Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • 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
• • 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
  • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/06—Fungi, e.g. yeasts
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present disclosure relates generally to bacterial strains having antimicrobial activity and the use of the same as biocontroi agents. Also provided are biocontroi compositions comprising said bacterial strains, in particular for inhibiting microbial plant pathogens.
• All plants are susceptible to attack by microorganisms, such as bacteri and fungi.
• microorganisms such as bacteri and fungi.
• microbial pathogens and diseases caused by them can result in significant cro damage, loss of yield and economic losses both preharvest and postharvest.
• Diseases caused by microbial pathogens can also lead to decreased shelf-life of produce, and to higher costs for consumers.
• fungi are known plant pathogens causing many di eases that harm or destroy crops worldwide.
• Most plant pathogenic fungi are ascomyeetes (including Fusarium spp., Thietaviopsis spp., Botrytis spp., VerticiUium spp,, and Magnaporthe spp.) and basidiomyeetes (including Rkiwctoma spp., Puccinia spp. and Armi aria spp.).
• ascomyeetes including Fusarium spp., Thietaviopsis spp., Botrytis spp., VerticiUium spp,, and Magnaporthe spp.
• basidiomyeetes including Rkiwctoma spp., Puccinia spp. and Armi aria spp.
• fungi of the genus Fusarium filamentous fungi widely distributed in soil.
• Fusarium oxysponmt affect plants including tomatoes, melons, ginger, bananas and legumes with a wilt disease (Fusarium Wilt) causing symptoms such as vascular wilt, necrosis, premature leaf drop and stunting of growth
• Fus riumdry rot of potatoes is caused by several species of the Fusarium genus. Fusariwn dry rot is an economically important problem in potatoes, both in the field and in storage, and is one of the leading causes of postharvest potato losses.
• bananas Fusarium is the causal agent of the highly destructive Panama disease.
• Fusarium oxyspo m i an imperfect asexual fungus that spreads by means of three types of spores: microconidia, macroconidia, and chlamydospores.
• Plant pathogenic bacteria also cause many damaging and economically significant diseases in plants. Examples include species of Erwtfiia, Pectabaciertum, P ntoea, Agrobacierium, Pseudommuis, Ralskmia. Birrkhoiderki. Acidovorax, X n thotnanas. Clavibacter, Streptomyces, Kylella, Spiroplasma, and Fhytoplasma. Plant pathogenic bacteria cause a range of symptoms including galls and overgrowths, wilts, leaf spots, specks and blights, soft rots, as well as scabs and cankers.
• Potato scab is a disease that infects potato tubers as well as other root crops such as radish, beet, carrot and parsnips, it causes unsightly necrotic lesions on the tuber surface resulting in huge economic losses.
• the primary causal pathogen is Streptomyces scabies found in the soil of potato growing regions worldwide, a Gram positive, aerobic filamentous bacteria producing grey mycelia on most solid media. The vegetative filaments break off to form spores enabling the bacteria to survive long periods of time and spread via water, wind and .soil. It is able to survive long periods of lime, even years, in tile soil surviving on decaying plant material, as well as surviving passage through animal digestive tracts.
• Various approaches- ave been used to reduce disease severity, however presently no effective control is available for potato scab.
• Fungicides and other pesticides applied to plants to combat pathogenic microorganisms and to treat or prevent disease caused by such pathogens are typically chemical in nature (often synthetic and non-naturafly occurring). These can be expensive to manufacture and bring with them unwanted side effects, includin toxicity to animals, and environmental concerns. There is a clear and continuing need for the development of alternative approaches. Bioeontrol agents and Compositions are an attractive alternative, being safer, more biodegradable, and les expensive to develop.
• a first aspect of the present disclosure provides a method for treating or preventing infection of a subject by a microbial pathogen, the method comprising administerin to the subject, or otherwise exposin the subject to, an effective amount of a composition comprising at least one strain of Lactobacillus- selected from Lactobacillus pamfarraginis, Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae, or a culture supernatant or cell free filtrate derived from culture media in which said strain has been cultured.
• a second aspect provide method for treating or preventing disease in a subject caused by, or associated with, a microbial pathogen, the method comprising administering to the subject, or otherwise exposing the subject to, an effective amount of a composition comprising at least one strain, of Lactobacillus selected from Lactobacillus pamfarraginis, Lactobacillus buchner Lactobacillus rapi and Lactobacillus ze e ⁇ or a culture supernatant or cell free filtrate deri ved from culture media in which said strain has been cultured,
• exposing the subject to the composition may comprise directly or indirectly exposing the subject to the composition.
• the plant may be exposed to the composition by application of the composition to a part of the plant or to the soil into which the plan is growing or is to be planted.
• the subject is an animal
• the animal may be exposed to the composition by application of the composition to pasture or other grass (or soil on which the pasture or other grass is grown) on which the animal feeds.
• the method may further comprise administering to the subject, or otherwise exposing the subject to, an. effective amount of one or more antimicrobial agents.
• a third aspect provides a method for inhibiting the growth of a microorganism, the method comprising exposing the microorganism, or an environment colonised by or capable of being colonised by the microorganism, to an effective amount of a composition comprisin at least one strain of Lactobacillus selected from Lactobacillus parafarragmis, Lactobacillus buchmri, Lactobacillus rapi and Lactobacillus zeae, or a culture supernatant or cell free filtrate deri ved from culture media in which said strain has been cultured.
• the subject is a plant
• the subject is a plant and the environment is soil, plant roots and/or plant foliage.
• Soil may be treated with the composition prior to planting of the plant, at the time of planting or after planting.
• plant roots may be treated with the composition prior to planting of the plant, at the time of planting or after planting.
• the method may comprise one treatment or multiple treatments of the environment or the subject with the composition.
• the method may further comprise exposing the microorganism to an effective amount of one or mote antimicrobial agents.
• a fourth aspect provides a bioeontr l composition for treating or preventing infection of a subject by a microbial pathogen, the composition comprising at least one strain of Lactobacillus selected from. Lactobacillus parajhrraginis, Lactobacillus buchmri, Lactobacillus rapi and Lactobacillus zeae. or a culture supernatant or cell free filtrate derived from culture media in which said strain has been cultured.
• a fifth aspect provides a biocontrol composition for the treatment or prevention of a disease in a subject caused ' by, or associated with, infection of the subject by a microbial pathogen, the composition comprising at least one strain of Lactobacillus- selected from Lactobacillus pamfarraginis, Lactobacillus buchneti. Lactobacillus rapi and. Lactobacillus z e, or a culture supernatant or cell free filtrate derived from culture media in which said strain has been cultured.
