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Title:
BACILLUS STRAINS AND AGENTS WITH BENEFICIAL PROPERTIES
Document Type and Number:
WIPO Patent Application WO/2017/081105
Kind Code:
A1
Abstract:
The invention provides a novel bacterial strain, identified herein as Bacillus subtilis HF1, which shows utility as an agent for controlling plants diseases and also for promoting the growth of animals. The invention provides agents, methods, processes, uses and compositions having utility for these things, based on the strain, or derivatives thereof.

Inventors:
HU, Zanmin (Chinese Academy of SciencesNO.1 West Beichen Road,Chaoyang District, Beijing 1, 100101, CN)
FAN, Chengming (Chinese Academy of SciencesNO.1 West Beichen Road,Chaoyang District, Beijing 1, 100101, CN)
CHEN, Yuhong (Chinese Academy of SciencesNO.1 West Beichen Road,Chaoyang District, Beijing 1, 100101, CN)
Application Number:
EP2016/077179
Publication Date:
May 18, 2017
Filing Date:
November 09, 2016
Export Citation:
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Assignee:
INSTITUTE OF GENETICS AND DEVELOPMENTAL BIOLOGY, CHINESE ACADEMY OF SCIENCES (No. 1 West Beichen Road, Chaoyang District, Beijing 1, 100101, CN)
International Classes:
C12N1/20; A01N63/00; A23K10/16; A23L33/135; B09C1/10; C12R1/125
Domestic Patent References:
WO2013029013A12013-02-28
WO2008041786A12008-04-10
Other References:
YUN CAO ET AL: "Bacillus subtilis SQR 9 can control wilt in cucumber by colonizing plant roots", BIOLOGY AND FERTILITY OF SOILS ; COOPERATING JOURNAL OF INTERNATIONAL SOCIETY OF SOIL SCIENCE, SPRINGER, BERLIN, DE, vol. 47, no. 5, 31 March 2011 (2011-03-31), pages 495 - 506, XP019913882, ISSN: 1432-0789, DOI: 10.1007/S00374-011-0556-2
"Pesticides in the Modern World - Pesticides Use and Management", 19 October 2011, INTECH, ISBN: 978-953-30-7459-7, article HELENE CAWOY ET AL: "Bacillus-Based Biological Control of Plant Diseases", pages: 273 - 297, XP055336926, DOI: 10.5772/17184
KE JIA ET AL: "Rhizosphere Inhibition of Cucumber Fusarium Wilt by Different Surfactinexcreting Strains of Bacillus subtilis", PLANT PATHOLOGY JOURNAL, vol. 31, no. 2, 1 June 2015 (2015-06-01), KR, pages 140 - 151, XP055336940, ISSN: 1598-2254, DOI: 10.5423/PPJ.OA.10.2014.0113
FANG CHEN ET AL: "The Effect of Biocontrol Bacteria on Rhizosphere Bacterial Communities Analyzed by Plating and PCR-DGGE", CURRENT MICROBIOLOGY, vol. 67, no. 2, 13 March 2013 (2013-03-13), Boston, pages 177 - 182, XP055335739, ISSN: 0343-8651, DOI: 10.1007/s00284-013-0347-0
SINOL SEN ET AL: "Effect of supplementation of Bacillus subtilis LS 1-2 to broiler diets on growth performance, nutrient retention, caecal microbiology and small intestinal morphology", RESEARCH IN VETERINARY SCIENCE., vol. 93, no. 1, 1 August 2012 (2012-08-01), GB, pages 264 - 268, XP055336914, ISSN: 0034-5288, DOI: 10.1016/j.rvsc.2011.05.021
LENINGER: "Biochemistry", 1972, WORTH PUBLISHERS INC.
JEFFREY H. MILLER: "Experiments in Molecular Genetics", 1972, COLD SPRING HARBOR LABORATORY
AUSUBEL, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1987
KRISHNA, RADHA E. ET AL.: "Strain Improvement of Selected Strain Bacillus subtilis (MTCC No. 10619) for Enhanced Production of Antimicrobial Metabolites", RESEARCH JOURNAL OF BIOTECHNOLOGY, vol. 6.3, 2011, pages 58 - 62
M P DEUTSCHER: "Methods in Enzymology - Guide to Protein Purification", vol. 182, ACADEMIC PRESS INC
"Protein Purification - principles and practice", 1982, PUB. SPRINGER-VERLAG
HARRIS; ANGAL: "Protein purification methods - a practical approach", 1989
Attorney, Agent or Firm:
KREMER, Simon et al. (Mewburn Ellis LLP, City Tower40 Basinghall Street, London Greater London EC2V 5DE, EC2V 5DE, GB)
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Claims:
Claims

