was done by initial denaturation at 95°C for 300s, followed by 32 cycles of denaturation at 95°C for 60s, annealing at 52°C for 60s and extension at 72°C for 120s with the final extension at 72°C for 7 min.
  • Genome sequencing of the three strains was carried out by Microbes NG, Birmingham, UK.
  • the downstream processing of sequenced data was accomplished using several bioinformatics tools:
  • the RAST annotation server and the SEED annotation environment were used to annotate the prokaryotic genome (Aziz et al., 2008; Overbeek et al., 2005).
  • BLAST Ring Image Generator (BRIG) has been employed for prokaryote genome comparisons (Alikhan et al., 2011).
  • the secondary metabolite profile of the sequenced strains has been evaluated by antiSMASH (Blin et al., 2019).
  • the draft genome is provided in the contigs as set out in SEQ ID NOs: 57-106, and it has 4,205,756 bp with 45.85% GC.
  • the 16S rRNA-encoding gene is set out in SEQ ID NO: 1.
  • the draft genome is provided in the contigs as set out in SEQ ID NOs: 107-144, and it has 4,203,670 bp with 45.84% GC.
  • the complete genome is provided in SEQ ID NO: 145.
  • the 16S rRNA-encoding gene is set out in SEQ ID NO: 2.
  • the draft genome is provided in the contigs as set out in SEQ ID NOs: 6-56 and it has 4,204,667 bp with 45.84% GC.
  • the 16S rRNA-encoding gene is set out in SEQ ID NO: 3.
  • Preliminary Sanger sequencing of the 16S rRNA gene identified the strains as Firmicutes belonging to the genera Bacillus (>90% of most frequent species). Draft genome sequencing clusters the tree strains as Bacillus velezensis (>50% of most frequent species).
  • the strains of Bacillus velezensis CM5, CM19 and CM35 isolated from the casing materials employed in the cultivation of Agaricus bisporus present antifungal properties against the mycoparasites Lecanicillium fungicola L1 and Mycogone perniciosa M1 (the causative agents of dry and wet bubble), both to prevent conidial germination and mycelium growth.
  • the strains do not show detectable toxicity against the host mycelium (considering that Agaricus is also a fungus) which suggest a non- detrimental effect on mushroom production when applied in the presence of the host. In this sense it is remarkable that most chemical fungicides are applied in the absence of the cited mycelium, for instance when the casing material is applied in the crop.
  • a prototype for a water-soluble powder has been designed. This powder is stable in the fridge, and corresponds to the pure strain selected with a concentration average of 10 10 - 10 12 ufc gr -1 .
  • the bacteria strains when introduced in the powder form, exhibited effective antagonism against the agents responsible for many of the major soil-borne diseases ( e.g .
  • the bacteria strains of the invention are natural inhabitants amongst the microbiome in crop casing material, and so they would thrive in the environmental niche where the crop develops.
  • biocontrol agents such as B. velezensis QST713, that are not natural inhabitants in crop casing material would be inferior because they may be less capable of adapting to the environment in which it is intended to be used, thereby less effective in exerting antagonism against pathogenic microorganisms in that environment.
  • the casing microbiome is a complex environment for which the microbiome structure has been characterised by the inventors (Carrasco et al., 2019; Carrasco et al., 2020).
  • B. velezensis QST713 has been shown to be effective in treating green mould in A. bisporus, caused by T. aggressivum.
  • T. aggressivum is a compost mould which firstly infects compost, and then later the fruiting bodies and casing. It would not have been expected that B. velezensis QST713 would be effective in the casing layer, let alone exerting antagonism that is as effective as the bacteria strains of the invention, as demonstrated herein.
  • the bacterial strains of the invention constitute the only tailor-made biocontrol solution for the mushroom industry, and also show biocontrol activity against plant parasites as shown in the figures in respect to the in vitro effect against mycoparasites of plants.
  • strains discovered can be an alternative to chemical treatments in crops to fight many of the most harmful diseases, such as dry bubble (caused by L. fungicola) and wet bubble (caused by M. perniciosa). Since the industry is facing limitations for pesticide use, and the nature of host and parasite (both fungi) requires both high selectivity and large investment, it is not expected the occurrence of new pesticides to reduce crops losses to substitute those ones approved (with evidences of resistance already detected). Therefore native strains with an active secondary metabolic pathway showing toxicity to fungi are a good alternative to promote greening with the industry and to minimize the impact of fungal diseases.
  • the bacteria strains CM5, CM 19 and CM35 were tested in crop trials with Lecanicillium fungicola (which causes dry bubble) and Mycogone perniciosa (which causes wet bubble). Materials and Methods
  • Compost and casing (a mixture of peat-based casing materials at a rate of 50% each (Euroveen B.V. + Spaghnum peat moss, Valimex KF)) physico-chemical characterization was carried out by measuring; pH, electrical conductivity (EC), moisture content, ash, organic matter, nitrogen, C/N ratio and water holding capacity according to Pardo- Gimenez et al. (2016).
  • Trial 1 and trial 2 were conducted in three separate rectangular growing rooms provided with climate control (automatic Fancom System®).
  • 90 blocks of Phase III compost (Germinados de compost SL, Lodosa, Spain) spawned at 1% with the A. bisporus commercial strain Silvan A15M were cultivated per growing room. The blocks were lined up in metal shelving at three levels in groups of 5 blocks (crop surface per group: 1 m 2 ).
  • the blocks were covered with a layer of 3-4 cm of the saturated casing materials in a completely randomized block design with 3 replicates per casing.
  • a conidial suspension of L. fungicola in Room 2 and M. perniciosa in Room 3 was sprayed onto the surface of the casing layer at a rate of 2xl0 2 conidia m-2 (Room 2- L. fungicola) and 2*10 2 aleutispores m -2 in trial 1 and 2*10 2 conidia m -2 in trial 2 (Room 3- M. perniciosa).
  • the solution employed for the suspension consisted of sterile distilled water plus a drop of Tween 80 to avoid spore clustering.
  • Table 2 collects the treatments applied on peat basedcasing material during the cropping trials to prevent losses from mycoparasites.
  • Biocontrol treatments were formulated as stable water soluble powder and applied in trial 1 at a dose of 10 9 cfu m 2 a) four days after applying the casing layer; b) after the first flush; c) after the second flush and in trial 2 at a dose of 10 9 cfu m 2 a) four days after applying the casing layer; b) at the end of the spawn-running (casing colonized by mycelium); c) after the first flush; d) after the second flush.
  • Healthy and diseased mushrooms were harvested daily during three flushes, and the potential suppressive effect was analysed by comparing disease incidence and mushroom yield between treated and control blocks. Mushroom production in the different treatments was compared by ANOVA. Fisher’s LSD (least significant difference) test, at 5% probability, was used to establish significant differences between means. Non-parametric tests including Kruskal- Wallis test and Mann- Whitney (Wilcoxon) W test to compare medians at the 95% level were also implemented for non-normal distributions.
  • the bacteria strains in the form of water-soluble powder prepared according to Example 5 was used in this experiment.
  • Microbiological field applications require the production of large batches of microbial cultures. It is possible to obtain these large batches by culturing the desired isolates in sterile flasks containing a liquid medium, but this methodology is time consuming and it can easily lead to contaminations. Furthermore, the only parameter that is possible to control is the temperature, and it is not possible to optimise important parameters such as pH, air saturation, carbon dioxide and oxygen content. Bioreactors are systems developed to make easier the growth of large microbial culture batches, limiting the contamination sources and allowing the control of all the parameters required to optimise the process and maximise the yield.
  • CM 19 and CM35 are as effective as those ones treated with pesticides.
  • Biocontrol treatments at the dosses applied, are as efficient as chemical treatments to prevent losses derived from the fungal diseases dry and wet bubble.
  • the application of a mixture of the three selected strains did not show a positive synergistic effect.
  • the results suggest that the biocontrol agents, in the form of the water soluble powder, can be effectively employed on crops, such as mushroom, to treat diseases, such as dry and wet bubble.
  • bacteria strains were deposited at the European Collection of Authenticated Cell Cultures (ECACC), Culture Collections, Public Health England, Porton Down, Salisbury, SP40JG, United Kingdom on 3 June 2021 by Gail Preston of Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, Oxfordshire, OX1 3RB, United Kingdom.
  • the bacteria strains were allocated the following accession numbers:
  • the depositor has authorised Oxford University Innovation Limited of Buxton Court, 3 West Way, Oxford, OX2 0JB, United Kingdom to refer to the deposited biological materials in this application and its priority application (GB 2009235.9), and has given her unreserved and irrevocable consent to the deposited materials being made available to the public in accordance with the relevant Patent Legislation of the country in which this Patent Application is filed.
  • Bacillus velezensis QST 713 GenBank Ac. No: CP025079
  • Bacillus velezensis M75 GenBank Ac. No: CP016395
  • Bacillus velezensis 157 GenBank Ac. No: CP022341.
  • colony forming units refer to individual colonies of bacteria.
  • Room 1 Control, non-inoculated;
  • Room 2 L. fungicola (200 conidia m -2 );
  • Room 3 M. pemiciosa (200 aleuriospores m -2 ).
  • Treatments Metrafenone (0.5 ml m -2 ); Chlorothalonil (2 ml m -2 ); CM5, CM19, CM35 (109 cfu m -2 ). Equal letters within the rows means non-statistical significant differences according to Fisher's LSD test at P ⁇ 0.05.
  • Room 1 Control, non-inoculated;
  • Room 2 L. fungicola (200 conidia m -2 );
  • Room 3 M. pemiciosa (200 conidia m -2 ).
  • Treatments Metrafenone ( 0.5 ml m -2 ); CM5+CM19+CM35, CM5, CM19, CM35 (109 cfu m -2 ).
  • Equal letters within the rows means non-statistical significant differences according to Fisher’s LSD test at P ⁇ 0.05.
  • SEQ ID NO: 1 is the DNA sequence encoding the 16S rRNA of Bacillus velezensis strain CM5 (1872bp).
  • SEQ ID NO: 2 is the DNA sequence encoding the 16S rRNA of Bacillus velezensis strain CM 19 (1832 bp).
  • SEQ ID NO: 3 is the DNA sequence encoding the 16S rRNA of Bacillus velezensis strain CM35 (1804 bp).
  • SEQ ID NO: 4 is the forward primer used in the Examples for amplifying the 16S rRNA gene in a PCR.
  • SEQ ID NO: 5 is the reverse primer used in the Examples for amplifying the 16S rRNA gene in a PCR.
  • SEQ ID NOs: 6-56 are contigs 1-51 of the draft genome sequence of Bacillus velezensis strain CM35.
  • SEQ ID NOs: 57-106 are contigs 1-49 of the genome sequence of Bacillus velezensis strain CM5.
  • SEQ ID NOs: 107-144 are contigs 1-38 of the genome sequence of Bacillus velezensis strain CM 19.
  • SEQ ID NO: 145 is the complete genome sequence of Bacillus velezensis strain
  • SEQ ID NO: 146 is the amino acid sequence of glycerol uptake facilitator (glpF) (Location: 983698-984525 in SEQ ID NO: 145).
  • SEQ ID NO: 147 is the amino acid sequence of dihydroxy-acid dehydratase (ilvD) (Location: 2176459-2178135 in SEQ ID NO: 145).
  • SEQ ID NO: 148 is the amino acid sequence of phosphotransacetylase (pta l) (Location: 2581178-2582071 in SEQ ID NO: 145).
  • SEQ ID NO: 149 is the amino acid sequence of phosphotransacetylase (pta_2) (Location: 3916727-3917698 in SEQ ID NO: 145).
  • SEQ ID NO: 150 is the amino acid sequence of phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH) (Location: 727571-729109 in SEQ ID NO: 145).
  • SEQ ID NO: 151 is the amino acid sequence of triose phosphate isomerase (tpiA) (Location: 3532919-3533680 in SEQ ID NO: 145).
  • SEQ ID NO: 152 is the amino acid sequence of protein translocase subunit SecA (SecA) (Location: 3691586-3694111 in SEQ ID NO: 145).
  • SEQ ID NO: 153 is the amino acid sequence of Carbamoyl -phosphate synthase arginine-specific (carA_l) (Location: 1153790-1154848 in SEQ ID NO: 145).
  • SEQ ID NO: 154 is the amino acid sequence of Carbamoyl -phosphate synthase small chain (carA_2) (Location: 1606969-1608063 in SEQ ID NO: 145).
  • SEQ ID NO: 155 is the amino acid sequence of Protein RecA (RecA) (Location: 1752657-1753700 in SEQ ID NO: 145).
  • SEQ ID NO: 156 is the amino acid sequence of Chaperone protein DnaK (DnaK) (Location: 2701328-2703166 in SEQ ID NO: 145).
  • SEQ ID NO: 157 is the amino acid sequence of ATP synthase subunit beta (atpD) (Location: 3839076-3840497 in SEQ ID N: 145).