• a sixth aspect provides a composition for inhibiting the growth of a microorganism, the composition comprising at least one strain of Lactobacillus selected from Lactobacillus pamfarraginis * Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae, or a culture supernatant or cell free filtrate derived from culture media in which said strain has been cultured.
• compositions disclosed herein may comprise one or more strains of Lactobacillus diaUvor n (N3) deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI 2/022847, Lactobacillus parafarraginis (Ni l ) deposited with the National Measurement Institute, Australia on 14 Decembe 2012 under Accession Number VI 2/022848.
• Lactobacillus brevis (TD) deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022851, ictobacillus paracas&i, strain designated ' ⁇ deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI 2/022849, Lactobacillus casei and strain designated herein B' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022850: or a culture supernatant or cell free filtrate derived from culture medi in which one or more of these strains have been cultured.
• Lactobacillus is selected from Lactobacillus parafarraginis, Lactobacillus buchnerL Lactobacillus rapi and Lactobacillus zeae, for the manufacture of a composition for treating or preventing infection of a subject by a microbial pathogen.
• Lactobacillus is selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi and Lactobacillus zeae, for the manufacture of a composition for treating or preventing a disease in a subject caused by, or associated with, infection, of the subject: by a microbial pathogen.
• Lactobacillu is selected from Lactobacillus parafarraginis, Lactobacillus buekneri, .Lactobacillus rapi and Lactobacillus ' zeae, for the manufacture of a composition for inhibiting the gro wth of a microorganism.
• Lactobacillus * wherei the Lactobacillus is selected from Lactobacillus parafarraglnls, Lactobacillus buch ri, Lactobacillus rapt, Lactobacillus zeae, Lactobacillus paracasei n strain designated herein T9' deposited with the National Measurement Institute. Australia on 14 December 2012 under Accession Number V.12 022849, Lactobacillus casei. and strain designated herein 'TB' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022850, for the manufacture of a composition for treating or preventing infection of a subject by a microbial pathogen.
• Lactobacillus i selected from Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi, Lactobacillus zeae, Lactobacillus paracuses, strain designated herein T9' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI 2 022849, Lactobacillus casei and strain designated herein ' B' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V I 2/022850, for the manufacture o a composition for treating or preventing a disease in a subject caused by, or associated with, infection of the subject by a microbial pathogen.
• Lactobacillus is selected from Lactobacillus parafarraginis, L ' actobacillus buchmri, Lactobacillus rapi, Lactobacillus zeae, Lactobacillus paracasei, strain designated herein T9' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession. Number VI 2/022849, Lactoi icillus casei and strain designated herein 'TB' deposited with the National Measurement Institute, Australi on 14 December 2012 under Accession Number V 12/022850, for the manufacture of a composition for inhibiting the growth of a microorganism.
• Lactobacillus diolivorans (N3) deposited with the National Measurement Institute, Australia on ⁇ 4 December 201 under Accession Number VI 2/022847, Lactobacillus parafarragm (Ni l ) deposited wit the National Measurement institute, Australia -on 14 December 2012 under Accession Number V12/022848, Lactobacillus brevt ' s (TD) deposited wit the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/02285 L Lactobacillus paracasei, strain designated herein T9 * deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V I 2/022849, Lactobacillu casei and strain designated herein ' ⁇ ' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI /022850.
• the Laciobadilus par jarraginis strain may be Lactobacillus parafarraginis L l 8, In a particular embodiment the Lactobacillus parafarraginis strain is Lactobacillus parafarmginis Lpl 8 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number I 1/022945.
• the Lactobacillus buchneri strain may be Lactobacillus buchneri Lb23. hi a particular embodiment the Lactobacillus buchneri, strain is Lactobacillus buchneri Lb23 deposited with National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022946.
• the Lactobacillus rapi strain may be Lactobacillus rapi Lr24.
• the Lactobacillus rapi strain is Lactobacillus rapi Lr24 deposited with National Measurement Institute, Australia on 27 Octobe 2011 under Accession Number V I 1/022947.
• the Lactobacillus z,eae strain may be Lactobacillus zeae Lz26.
• the Lactobacillus z&ie strain is Lactobacillu z ⁇ c Lz26 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number VI 1/022948.
• the composition may further comprise strain of Acetobacter fabarum.
• the Acetobacter fabarum strain ma be Acetobacter fabarum Afl5.
• the Acetobacter fabarum strain is Acetobacter fabarum ⁇ 5 deposited with the National Measurement Institute, Australia on 27 October 201. 1 under Accession Number V I 1/022943.
• the composition may further comprise yeast The yeast may be a strain of Candida ethanolica.
• the Candida ethanolica strain may be Candida ethanolica Ce31 , In a particular embodiment, the Candida ethanolica strain is Candida ethanolica Ce31 deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI /022944.
• the composition may comprise two or more of said Lactobacillus species, three of said Lactobacillus species o all of said Lactobacillus species.
• the composition may represent a symbiotic combination of two or more or thre or more of said Lactobacillus species.
• strains in the composition may be encapsulated. Where multiple strains are encapsulated- th strains may be individually encapsulated or combined in a single encapsulation.
• composition may further comprise one or more antimicrobial agents.
• the subject may be a plant or an animal.
• the subject is a plant.
• the plant ma be an agricultural crop species, a horticultural crop species or a crop species for fuel or pharmaceutical production.
• the subject is a non-human animal, such as a milk-producing mammal (for ex ample a cow or goal) .
• the microbial pathogen may be a causative agent of a plant diseas or animal disease.
• the disease may be selected from a rot, wilt, rust, spot, blight, canker, mildew, mould, gall, scab or mastitis.
• the microbial pathogen may he fungus or bacteria.
• the fungus may be selected fro a Fusarium sp., a Pseudoc rcospom sp., a Phialemonium sp, a Botrylis sp. or a Rhizocionia sp.
• the Fusarium sp. may be Fusarium oxysporum, such as Fusarium Qjcyspvrum f. sp. zingiberi, Fusarium oxyspommi. sp. mveum or Fusarium oxyspo um f. sp. cubens .
• the Phialemonium sp. may be Phialemonium dimorphosporum.
• the Botrytis sp. may be Boirytix cinerea.
• the Rhizocionia sp. may be RhizwMniasohmi. The skilled addressee will appreciate that this list is not exhaustive. Additional suitable fungal species are disclosed hereinbelow.