1 An isolate of Bacillus subtilis HF1 deposited under accession number CGMCC No. 1 1487 or a mutant thereof.

2 A spore of the Bacillus subtilis isolate of claim 1

3 A culture broth of the Bacillus subtilis isolate of claim 1

***

4 A process for culturing the Bacillus subtilis isolate of claim 1 which process comprises growing the isolate in a suitable medium, optionally under aerobic conditions between 15 to 37°C for between one and four days.

***

5 A process for producing Bacillus subtilis isolate of claim 1 comprising performing a process as claimed in claim 4 and further comprising recovering the Bacillus cells from medium.

6 A process as claimed in claim 5 wherein the recovering is by one or more of washing, filtering and\or sedimentary techniques.

7 A process as claimed in claim 5 or claim 6 wherein the recovered cells are stored under chilled conditions or freeze dried.

***

8 A Bacillus subtilis cell obtained or obtainable by the process of any one of claims 5 to 7.

***

9 A cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of the Bacillus subtilis isolate of claim 1 or claim 8.

10 A composition comprising the Bacillus subtilis of claim 1 , the spore of claim 2, the culture broth of claim 3, the cell of claim 8, or the cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of claim 9 in, or including, a suitable carrier, optionally in dried form.

1 1 A composition as claimed in claim 10 which comprises Bacillus subtilis HF1 intact cells in the concentration range from 1 x 103 to 1 x 1014.

***

12 A process for producing a Bacillus subtilis HF1 supernatant preparation comprising performing a process as claimed in claim 4 and further comprising recovering the supernatant from medium. 13 A process as claimed in claim 12 wherein the recovering is by one or more of washing, filtering and\or sedimentary techniques.

14 A process as claimed in claim 13 wherein the supernatant preparation is recovered by centrifugation.

15 A process as claimed in any one of claims 12 to 14 wherein the supernatant preparation is heated to at least 100°C.

16 A process as claimed in any one of claims 12 to 15 wherein the supernatant preparation is filtered using a biofilter having a diameter of equal to more than 0.22 μηη.

17 A process as claimed in any one of claims 12 to 16 wherein the supernatant preparation is diluted to between 0.1 % and 5%.

***

18 A Bacillus subtilis HF1 supernatant preparation obtained or obtainable by the process of any one of claims 12 to 17, optionally in dried form.

***

19 A process for producing a Bacillus subtilis HF1 agent comprising isolating an active agent from a Bacillus subtilis HF1 source material which is a supernatant preparation as claimed in claim 18 or a cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of claim 9,

the process comprising fractionation using one or more of: selective precipitation, proteolysis, ultrafiltration, ion-exchange chromatography, normal or reversed phase chromatography, followed by assay of fractions for the ability to suppress the hyphal growth of a soil-borne phytopathogenic fungus or for promoting the growth or health of an animal.

***

20 Use of a Bacillus subtilis HF1 agent selected from the list consisting of:

(i) the Bacillus subtilis isolate of claim 1 ;

(ii) the spore of claim 2;

(iii) the culture broth of claim 3

(iv) the cell of claim 9;

(v) the cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of claim 10;

(vi) the composition of claim 10 or claim 1 1 ;

(vii) the Bacillus subtilis HF1 supernatant preparation of claim 18, which is optionally partially purified, fractionated, or concentrated;

for the control of plant disease.

***

21 A method for treating or controlling disease in a plant, the method comprising contacting the plant with a Bacillus subtilis HF1 agent of claim 20.