  • SEQ ID NO: 158 is the amino acid sequence of DNA gyrase subunit B (gyrB) (Location: 4454-6376 in SEQ ID NO: 145).
  • SEQ ID NO: 159 is the amino acid sequence of Translation initiation factor IF-2 (inffi) (Location: 1718422-1720569 in SEQ ID NO: 145).
  • PurR is the amino acid sequence of transcription regulator associated with purine metabolism (PurR) (Location: 55037-55867 in SEQ ID NO: 145)
  • PurR is the amino acid sequence of transcription regulator associated with purine metabolism (PurR) in CM5 and CM35.
  • SEQ ID NO: 162 is the amino acid sequence of Carbamoyl-phosphate synthase small chain (carA) in CM5 and CM35.
  • SEQ ID NO: 163 is the amino acid sequence of glycerol uptake facilitator (glpF) (Location: 983695-984525 in SEQ ID NO: 145).
  • SEQ ID NO: 164 is the nucleotide sequence of glycerol uptake facilitator (glpF).
  • SEQ ID NO: 165 is the nucleotide sequence of phosphotransacetylase (pta l).
  • SEQ ID NO: 166 is the nucleotide sequence of phosphotransacetylase (pta_2).
  • SEQ ID NO: 167 is the nucleotide sequence of phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH).
  • SEQ ID NO: 168 is the nucleotide sequence of triose phosphate isomerase (tpiA).
  • SEQ ID NO: 169 is the nucleotide sequence of protein translocase subunit SecA
  • SEQ ID NO: 170 is the nucleotide sequence of Carbamoyl-phosphate synthase arginine-specific (carA l).
  • SEQ ID NO: 171 is the nucleotide sequence of Carbamoyl-phosphate synthase small chain (carA_2).
  • SEQ ID NO: 172 is the nucleotide sequence of Protein RecA (RecA).
  • SEQ ID NO: 173 is the nucleotide sequence of Chaperone protein DnaK (DnaK).
  • SEQ ID NO: 174 is the nucleotide sequence of ATP synthase subunit beta (atpD).
  • SEQ ID NO: 175 is the nucleotide sequence of DNA gyrase subunit B (gyrB).
  • SEQ ID NO: 176 is the nucleotide sequence of Translation initiation factor IF-2 (inffi).
  • PurR is the nucleotide sequence of transcription regulator associated with purine metabolism (PurR).
  • SEQ ID NO: 178 is the nucleotide sequence of transcription regulator associated with purine metabolism (PurR) in CM5 and CM35.
  • SEQ ID NO: 179 is the nucleotide sequence of Carbamoyl-phosphate synthase small chain (carA) in CM5 and CM35.

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Abstract

The present invention relates to bacteria strains which are, or are derived from, natural inhabitants in the microbiome of growth medium for crops. Such bacteria strains are capable of selective suppressive effects on crop pathogenic microorganisms, in particular mushroom crops, and are therefore useful for controlling the growth of crop pathogenic microorganisms and improving crop growth.

Description

BACTERIA
Field of the Invention
The invention relates to bacteria strains for controlling the growth of crop pathogenic microorganisms and improving crop growth. Background to the Invention
Pathogens and pests affect crops, causing substantial economic losses and threatening food security. For example, mycoparasites are responsible for the largest crop losses in commercial mushroom production (Fletcher & Gaze, 2008), causing diseases such as dry bubble ( Lecanicillium fungicola), wet bubble ( Mycogone perniciosa) and green mould ( Trichoderma spp.), which are major challenges for mushroom cultivation, directly affecting growers’ profit margins. Dry bubble disease accounts for estimated 20% of button mushroom crop losses globally (Tasmanian Institute of Agriculture, 2018). Losses of production up to 40%, with 70% of profit shortcuts due to quality depreciation have been described in Spain. The control of pests and pathogens currently relies on the application of integrated management programs in addition to the use of chemical pesticides, mostly fungicides. However, reports of the emergence of resistant strains and the increase in concern for the impact on environment and human health restrict the use of chemical fungicides.
Developing new biocontrol agents for crops is a challenge for the agricultural industry, especially the food industry. It is notable that frequently in vitro results do not translate into efficiency in the field, mostly due to the limited knowledge about the ecology of the crop and the difficulties of reproducing a reliable microcosm in the lab.
Development of biocontrol agents is an active field. For example, bacteria isolated from mushroom compost were tested for activity against Trichoderma spp., the causal agents of green mould in A. bisporus and one isolate was identified to be a Bacillus subtilis strain (Milijasevic-Marcic et al., 2017). Another example of a biocontrol agent is Bacillus velezensis strain QST713, the active ingredient in a commercially available biofungicide, Serenade®, which has been registered for use against many crop pathogens. B. velezensis QST713 has been used industrially in France for the protection of button mushrooms, Agaricus bisporus, against green mould disease. Reports on the biocontrol mechanism of this bacteria are underway (Pandin et ah, 2018 and 2019).
It is an object of the invention to identify further biocontrol agents, such as bacteria strains, for controlling the growth of crop pathogenic microorganisms and improving crop growth.
Summary of the Invention
The inventors have surprisingly identified Bacillus velezensis strains in the casing material employed in mushroom cultivation that are capable of exerting antagonism against pathogenic microorganisms responsible for many of the major soil-borne diseases, such as mycoparasites. These strains may be useful as biocontrol agents for controlling the growth of crop pathogenic microorganisms and improving crop growth.
The inventors hypothesised that some mushroom-associated microorganisms can specifically inhibit mycoparasitic species without affecting mushroom growth, and the identified microorganisms would have useful antagonistic properties against pathogenic microorganisms in crops, and hence they would be useful biocontrol agents. The inventors therefore looked for possible biocontrol agents in the casing inhabitants that occur naturally in high densities and do not have deleterious effects on mushrooms such as Agaricus bisporus. The casing is the material applied to cover the fully colonized compost where the following takes place: the transition from vegetative growth (mycelium) to reproductive growth (mushrooms), also known as mushroom fructification. The inhabitants in raw casing material are thought to provide natural suppressive effects against mycoparasites such as L.fungicola, M. perniciosa and Cladobotryum mycophilum, although these suppressive effects are weakened during the colonization of the casing material by the mycelium of A. bisporus, resulting in the contribution of disease outbreaks such as cobweb (caused by Cladobotryum spp.), bubble diseases (caused by L. fungicola or M. perniciosa) or green mould (caused by Trichoderma spp.).
The inventors identified Bacillus velezensis strains CM5, CM19 and CM35, each of which is capable of exerting antagonism against the mycoparasites Lecanicillium fungicola L1 and Mycogone perniciosa M1, to prevent conidial germination and mycelium growth. Furthermore, the strains do not show detectable toxicity against the host mycelium ( Agaricus bisporus) which suggests a non-detrimental effect on mushroom production when applied as an inoculant.
The inventors were surprised to find that CM5, CM19 and CM35 were effective in exerting antagonism against mycoparasites when the strains were introduced into casing material during mushroom fruitbody formation, because as mentioned above A. bisporus tends to release the mycoparasites from fungistasis in the casing layer. The data from crop trials show that no deleterious effect on mushroom production has been detected due to the application of the bacterial treatments.
Furthermore, the inventors have formulated the bacteria strains into powder formulations that would be useful for applying the bacteria to crops. The bacteria strains, when introduced in the water-soluble powder form, exhibited effective antagonism against the agents responsible for many of the major soil-borne diseases (e.g. L. fungicola, M. perniciosa, Trichoderma atroviride (mushroom mycoparasites and competitors), Fusarium commune, F. oxysporum, Botrytis cinerea, Alternaria spp. (plant mycoparasites)). The genome sequencing and the metabolism studies of the bacteria strains suggested the production of metabolites with an active antifungal profile. The results therefore suggest that the bacteria strains discovered can be used to control the growth of crop pathogens and improve crop growth, not only for button mushrooms, Agaricus bisporus, but also for other plant crops.
Thus, the invention provides a bacteria strain which: (a) comprises a 16S rRNA- encoding gene having ≥95% sequence identity with SEQ ID NO: 1, 2 or 3, (b) comprises a genome having ≥90% identity to the genome sequence of B. velezensis CM5, CM 19 or CM35, (c) is Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301, or a variant thereof, (d) is Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302, or a variant thereof, or (e) is Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303, or a variant thereof, wherein said strain is capable of exerting antagonism against a crop pathogenic microorganism.
The invention also provides a bioinoculant composition comprising the bacteria strain of the invention. The invention also provides a crop growth medium comprising the bacteria strain of the invention.
The invention also provides a method for controlling the growth of a crop pathogenic microorganism, improving crop growth and/or treating or preventing a disease caused by a crop pathogenic microorganism, comprising administering to a crop and/or the environment surrounding the crop, the bacteria strain of the invention, the bioinoculant composition of the invention, or the growth medium composition of the invention.
The invention also provides a method of treating and/or preventing a disease, such as dry bubble disease, wet bubble disease or cobweb, comprising administering to a crop and/or the environment surrounding the crop, the bacteria strain of the invention, the bioinoculant composition of the invention, or the growth medium composition of the invention. For example, the method involves applying to a crop the B. velezensis strain, or a variant thereof, of the invention, or the composition of the invention.
The invention also provides the use of the bacteria strain of the invention for controlling the growth of a crop pathogenic microorganism and/or improving crop growth, such as mushroom growth.
The invention also provides a growth medium for crops obtainable by treating with the bacteria strain of the invention.
The invention also provides a method of isolating a microorganism with antagonistic properties against one or more crop pathogenic microorganisms, comprising the steps of obtaining a sample from casing material, testing the sample for antagonistic properties and isolating microorganism with said antagonistic properties.
Brief Description of the Figures
Figure 1 shows healthy and diseased mushrooms a, b) healthy crop (control); c, d) symptoms of mushrooms infected with L. fungicola (dry bubble); e, f) undifferentiated mass of A. bisporus tissue infected by M. perniciosa (wet bubble).
Figure 2 shows mycotoxicity of the strains against the mycoparasites evaluated. Circles indicate strains with antifungal activity towards L. fungicola and M. perniciosa.
Figure 3 shows the effect of CM5 co-cultured with mycoparasites in LBA: a) inhibition of L. fungicola,· b) Inhibition of T. aggressivum; c) inhibition of C. mycophilum; d) inhibition of conidial germination (L. fungicola) compared to non-active strains (circles). In a)-c) the mycoparasite is inoculated in the centre of the plate, and candidate biocontrol agents (CBAs) are inoculated adjacent to the growing mycelium. In d) the conidial suspension is spread on the agar and the CBAs inoculated afterwards.
Figure 4 shows two time points of mycotoxicity of the selected strains against bisporus Amycel XXX® in agar compost.
Figure 5 shows radial inhibition generated by strains CM5, CM 19 and CM35 respectively on the mycelial growth of a) L. fungicola and b) M. perniciosa.
Figure 6 shows mycoparasites, L. fungicola L1 and M. perniciosa M1, co-cultured with strains CM5, CM19 and CM35. Negative control with four non-fungitoxic strains was included.
Figure 7 shows the Inhibition Zone Diameter (IZD) generated by the strains CM5, CM 19 and CM35, respectively, in the conidial germination of L. fungicola L1 (109 conidia/plate).
Figure 8 shows L. fungicola L1 co-cultured with the strains CM5, CM19 and CM35 introduced as a water-soluble bacterial powder (50 μl of 109 ufc/ml) against 102, 103, 104 and 105 conidia per plate (PDA). Top row shows growth of L. fungicola in the absence of the bacterial strains. The second, third, and fourth rows correspond to strains CM5, CM19, and CM35, respectively.
Figure 9 shows Trichoderma atroviride TA1 co-cultured with the strains CM5, CM19 and CM35 introduced as a water-soluble bacterial powder (50 μl of 109 ufc/ml) against 102, 103, 104 and 105 conidia per plate (PDA). Top row shows growth of L. fungicola in the absence of the bacterial strains. The second, third, and fourth rows correspond to strains CM5, CM19, and CM35, respectively.
Figure 10 shows Fusarium oxysporum CEBAS-CSIC co-cultured with the strains CM5, CM 19 and CM35 introduced as a water-soluble bacterial powder (50 μl of 109 ufc/ml) against 102, 103 and 104 conidia per plate (PDA). The first, second, and third rows correspond to strains CM5, CM19, and CM35, respectively.
Figure 11 shows Fusarium commune CEBAS-CSIC co-cultured with the strains CM5, CM 19 and CM35 introduced as a water-soluble bacterial powder (50 μl of 109 ufc/ml) against 102, 103, 104 and 105 conidia per plate (PDA). The first, second, and third columns correspond to strains CM5, CM19, and CM35, respectively. Figure 12 shows Botrytis cinerea CEBAS-CSIC co-cultured with the strains CM5, CM19 and CM35 introduced as a water-soluble bacterial powder (50 μl of 109 ufc/ml) against 102, 103, 104 and 105 conidia per plate (PDA). The first, second, and third columns correspond to strains CM5, CM19, and CM35, respectively.