• the bacteria may be Gram positive or Gram negative.
• the bacteria may be a Streptomyc& sp., a Staphylococcus sp., an Escherichia sp.. a Pseudomonas sp.. a Pantoea sp, or a Streptococcus sp.
• the Streptomyces sp. may be Streptomyces scabies.
• the Staphylococcus sp may be Staphylococcus aureus.
• the Streptococcus sp. may be # uheris.
• the Escherichia sp. may be Escherichia coli.
• the Pseudomonas sp. may be Pseudomonas savastani.
• the Pantoea sp may be Pantoea agglomerons, The skilled addressee will appreciate mat this list is not exhaustive. Additional suitable bacterial species are disclosed herembelow.
• FIG. 1 Root height,, plant height and plant weight of watermelon plants following three weeks of treatment as described in Example 3.
• the bars represent, from left to right, watermelon seedlings from experiments A, B, C and D.
• antimicrobial agent refers to any agent that, alone or in combination with another agent, is capable of killin or inhibiting the growth of one o more species of microorganism.
• Antimicrobial agents include, but are not limited to, antibiotics, detergents, surfactants, agents that induce oxidative stress, bacteriocins and antimicrobial enzymes (e.g. lipases, promises and lyases) and various other proteolytic en3 ⁇ 4ymes and nucleases, peptides and phage.
• Reference to an antimicrobial agent includes reference to both natural and synthetic antimicrobial agents,
• exposing means generally bringing into contact with. Exposure of a subject to a composition or agent as described herein includes administration of the composition or agent to the subject, or otherwise bringing the composition or agent into contact with the subject, whether directly or indirectly. For example, exposing a subject to a composition or agent may include applyin or administering the composition or agent to an environment Inhabited by the subject or to a feed, liquid or other nutrient composition to be administered by the subject. In the present disclosure the terms “exposing”, “administering” and “contacting” and variations thereof may, in some contests, be used interchangeably.
• inhibiting and variations thereof such as “inhibition” and “inhibits” as used herein in relation to- microbial growth refers to any microcidal or mier statie activity of a composition or agent, Such inhibition ma be in magnitude and/or be temporal or spatial in nature. Inhibition of the growth of bacteria or fungi by a composition or agent can be assessed by measuring microbial growth in the presence and absence of the composition or agent. The microbial growth may be inhibited by the composition or agent by at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. 75%, 80%, 85%, 90%, 95% or more compared to the growth of the same microbe that is not exposed to the composition or agent.
• subject refers to any plant or animal infected or suspected of bein infected by, or susceptible to infection from, a. microbial pathogen.
• Plants include, without limitation, plants that produce -fruits, vegetables, grains, tubers, legumes, flowers, and leafs or any other economically or environmentally important plant. Thus the plant may be a crop species.
• crop as used herein refer to any plant grown to be harvested or used for any economic purpose, including for example huma foods, livestock fodder, fuel or pharmaceutical production (e.g. poppies).
• Animals include, for example, mammals, birds, fish, reptiles, amphibians, and any other vertebrates or invertebrates, such as those of economic, environmental, and/or other significant importance.
• Mammal include, but are not limited to, livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans.
• the term "effective amount” refers to a amount: of microbial inoculant or fertilizer composition applied to a given area of soil or vegetation that is sufficient to effec one or more beneficial or desired outcomes, for example, in terms of plant growth rates, crop yields, or nutrient availability in the soiL
• An "effective amount” can be provided in one o more administrations.
• treating refers to any and all applications which remedy, or otherwise hinder, retard, or reverse the progression of, an infection or disease or at least one symptom of an infection or disease, including reducing the severity of an infection or disease, Thus, treatment doe not necessarily imply that a subject is treated until complete elimination of the infection or recovery from a disease.
• preventing refers to any and all applications which prevent the establishment of an infection or disease or otherwise delay the onset of an infection or disease.
• kits for treating or preventing infection of a subject by a microbial pathogen comprising administering t the subject, or otherwise exposing the subject to, an effective amount of a composition comprising at least one strain of Lactobacillus selected from Lactobacillus parafarmginti, Lactobacillus buchnerL Lactobacillus rapt and Lactobacillus zea e .
• Also provided are methods for treating or preventing a disease in a subject caused by, or associated with, a microbial pathogen comprisin administering to the subject, or otherwise exposin the subject to, an effective amount of a composition comprising at least one strain of Lactobacillus selected from Lactobacillus pamfarmgims, Lactobacillus buchneri, Laciobacilius rapt and Lactobacillus zeae.
• kits for inhibitin the growth of a microorganism comprising exposing the microorganism, or an environment colonised by or capable of being colonised by the microorganism, to an effective amount of a composition comprising at least one strain of Lactobacillus selected fro Lactobacillus parafarmginis, Lactobacillus buchneri, Lactobacillus rapt and Laciobacilius zeae.
• Also provided are methods for treating or preventing a disease in a subject caused by, or associated with, a microbial, pathogen comprising administering to the subject, or otherwise exposin the subject to, an effective amount of a composition comprising at least one strain of Lactobacillus selected from Lactobacillu pamfarraginis, Lactobacillus buchnerii Lactobacillus rapt, Lactobacillus zeae, Lactobacillus casei, Lactobacillus paracasei, strain designated herein ⁇ 9' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI 2/022849, and strain designated herein ' ⁇ ' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022850.
• a microorganism comprising exposin the microorganism, or an environment colonised by or capable of being colonised by the microorganism, to an effective amount of a composition comprising at least, one strain of Lactobacillus selected from Lactobacillus parafarraginis,
• Lactobacillus buchneri Lactobacillus rapi, Lactobacillus zeae, Lactobacillus casei, Lactobacillus p racas i, strain designated herein 9' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022849, and strain designated herein TB' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V I 2/022850.
• Novel biocontrol compositions are. also provided for treating and preventing infections caused by microbial pathogens, and disease caused by, or associated with, such infections, and for inhibiting the growth of microorganisms.
• the microbial pathogen or microorganism may be a fungal or bacterial pathogen capable of infecting and/or causing disease in any plant or animal species.
• Methods and compositions of the present disclosure therefore find application in the treatment and prevention of fungal diseases of plants and animals, such as rots, wilts, rusts, spots, blights, cankers, mildews and moulds.
• Methods and compositions of the present disclosure also find application in the treatment and prevention of bacterial diseases of plants and animals, including galls, scabs and other diseases such as mastitis.