22 A method as claimed in claim 21 wherein the disease is a soil borne or air borne disease. ***

23 A method as claimed in claim 21 or claim 22 wherein the agent is applied to the plant by applying to the soil in which the plant is or will be planted.

24 A method as claimed in claim 21 or claim 22 wherein the agent is applied to the the seed of a plant.

25 A method as claimed in claim 21 or claim 22 wherein the agent is applied to the plant by spraying, dusting, foliar spraying, misting, aerosolizing or fumigation.

26 A method as claimed in claim 21 or claim 22 or claim 25 wherein the agent is applied to the plant as a seedling.

27 A method as claimed in claim 21 or claim 22 or claim 25 wherein the agent is applied to the plant at harvest or after harvest to prevent or minimize post-harvest disease.

28 A method as claimed in any one of claims 21 to 27 wherein the agent is applied to the plant repeatedly at different times in the plant life cycle.

29 A method as claimed in any one of claims 21 to 28 wherein the agent is applied to the plant in conjunction with other disease control agents.

30 A method for preservation of an agricultural plant product, the method comprising contacting the plant product with a Bacillus subtilis HF1 agent of claim 20.

31 A method as claimed in any one of claims 21 to 30 wherein the plant is selected from the list consisting of: cereal, vegetable and arable crops, grasses, lawns, pastures, fruit trees; ornamental trees and plants; flowering plants; medical plants; tobacco.

32 A method as claimed in any one of claims 21 to 31 wherein the disease is one caused by a phytopathogenic fungus.

33 A method for controlling a phytopathogenic fungus, the method comprising contacting the fungus with a Bacillus subtilis HF1 agent of claim 20.

34 A method as claimed in claim 33 wherein the agent is applied to the environment of the fungus.

35 A method as claimed in any one of claims 32 to 34 wherein the agent suppresses hyphal growth or spore germination of the fungus. 36 The method of any one of claims 32 to 35 wherein the phytopathogenic fungus is selected from Fusarium spp., Colletot chum spp., Rhizoctonia spp., Phytophthora spp., Alternaria spp. Gaeumannomyces graminis, Verticillium dahliae, Calonectria nivale, Ceratobasidium cornigerum, Botryospuaeria berengeriana, Stemphylium solani,

Coniothyrium spp, Phoma crystallifera, Myrmecridium schulzeri, Corynespora cassiicola.

37 A method for soil remediation, the method comprising contacting the soil with a Bacillus subtilis HF1 agent of claim 20.

38 A composition comprising at least one Bacillus subtilis HF1 agent of claim 20 and an agriculturally acceptable carrier.

39 A composition as claimed in claim 38 wherein the agriculturally acceptable carrier comprises one or more humectants, spreaders, stickers, stabilisers, penetrants, emulsifiers, dispersants, surfactants, buffers or binders.

40 A composition as claimed in claim 38 or claim 39 which is a spray, suspension, concentrate, foam, drench, slurry, gel, dip or paste.

***

41 Use of a Bacillus subtilis HF1 agent selected from the list consisting of:

(i) the Bacillus subtilis isolate of claim 1 ;

(ii) the spore of claim 2;

(iii) the culture broth of claim 3

(iv) the cell of claim 9;

(v) the cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of claim 10;

(vi) the composition of claim 10 or claim 1 1 ;

(vii) the Bacillus subtilis HF1 supernatant preparation of claim 18, which is optionally partially purified, fractionated, or concentrated;

for improving animal weight, or animal weight where the animal is optionally a human.

42 Use of a Bacillus subtilis HF1 agent selected from the list consisting of:

(i) the Bacillus subtilis isolate of claim 1 ;

(ii) the spore of claim 2;

(iii) the culture broth of claim 3

(iv) the cell of claim 9;

(v) the cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of claim 10;

(vi) the composition of claim 10 or claim 1 1 ;

(vii) the Bacillus subtilis HF1 supernatant preparation of claim 18, which is optionally partially purified, fractionated, or concentrated;

in the preparation of a composition for improving animal weight, or animal health where the animal is optionally a human.