Figure 13 shows the production (kg m-2) of healthy mushrooms in the first trial in crop: a) control room, non-inoculated; b) room infected with Lecanicillium fungicola L1; c) room infected with Mycogone perniciosa M1.
Figure 14 shows the production (kg m-2) of healthy mushrooms in the second trial in crop: a), control room, non-inoculated; b) room infected with Lecanicillium fungicola L1; c) room infected with Mycogone perniciosa M1.
Detailed Description of the Invention
Bacteria of the invention
A bacteria strain of the invention may comprise a 16S rRNA-encoding gene having ≥95% (i.e. equal or greater than 95%), ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the 16S rRNA-encoding gene of Bacillus velezensis CM5 (SEQ ID NO: 1). For example, the 16S rRNA-encoding gene may have ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9% or 100% sequence identity with SEQ ID NO: 1. The 16S rRNA-encoding gene of the bacteria strain of the invention may differ from SEQ ID NO: 1 by 1, ≤2 (i.e. equal to or less than 2), ≤3, ≤4, ≤5, ≤6 or ≤7 bases. The bacteria strain may be a Bacillus velezensis strain.
A bacteria strain of the invention may comprise a genome having ≥90% (i.e. equal or greater than 90%), ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9% or 100% sequence identity with the genome sequence of Bacillus velezensis CM5. The draft genome of Bacillus velezensis CM5 is provided in SEQ ID NOs: 57-106, and it has 4,205,756 bp with 45.85% GC. Thus, the genome sequence of Bacillus velezensis CM5 may be obtainable by the assembly of the contigs provided in SEQ ID NOs: 57-106. The bacteria strain may be a Bacillus velezensis strain.
A bacteria strain of the invention may be Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301. A bacteria strain of the invention may comprise a 16S rRNA-encoding gene having ≥95% (i.e. equal or greater than 95%), ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the 16S rRNA-encoding gene of Bacillus velezensis CM19 (SEQ ID NO: 2). For example, the 16S rRNA-encoding gene may have ≥98.4%, ≥98.5, ≥98.6%, ≥98.7%, ≥98.8%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9% or 100% sequence identity with SEQ ID NO: 2. The 16S rRNA-encoding gene of the bacteria strain of the invention may differ from SEQ ID NO: 2 by 1, ≤2 (i.e. equal to or less than 2), ≤3, ≤4, ≤5, ≤6, ≤7, ≤8, ≤9, ≤10, ≤15, ≤20, ≤25 or ≤29 bases. The bacteria strain may be a Bacillus velezensis strain.
A bacteria strain of the invention may comprise a genome having ≥90% (i.e. equal or greater than 90%), ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9% or 100% sequence identity with the genome sequence of Bacillus velezensis CM 19. The complete genome of Bacillus velezensis CM19 is provided in SEQ ID NO: 145, and it has 4,203,670 bp with 45.84% GC. The draft genome is provided in SEQ ID NOs: 107-144. Thus, the genome sequence of Bacillus velezensis CM 19 may be obtainable by the assembly of the contigs provided in SEQ ID NOs: 107-144. The bacteria strain may be a Bacillus velezensis strain.
A bacteria strain of the invention may be Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302.
A bacteria strain of the invention may comprise a 16S rRNA-encoding gene having ≥95% (i.e. equal or greater than 95%), ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the 16S rRNA-encoding gene of Bacillus velezensis CM35 (SEQ ID NO: 3). For example, the 16S rRNA-encoding gene may have ≥98.7%, ≥98.8%, ≥98.9%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9% or 100% sequence identity with SEQ ID NO: 3. The 16S rRNA-encoding gene of the bacteria strain of the invention may differ from SEQ ID NO: 3 by 1, ≤2 (i.e. equal to or less than 2), ≤3, ≤4, ≤5, ≤6, ≤7, ≤8, ≤9, ≤10, ≤15, ≤20 or ≤23 bases. The bacteria strain may be a Bacillus velezensis strain.
A bacteria strain of the invention may comprise a genome having ≥90% (i.e. equal or greater than 90%), ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, ≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9% or 100% sequence identity with the genome sequence of Bacillus velezensis CM35. The draft genome of Bacillus velezensis CM35 is provided in SEQ ID NOs: 6-56 and it has 4,204,667 bp with 45.84% GC. Thus, the genome sequence of Bacillus velezensis CM35 may be obtainable by the assembly of the contigs provided in SEQ ID NOs: 6-56. The bacteria strain may be a Bacillus velezensis strain.
A bacteria strain of the invention may be Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303.
A bacteria strain of the invention includes Bacillus velezensis CM5, CM 19 or CM35, and variants thereof. Bacillus velezensis CM5, CM19 and CM35 were initially isolated from casing materials for growing A. bisporus. Hence, a bacteria strain of the invention may be a natural inhabitant in a casing material, e.g. for growing A. bisporus.
A bacteria strain of the invention may be wild type, which is the unaltered form of the bacterial strain Bacillus velezensis CM5, CM19 or CM35. Hence, a bacteria strain of the invention may be a strain selected from:
Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301;
Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302; and
Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303.
A bacteria strain of the invention may be a variant of Bacillus velezensis CM5, CM19 or CM35. Hence, a bacteria strain of the invention may be a variant of a strain selected from:
Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301;
Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302; and
Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303. The variant typically retains the properties of its corresponding wild type strain, such as being capable of exerting antagonism against a crop pathogenic microorganism, e.g. inhibiting the growth of Lecanicillium fungicola and/or Mycogone perniciosa.
A bacteria strain of the invention may have ≥1 ( i. e. one or more), ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8 or ≥9 housekeeping gene having ≥98% ( i.e . equal or greater than 98%), ≥99% or 100% sequence identity with the corresponding housekeeping gene in B. velezensis CM5, CM 19 or CM35, wherein the housekeeping gene is selected from glycerol uptake facilitator ( glpF) (e.g. SEQ ID NO: 146 or 163), dihydroxy-acid dehydratase {ilvD) (e.g. SEQ ID NO: 147), phosphotransacetylase (pta ) (e.g. SEQ ID NO: 148 or 149), phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH) (e.g. SEQ ID NO: 150), triose phosphate isomerase (tpiA) (e.g. SEQ ID NO: 151), protein translocase subunit SecA ( secA ) (e.g. SEQ ID NO: 152), carbamoyl-phosphate synthase arginine- specific ( carA_l ) (e.g. SEQ ID NO: 153), carbamoyl-phosphate synthase small chain 0 carAJ ) (e.g. SEQ ID NO: 154 for CM19 or SEQ ID NO: 162 for CM5 or CM35), protein RecA ( recA ) (e.g. SEQ ID NO: 155), chaperone protein DnaK ( dnaK) (e.g. SEQ ID NO: 156), ATP synthase subunit beta ( atpD ) (e.g. SEQ ID NO: 157), DNA gyrase subunit B igyrB) (e.g. SEQ ID NO: 158) and/or translation initiation factor IF-2 ( infB ) (e.g. SEQ ID NO: 159). The names of these genes are in accordance with the pubMLST scheme for Bacillus spp. (https://pubmlst.org/).
A bacteria strain of the invention may have ≥1 {i.e. one or more), ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8 or ≥9 housekeeping gene having ≥98% {i.e. equal or greater than 98%), ≥99% or 100% sequence identity with the corresponding housekeeping gene in B. velezensis CM5, CM 19 or CM35, wherein the housekeeping gene is selected from glycerol uptake facilitator ( glpF) (e.g. SEQ ID NO: 146 or 163), dihydroxy-acid dehydratase {ilvD) (e.g. SEQ ID NO: 147), phosphotransacetylase {pta ) (e.g. SEQ ID NO: 148 or 149), phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH) (e.g. SEQ ID NO: 150), triose phosphate isomerase {tpiA) (e.g. SEQ ID NO: 151), protein translocase subunit SecA {secA) (e.g. SEQ ID NO: 152), carbamoyl-phosphate synthase arginine- specific {carA_1) (e.g. SEQ ID NO: 153), carbamoyl-phosphate synthase small chain {carA_2) (e.g. SEQ ID NO: 154 for CM19 or SEQ ID NO: 162 for CM5 or CM35), protein RecA {recA) (e.g. SEQ ID NO: 155), chaperone protein DnaK ( dnaK) (e.g. SEQ ID NO: 156), ATP synthase subunit beta {atpD) (e.g. SEQ ID NO: 157), DNA gyrase subunit B igyrB) (e.g. SEQ ID NO: 158), translation initiation factor IF-2 ( infB ) (e.g. SEQ ID NO: 159) and/or purine nucleotide synthesis repressor (purR ) (e.g. SEQ ID NO: 160 for CM19 or SEQ ID NO: 161 for CM5 or CM35).
A bacteria strain of the invention may have ≥1 ( /. e. one or more), ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8 or ≥9 housekeeping gene, such as the ones listed above, wherein the nucleic acid sequence encoding the housekeeping gene may have ≥90% ( i.e . equal or greater than 90%), ≥95% or 100% sequence identity with the nucleic acid encoding the corresponding housekeeping gene in B. velezensis CM5, CM19 or CM35 (e.g. any of the SEQ ID NOs: 164 to 179).
A bacteria strain of the invention may contain gene modifications relative to the wild type strain (e.g. Bacillus velezensis CM5, CM19 or CM35), such as gene disruptions or gene additions. Using known methods in the art, variants may be generated to improve or alter the characteristics of the bacteria of the present invention. A variant typically retains the crop pathogenic microorganism antagonistic properties of its corresponding wild type strain, e.g. inhibiting the growth of Lecanicillium fungicola and/or Mycogone perniciosa.
Such variants include deletions, insertions, inversions, repeats and substitutions in the genome of the bacteria selected according to general rules known in the art. For example, the variants may contain modified genes with codon optimisation. The gene modifications may increase the efficiency of pathogen antagonistic effects, expand antagonistic spectrum, expand the host range, for pest resistance, increase their production or to allow them to be grown outside their original habitat. Methods of codon optimisation are well known in the art.
A bacteria strain of the invention may express additional genes relative to the wild type strain (CM5, CM19 or CM35). The additional genes may increase the efficiency of pathogen antagonistic effects, expand antagonistic spectrum, expand the host range, for pest resistance, increase their production or to allow them to be grown outside their original habitat. Methods to express additional proteins in bacteria are known in the art.
A bacteria strain of the invention is particularly effective in growing in growth medium for crops, such as casing material for growing mushrooms. A bacteria strain of the invention may be in a culture in the form of a stock (e.g. frozen at -80°C or freeze-dried), in a liquid broth or in a formulation. Methods for reviving bacteria from frozen or freeze-dried cultures are known in the art.
The invention also provides a method of isolating a microorganism with antagonistic properties against one or more crop pathogenic microorganisms, comprising the steps of obtaining a sample from casing material for growing mushrooms, testing the sample for antagonistic properties and isolating microorganism with said antagonistic properties. The isolated microorganism may be a bacteria of the invention.
For isolation of the microorganism, e.g. bacteria, a sample may be suspended in a liquid, such as a bacterial growth broth. The suspension may then be centrifuged in order to pellet debris. The supernatant may be serially diluted and grown on nutrient agar plates. Individual colonies can then be isolated and they can then be, for example, transferred directly to storage or further cultured, such as in a fresh nutrient agar plates or in a growth broth, such as LB. Preliminary characterisation can be carried out by microscopic appearance, Gram reaction and catalase test, all of which are well known in the art. Bacterial strains can be identified based on the analyses of the 16S rDNA sequence, e.g. PCR assay. The individual colonies can be screened for desirable properties, such as antagonistic effects against pathogenic microorganisms in crops, e.g. fungal pathogens.
A bacteria strain of the invention is selected on the basis of it having particular advantageous properties for controlling the growth of crop pathogenic microorganism and/or improving crop growth. The bacteria strains of the invention are surprisingly effective in exerting antagonism against pathogenic microorganisms in crops, such as fungal crop pathogens, and in particular exhibiting selective suppressive effects against fungal crop pathogens. A bacteria strain of the invention may be superior in exerting antagonism against pathogenic microorganisms in crops compared to other bacteria strains isolated from the casing material for growing mushrooms.
Exerting antagonism against pathogenic microorganisms in crops
A bacteria strain of the invention is capable of exerting antagonism against pathogenic microorganisms in crops. A bacteria strain of the invention may kill, deter, suppress growth or inhibit reproduction of the pathogenic microorganism. A bacteria strain of the invention may exert antagonism by producing specific substances that affect pathogen cell membranes such as those described below, by inducing resistance, and may act as competitors to pathogenic microorganism for nutrient sources and space. Examples of pathogenic microorganisms are provided below.