• method and compositions disclosed herein may be employed in the treatment and prevention of fungal diseases selected from Fusarium dry rot, Fusarium wilt, black dot, late blight, black scurf, Rhizoctonia, pink rot, target spot, Panama disease, stripe rust (yellow rust), soft rot, stem rust (black rust), grey mould, Phytophthora heart rot, smut, Phytophthora rot, peanut rust, Rhizoctonia stem rot, rhizome rot, fungal husk spot, trunk canker, white root rot, verticillium wilt and ginger yellows, and of infections by the causative agents o these diseases.
• fungal diseases selected from Fusarium dry rot, Fusarium wilt, black dot, late blight, black scurf, Rhizoctonia, pink rot, target spot, Panama disease, stripe rust (yellow rust), soft rot, stem
• the plant affected by the disease may be, for example, a food crop (for humans or other animals) such as any fruit, vegetable, nut. seed or grain producing plant.
• Exemplary crop plants include, but are not limited to, tubers and other below-ground vegetables (such a potatoes, beetroots, radishes, carrots, onions, etc.), ground-growing or vine vegetables (such as pumpkin and other members of the squash family, beans, peas, asparagus, etc.), leaf vegetables (such as lettuces, chard, spinach, alfalfa, etc.), othe vegetables (such as tomatoes, brassic including broccoli, avocadoes, etc.), fruit (such as berries, olives, stone fruits including nectarines and peaches, tropical fruits including mangoes and bananas, apples, pears, watermelon, mandarins, oranges, mandarins, kiwi fruit, coconut, etc.), cereals (such as rice, maize, wheat, barley, millet, oats, ry
• the animal may be exposed to a composition disclosed herein indirectly by application of the composition to pasture, grass or other plant on which the animal feeds.
• exemplary animals are dairy cattle.
• methods and compositions disclosed herein may be employed in the treatment and prevention of bacterial diseases selected from common scab, bacterial spot, bacterial speck, potato scab, bacterial soft rot, crown gall disease and mastitis, and of infections by the causative agents of these diseases.
• Fusarium spp. such Fusarium oxysporum and special forms thereof including Fusarium oxyspomm f, sp. zingiberi, Fusarium oxyspomm f.sp. niv utTh and Fusarium oxyspomm f.sp, cubense; CoUeclotrichum coccodes: Phytophthora spp.
• Phytophthora itifest such as Phytophthora itifest , Phytophthora eiythroseptica, Phytophthora cinnamomi; Rhizocttmi solani; Coryne pora casSiicala; Puccmia spp. such a Puctini arachidus, Puccmia striiformis, Puccini graminis f. sp. triiici; Botrytis cinerea; Rhizoctonia; Pythium mmotylum; Fsuedocercospara macadamiae; RoseUmia necanrix; Verticiltum spp. such as Verticiltu deiilim; Phiaiemonium dimorphosporum; TMelaviop spp. ; Magnaporthe grisea,
• Bacterial pathogens against which methods and compositions disclosed herein find application include, but are not limited to, Streptomyc.es scabies; Xanthomonas spp. such as Xanthonianascampestris pv- Vesicatoria; Pseiidamonas spp. such as P. savastanoL P. syringae, P. aeruginosa, and P. fluarescens; E. coli; Listeria-monocytogenes; Staphylococcus spp. such as S. aureus: Streptococcus spp. such as S.
• parahaemoUticus Aciinobacillus pleuropn.euHiomae;Chromob Cter nokiceum; Caxiella h.urnetti;.FrdftciseUa tular sis; Haemophilus influenza; Pasteurella multockki; Shigella flexneri; Salmonella typhi; Salmonella typhimurium; Yersinia pes lis; and Yersinia pseudotuberculosis.
• fungal pathogens and diseases and bacterial pathogens and diseases disclosed herein are exemplary only, and the scope of the present disclosure is not limited thereto. Numerous other fungal pathogens and diseases, and bacterial pathogens and diseases will be known to those skilled in the ail, and methods and compositions of the present disclosure may also be used to combat these.
• compositions disclosed herein comprise strains: of one or more bacterial species selected from Lactobacillus pm-qfarraginis, Lactobacillus buchneri. Lactobacillus r pi, Lactobacillus zeae, Lactobacillus hrevis and Lactobacillus diolimrans.
• compositions may comprise a culture supernatant or cell free filtrate derived from culture media in which the above referenced strains have been cultured.
• compositions disclosed herein comprise strains of one or more bacterial species selected from Lactobacillus parajarragmis.
• the Lactobacillus parafar ginis strain may be Lactobacillus pamfarmginisLp 18.
• the Lactobacillus pamfarmgmis strain is Lactobacillus parafarmgims Lp l8 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number VI 1/022945.
• the Lactobacillus buchneri strain may be Lactobacillus buchneri Lb23.
• the Lactobacillus buchneri strain i Lactobacillus buchneri Lb23 deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number VI 1/022946.
• the Lactobacillus rapi strain may be Lactobacillus rapi Lr24.
• the UtctobaciHus rapi strain is Lactobacillus rapi Lr24 deposited with National Measurement. Institute, Australia oft 27 October 2011 under Accession Number VI .1/022947.
• the Lactobacillus zea strain may be Lactobacillus zeae Lz26.
• the Lactobacillus zeae strain is Lactobacillus zeae Lz26 deposited with National Measurement Institute, Australi on 27 October 2011 under Accession Number VI 1/022948.
• compositions disclosed herein may comprise one or more strains selected from Lactobacillus diolhmrcms (N3) deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI 2/022847, Lactobacillus parafarraginis (Ni l) deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V12/022848, iMCfohacUlus hrevi (TD) deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number VI 2/022851, Lactobatillus paracasein strain designated herein 'T9' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022849, Lactobacillus ca ei and strain designated herein ' ⁇ ' deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V 12/022850; or a culture supernatant or cell free filtrate derived from culture media in which one or more
• compositions of the present disclosure may further comprise a strain of Acetobacter fabarum, or a culture supernatant or cell free filtrate derived from culture medi in which a strain of ' Acetobacter fabarum has been cultured.
• the Acetobacter fabarum. strain may be Acetobacter f aba rum Af!.5.
• the Acetobacter fabarum strain is Acetobacter fabarum Afl5 deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022943.
• Compositions may further comprise a yeast, or culture supernatant or cell free filtrate derived from culture media in which a yeast has been cultured.
• the yeast may be a strain of Candid eihanoUca.