43 A method for promoting the growth or health of an animal, which is optionally a human, the method comprising administering to the animal a Bacillus subtilis HF1 agent of claim 41 or claim 42. 44 A method as claimed in claim 43 to increase a production parameter for a group of treated animals as compared to control animals, which production parameters are selected from average daily weight gain, feed conversion rates, and consistency of growth among the group of treated animals.

45 A method as claimed in claim 43 or claim 44 wherein the agent is administered orally to the animal, optionally to improve the gastrointestinal health of the animal.

46 A method as claimed in any one of claims 43 to 45 wherein animal is a poultry animal or a farmed aquatic organism, preferably a fish or crustacean.

47 A method as claimed in any one of claims 43 to 46 wherein animal is a poultry animal, preferably a chicken or turkey, and the agent is administered in drinking water or dried onto a solid foodstuff.

***

48 A Bacillus subtilis HF1 agent selected from the list consisting of:

(i) the Bacillus subtilis isolate of claim 1 ;

(ii) the spore of claim 2;

(iii) the culture broth of claim 3

(iv) the cell of claim 9;

(v) the cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet of claim 10;

(vi) the composition of claim 10 or claim 1 1 ;

(vii) the Bacillus subtilis HF1 supernatant preparation of claim 18, which is optionally partially purified, fractionated, or concentrated;

for use in a method of improving animal health or weight.

49 An agent for use of claim 48, wherein the method is a method as defined in any one of claims 43 to 47.

50 A human foodstuff or other animal feed or supplement comprising the agent of claim 48, which is optionally an aquaculture feed additive for fisheries, or a solid food additive, or an additive for drinking water.

*** 51 Use of the growth medium having the following composition: Glucose 10 to 28 g/L, KNOs 6 g/L, KH2P04 1.2 g/L, MgS04-7H201 .2 g/L, Na3C6H507 0.2 g/L, CaCI2-2H20 105 mg/L, FeS04«7H20 16 mg/L, EDTA 2.1 mg/L, H3BO3 2.86 mg/L, ZnS04«7H20 0.222 mg/L, MnCI2«4H20 1.81 mg/L, Na2Mo04 0.021 mg/L and CuS04«5H20 0.07 mg/L, for growing, culturing or fermenting a Bacillus strain.

52 Use of the growth medium of claim 51 for growing, culturing or fermenting the Bacillus subtilis isolate of claim 1 .

***

53 A process preparing a mutant of the Bacillus subtilis HF1 isolate deposited under accession number CGMCC No. 1 1487, which process comprises subjecting said strain to mutagenesis. 54 A process as claimed in claim 53 wherein the mutagenesis is UV mutagenesis.

55 A process as claimed in claim 53 or claim 54 wherein the mutagenesis is followed by screening and selection of mutant Bacillus subtilis HF1 strains exhibiting improved or modified production of antimicrobials or growth enhancement in an animal.

Description:
Bacillus strains and agents with beneficial properties

TECHNICAL FIELD

The present invention relates to agents comprising or derived from Bacillus species which have utility in for controlling plant diseases and promoting the health and growth of animals, including humans.

The invention further relates to materials and methods for identifying, preparing or using such agents.

BACKGROUND ART

Reducing the usage of artificial crop protection products and their residues, and plant preservatives, are important aims in improving the sustainability of the agricultural production system.

There is therefore an ongoing requirement for agents and related materials which have activity against plant diseases, for example those caused by filamentous phytopathogenic fungi. Novel agents are required for instance to overcome the problem of resistance to existing compounds, or to expand the range of diseases which can be controlled, or the ways in which existing agents are compounds. Novel agents having a biological origin may be preferable since they are typically less persistent in the environment.

Additionally, the use of antibiotics in animal and human health, in particular poultry, fish and shellfish production, is coming under increasing pressure from both consumers and government regulatory agencies. This has created a need for effective antibiotic alternatives. The use of direct-fed microbials, or active agents derived therefrom, may be one such potential alternative.