Methods of determining antagonistic effects against a pathogen are known in the art and described in the examples. For example, antagonism may be determined by assessing the degree of inhibition on nutrient agar plates, e.g. in a dual culture assay as described by Sakthivel and Gnanamanickam (1986). A bacteria strain may be inoculated at a set distance from a pathogen, and the difference in the growth of the pathogen in the absence and presence of the bacterial strain after a certain amount of time can be measured in order to indicate antagonistic activity.
In another method, bacterial strains to be tested can be placed on a plate containing a uniform spread of pathogen. The presence of a halo surrounding the bacteria is indicative of anti-pathogen activity.
A bacteria of the invention may inhibit growth of a pathogenic microorganism by ≥50% (i.e. equal or greater than 50%), ≥60%, ≥70%, ≥80%, ≥90%, ≥95% or 100%. For example, the percentage growth inhibition (PGI) may be calculated using the formula: PGI (%) = (KR-R1)/KR x 100, where KR is the distance of pathogenic microorganism from the point of inoculation to the colony margin on control dishes, and R1 is the distance of pathogenic microorganism growth from the point of inoculation to the colony margin on treated dishes in the antagonist’s direction.
For example, a bacteria strain of the invention may inhibit the growth of Lecanicillium fungicola L1 and/or Mycogone perniciosa M1 by ≥50% {i.e. equal or greater than 50%), ≥60%, ≥70%, ≥80%, ≥90%, ≥95% or 100% in an in vitro dual culture test. An in vitro dual culture test for mycelial growth inhibition typically involves culturing a microorganism (e.g. Lecanicillium fungicola L1 or Mycogone perniciosa M1) on an appropriate medium (e.g. PDA or MEA) at 22°C for 12 days; adding the bacteria colonies of interest (e.g. B. velezensis CM5, CM9 or CM17) at 10 mm from the margin of the vegetative mycelium; incubating the plates at 22°C for a further 5 days; and determining the amount of inhibition of the microorganism caused by the bacteria of interest by measuring the radial growth rate of the colonies and confronting to control plates. An in vitro dual culture test for spore germination inhibition typically involves plating 50 μl of a conidial suspension (102- 104 conidia ml-1) of the selected mycoparasite thoroughly distributed on an appropriate medium (e.g. PDA or MEA); in the middle of the plate a bacterial plug is disposed and the plates are incubated for 5-7 days, to subsequently measure the inhibition halo.
In the case where the pathogenic microorganism is a fungus, the bacteria of the invention may exert antagonism by preventing conidial germination and/or by inhibiting mycelium growth.
Secondary metabolites
A bacteria strain of the invention may exert antagonism against a pathogenic microorganism through chemicals produced by them. For example, a bacteria strain of the invention may express secondary metabolites, such as non-ribosomal peptides and/or polyketides, which may have antifungal activity. For example, the bacteria strain may contain gene clusters encoding the enzymatic machinery for synthesis of secondary metabolites. Ways to identify and detect secondary metabolites produced by bacteria are known in the art. Furthermore, ways to identify gene clusters encoding the enzymatic machinery for synthesis of peptides are also known in the art and described in the examples.
A bacteria strain of the invention may express antibiotics (e.g. bacilysin, bacillomycin, iturin A, surfactin or fengycin), siderophones or lytic enzymes (e.g. β-1,3- glucanase, chitinase, protease, lipase or amylase) or polyketides (e.g. bacillaene, diffidin or macrolactin). Macrolactin has been described to exhibit antagonistic effects against the soil-borne plant pathogen Ralstonia solanacearum (Yuan et al., 2012). Bacillaene has been shown to suppress the growth of fungi such as Pseudoxylaria and Trichoderma (Um et al., 2013). Fengycin was the first discovered antifungal from Bacillus strains (Vanittanakom et al., 1986), and has been shown to exhibit antifungal activity for instance against Fusarium graminearum or Sclerotinia sclerotiorum (Ramarathnam et al., 2007).
A bacteria strain of the invention may be capable of expressing one or more metabolites selected from: macrolactin, bacillaene, fengycin, difficidin, bacilysin, teichuronic acid, butirosin, subtilin, bacillibactin, rhizocticin and/or surfactin. For example, the bacteria strain may be capable of expressing subtilin.
A bacteria strain of the invention may be capable of expressing macrolactin, bacillaene, fengycin, difficidin, bacilysin, teichuronic acid, butirosin, subtilin, bacillibactin, rhizocticin and surfactin. For example, each of Bacillus velezensis CM5, CM 19 and CM35 is capable of expressing all of these metabolites (see Table 1 at Example 7).
A bacteria strain of the invention may have plant growth promoting traits, including the ability to synthesise or degrade plant and fungal hormones and intermediates in plant and fungal hormone biosynthesis, to produce chemicals that modulate plant and fungal hormone biosynthesis, to secrete compounds that promote mobilisation of soil minerals, and to produce volatile or diffusible chemicals that induce plant and fungal immune responses.
Pathogenic microorganisms, diseases and treatment/prevention of diseases
The invention refers to any pathogenic microorganisms that cause diseases or disease symptoms in crops. Such pathogenic microorganisms include bacterial, viral, fungal, nematode, phytomyxean, protozoan, algal and parasite crop pathogens.
The microorganism may be a fungal pathogen, such as a mycoparasite. A bacteria strain of the invention is capable of specifically inhibiting a mycoparasite without affecting crop growth ( e.g . A. bisporus).
A pathogenic microorganism that a bacteria strain of the invention is capable of exerting antagonism against may be one or more microorganisms selected from: Lecanicillium fungicola, Mycogone perniciosa, Trichoderma aggresivum., Cladobotryum mycophilum, Trichoderma atroviride, Fusarium commune, Alternaria solani, Alternaria brassiciola, Botrytis cinerea, Fusarium oxysporum, Phytophthora infestans, Sclerotinia sclerotorium and Cladosporium fulvum.
A bacteria strain of the invention may have a broad spectrum antifungal effect. For example, the bacteria strain may have antifungal effect against a number of mycoparasites.
A bacteria strain of the invention is particularly capable of exerting antagonism against Lecanicillium fungicola, Mycogone perniciosa, Trichoderma aggresivum, Cladobotryum mycophilum, Trichoderma atroviride, Fusarium commune, and/or Botrytis cinerea, as demonstrated in the examples herein.
In particularly, a bacteria strain of the invention may exert antagonism against the mycoparasite, Lecanicillium fungicola, which causes dry bubble disease in crops, such as mushrooms. The bacteria of the invention may prevent conidial germination and mycelium growth of the Lecanicillium fungicola, as shown e.g. in Figures 2, 3, 5-8. Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Lecanicillium fungicola, such as dry bubble disease in crops, for example, mushrooms, e.g. Agaricus bisporus. The bacteria of the invention is particularly effective in preventing such diseases.
A bacteria strain of the invention may exert antagonism against the mycoparasite, Mycogone perniciosa which causes wet bubble disease in crops, such as mushrooms. The bacteria of the invention may prevent conidial germination and mycelium growth of the Mycogone perniciosa, e.g. see Figures 2 and 5-6. Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Mycogone perniciosa, such as wet bubble disease in crops, for example, mushrooms, e.g. Agaricus bisporus. The bacteria of the invention is particularly effective in preventing such diseases.
A bacteria strain of the invention may exert antagonism against mycoparasites, Trichoderma spp, such as Trichoderma aggresivum, Trichoderma atroviride, which cause green mould in crops, such as mushrooms (e.g. see Figures 3 and 9). Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Trichoderma spp, such as green mould in crops, for example, mushrooms, e.g. Agaricus bisporus. The bacteria of the invention is particularly effective in preventing such diseases.
A bacteria strain of the invention may exert antagonism against the fungus, Rhizoctonbia solani. R. solani causes a wide range of commercially significant crop diseases. It is one of the fungi responsible for brown patch (a turfgrass disease), damping off (e.g. in soybean seedlings), black scurf of potatoes, bare patch of cereals, root rot of sugar beet, belly rot of cucumber, sheath blight of rice, and many other pathogenic conditions. Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by R. solani. The bacteria of the invention is particularly effective in preventing such diseases.
A bacteria strain of the invention may exert antagonism against Cladobotryum spp (e.g. see Figure 3). Several species of Cladobotryum, including C. dendroides, C. mycophilum, C. varium, C. multiseptatum, and C. verticillatum, are known to be the causal agents of cobweb disease in the mushroom A. bisporus and are found in mushroom- growing countries worldwide. Cladobotryum varium was reported to be a causal agent of fungal disease in mushrooms P. eryngii and F. velutipes. C. mycophilum has also been reported as a causal agent of cobweb disease in mushroom P. eryngii. Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Cladobotryum spp, such as cobweb disease in crops, for example mushrooms, e.g. Agaricus bisporus. The bacteria of the invention is particularly effective in preventing such diseases.
A bacteria strain of the invention may exert antagonism against Botrytis cinerea (e.g. see Figure 12), which causes grey mould. Botrytis cinerea is a necro trophic fungus that affects a wide variety of hosts including protein crops, fiber crops, oil crops, and horticultural crops. Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Botrytis cinerea, such as grey mould. The bacteria of the invention is particularly effective in preventing such diseases.
The bacteria of the invention may exert antagonism against Fusarium spp, such as F. commune and F. oxysporum (e.g. see Figures 10 and 11). Fusarium species can cause damping-off and root rot of young conifer seedlings, resulting in severe crop and economic losses in forest nurseries. It is one of the most important soil-borne pathogens within nurseries throughout western North America. This fungal genus is ubiquitous in most container and bareroot nurseries on healthy and diseased conifer seedlings, in nursery soils, and on conifer seeds of several species, especially Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), western white pine (Pinus monticola Dough), and ponderosa pine (Pinus ponderosa Dough ex Laws.). Thus, a bacteria strain of the invention is particularly useful for treating and/or preventing any of the diseases caused by Fusarium spp. The bacteria of the invention is particularly effective in preventing such diseases.
Crops
All crops are contemplated for use with the invention. Examples of crops include food crops, such as fungal crops and horticultural crops.
Crops that are useful with the invention include fungal crops, e.g. mushroom. Examples of mushrooms include: Agaricus bisporus (commonly known as button mushroom), Pleurotus spp. and in particular Pleurotus ostreatus (commonly known as oyster mushroom), Lentinula edodes (commonly known as shiitake mushroom), Auricularia auricula-judae (commonly known as Jew's ear mushroom), Volvariella volvacea (commonly known as paddy straw mushroom), Flammulina velutipes (commonly known as enoki mushroom), Tremella fuciformis (commonly known as snow fungus mushroom), Hypsizygus tessellatus (aka Hypsizygus marmoreus, commonly known as the beech mushroom), Ganoderma lucidum (commonly known as reishi), Stropharia rugosoannulata (commonly known as wine cap mushroom), Calocybe gambosa (St. George's mushroom), Agrocybe aegerita (poplar mushroom), Pholiota nameko (commonly known as butterscotch mushroom) and Hericium erinaceus (commonly known as monkey head mushroom).
Horticultural crops may be useful with the invention and these include vegetables (examples are chickpeas, lettuce, broccoli, and beans) and small fruit crops (examples are grape, strawberry, and raspberry).
The following crops may also be useful with the invention: rice, wheat, sugarcane and other sugar crops, maize (com), soybean oil, potatoes, palm oil, cassava, legume pulses, sunflowerseed oil, rape and mustard oil, sorghum, millet, groundnuts, beans, sweet potatoes, bananas, soybeans, cottonseed oil, groundnut oil and/or yams.
The bacterial strains of the invention can be applied to crops to improve crop growth and control pathogenic microorganism growth in crops. Therefore, the bacterial strains can be used to benefit any crop, including healthy crops and crops affected by a crop disease or crop disease symptoms ( e.g . the detectable presence of a known crop pathogen, or the presence of rot, mottling, galls, discoloration such as yellowing or browning, fruit greening, stunted growth, crop death, cellular death, cell wall breakdown, and/or the presence of spots, lesions, dieback, wilting, dwarfing, Witch's broom and/or knots).
Administration of the bacteria strains of the invention to the crop
The invention involves administering the bacteria strains and bacterial compositions of the invention to a crop and/or the environment surrounding the crop, e.g. the soil, casing material, and/or compost surrounding the crop, the water applied to the crop, or the hydroponic system in which the crop is grown.
Methods for administering the bacteria strains and compositions of the invention include application to the surface of the growth medium (e.g. casing material, substrate or compost), to the crop or parts of the crop, such as fruiting body, to the substrate, to the compost and/or to the environment of the crop. Administration methods may include spraying, such as by foliar spraying, injection or soaking. However, in embodiments where the crop is a mushroom, the bacteria strains and compositions of the invention are not applied onto the mushroom. For example, the strains and compositions of the invention are not applied onto the mushroom by foliar application.
For example, the strains and compositions of the invention may be applied to the surface of the casing material, not the compost.
The bacteria strains and compositions of the invention may also be administered by mixing with the irrigation water of the crop and/or with the growth medium ( e.g . casing material).