• the Candida eihanoUca strain may be Candida ethanolica Ce31.
• the Candida eihanoUca strain is Candida ethanolica Ce31 deposited with the National Measurement Institute. Australia on 27 October 201 1 under Accession Number V I 1/022944.
• concentrations of individual microbial strains to be added to compositions disclosed herein will depend on a variety of factors including the identity and number of individual strains employed, the microbial pathogen, infection or disease to be treated, the form in which a composition is applied and the means by which it is applied. For any given case, appropriate concentrations may be determined by one of ordinary skill in the art using only routine experimentation. By way of example only, the concentration of each strain present in the composition may be from about 1 x lO 2 cfu/ml to about 1 x 10 10 cfu/ml, and.
• x 10 3 cfu/ml may he about 1 x 10 3 cfu/ml, about 2,5 x 1Q 3 cfu/ml, about, 5 ⁇ 10 3 cfu/ml, 1. x 10 4 cfu/ml, about 2.5 x 10 4 cfu/ml, about 5 x 10 4 cfu/ml, 1 x 1.0 5 cfu/ml, about 2.5 x 1.0 5 cfu/ml, about 5 x 10?
• the final concentration of the Lactobacillus strains i about 2,5 x lif cfu/ml.
• the final concentration of Acetobacter fabarum may be about 1 x 10' J cfu/ml and the final concentration of Candida ethanolica may be about 1 x lO ⁇ cfu/ml.
• variants of the microbial strains described herein As used herein, the term "variant" refers to both naturally occurring and specifically developed variants or mutants of the.
• Variants may or may not have the same identifying biological characteristics of the specific strain exemplified herein, provided they share similar advantageou properties in terms of treating or preventing infections caused by, or treatin or preventing diseases caused by or associated with, microbial pathogens.
• Illustrative examples of suitable methods for preparing variants of the microbial strains exemplified herein include, but are not limited to. gene integration techniques such as those mediated by insertionai.
• variants are microbial strains phylogenetieally closely related to strains disclosed herein and strains possessing substantial sequence identity with the strains disclosed herein at one or more phylogenetieally informative markers such as rRNA genes, elongation and initiation factor genes, RNA polymerase subunit genes, DNA gyrase genes, heat shock protein genes and recA genes.
• the 16S rRNA genes of a "variant 1 ' strain as contemplated herein may share about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identit with a strain di closed herein.
• Methods of the present disclosure may further comprise administering to a subject in need of treatment, or otherwise exposing the subject to, one or more antimicrobial agents.
• Administration or exposure to a composition disclosed herein and an antimicrobial agent may be at the same time or at: different times, i.e. simultaneous or sequential.
• Antimicrobial agents may be co-formulated with microbial strains used in the methods.
• Compositions disclosed herein may therefore comprise one or more antimicrobial agents, in instances where the microbial strains and antimicrobial agents are formulated in different compositions, they can be administered or delivered by the same or different routes or means.
• antimicrobial agents suitable for the methods described herein include, but are not limited to, antibiotics, detergents, surfactants, agents that induce oxidative stress, bacleriocins and antimicrobial enzymes (e.g. lipases, pronases and lyases) and various other proteolytic enzymes and nucleases, peptides and phage.
• the antimicrobial agents may be natural o synthetic.
• the antimicrobial agent employed may be selected for the particular application of the invention on a case-by-case basis, and those skilled in the art will appreciate that the scope of the present invention is not limited by the nature or identity of the particular antimicrobial agent.
• Non-limiting examples of antimicrobial agents include fluoroquinolones, aminoglycosides, glycopeptides, lincosamides, cephalosporins and related beta-lactams, macralides, nitroimidazoles, penicillins, polymyxins, tetracyclines, and any combination thereof.
• the methods of the present invention can employ acedapsone; aeetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil; amicycline; amitloxacin;- amifloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; aminos alicylate sodium; amoxicillin; amphomycin; ampieillin; ampicillin sodium; apalcillin sodium; apramycin; aspartocm; astromicin Sulfate; vilamycin; ayoparein; azithromycin; azlocillin; a/Jocil!in sodium; bacampieillin hydrochloride; bacitracin; bacitracin methylene disalieylate; bacitracin zinc; bamhermyeins; benzoylpas calcium; bcrythromycin; betamicin sulf
• compositions disclosed herein may optionally further comprise one or more additional microbial organisms, fo example, agronomically beneficial microorganisms.
• agronomically beneficial microorganisms may act in synergy or concert with, or otherwise cooperate with the organisms of the present disclosure.
• agronomically beneficial microorganisms include Bacillus sp., Pseudamonas sp., Rhizobium sp., Azospirittum sp.. Azatohacter sp residential phototrophie and cellulose degrading bacteria, Clostridium sp., Trichoderm sp. and the like.
• inoculated bacteria can find survival difficult among naturall occurring competitor and predator organisms.
• one or more of the strains may be encapsulated in. for example, a suitable polymeric matrix.
• encapsulation may comprise alginate beads such as has been described by Young et i, 2006, Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humic acid. Biotechnology and Bio ngmeering 95:76-83, the disclosure of whic is incorporated herein by reference in its entirety.
• any suitable encapsulation material or matrix may be used. Encapsulation may be achieved using methods and techniques known to those skilled in the art.
• Encapsulated microorganisms can include nutrients or other components of the composition in addition to the microorganisms.
• compositions disclosed herein may be applied or administered directly or indirectly to any plant or animal in need of treatment.
• composition may be applied to plant parts (such as foliage) or seeds, or alternatively may be applied to soil in which the plant are growing or to be. grown or in which seeds have been or are to be sown.
• Application may be by any suitable means and may be on any suitable scale.
• application may comprise pouring, spreading or spraying, including broad scale or bulk spreading or spraying, soaking of seeds before planting, and/or drenching of seeds after planting or seedlings.
• seeds, seedlings or mature plant may be treated as many times as appropriate.
• the number of applications required can readily be determined by those skilled in the art depending on, for example, the plant in question, pathogen or disease to be treated, the stage of development of the plant at which treatment is initiated, the state of health of the plant, the growth, environmental and/or climatic conditions in which the plant is grown and the purpose for which the plant is gro wn.
• compositions disclosed herein may be prepared in any suitable form depending on the means by which the composition is to be applied to the soil or to plant seeds or vegetation.
• suitable forms- can include, for example, slurries, liquids, and solid forms.
• Solid forms include powders, granules, larger particulate form and pellets.