DISCLOSURE OF THE INVENTION

The present inventors have identified a novel bacterial strain, identified herein as Bacillus subtilis HF1 , which shows utility as an agent for controlling plant diseases and also for promoting the health and growth of humans and other animals.

Thus the invention relates, inter alia, to methods and materials for controlling plant diseases which are based on, or related to, Bacillus subtilis HF1 , or active compounds (for example fungicidal compounds) or mutants derived therefrom.

The invention further relates, inter alia, to methods and materials for controlling, inhibiting, or suppressing microorganisms, particularly fungi, which are based on, or related to, Bacillus subtilis HF1 , or active compounds (for example fungicidal compounds) or mutants derived therefrom. The microorganisms will preferably be pathogens e.g.

phytopathogens.

In the Examples presented hereinafter, HF1 showed effect against a wide spectrum of fungal diseases, which suggests a utility in combatting fungal resistance. It is believed that HF1 can be used for controlling fungi spread by both soil and air.

Furthermore, as demonstrated below, the utility of Bacillus subtilis HF1 is not limited to the live strain. The fermentation broth can be used either directly, dried, concentrated or in treated form, as well as a source of isolated or enriched active agents. In other aspects, Bacillus subtilis HF1 can be used as a source of derived-strains, such as mutants having similar or improved properties.

The HF1 products described herein, which are based on, or related to, Bacillus subtilis HF1 , or active compounds or mutants derived therefrom, can be used as biofertilizers or biopesticides for combating plant diseases and improving plant health, or for preserving plant products, including derived food products, at or after harvest.

The present invention further relates to methods and materials for improving the growth or health of agricultural or other animals (including humans, poultry, fish and shellfish) using the HF1 products described herein. For example they can be used as feed additives or veterinary or medicinal drugs for health improvement. In certain embodiments the invention provides bacterial isolates, probiotic formulations comprising the isolates and methods of using the probiotic formulations and isolates to improve the health or growth of animals.

Some of these aspects and embodiments will now be described in more detail.

Deposited strains and mutants

A particular strain of Bacillus subtilis HF1 used to demonstrate the agents of the invention has been deposited (under the terms of the Budapest Treaty) at China General

Microbiological Culture Collection Center, CGMCC, in Institute of Microbiology, Chinese Academy of Sciences, N0.1 West Beichen Road, Chaoyang District, Beijing 100101 , China under the accession number CGMCC No. 1 1487.

The strain was identified as belonging to the species Bacillus subtilis based on the 16S rDNA analysis as set out in the Examples below.

Certain characteristics of the strain are set out in Example 2.

This strain, for instance in isolated or substantially isolated forms or cultures, form one aspect of the present invention.

Accordingly in one aspect, the invention provides Bacillus subtilis HF1 strain having accession number CGMCC No. 1 1487 or a mutant thereof.

By "mutant thereof" is meant a further strain derived from the deposited strain which has been altered in respect of one or more characteristics compared to the deposited strain. Methods of producing mutant strains are described below.

Typically the mutants will share one or more of the characteristics of deposited Bacillus subtilis HF1 strain described in Example 2 below, as well as the activities described herein.

In one embodiment the Bacillus subtilis HF1 strain, or mutant thereof, is in the form of a biologically pure culture.

Unless context demands otherwise, where the term "Bacillus subtilis HF1 strain" is used herein, it will be understood to apply to both the deposited strain, and derived mutants thereof. Bacillus subtilis is known for its ability to form small, tough, protective and metabolically dormant endospores. These spores of Bacillus subtilis HF1 strains, and their uses in the methods and processes described herein, form other aspect of the present invention. In one aspect, the invention provides a composition comprising at least one Bacillus subtilis HF1 strain of the invention, in a suitable carrier. Such carriers are discussed in more detail hereinafter, but will be appropriate to the required utility e.g. controlling plant pathogens (an agriculturally acceptable carrier) or for improving animal health (e.g. a feed or additive to drinking water).