The administration method ideally provides an effective amount of the bacteria to the crop for controlling the growth of a crop pathogenic microorganism and improving crop growth. By way of example, an effective amount of bacteria may be an amount of bacteria which is sufficient to improve health, growth or productivity of the crop, and/or which reduces the effects, titre or symptoms of the crop disease, or prevents worsening of the crop disease, symptoms or infection of the crop. For example, an effective amount may be ≥10, ≥50, or ≥100 cfu of the bacterial strain per gram of crop, e.g. at one week after administration.
The bacteria strains and compositions of the invention may be applied to the crop one or more times throughout the growth of the crop. For example, where the crop is mushroom, a bacteria strain or a composition of the invention may be applied: (1) after applying the casing layer, e.g. four days after applying the casing layer, (2) after the first flush and/or (3) after the second flush.
Improve crop growth and control the growth of crop pathogenic microorganism
The invention relates to improving crop growth and controlling the growth of crop pathogenic microorganism.
The improvement in crop growth may be due to improved resistance to disease. Hence, the invention may also provide a method of improving resistance to disease. This may be an increase of defence in a healthy crop or a decrease in disease severity in a crop or in a population of crops, or in the number of diseased crops in a crop population.
The improvement in crop growth may be due to improved ability to defend against disease. Hence, the invention may also provide a method of improving the ability of a crop to defend against disease. This may be a measurable increase in crop defence against a disease, e.g. measured in terms of a measurable decrease in disease symptoms, pathogen titre, or loss of crop yield and/or quality, or a measurable increase in growth, crop quantity or quality.
The improvement in crop growth may be due to a reduction of disease symptoms. Hence, the invention may also provide a method of reducing disease symptoms. This may be a measurable decrease in the number or severity of disease symptoms.
The improvement in crop growth may be due to faster growth. Hence, the invention may also provide a method of inducing faster growth of a crop. This may be a measurable increase in the rate of growth of a crop, including seedlings, stems, roots, seeds, flowers, fruits, leaves and shoots thereof.
The improvement in crop growth may be due to improved crop productivity and/or quality. Hence, the invention may also provide a method of improving crop productivity and/or quality. This may be a measurable increase in the quantity or quality of a crop in terms of numbers, size, or weight of crop seeds, fruits, vegetable matter, fibre, grain, and the like, or in terms of sugar content, juice content, unblemished appearance, colour, and/or taste.
The improvement in crop growth may be due to improved germination. Hence, the invention may also provide a method of improving germination. This may be a measurable increase of the chance of successful germination of an individual seed (or mycelium in the case of mushrooms), a measurable increase in the percentage of seeds/active mycelium successfully germinating, and/or a measurable increase in the speed of germination.
The improvement in crop growth may be due to improved seedling emergence, or improved pinning in the case of mushrooms. Hence, the invention may also provide a method of improving seedling emergence. The invention may also provide a method of improving pinning in mushrooms. For example, a measurable increase in the speed of growth and/or development of successfully germinated individual seeds or spores can be observed. Bioinoculant
The invention provides a composition comprising a bacteria strain of the invention.
The composition may be a bioinoculant. The bioinoculant may comprise an excipient. In case of a liquid formulation, the excipient may be water or an organic solvent (. e.g . xilene, methanol, ethylene-glycol or mineral oil), a dispersion stabilizator, a surfactant (. e.g . calcium-dodecyl-benzene-sulphonate, polyglycol-ether, etoxylated alkyl-phenol or alkyl-aryl-sulphonates), optionally waxes. In case of a granular formulation, the excipient may be montmorillonite, bentonite, wood flour, starch, cellulose and a binder, such as e.g. a mineral oil, polyvinyl-alcohol or saccharose. The excipient may be selected by the skilled person without undue experimentation.
A bioinoculant composition of the invention may comprise cryoprotector, such as milk and/or sacarose solution.
A bioinoculant composition of the invention may be in the form of a water-soluble powder. Alternatively, the inoculant composition of the invention may be in the form of an aqueous suspension, suspension concentrate, capsulated concentrate, emulsion forming liquid spray, granule, granule dispersible in water, or microgranule. The formulation which may be used according to the invention may be selected by the skilled person without undue experimentation.
The concentration of each of the bacteria strain of the invention in the formulation maybe between 1010 to 1012 cfu gr-1 (colony forming units per gram of bacteria), e.g. 1010, 1011 or 1012 cfu gr-1.
Growth medium
The invention also provides a growth medium comprising a bacteria strain of the invention. The invention also provides a growth medium obtainable by treating with a bacteria strain of the invention.
A growth medium may be any medium that provides nutrients for a crop to grow, e.g. casing material, substrate, compost or soil. Casing materials and substrates are typically used for growing mushrooms. Casing materials useful with the invention may be black peat with sugar beet lime, blonde peat from Spaghnum peat moss, or a mixture of the two. When a bacteria of the invention is applied to a growth medium, it may influence the microbial ecology of the growth medium, and so a growth medium that has been treated with a bacteria strain of the invention may have a distinctive environment that would be capable of supporting crop growth and controlling the growth of pathogenic microorganisms. The distinctive environment may be the result of having treated the growth medium, e.g. having treated the growth medium with 1, ≤2, ≤3, ≤4, ≤5 or ≤6 days, or 1, ≤2, ≤3, ≤4, ≤5, ≤6, ≤7 or ≤8 weeks.
A growth medium of the invention typically has an enriched population of a bacteria strain of the invention compared to the amount that is naturally present in the medium. For example, the growth medium of the invention may have a population of a bacteria of the invention that is ≥10%, ≥20%, ≥30%, ≥40%, >50% compared to the amount that is naturally present in the medium. The amount of bacteria that is naturally present in the medium can be determined using routine methods, such as using PCR or labelling techniques for DNA that is specific to the bacteria of interest.
Combination approach
A bacteria strain of the invention may be combined with further agents in an integrated strategy for controlling the growth of crop pathogenic microorganism and/or improving crop growth. The further agent may be further bacteria strains of the invention. The further agent may be a biocontrol agent, such as Bacillus velezensis strain QST713. The further agent may be a chemical fungicide, such as prochloraz -manganese, chlorothalonil or metrafenone, or a plant or fungal growth promoting chemical, and/or an elicitor of plant or fungal immune responses.
Other
It is to be understood that different applications of the disclosed bacteria strains, bioinoculants and/or growth medium of the invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a bacteria strain” includes two or more “bacteria strains”.
“A bacteria strain of the invention” as used herein is a collective term referring to any bacteria strain described herein, i.e. including CM5, CM19, CM35, and variants thereof.
Furthermore, when referring to “≥x” herein, this means equal to or greater than x. When referring to “≤y” herein, this means equal to or less than y.
When referring to sequence identity between two sequences, their sequences are compared and the amount of identical nucleotides between the two sequences is determined. For example, a first nucleic acid sequence having at least 70% nucleic acid sequence identity with a second sequence requires that, following alignment of the first nucleic acid sequence with the second sequence, at least 70% of the nucleotides in the first nucleic acid sequence are identical to the corresponding nucleotides in the second sequence.
Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 3 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 3, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, the gaps or missing positions should be considered to be non-identical positions.
Sequences are typically aligned for identity calculations using a mathematical algorithm, such as the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87 (1990): 22642268), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90 (1993): 5873 5877). Such an algorithm is incorporated into the XBLAST programs of Altschul et al. (J. Mol. Biol. 215 (1990): 403 410). To obtain gapped alignments, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25 (1997): 3389 3402). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. The BLAST algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
The following examples illustrate the invention. Examples
Example 1 - Isolation of antagonistic firmicutes
Bacteria strains were isolated from casing materials for growing mushroom crops in the following way.
Contamination of the casing material for growing mushroom (A. bisporus) is considered the primary source of infection for many diseases. The casing is the material applied to cover the fully colonized compost where the following takes place: mushroom fructification, and transition from vegetative growth (mycelium) to reproductive growth (mushrooms). For example, dry and wet bubble diseases are caused by Lecanicillium fungicola and Mycogone perniciosa, respectively, in the casing layer (Figure 1). In raw casing material, natural suppressive effects against mycoparasites such as L. fungicola, M. perniciosa and Cladobotryum mycophilum can be observed in the absence of the host, and the native microbiota are thought to be responsible for these effects. However, the colonization of the casing material by the mycelium of A. bisporus has been described to break the casing fungistasis (which consists of the state of latency of fungi in substrates such as soil or casing that inhibits spore germination, fungal growth or sporulation), thereby contributing to the disease outbreaks such as cobweb, bubble diseases or green mould (Berendsen et ah, 2012a; 2012b).
Mycoparasites are responsible for the largest crop losses in commercial mushroom production (Fletcher & Gaze, 2008), causing such diseases as dry bubble (Lecanicillium fungicola), wet bubble (Mycogone perniciosa) and green mould (Trichoderma spp.), which are major challenges for mushroom cultivation, directly affecting growers' profit margins. For example, dry bubble disease accounts for estimated 20% of button mushroom crop losses globally (Tasmanian Institute of Agriculture, 2018). Losses of production up to 40%, with 70% of profit shortcuts due to quality depreciation have been described in Spain.
Casing materials
Three casing materials used by the industry in commercial fungal crops have been used:
1) Black peat amended with sugar beet lime (Euroveen B.V.), used mainly for the cultivation of mushrooms for the fresh market. 2) Blonde peat from Spaghnum peat moss with little contribution of organic matter, used mainly to produce mushrooms for canning.
3) Mixture of both commercial in a percentage of 70% black peat and 30% blonde peat.
The materials were analysed physico-chemically, registering the content in humidity, total nitrogen, ash, pH, conductivity, content in organic matter, C / N ratio and saturation percentage (Pardo-Gimenez and Pardo-Gonzalez, 2008).
Cultivation and counting of colonies
10 g of casing materials were suspended in 100 ml of 0.1% sterile broth of bacteriological pectone (Sigma). To do this, Erlenmeyer flasks were placed in an orbital shaker- incubator (Ovan 1000001087) with 120 rpm at 25°C for 10 min. The supernatant of the suspension was transferred to a microtitre plate where successive 10-fold dilutions were made from 10-1 to 10-4. Then 100 pL of each of the solutions corresponding to the different casing materials evaluated were transferred to different culture media in 90 mm Petri dishes, the solution was homogeneously distributed over the culture medium with a sterile rake for microbiology.
The growing media used in the examples are the following:
- Agar-casing: 150 gr of dried and milled fresh casing are autoclaved with approx. 800 mL of distilled water at 121°C and 1 at. The supernatant is filtered through a double layer of muslin to remove the bulk of the substrate, and 10 g of tryptone, 5 g of yeast extract and 15 g of agar are added to the supernatant liquid up to 1 L with distilled water.
- Agar-compost: 50 gr of dried and ground phase II compost are autoclaved with approx. 800 mL of distilled water at 121°C and 1 at. The supernatant is filtered through a double layer of muslin to remove the bulk of the substrate, and 15 g of agar is added up to 1 L with distilled water.
- King's agar with CFC (selective medium for Pseudomonas): Peptone-protease No.3, 20 g / L; K2HP04, 1.5 g / L; MgS04, 1.5 g / L; Agar, 15 g / L; Glycerol, 10 mL / L, plus CFC Pseudomonas supplement (content of a vial for 500 mL of medium): cetrimide (5 mg); Fusionic acid (5 mg); Cephalosporin (25 mg).
- LBA: 10 g Tryptone (Cultimed) + 5 g Yeast extract (Cultimed) + 10 g NaCl + 15 g Agar for 1L. - TSA (Cultimed): Tryptone soya agar, prepared according to the manufacturer's instructions.
- HiCrome™ Bacillus Agar (HimediaLAbs).
The plates were incubated at 25 and 28 ° C for 72h, then the colony forming units (c.f.u.) per plate in each of the media were counted, to estimate the differences between isolated microbial populations of each growing medium. An analysis of variance (ANOVA) was performed using the Statgraphics Centurium XVI software, analyzing the significant differences by Fisher's LSD test (P <0.05).
Isolation and preservation of individual colonies
Individual colonies were re-isolated from the original plates based on the phenological and morphological characteristics of the colony observed in the binocular microscope (Nikon C-LEDS) by means of a sterile applicator, the individual colonies were transferred to different Petri dishes on Luria-Bertani Broth-agar (LBA) medium. The re- isolated colonies were incubated at 28°C for 24-48h.
To prepare the biobank stock, a fermentative process was carried out for each of the individual colonies by sowing them in Luria Broth, (LB) (10 g Tryptone (Cultimed) + 5 g Yeast extract (Cultimed) + 10 g NaCl). The colonies were sown by means of a sowing loop from the original cultures reisolated in LBA. Taking 5 plugs of colony in 100 mL of sterile broth (LB) arranged in erlenmeyers. The erlenmeyers were incubated between 25 and 28 °C in an orbital shaker at 150 rpm between 5 and 18 hours. The colony multiplication ratio was determined by spectrophotometer reading (Thermo Fisher Scientific - Genesys 10-S) of the optical density of the culture broth at a wavelength of 600 nm (OD600). The fermentation was stopped when the absorbance at OD600 reached a value of 1. The culture broth fermented with the bacteria was resuspended in a 20% glycerol solution. Glycerol is a low molecular weight cryoprotective agent that penetrates cells. Finally, the tubes were stored in ultra-freezers at -80°C.