• Solid forms can be encapsulated in water soluble coatings (for example dyed or undyed gelatin spheres or capsules), extended release coatings, or by microencapsulation to a free flowing powder using one or more of. for example, gelatin, polyvinyl alcohol, elhylceliulose, cellulose acetate phthalate, or styrene maleic anhydride.
• Liquids may include aqueous solutions and aqueous suspensions, and emuisifiable concentrates.
• suitable carrier components that aid dispersion, adhesion and conservation/stability.
• suitable carriers will be known to those skilled in the ar and include, for example, chitosan, vermieulite, compost, talc, milk powder, gels and the like.
• compositions of the present disclosure may be incorporated into compositions of the present disclosure, such as humie substances, trace elements, organic material, penetrants, macron utrients, raicronutrients and other soil and/or plant additives.
• Humus or bumic substances tha may be incorporated may include, but are not limited to, humic acid derived from, for example oxidised lignite or leonardite, fulvic acid and hurnates such as potassium humate.
• Organic material added may include, but is not limited to. biosolids, animal manure, compost or composted organic byproducts, activated sludge or processed animal or vegetable byproducts (including blood meal, feather meal, cottonseed meal, ocean kelp meal, seaweed extract, fish emulsions and fish meal).
• Penetrants include, but are not limited to, non-ionic wetting agents, detergent based surfactants, silicones, and/or organo-silicones. Suitable penetrants will be known to those skilled in the art, non-limiting examples including polymeric polyoxyalkylenes, allinol, nonoxynoL octoxynol, oxycastrol, TRITON, TWEEN, Sylgard 309, Silwct L-77, and Herbex (silicone surfactant blend),
• compositions of the present disclosure include, for example, water trapping agents such as zeolites, enzymes, plant growth hormone such as gibbercllins, and pest control agents such as acaracides, insecticides, fungicides and nematoeides.
• water trapping agents such as zeolites, enzymes, plant growth hormone such as gibbercllins
• pest control agents such as acaracides, insecticides, fungicides and nematoeides.
• compositions of the present disclosure may be freeze dried to extend shelf life and/or to aid in agricultural application such as field dispersal.
• compositions comprising encapsulated strain may be freeze dried to extend shelf life and/or to aid in agricultural application such as field dispersal.
• Example 1- Microbial strains and ' strain combinations
• Lactobacillus parafarraginis Lp18 was isolated from an. environmental source. Partial 16S rRNA sequencin indicated 100% ⁇ similarity t Lactobacillus, parafarraginis AB 262735 which has a risk group of 1 (TRBA), When cultured on MRS medi for 3 days at 34°C, anaerobically, Lpl8 produces cream, round, slight sheen, convex, colon diameter 1- 2mm (facultative anaerobe). Its microscopic appearance is Gra positive, non-motile, short rods rectangular, mainly diploid. Lactobacillus parafarraginis L l8 was deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022945.
• Lactobacillus buckneri Lb23 wa isolated from an environmental source. Partial 16S rRNA sequencin indicated 99% similarity to Lactobacillus buckneri AB 429368 which has a risk group of 1 (TRBA). When cultured on MRS media for 4 days at 34°C, anaerobically, Lb23 produces cream, shiny, convex, colony diameter l-2mm (facultative anaerobe). Its microscopic appearance is Gram positive, non-motile, rods in chains. Lactobacillus buckneri Lh23 was deposited with the National Measurement Institute, Australi on 27 October 2011 under Accession Number V11 /022946.
• Lactobacillus mpi Lr24 was isolated from an environmental source. Partial 16S rRNA sequencing indicated 99% similarity to Lactobacillus rapi AB 366389 which has a risk group of 1 (DS ' MZ). When cultured on MRS media for 4 days at 34°C, anaerohieally, Lr24 produces cream, round, shiny colonies with a diameter of 0.5mm .(facultative anaerobe), Its microscopic appearance is Gram positive, non-motile, short rods single or diploid. Lactobacillus rapi Lr24 was deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022947.
• Lactobacillus zeae L/,26 was isolated from an environmental source. Partial 16S rRNA sequencing indicated 99% similarity to Lactobacillus z.eac AB 008213.1 which has a risk group of 1 (TRBA). When cultured on MRS media for 48 hours at 34°C, anaerobieally. Lz26 produces white, round, shiny, convex, colonies with a diameter of 1mm (facultative anaerobe). Its microscopic appearance is Gram positive, non-motile, short rods almost coccoid, diploid and some chains. Lactobacillus z,eae L/26 was deposited with the National Measurement: Institute, Australia on 27 October 201. 1 under Accession Numbe V 1 1 /022948.
• Acetobacier fabarum Afl.5 was isolated from an environmental source. Partial 16S rRNA sequencing indicated 100%. similarity to Acetobacier fabarum AM 905849 which has a risk group of 1 (DSMZ). When cultured on Malt extract media for 3 days at 34°C, AF15 produces opaque, round, shiny, convex, colony diameter 1mm (aerobic). Its microscopic appearance is Gram negative, rods single or diploid. Acetobacier fabarum Afl.5 was deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI.1/022943.
• Candida ethanotica Ce31 was isolated from an environmental source. Partial 16S rRNA sequencing indicated 89% similarity to Candida ethanolica AB 346.18. When cultured on Malt extract media for 2 days at 34°C, Ce 1 produces cream, flat dull, roundish, colony diameter 2-3mm. (aerobic). Its microscopic appearance is budding, ovoid yeast.
• Candida ethanolica Ce31 was deposited with the National Measurement Institute, Australia on 27 October 201 1 under Accession Number VI 1/022944.
• strains used in experiments described herein were: wi hacillus dw!tvorms (N3) deposited with the National Measm3 ⁇ 4ment Institute, Australia on 14 December 2012 under Accession Number VI 2/022847; Lactobacillus p rafarraginis (Nil) deposited with the National Measurement Institute, Australia on 1.4 December 2012 unde Accession Number VI 2/022848; Lactobacillus brevis (TD) deposited with the National Measurement Institute.
• API ® 50 CH strips show TB ferments D-Galactose, D-Glucose, D-Fructose, D-Mannose, Methyl - aD-Mannopyranoside, Methyl-aD-Glucopyranoside, N-Aeetylglueosamine, Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-CeHobiose, D-Maltose, D-Lactose, Sucrose, D- Mele3 ⁇ 4itose, D-Raffinose, Gentiohiose, D-Tagatose and Potassium gluconate.