Growth and culture of Bacillus strains

The inventors have established that a growth medium (termed "Endo" herein) having the following composition is particularly suitable for growing the strains of the invention:

Glucose 10 to 28 g/L, KN0 3 6 g/L, KH 2 P0 4 1.2 g/L, MgS0 4 -7H 2 01 .2 g/L, NasCeHsCv 0.2 g/L, CaCI 2 -2H 2 0 105 mg/L, FeS0 4 -7H 2 0 16 mg/L, EDTA 2.1 mg/L, H 3 B0 3 2.86 mg/L, ZnS0 4 « 7H 2 0 0.222 mg/L, MnCI 2 « 4H 2 0 1 .81 mg/L, Na 2 Mo0 4 0.021 mg/L and

CuS0 4 « 5H 2 0 0.07 mg/L.

Use of this medium in growing, culturing or fermenting Bacillus strains, such as the strains of the invention, forms another aspect of the invention.

Mutants of deposited strain

Mutants can be provided from the deposited Bacillus subtilis HF1 strain by those skilled in the art using standard methodologies.

For example methods of DNA mutagenesis and screening for mutants are well known to these skilled in the art and include a variety of chemical and enzymatic methods.

Examples of such methods include but are not limited to exposure to ultraviolet light (UV), nitrous acid, N-methyl-N'-nitro-N-nitrosoguanidine (NG), and 4-nitroquinoline-N-oxide (4NQO). (Leninger (1972) Biochemistry. Worth Publishers Inc., NY; Jeffrey H. Miller (1972) "Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Springs Harbor, New York). Preferred methods, include UV and NG.

By way of example, mutant strains of Bacillus subtilis HF1 strain capable of producing anti-microbial compounds or enhancing the growth of animals may be generated by UV mutagenesis. By way of examples, mutants may be produced by exposure of the spores to UV light for a period of about 10 to about 180 seconds, preferably 45 to about 90 seconds, and most preferably 65 to 75 seconds. Mutageneses involving NG may involve varying the concentration of the NG and exposure time. By way of example NG, at about 13 milligrams/liter (mg/L) to about 400mg/L of NG may be used for a period of exposure of about 15 minutes to about 120 minutes. Alternative methods for generating mutants include techniques in the field of molecular biology. Examples of such techniques include, but are not limited to, oligonucleotide directed mutagenesis, linker scanning mutations or oligonucleotide directed mutagenesis using polymerase chain reaction (Ausubel (1987) Current Protocols in Molecular Biology).

Screening and selection of mutant Bacillus subtilis HF1 strains exhibiting improved or modified production of antimicrobials or growth enhancement in animals can be performed by conventional assay methodology such as those described herein.

Examples of selection criteria for screening mutants includes, but is not limited, to the ability of the bacillus colony to inhibit hyphal growth on a petri dish. An non-limiting example of strain improvement via UV irradiation of a selected Bacillus subtilis strain to enhance production of antimicrobial metabolites is given by Krishna, Radha E., et al. "Strain Improvement of Selected Strain Bacillus subtilis (MTCC No. 10619) for Enhanced Production of Antimicrobial Metabolites." RESEARCH JOURNAL OF BIOTECHNOLOGY 6.3 (201 1 ): 58-62.

Thus aspects of the present invention include derived mutants of the deposited Bacillus subtilis HF1 strain, plus processes for preparing these by subjecting it to one or more rounds of mutagenesis, and culturing the resulting strains.

Compositions and related products

Bacillus subtilis HF1 cells may be prepared for use in the compositions using standard drying and fermentation techniques known in the art. Growth is commonly effected under aerobic conditions in a bioreactor at suitable temperatures and pH for growth. Typical growth temperatures are from 15 to 37°C, commonly 27°C to 32°C. Agitation may be used e.g. about 170 rpm.

Cultured material greater than or equal to about one, two, three or four days old may be used.

Typical concentration ranges for the Bacillus subtilis HF1 intact cells in the composition in the form of intact cells, is from 1 x 10 3 to 1 x 10 14 , preferably 1 x 10 4 to 1 x 10 10 , more preferably 1 x 10 6 to 1 x 10 8 cells/mg. It will be appreciated that compositions with cell concentrates in order of 1 x 10 11 to 1 x 10 14 may be prepared and diluted before application if required.