Example 2 - Selection of strains with antifungal activity
The pathogenic fungi used in the current work were isolated from a commercial crop showing symptoms of disease.
Two species, Lecanicillium fungicola L1 and Mycogone perniciosa M1 (available at the UOX Biobank), causing dry bubble and wet bubble disease in Agaricus bisporus crops were chosen for in vitro dual culture testing. Fungal cultures were maintained on glycerol stock solutions at -80°C. Agar discs of fungal isolates were grown on PDA for 3 days at 22°C to obtain inoculum.
To initially screen the isolated strains, a conidial suspension of L. fungicola L1 and M. perniciosa M1 was prepared from a 9-days-old sporulating culture plated on PDA according to Carrasco et al. (2016). 50 μl of a 106 conidia/ml suspension was applied to PDA plates. 106 cfu of the bacterial strains previously cultured in Luria-Broth overnight (28°C, 200 rpm) were plated at intervals on the plates and the plates were incubated at 25 °C for 5-8 days.
Figure 2 shows an example of the screening plates from which the strains CM5, CM19 and CM35 were identified. In particular, Figure 2 shows mycotoxicity of the strains CM5 and CM 19 against L. fungicola and M. perniciosa.
To confirm the inhibition of parasitic mycelium by CM5, the strain was co-cultured in LBA with L. fungicola, T. aggressivum, C. mycophilum by plating a 5 mm agar plug of a 7-days-old PDA culture of mycoparasites. The cultures were incubated at 22°C for 4-12 days (depending of the targeted mycoparasite) and subsequently four plugs of strains CM5 (incubated in LBA overnight) were disposed at 10 mm from the margin of the fungal colony in a cross shape. The plates were incubated for a further 5 days.
The results are shown in Figure 3(a)-(c). It can be seen that CM5 was effective in inhibiting L. fungicola (a), T. aggressivum (b) and C. mycophilum (c).
A conidial germination of L. fungicola prepared as above was used to compare fungitoxic activity of CM5 to non-active strains. The conidial suspension was spread on the agar and the CBAs inoculated afterwards. The results are shown in Figure 3(d). It can be seen that CM5 was effective in inhibiting conidial germination of L. fungicola.
Next, fungitoxic activity of the candidate stains against bisporus was investigated. The candidate strains were streaked in agar compost vertically next to the culture of the commercial A. bisporus strain Amycel XXX®. The results are shown in Figure 4, which shows two time points of the experiment. It can be seen that A. bisporus growth was not inhibited by proximity to the candidate strains and was even able to overgrow the bacterial culture. Thus, the candidate strains show fungicidal selectivity in- vitro. Example 3 - Inhibition of my coparasites: germination and mycelium growth
Next, the effects of the candidate strain on inhibition of the parasitic mycelium was evaluated. A 5 mm agar plug of a 7-days-old PDA culture of each of L. fungicola L1 and M. perniciosa M1 was plated. The cultures were incubated at 22°C for 12 days and subsequently four plugs of the bacterial strains (incubated in LB A overnight) were deposited 10 mm from the margin of the fungal colony in a cross shape. The plates were incubated for a further 5 days and the radial growth of the colony was measured and compared to the non-inoculated with bacteria as control.
The results are shown in Figure 5. Figure 5 shows that all three candidate strains, CM5, CM19 and CM35, generated radial inhibition on the mycelial growth of L. fungicola and M. perniciosa. The mycelium plug co-cultured with the strains showed a radial inhibition rate of 56.5% (with CM5), 53.6 (with CM19) and 56.7 (with CM35) for L. fungicola L1 and of 54.5% (with CM5), 55.5% (with CM19) and 55.6% (with CM35) for M. perniciosa ML
Example 4 - Inhibition of mycoparasites
Inhibition of the candidate strains against the mycoparasites, L. fungicola L1 and M. perniciosa M1, when co-cultured in vitro was further investigated.
A conidial suspension of L. fungicola L1 and M. perniciosa M1 was prepared from a 9-days-old sporulating culture plated on PDA according to Carrasco et al. (2016). 50 μl of a 106 conidia/ml suspension was applied to PDA plates. 106 cfu of the bacterial strains previously cultured in Luria-Broth overnight (28°C, 200 rpm) was plated either at intervals or at the center of the plate and the plates were incubated at 25 °C for 5-8 days.
Figure 6 shows growth of L. fungicola L1 and M. perniciosa M1 on plates without a bacterial strain (control) and with CM5, CM 19 and CM35 strains, and a non-fungitoxic bacterial strain. It can be seen that the growth of L. fungicola L1 and M. perniciosa M1 was unaffected in the plate with the non-fungitoxic bacteria. In contrast, CM5, CM 19 and CM35 all inhibited growth of L. fungicola L1 and M. perniciosa ML It can be seen that strains CM19 and CM35 exhibit greater antagonism of M. perniciosa M1 than CM5. The strains show clear inhibition of mycoparasites when co-cultured in vitro.
Figure 7 shows the plates from which the radial inhibition rate of the strains against L. fungicola L1 and M. perniciosa M1 was determined. An inhibition halo equal to 25.8 mm (with CM5), 17.1 (with CM19) and 23.4 (with CM35) for L. fungicola L1 and
33.8 mm (with CM5), 27.3 (with CM19) and 35.16 for M. perniciosa M1 was determined.
Example 5 - Design of a stable water-soluble bacterial powder as a commercial formulation
A water-soluble powder has been designed as prototype of commercial formulation to apply computed cfu (colony forming units of bacteria) in crop while watering through spraying systems.
The candidate strains grow efficiently in aerated Luria-Bertani broth (LB) media at 28°C and neutral pH (pH= 7). Each strain recovered from the biobank stock was fermented overnight in LB broth and the broth, with OD600> 0.7 was used to inoculate a bioreactor (a 5 L Biostat A fermenter (Sartorius)).
Growth conditions in the bioreactor are as follows: T= 28°C; pH= 7; the critical oxygen concentration is 10-50% of air saturation above which the bacteria grow efficiently; time: 7-8 h (to a OD600= 1). When the fermentation is complete (check bacterial growth in spectrophotometer), the broth is removed from the vessel and centrifuged at 4500 rpm for 10 min at 10°C. The supernatant is removed and the bacterial cell pellet is washed twice with phosphate buffer and resuspended in 100 ml of 10% skim milk and 10% sacarose solution as cryoprotector. The frozen solution is freeze-dried and the resulting product is pulverized into a ready-to-use water soluble powder. This powder is stable in the fridge.
A 10-fold serial dilution of the water-soluble powder in sterile water is prepared and plated on LB A plates to estimate the concentration of bacteria (cfu) in the powder and to adjust treatments.
It was found that the powder corresponding to the pure strain selected with a concentration average of 1010-1012 ufc/g would be most useful for applying to crops.
Example 6 - Antagonism against causal agents for major soil-borne diseases
The water-soluble bacterial powder prepared in Example 5 was used to test the potential of the candidate strains to fight the causal agents for major soil-borne diseases.
A sterile 5 mm paper inoculated with 106 cfu/ml of each strains have been confronted to increasing doses of mycoparasites (102, 103, 104, 105 conidia per plate) and incubated at room temperature. The mycoparasites tested were: L. fungicola L1, M. perniciosa M1, Fusarium commune CEBAS-CSIC, Fusarium oxysporum CEBAS- CSIC, Botrytis cinerea 447, Alternaria spp. CEBAS-CSIC, Trichoderma atroviride Tal.
It was found that a remarkable halo of inhibition has been detected in each of the colonies of the mycoparasites when co-cultured with each of the candidate strains. In particular, Figures 8-12 show the inhibition halos produced by strains CM5, CM19 and CM35 against L. fungicola L1, Trichoderma atroviride TA1, Fusarium oxysporum CEBAS-CSIC, Fusarium commune CEBAS-CSIC and Botrytis cinerea 447, respectively.
It can be seen that each of the candidate strains exhibited inhibition halos even at high concentrations of the different mycoparasites in the plates. The candidate strains appear to have differing activity against different pathogenic fungi.
Example 7 - Genome Sequencing, Assembly and Annotation
The candidate bacteria strains were identified based on the analyses of the 16S rDNA sequence. gDNA from the candidate bacterial strains was obtained by centrifuging an overnight fermented LB broth at 28°C was extracted using an NucleoSpin® Microbial DNA (Macherey-Nigel). The concentration and purity of the extracted DNA was assessed using a NanoDrop ND1000 spectrophotometer. Sanger sequencing of a partial fragment of the 16S rRNA gene was carried out by PCR using the primers: 8f or 27f: 5’- GAGTTTGATYMTGGCTCAG-3 ’ (SEQ ID NO: 4) and 1492r slightly modified: 5’- GGYTACCTTGTTACGACTT-3 ’ (SEQ ID NO: 5) (Lane, 1991). Each 40 μl reaction mixture consisted of 5 μl of buffer, 1.5 μl MgCl2, 1 μl dNTP, 1 μl of each primer, 10 μl Taq Polymerase DNA (1-10 ng/μl, from Bioline (BIOTAQ™ DNA Polymerase)) and 20.2 μl H2O. Amplification was done by initial denaturation at 95°C for 300s, followed by 32 cycles of denaturation at 95°C for 60s, annealing at 52°C for 60s and extension at 72°C for 120s with the final extension at 72°C for 7 min.
Reads coupled to BLASTn search allowed for initial identification of the isolate
Genome sequencing of the three strains was carried out by Microbes NG, Birmingham, UK. The downstream processing of sequenced data was accomplished using several bioinformatics tools:
Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 (Bolger et al., 2014). De novo assembly was performed on samples using SPAdes version 3.7 (Bankevich et al., 2012), and contigs were annotated using Prokka 1.11 (Seeman, 2014). Artemis genome browser was used for visualization and annotation (Rutherford et al., 2000). Assembly metrics were calculated by Quast (Gurevich et al., 2013). A standard analysis pipeline assigned reads to the closest available reference genome using Kraken (Wood et al., 2014).
The RAST annotation server and the SEED annotation environment were used to annotate the prokaryotic genome (Aziz et al., 2008; Overbeek et al., 2005). BLAST Ring Image Generator (BRIG) has been employed for prokaryote genome comparisons (Alikhan et al., 2011). The secondary metabolite profile of the sequenced strains has been evaluated by antiSMASH (Blin et al., 2019).
For the CM5 strain, the draft genome is provided in the contigs as set out in SEQ ID NOs: 57-106, and it has 4,205,756 bp with 45.85% GC. The 16S rRNA-encoding gene is set out in SEQ ID NO: 1.
For the CM 19 strain, the draft genome is provided in the contigs as set out in SEQ ID NOs: 107-144, and it has 4,203,670 bp with 45.84% GC. The complete genome is provided in SEQ ID NO: 145. The 16S rRNA-encoding gene is set out in SEQ ID NO: 2.
For the CM35 strain, the draft genome is provided in the contigs as set out in SEQ ID NOs: 6-56 and it has 4,204,667 bp with 45.84% GC. The 16S rRNA-encoding gene is set out in SEQ ID NO: 3.
Preliminary Sanger sequencing of the 16S rRNA gene identified the strains as Firmicutes belonging to the genera Bacillus (>90% of most frequent species). Draft genome sequencing clusters the tree strains as Bacillus velezensis (>50% of most frequent species).
In the context of phytopathogen biocontrol, the antiSMASH analysis allowed to identify gene clusters predicted to encode the enzymatic machinery for synthesis of nonribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) and polyketides (bacillaene, difficidin, and macrolactin). These results are summarised in Table 1.
Secondary metabolic profile of the genomes of the isolated strains presents pks2 clusters associated to the clusters polyketide biosynthesis of antifungal compounds such as Macrolactin, which has been described to exhibit antagonistic effects against the soilbome plant pathogen Ralstonia solanacearum (Yuan et al., 2012); Bacillaene, that suppresses the growth of fungi such as Pseudoxylaria and Trichoderma (Um et al., 2013); Fengycin, the first discovered antifungal from Bacillus strains (Vanittanakom et al., 1986), lipopeptide with antifungal activity for instance against Fusarium graminearum or Sclerotinia sclerotiorum (Ramarathnam et al., 2007).
Therefore, genome sequencing and the metabolism study suggest the production of metabolites with an active antifungal profile.
Discussion
In summary, the strains of Bacillus velezensis CM5, CM19 and CM35 isolated from the casing materials employed in the cultivation of Agaricus bisporus present antifungal properties against the mycoparasites Lecanicillium fungicola L1 and Mycogone perniciosa M1 (the causative agents of dry and wet bubble), both to prevent conidial germination and mycelium growth. The strains do not show detectable toxicity against the host mycelium (considering that Agaricus is also a fungus) which suggest a non- detrimental effect on mushroom production when applied in the presence of the host. In this sense it is remarkable that most chemical fungicides are applied in the absence of the cited mycelium, for instance when the casing material is applied in the crop.