• N-Acetylglucosamine Amygdalin, Arbutin, Esculin ferric citrate, Salicin, D-Cellobiose, D-Maliose, Sucrose, D-Trehalose, Inulin, Gentiobiose, D- Turanose, D-Tagatose L-ArabitoL Potassiu gluconate and Potassium 2-Ketoglueonate.
• the composition referred to herein below a the OL composition * comprises six microbial strains described above (namely Acetobacter fabarum Af 15, Lactobacillus parafarragi is Lpl8, Lactobacillus buchneri Lb23, Lactobacillus rapi Lr24, Lactobacillus zeae Lz26, and Candida ethanolica Ce31) at final concentrations of 2.5 x 10 5 cfu/ml for each of the. Lactobacillus strains, 1.0 x 10 ' cfu/ml for Candida ethanolica Ce31 and 1.0 x 10" cfti/ml for Acetohacter fabarum At ⁇ 5.
• composition referred to herein below as 'Mix G' comprises three of the microbial strains described above, specifically Lactobacillus zeae Lz26, strain TB ( ⁇ Accession Number VI /022850) and T9 (NMI Accession Number V 12/022849). Fresh cultures of bacteria were added at the respective final concent rations 1 x 10 7 cfu/ml Lactobacillus zeae Lz26J. x 10 ' cfu/ml TB and 1 .10 efii/ml T9, to a mix of 2% trace elements, 0.3% humie. 4% molasses and water. The composition was adjusted to pH 3.8-4.2 with phosphoric acid.
• T Mix ⁇ comprises five of the microbial strains described above, specifically Lactobacillus zeae Lz26. Lactobacillus buchneri Lh23. Lactobacillus parafarraginis Lpl8, Candida ethanolica Ce3I, and Acetobacter fabarum Afl5. Fresh cultures of bacteria were added at the respective final concentrations 1 x lO'cfu ml Lactobacillus ze e L3 ⁇ 426, 1 x lO 3 etu/mlLactobacill s buchneri Lb23.1 x lO'cfu/ml Lactobacillus parafarraginis Lpl8.
• the composition referred to herein below as "Mix 2' comprises five of the microbial strains described above, specifically Lactobacillus zeae Lz26, Lactobacillus parafarraginis L l.8, Lactobacillus buchneri ' Lb23, Lactobacillus rapi Lr24, and Acetohacter fabarum Af 15. Fresh cultures of bacteria were added at the respective final concentrations!
• composition referred to herein below as 'Mix 3' comprises four of the microbial strains described above, specifically Lactobacillus zeae Lz26, Lactobacillus parafarraginis Lpl 8. Lactobacillus buchneri Lb23 and Lactobacillus rapi Lr24, Fresh cultures of Lactobacillus sp.
• glycerol stocks were made of each isolate and maintained at -80°C for long- term culture storage. Short-term storage of the cultures were maintained at 4°C on agar slopes (3 month storage) and on agar plates which are subcultured monthly. To maintain the isolates original traits, a fresh plate is made from the -8Q°C stock following three plate subcultures.
• Lactobacillus strains were grown with or without air (L rapi prefers anaerobic) either in MRS broth (Difco) o on MRS agar plates depending on application. The cultures were routinely grown for 2 days at a mesophiljc temperature of 30-34°C. The Acetobacier and Ethanotica strains were -grown aerobically either in Malt extract broth (Oxoid) or on Malt extract agar plates depending on application. The cultures were routinely grown for 2 days at a mesophilic temperature of 3()-34°C.
• the inventor conducted a laboratory experiment in which the plant, pathogen Fu r ifti oxyspamm f. sp zingiheri was challenged with individual bacterial strains described in Example 1 (hereinafter "GL" strains) to determine if they show an antagonistic effect against the Fusarium ginger pathogen.
• GL bacterial strains described in Example 1
• a pure isolate of Fusarium oxysporum f. sp zmgiberi (hereinafter u foz") was purchased fro the Herbarium (BRIP) Queensland DPI culture collection. Foz was routinel grown on PDA solid medi aerobically at room temperature. Alter a few days a pink growth is seen and after around 5 days white aerial mycelium develop, it also grows well on malt extract (ME) and MRS solid media. Lactobacillus. par farraginis L l8, Lactobacillus buchneri Lh23, Lactobacillus rapt Lr24, and Lactobacillus zeae Lz26 were routinely grown anaerobically on MRS media, at 34°C.
• Acetahacte ' r fabarwn Afl5 and Candida, ethanolica Ce31 were routinely grown aerobically o ME agar at 34°C.
• the antifungal activity of the GL bacterial strains was determined using four different methods, well plates, cross streak plates, a dual plate screen and a culture drop method according to the following experimental protocols.
• Thi method was developed to circumvent the difficulties associated with different growth requirements of the GL strains and the pathogens.
• the screen was performed in. a two section petri dish using two different growth media per section. • Using sterile petri dishes with two sections the GL bacteria culture agar was poured in both sections.
• the inventors investigated the effect of a composition comprising the bacterial strains described in Example 1 on the plant pathogen Fusarium oxysporum in Huntsman watermelon seedlings.
• Fusarium oxysporum has many specialized forms (f. sp).
• the form affecting watermelons causing Fusarium wilt is Fusarium oxyspomm t. s niveum.
• the seedlings used in this study were infected with Fusarium oxysporum and obtained from a watermelon farm with a significant Fusarium problem in the soil (provided by Jason Kiot3 ⁇ 4),
• composition (referred to below as 'GL composition') comprised six microbial •strains listed in. Example 1. (namely Acetohacter faharum. ⁇ 5, Lactobacillus parafarraginis Lp.1.8, LactobticiU s huchneri Lb 23, Lactobacillus rcipi Lr24, Lactobacillus zeae Lz26, and Candida ethancUca Ce3.1) at final concentrations of 2.5 x 10 ' ' cfu/tnl for each of the Lactobacillus- strains, 1.0 x cfu/m! for Candida ethanalica and 1.0 10 6 cfu/ml for Acetobacter fabarum.
• the strains were grown as described in Example 1 and mixed with 2% trace elements, 0,3% humate (Soluble Humate, LawrieCo), 3% molasses and 0.1 -0.2% phosphoric acid. Phosphoric acid was added to the point where pH was in the range 3.8 to 4.0.
• the trace elements component typically comprised the following (per 1 ,000L):
• Experiment A 4/4 plants survived the Fusarium ax sporum infection. The plants had larger and more vibrant leave in comparison to the other treatments. The plants had a much stronger main stem. The root system was significantly compact and. dense in all replicates. The average weight of the plants was the highest and was a good indication of overall plant growth.