A prototype for a water-soluble powder has been designed. This powder is stable in the fridge, and corresponds to the pure strain selected with a concentration average of 1010- 1012 ufc gr-1. The bacteria strains, when introduced in the powder form, exhibited effective antagonism against the agents responsible for many of the major soil-borne diseases ( e.g .
L. fungicola, M. perniciosa, Fusarium commune, F. oxysporum, Botrytis cinereai, Alternaria spp. and Trichoderma atroviride).
The bacteria strains of the invention are natural inhabitants amongst the microbiome in crop casing material, and so they would thrive in the environmental niche where the crop develops. In contrast, biocontrol agents, such as B. velezensis QST713, that are not natural inhabitants in crop casing material would be inferior because they may be less capable of adapting to the environment in which it is intended to be used, thereby less effective in exerting antagonism against pathogenic microorganisms in that environment. The casing microbiome is a complex environment for which the microbiome structure has been characterised by the inventors (Carrasco et al., 2019; Carrasco et al., 2020). In this environment the native microbiota evolves along the process of cultivation due to inhabitant competition and the action of the host that colonized the casing provoking the breakdown of the native fungistasis described in literature (Berendsen et al., 2012a). Bacillus velezensis is widespread in soil substrates, however, compared to other biocontrol agents in the market the bacterial strains of the invention are native to the substrate employed in mushroom cultivation. This means that they are biologically adapted to the environment where they would be introduced as treatment which is likely to increase persistence, proliferation and survival of these strains to lengthen protection.
Furthermore, B. velezensis QST713 has been shown to be effective in treating green mould in A. bisporus, caused by T. aggressivum. T. aggressivum is a compost mould which firstly infects compost, and then later the fruiting bodies and casing. It would not have been expected that B. velezensis QST713 would be effective in the casing layer, let alone exerting antagonism that is as effective as the bacteria strains of the invention, as demonstrated herein. Overall, the bacterial strains of the invention constitute the only tailor-made biocontrol solution for the mushroom industry, and also show biocontrol activity against plant parasites as shown in the figures in respect to the in vitro effect against mycoparasites of plants.
The results suggest that the strains discovered can be an alternative to chemical treatments in crops to fight many of the most harmful diseases, such as dry bubble (caused by L. fungicola) and wet bubble (caused by M. perniciosa). Since the industry is facing limitations for pesticide use, and the nature of host and parasite (both fungi) requires both high selectivity and large investment, it is not expected the occurrence of new pesticides to reduce crops losses to substitute those ones approved (with evidences of resistance already detected). Therefore native strains with an active secondary metabolic pathway showing toxicity to fungi are a good alternative to promote greening with the industry and to minimize the impact of fungal diseases.
Example 8
The bacteria strains CM5, CM 19 and CM35 were tested in crop trials with Lecanicillium fungicola (which causes dry bubble) and Mycogone perniciosa (which causes wet bubble). Materials and Methods
Crop Trials
Compost and casing (a mixture of peat-based casing materials at a rate of 50% each (Euroveen B.V. + Spaghnum peat moss, Valimex KF)) physico-chemical characterization was carried out by measuring; pH, electrical conductivity (EC), moisture content, ash, organic matter, nitrogen, C/N ratio and water holding capacity according to Pardo- Gimenez et al. (2016). Trial 1 and trial 2 were conducted in three separate rectangular growing rooms provided with climate control (automatic Fancom System®). 90 blocks of Phase III compost (Germinados de compost SL, Lodosa, Spain) spawned at 1% with the A. bisporus commercial strain Silvan A15M were cultivated per growing room. The blocks were lined up in metal shelving at three levels in groups of 5 blocks (crop surface per group: 1 m2).
On day 0 of cropping, the blocks were covered with a layer of 3-4 cm of the saturated casing materials in a completely randomized block design with 3 replicates per casing. Seven days after casing, a conidial suspension of L. fungicola in Room 2 and M. perniciosa in Room 3 was sprayed onto the surface of the casing layer at a rate of 2xl02 conidia m-2 (Room 2- L. fungicola) and 2*102 aleutispores m-2 in trial 1 and 2*102 conidia m-2 in trial 2 (Room 3- M. perniciosa). The solution employed for the suspension consisted of sterile distilled water plus a drop of Tween 80 to avoid spore clustering. Disease inoculum was prepared on the day of inoculation as described by Carrasco et al. (2016) from strains of L. fungicola LI and M. perniciosa Ml previously isolated from diseased commercial crops. Room 1 was not infected and was used as a negative control (the control blocks were sprayed with water/Tween 80 solution).
Table 2 collects the treatments applied on peat basedcasing material during the cropping trials to prevent losses from mycoparasites.
The chemical fungicides approved for mushroom used, Banko Champinon ® (Arysta LifeScience) formulated as chlorthalonil 50% 50% [SC] P/V and Vivando ® (BASF) formulated as metrafenone 50% p/v. SC, have been used as control treatments. Chlorothalonil was applied in Trial 1 and Trial 2 four days after applying the casing layer at a dose of 2 ml m-2, Metrafenone was applied only in trial 1 at a dose of 0.5 ml m-2 a) four days after applying the casing layer; b) after the first flush; c) after the second flush. Biocontrol treatments were formulated as stable water soluble powder and applied in trial 1 at a dose of 109 cfu m2 a) four days after applying the casing layer; b) after the first flush; c) after the second flush and in trial 2 at a dose of 109 cfu m2 a) four days after applying the casing layer; b) at the end of the spawn-running (casing colonized by mycelium); c) after the first flush; d) after the second flush.
Healthy and diseased mushrooms were harvested daily during three flushes, and the potential suppressive effect was analysed by comparing disease incidence and mushroom yield between treated and control blocks. Mushroom production in the different treatments was compared by ANOVA. Fisher’s LSD (least significant difference) test, at 5% probability, was used to establish significant differences between means. Non-parametric tests including Kruskal- Wallis test and Mann- Whitney (Wilcoxon) W test to compare medians at the 95% level were also implemented for non-normal distributions.
Formulation of bacterial treatments
The bacteria strains in the form of water-soluble powder prepared according to Example 5 was used in this experiment.
Microbiological field applications require the production of large batches of microbial cultures. It is possible to obtain these large batches by culturing the desired isolates in sterile flasks containing a liquid medium, but this methodology is time consuming and it can easily lead to contaminations. Furthermore, the only parameter that is possible to control is the temperature, and it is not possible to optimise important parameters such as pH, air saturation, carbon dioxide and oxygen content. Bioreactors are systems developed to make easier the growth of large microbial culture batches, limiting the contamination sources and allowing the control of all the parameters required to optimise the process and maximise the yield.
Results
The results from the first trial are shown in Table 3 and Figure 13. The results from the second trial are shown in Table 4 and Figure 14.
No deleterious effect on crop production has been detected due to the application of the bacterial treatments at the application dosses, as reflected while comparing treated and non- treated blocks in room 1. In the first trial, three applications (1- during casing germination, and during interflush period: 2- after the 1st flush and 3- after the 2nd flush) have been conducted (Table 2) while in the second trial four applications (1- during casing germination, 2- on colonized casing, and during interflush period: 3- after the 1st flush and 4- after the 2nd flush) were provided; treatments showed efficiency both at three and four application rates. Only those blocks treated with a mixture of CM5+CM19+CM35 at a rate of 109 cfu m-2 per strain showed a significant detrimental effect in the second trial and room 1 , suggesting that the synergistic effect associated to the use of a mixture of the three strains could be deleterious for the production.
Comparing the production harvested from the blocks treated with biocontrol agents with approved chemicals, both chlorothalonil and metrafenone, no-significant differences were detected. The results suggest that the treatment with the individual strains CM5,
CM 19 and CM35 are as effective as those ones treated with pesticides.
In trial 1 (Figure 13; Table 3), the room 3, infected with aleurispores from M. perniciosa was affected severely with wet bubble, and a drastic yield decrease was noted. Although no significant differences in production was detected during the trial, the blocks treated with the strain CM5 showed good performance, meanwhile in room 2 (infected with dry bubble) this was the least productive treatment showing significant differences with the rest. This fact suggests that even when the strains showed antifungal activity against both mycoparasites in vitro, the in vivo response could be different against both organism. Although it is noteworthy that in the second trial, none significant differences among trial were detected in room 2 or room 3.
Since fungal parasites affect hardly during late flushes (Carrasco et al,.2017), the production parameters during late flushes can be considered as an overview to evaluate fungicide and biocontrol efficiency, therefore a good indicator for our trials is to analyze the production data in third flush. In room 2 and 3 the third flush collected has been lower than in control room during both trials due to the widespread infection. Only room 2 in trial 1 showed significant differences in between chlorothalonil and biocontrol treated groups, while none significant differences were detected among control and biocontrol treated blocks.
Conclusion
In conclusion, individual treatment with the strains CM5, CM 19 and CM35, at a rate of 109 cfu m-2, did not show any deleterious effect on crop production when applied at three or four separate times during the crop cycle. No effect on cap pitting has been detected associated to the bacteria application, since this kind of harmful symptoms are described for pathogenic bacteria such as bacterial blotch (Osdaghi et al,.2019).
Biocontrol treatments, at the dosses applied, are as efficient as chemical treatments to prevent losses derived from the fungal diseases dry and wet bubble. The application of a mixture of the three selected strains did not show a positive synergistic effect. The results suggest that the biocontrol agents, in the form of the water soluble powder, can be effectively employed on crops, such as mushroom, to treat diseases, such as dry and wet bubble.
Example 9
The housekeeping genes from CM5, CM 19 and CM35, respectively, were identified and sequenced, and are summarised in Tables 5 to 7.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 742966.
Deposit information
The following bacteria strains were deposited at the European Collection of Authenticated Cell Cultures (ECACC), Culture Collections, Public Health England, Porton Down, Salisbury, SP40JG, United Kingdom on 3 June 2021 by Gail Preston of Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, Oxfordshire, OX1 3RB, United Kingdom. The bacteria strains were allocated the following accession numbers:
Bacillus velezensis CM5 - Accession Number 21060301;
Bacillus velezensis CM19 - Accession Number 21060302; and
Bacillus velezensis CM35- Accession Number 21060303.
The depositor has authorised Oxford University Innovation Limited of Buxton Court, 3 West Way, Oxford, OX2 0JB, United Kingdom to refer to the deposited biological materials in this application and its priority application (GB 2009235.9), and has given her unreserved and irrevocable consent to the deposited materials being made available to the public in accordance with the relevant Patent Legislation of the country in which this Patent Application is filed.
References
Alikhan et al. (2011) BMC genomics, 12(1), 402. Bankevich et al. (2012) J Comput Biol, 19:455-77.
Berendsen et al. (2010) Molecular plant pathology, 11(5), 585-595.
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Berendsen et al. (2012b) Biol Control, 63: 210-221.
Bolger et al. (2014). Bioinformatics, 30(15), 2114-2120. Carrasco J. (2016) PhD thesis, Universidad de Castilla-La Mancha. 168 pp. http://hdl.handle.net/10578/9752.
Carrasco et al. (2016) Ann Appl Biol, 168:214-224.
Carrasco et al. (2017) Span J Agric Res; 15(2), 19.
Carrasco et al. (2019) Microbiology, 165(6). Carrasco et al. (2020) Microbial Biotechnology; Under revision.
Fletcher & Gaze (2008) Mushroom pest and disease control 192 pp., Intercept, Newcastle upon Tyne, UK
Gurevich et al. (2013) Bioinformatics, 29(8), 1072-1075.
Lane (1991) 16S/23S rRNA sequencing. 115-175, John Willey and Sons Ltd., Chichester, UK.
Milijasevic-Marcic etal. (2017) Euro J Plant Path, 148(3):509— 519.
Osdaghi et al. (2019) Plant disease, 103.11: 2714-2732.
Pandin et al. (2019) Appl Environ Microbiol, 85(12).
Pandin et al. (2018) J Biotechnol, 278:10-19. Pardo-Gimenez et al. (2016) J Sci Food Agric, 96:3838-3845.
Ramarathnam, et al. (2007) Canadian Journal of Microbiology, 53(7):901 -911. Rutherford et al. (2000). Bioinformatics, 16(10), 944-945.
Vanittanakom (1986) The Journal of antibiotics 39, 888-901.
Wood et al. (2014) Genome Biol, 15:R46-R. Tasmanian Institute of Agriculture. (2018). Defending the mushroom industry against disease (https://www.utas.edu.au/tia/news-events/news-items/defending-the-mushroom- industry-against-disease)
Table 1. Secondary metabolism of draft genomes from the strains selected against strains from GenBank (conducted by AntiSmash).
Figure imgf000040_0001
Figure imgf000041_0001
* Bacillus velezensis QST 713 (GenBank Ac. No: CP025079); Bacillus velezensis M75 (GenBank Ac. No: CP016395); Bacillus velezensis 157 (GenBank Ac. No: CP022341).
Table 2. Treatments and dose applied on crop trials, from chemical fungicides (chlorothalonil or metrafenone) and biocontrol agents.
Figure imgf000042_0001
*cfu: colony forming units refer to individual colonies of bacteria.
Table 3. Production (kg m-2) of healthy mushrooms (first pilot trial in crop).
Figure imgf000043_0001
Room 1: Control, non-inoculated; Room 2: L. fungicola (200 conidia m-2); Room 3: M. pemiciosa (200 aleuriospores m-2). Treatments: Metrafenone (0.5 ml m-2); Chlorothalonil (2 ml m-2); CM5, CM19, CM35 (109 cfu m-2). Equal letters within the rows means non-statistical significant differences according to Fisher's LSD test at P<0.05.
Table 4. Production (kg m-2) of healthy mushrooms (second pilot trial in crop).
Figure imgf000044_0001
Room 1: Control, non-inoculated; Room 2: L. fungicola (200 conidia m-2); Room 3: M. pemiciosa (200 conidia m-2). Treatments: Metrafenone ( 0.5 ml m-2); CM5+CM19+CM35, CM5, CM19, CM35 (109 cfu m-2). Equal letters within the rows means non-statistical significant differences according to Fisher’s LSD test at P<0.05.
Table 5. Housekeeping genes of CM5.
Figure imgf000045_0001
Table 6. Housekeeping genes of CM19.
Figure imgf000046_0001
Table 7. Housekeeping genes of CM35.
Figure imgf000047_0001
Brief Description of the sequence listing
SEQ ID NO: 1 is the DNA sequence encoding the 16S rRNA of Bacillus velezensis strain CM5 (1872bp).
SEQ ID NO: 2 is the DNA sequence encoding the 16S rRNA of Bacillus velezensis strain CM 19 (1832 bp).
SEQ ID NO: 3 is the DNA sequence encoding the 16S rRNA of Bacillus velezensis strain CM35 (1804 bp).
SEQ ID NO: 4 is the forward primer used in the Examples for amplifying the 16S rRNA gene in a PCR.
SEQ ID NO: 5 is the reverse primer used in the Examples for amplifying the 16S rRNA gene in a PCR.
SEQ ID NOs: 6-56 are contigs 1-51 of the draft genome sequence of Bacillus velezensis strain CM35.
SEQ ID NOs: 57-106 are contigs 1-49 of the genome sequence of Bacillus velezensis strain CM5.
SEQ ID NOs: 107-144 are contigs 1-38 of the genome sequence of Bacillus velezensis strain CM 19.
SEQ ID NO: 145 is the complete genome sequence of Bacillus velezensis strain
CM19.
SEQ ID NO: 146 is the amino acid sequence of glycerol uptake facilitator (glpF) (Location: 983698-984525 in SEQ ID NO: 145).
SEQ ID NO: 147 is the amino acid sequence of dihydroxy-acid dehydratase (ilvD) (Location: 2176459-2178135 in SEQ ID NO: 145).
SEQ ID NO: 148: is the amino acid sequence of phosphotransacetylase (pta l) (Location: 2581178-2582071 in SEQ ID NO: 145).
SEQ ID NO: 149: is the amino acid sequence of phosphotransacetylase (pta_2) (Location: 3916727-3917698 in SEQ ID NO: 145).
SEQ ID NO: 150: is the amino acid sequence of phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH) (Location: 727571-729109 in SEQ ID NO: 145).
SEQ ID NO: 151: is the amino acid sequence of triose phosphate isomerase (tpiA) (Location: 3532919-3533680 in SEQ ID NO: 145). SEQ ID NO: 152: is the amino acid sequence of protein translocase subunit SecA (SecA) (Location: 3691586-3694111 in SEQ ID NO: 145).
SEQ ID NO: 153: is the amino acid sequence of Carbamoyl -phosphate synthase arginine-specific (carA_l) (Location: 1153790-1154848 in SEQ ID NO: 145).
SEQ ID NO: 154: is the amino acid sequence of Carbamoyl -phosphate synthase small chain (carA_2) (Location: 1606969-1608063 in SEQ ID NO: 145).
SEQ ID NO: 155: is the amino acid sequence of Protein RecA (RecA) (Location: 1752657-1753700 in SEQ ID NO: 145).
SEQ ID NO: 156: is the amino acid sequence of Chaperone protein DnaK (DnaK) (Location: 2701328-2703166 in SEQ ID NO: 145).
SEQ ID NO: 157: is the amino acid sequence of ATP synthase subunit beta (atpD) (Location: 3839076-3840497 in SEQ ID N: 145).
SEQ ID NO: 158: is the amino acid sequence of DNA gyrase subunit B (gyrB) (Location: 4454-6376 in SEQ ID NO: 145).
SEQ ID NO: 159: is the amino acid sequence of Translation initiation factor IF-2 (inffi) (Location: 1718422-1720569 in SEQ ID NO: 145).
SEQ ID NO: 160: PurR: is the amino acid sequence of transcription regulator associated with purine metabolism (PurR) (Location: 55037-55867 in SEQ ID NO: 145)
SEQ ID NO: 161: PurR: is the amino acid sequence of transcription regulator associated with purine metabolism (PurR) in CM5 and CM35.
SEQ ID NO: 162: is the amino acid sequence of Carbamoyl-phosphate synthase small chain (carA) in CM5 and CM35.
SEQ ID NO: 163 is the amino acid sequence of glycerol uptake facilitator (glpF) (Location: 983695-984525 in SEQ ID NO: 145).
SEQ ID NO: 164 is the nucleotide sequence of glycerol uptake facilitator (glpF).
SEQ ID NO: 165: is the nucleotide sequence of phosphotransacetylase (pta l).
SEQ ID NO: 166: is the nucleotide sequence of phosphotransacetylase (pta_2).
SEQ ID NO: 167: is the nucleotide sequence of phosphoribosylamino-imidazole carboxy formyl formyltransferase (purH).
SEQ ID NO: 168: is the nucleotide sequence of triose phosphate isomerase (tpiA).
SEQ ID NO: 169: is the nucleotide sequence of protein translocase subunit SecA
(SecA). SEQ ID NO: 170: is the nucleotide sequence of Carbamoyl-phosphate synthase arginine-specific (carA l).
SEQ ID NO: 171: is the nucleotide sequence of Carbamoyl-phosphate synthase small chain (carA_2). SEQ ID NO: 172: is the nucleotide sequence of Protein RecA (RecA).
SEQ ID NO: 173: is the nucleotide sequence of Chaperone protein DnaK (DnaK).
SEQ ID NO: 174: is the nucleotide sequence of ATP synthase subunit beta (atpD).
SEQ ID NO: 175: is the nucleotide sequence of DNA gyrase subunit B (gyrB).
SEQ ID NO: 176: is the nucleotide sequence of Translation initiation factor IF-2 (inffi).
SEQ ID NO: 177: PurR: is the nucleotide sequence of transcription regulator associated with purine metabolism (PurR).
SEQ ID NO: 178: PurR: is the nucleotide sequence of transcription regulator associated with purine metabolism (PurR) in CM5 and CM35. SEQ ID NO: 179 is the nucleotide sequence of Carbamoyl-phosphate synthase small chain (carA) in CM5 and CM35.

Claims

Claims
1. A bacteria strain which:
(a) comprises a 16S rRNA-encoding gene having ≥95% sequence identity with SEQ ID NO: 1, 2 or 3;
(b) comprises a genome having ≥90% identity to the genome sequence of Bacillus velezensis CM5, CM19 or CM35;
(c) is Bacillus velezensis CM5 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060301, or a variant thereof;
(d) is Bacillus velezensis CM 19 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060302, or a variant thereof; or
(e) is Bacillus velezensis CM35 deposited at the European Collection of Authenticated Cell Cultures (ECACC) under accession number 21060303, or a variant thereof; wherein said strain is capable of exerting antagonism against a crop pathogenic microorganism.
2. The bacteria strain of claim 1, wherein the genome of B. velezensis CM5 is obtainable by the assembly of the contigs provided in SEQ ID NOs: 57-106; the genome of B. velezensis CM19 is provided in SEQ ID NO: 145 or is obtainable by the assembly of the contigs provided in SEQ ID NOs: 107-144; or the genome of B. velezensis CM35 is obtainable by the assembly of the contigs provided in SEQ ID NOs: 6-56.
3. The bacteria strain of claim 1 or claim 2, wherein the bacteria comprises one or more housekeeping gene having ≥98% sequence identity with the corresponding housekeeping gene in B. velezensis CM5, CM19 or CM35, wherein the housekeeping gene is selected from: glpF, ilvD, pta, purH, purR, tpiA, secA, carA, recA, dnaK, atpD, gyrB and/or inffi.
4. The bacteria strain of any preceding claim, wherein the bacteria strain is capable of expressing one or more metabolites selected from: macrolactin, bacillaene, fengycin, difficidin, bacilysin, teichuronic acid, butirosin, subtilin, bacillibactin, rhizocticin and/or surfactin.
5. The bacteria strain of any preceding claim, which is capable of inhibiting the growth of Lecanicillium fungicola and/or Mycogone perniciosa by ≥50% in an in vitro dual culture test.
6. A bioinoculant composition comprising the bacteria strain of any preceding claim.
7. The bioinoculant composition of claim 6, which is a water-soluble powder.
8. The bioinoculant composition of claim 7, wherein the bacteria strain is present in the concentration of between 1010 and 1012 cfu gr-1 (colony forming units per gram of bacteria).
9. A crop growth medium comprising the bacteria strain of any of claims 1-5.
10. The crop growth medium of claim 9, wherein the growth medium is a casing material.
11. A method for controlling the growth of a crop pathogenic microorganism, improving crop growth and/or treating or preventing a disease caused by a crop pathogenic microorganism, comprising administering to a crop and/or the environment surrounding the crop the bacteria strain of any of claims 1-5, the bioinoculant composition of any of claims 6-8, or the crop growth medium composition of claim 9 or claim 10.
12. The method of claim 11, comprising the steps of: a) applying the bacteria strain of any of claims 1-5 or the inoculant composition of any of claims 6-8, to the surface of the culturing medium, to the fruiting body, to the casing material, to the substrate, to the compost and/or to the environment of the crop; and/or b) mixing the bacteria strain, of any of claims 1-5 or the inoculant composition of any of claims 6-8 with the water and/or a growth medium of the crop.
13. The bacteria strain, the bioinoculant composition, the crop growth medium or the method of any preceding claim, wherein the crop is a mushroom, such as Agricola bisporus.
14. The bacteria strain, the inoculant composition, the crop growth medium or the method of any preceding claim, wherein the pathogenic microorganism is a fungal pathogen, such as a mycoparasite.
15. The bacteria strain, the inoculant composition, the crop growth medium or the method of claim 14, wherein the bacteria strain is capable of preventing conidial germination and/or mycelium growth of the fungal pathogen.
16. The bacteria strain, the inoculant composition, the crop growth medium or the method of any preceding claim, wherein the pathogenic microorganism is selected from: Lecanicillium fungicola, Mycogone perniciosa, Trichoderma aggresivum., Cladobotryum mycophilum, Trichoderma atroviride, Fusarium commune, Alternaria solani, Alternaria brassiciola, Botrytis cinerea, Fusarium oxysporum, Phytophthora infestans, Sclerotinia sclerotorium, and Cldosporium fulvum.
17. The bacteria strain, the inoculant composition, the crop growth medium or the method of claim 16, wherein the pathogenic microorganism is selected from: Lecanicillium fungicola, Mycogone perniciosa, Cladobotryum mycophilum and/or Trichoderma aggressivum.
18. Use of the bacteria strain of any of claims 1-5 for controlling the growth of a crop pathogenic microorganism and/or improving crop growth, such as mushroom growth.
19. Growth medium for crops obtainable by treating with the bacteria strain of any of claims 1-5.
20. The growth medium of claim 19, wherein the growth medium is casing material.
21. A method of isolating a microorganism with antagonistic properties against one or more crop pathogenic microorganisms, comprising the steps of obtaining a sample from casing material, testing the sample for antagonistic properties and isolating microorganism with said antagonistic properties.
22. The method of claim 21, wherein the test comprises an in vitro dual culture test with Lecanicillium fungicola L1 and/or Mycogone perniciosa M1.
23. The method of claim 21 or claim 22, wherein the isolated microorganism is a bacteria strain.
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CN114634891A (en) * 2022-03-24 2022-06-17 厦门萤星海科技有限公司 Bacillus belgii ZH3 and application thereof
CN114634891B (en) * 2022-03-24 2023-06-02 厦门萤星海科技有限公司 Bacillus bailii ZH3 and application thereof

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