• Experiment B 3/4 plants survived the Fusarium axysporum infection. One plant was affected by the pathogen after the first week. The leaf size was average and the plant health was good. The root system was less dense than in A. Overall plant health was satisfying. The mai stem of the plants was strong.
• Experiment C 1/4 plants survived the Fusarium oxysporum. infection. The overall plant growth and health was very poor.
• Experiment D All replicates were affected by the Fusarium oxysporum and died within a week.
• the inventors investigated whether a microbial strain composition described herein can protect watermelon seedlings against Fusarium sp. infected soil.
• Each seed was planted to a similar depth and immediately after planting each cell was dosed with 6ml of sterile water or 1ml of Mix G (1:10) plus 5ml sterile water.
• the propagation houses were sealed and left at ambient, temperature out of direct sunlight for 12 days.
• Experiment 2 was essentially set up in the same way as Experiment 1 with two exceptions. Seed were soaked for 30 mins and the initial microbial composition dose was reduced by a third (3000 of MixG diluted 1 : 10 was added to 3ml of sterile water) but repeated afte 1 week. 3ml sterile water was added to the water controls.
• Fusarium oxysporum f. sp cuhense (races 1-4) is the causal pathogen of the destructive Panama disease in bananas. Due t stric quarantine containment the causal pathogen could not be directly tested in the laboratory, however, less virulent strain was permitted ⁇ foe accession no. 24322).
• the dual plate assay was performed which challenged the pathogen to grow against the GL composition (see Example 3), and the individual GL strains described in Example 1. The experiment was performed in duplicate and repeated. The results are shown in Table 5.
• Pseudocercospom macadami is the causal pathogen of husk spot of macadamia nuts. It causes the nuts to drop from the trees prematurely creating great economic loss to the macadamia industry. Some varieties of macadamia tree are more susceptible than others, sueh as A 16.
• a pure isolate of Pseudocercospara mac damiae was acquired from the Queensland Department of Primary Industries culture collection. The ability of GL strains described in Example 1 to inhibit growth of this fungal pathogen was tested as deseribed in Example 2. The results are shown below in Table 6. Lactobacillus pamfarraginis Lpl8 Lactobacillus buchmri Lb23.
• Lactobacillus- rapi Lr24, Lactobacillus zea Lz26, Lactobacillus dioUvoram (N3), Lactobacillus brevis (TD), TB and T9 were able to inhibit growth of the pathogen, using bot actively growing culture and growth media (filtrate).
• the inventors conducted a laboratory experiment to determine the ability of GL strains described in Bxamplc 1 to inhibit growth of the Gram positive bacterium Strepiomyces scabies, the causati ve agent of potato scab.
• Lactobacillus parafarraginis Lp-18, Lactobacillus bachneri Lb23, Lactobacillus rapt Lr24, and Lactobacillus zeae Lz26 were routinely grown anaerobically on MRS medi a 34°C, Acetobacter fabarum A l5 and Candida ethanotica Ce31 were routinely grown aerobically on ME agar at 34°C.
• the antibacterial activity of the bacterial strains was determined using three different methods, well plates, cross streak plates and a dual plate method as described in Example 2,
• Results are shown below in Table 10. Actively growing Lactobacillus parafarraginis Lpl8, Lactobacillus buchneri Lb23 and Lactobacillus zeae Lz26 and strain TB displayed best growth inhibition of S. scabies. The fact that cell free supernatant derived from cultures of these organisms did not inhibit S. scabie growth suggests an interaction between the GL strains and the pathogen. Table 10. Zone. Of inhibition of growth (mm) of Streptamyces scabies 24 hours after addition of GL strains
• Fresh overnight cultures were grown of individual Lactobacillus GL strains. 3ml of the overnight culture was spun at 4,000rpm for lOmins. The supernatant was decanted and filtered through a 0.45 ⁇ 1. syringe filter unit into a sterile /5 bottle. The sterile filtrate from each bacterial culture as well as a control of MRS growth media was diluted 1 : 1, 1 :3, 1 :5, 1:10 usin sterile 0.85% saline. Using the wide end of a sterile .1 ml tip, si well spaced, holes were cut in a fres nutrient agar plate.
• the plate was swabbed three times with one of the three mastitis pathogens (diluted to a 0.5 MacFarlands std). 80ul of each diluted filtrate (as well as undiluted and MRS) was added to each of the six. wells. This was repeated for each pathogen and each bacterial filtrate. The plates were incubated overnight at 34°C. The diameter of clear zones of inhibited growth were measured and recorded.
• Results are shown in Tables 11 , 12 and 13.
• Growt culture filtrates from Lactobacillus zeae Lz26, TB and T9 demonstrated antimicrobial activity against all three mastitis pathogens.
• the filtrates remained effective up to a 1:3 dilution. All three pathogens grew well in the absence of GL strains.
• Each strain (or mix) was diluted in sterile MRS media 1: 100, 1:1,000. and 1 :10,000.
• Nutrient agar/MRS dual plates were poured (described above). ⁇ of diluted culture was spread onto each MRS quarter of the dual plate (duplicates). A small glass 'hockey stick' was used t spread the ⁇ over the media.
• the plates were incubated anaerobicali at. 34°C for 48 hours.
• the three pathogens were diluted to a 0.5 MacFarlands std and using a sterile swab each was swabbed across both nutrien agar quarters of the dual plate. The plates were reincubated, for 2 days at 34°C.
• Results are shown in Tables 15, 16 and 17. Viable cultures of T9 and Mix 3 were the most effective requiring less than 100 colonies lo cause an inhibitory effect against all three mastitis causing bacterial species. Lactobacillus Lx26, Lb23 and T9 will be used to formulate a third anti-mastitis mix. All three pathogens gre well in the absence of GL strains compositions.
• Zones of inhibition of growth (mm) of bacterial isolates 24 hours after addition of GL strains were also determined (as described in above examples). The results are shown in Tables 18, 19 and 20.
• the inventors also investigated the ability of GL strains described in Example 1 to inhibit growth of Pseudomonas sav stanoi (causative agent of, inter alia, olive gall disease) using the dual plates screen method as described in Example 2. The results are shown in Table 21 .

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  • 2014-04-22 WO PCT/AU2014/050019 patent/WO2014172758A1/en not_active Ceased
• 2015
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