The strains may be harvested using conventional washing, filtering or sedimentary techniques such as centrifugation, or may be harvested using a cyclone system.

Harvested cells can be used immediately or stored under chilled conditions (for example at 4°C). Preferably cells should be used soon after harvest.

In other embodiments cells or broth products (with or without cells) may be lyophilised or dried using conventional methods, for example spray dried under heated or vacuum conditions. A preferred method of drying is freeze drying. Non-limiting examples of freeze drying techniques suitable for Bacillus species are described in WO 2008041786 A1.

Typical products may comprise cultured material of Bacillus subtilis HF1 without separation of spores and cells.

The practice of the invention is not limited to Bacillus subtilis HF1 strains, but also extends to products therefrom, either directly or via processing.

For example in Examples below culture broth was used - for example after one or more of centrifugation (e.g. 10,000 rpm), heating (e.g. to 100°C for about 10 minutes), and also dilution (e.g. with water to between 0.1 % and 5% e.g. about 1 %, 0.5% or 0.25%).

Thus in different aspects the Bacillus subtilis HF1 cells may be processed prior to use to produce active cell extracts, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates, cell pellets or may be used as whole cell preparations. Other agents

If desired, Bacillus subtilis HF1 agents can be purified, or enriched (for instance using the methods discussed below) from cells and cultures of Bacillus subtilis HF1 or mutants thereof.

Such enrichment can be, for example, be based on bio-filtration using particularly diameter or molecular weight cut-off filters. For example in experiments below the supernatant was filtered using a biofilter having a diameter of 0.22 μηη.

In other embodiments the supernatant was sterilized at high temperature (121°C for 10 minutes). Thus methods of providing, or isolating, agents described herein may include heating to this temperature to confirm that the agent is substantially heat stable at this temperature.

By 'substantially heat stable' is meant that the agent retains the relevant activity when tested after heating the agent at the relevant temperature for 10 minutes, and preferably retains at least 50% of the untreated activity.

If desired, this and the other properties of the agents described herein can be utilised to purify them from, or enrich their concentration in, compositions derived from Bacillus subtilis strains and culture medium supernatants therefrom.

The activity assays described herein provide a convenient method of assaying the level of active agent after each stage, or in each sample of eluent.

Methods of purifying peptides or other compounds from heterogenous mixtures are well known in the art (eg. selective precipitation, proteolysis, ultrafiltration with known molecular weight cut-off filters, ion-exchange chromatography, gel filtration, etc.). A particularly useful technique in this regard is ultracentrifugation (e.g. 150,000 x g for over one hour). Further methods which are known to be suitable for protein purification are disclosed in "Methods in Enzymology Vol 182 - Guide to Protein Purification" Ed. M P Deutscher, Pub. Academic Press Inc. Typical protocols are also set out in "Protein Purification - principles and practice" Pub. Springer-Verlag, New York Inc (1982), and by Harris & Angal (1989) "Protein purification methods - a practical approach" Pub. O.U.P. UK, or references or subsequent editions thereof.

Thus one aspect of the invention provides a process for producing an isolated or encirched Bacillus subtilis HF1 agent, the process comprising isolating or enriching an active agent from a Bacillus subtilis HF1 source material, for example from a supernatant preparation, cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet as described herein. The process may comprise using one or more of the isolation techniques described above. The activity of the agent can be assayed during the process e.g. for the ability to suppress the hyphal growth of a soil-borne phytopathogenic fungus or for promoting the growth or health of an animal.

Activity against pathogenic fungi can be assessed by ability of the agent to suppress the hyphal growth or fungal spore germination of a soil-borne filamentous phytopathogen fungus from one, two, three, four, five or preferably each of the following species:

Fusarium spp., Colletot chum spp., Rhizoctonia spp., Phytophthora spp., Alternaria spp. Gaeumannomyces graminis, Verticillium dahliae, Calonectria nivale, Ceratobasidium cornigerum, Botryosphaeria berengeriana, Corynespora cassiicola. The agents of the invention may lead to increase in production parameters for the treated animals as compared to controls. Production parameters include but are not limited to average daily weight gain, feed conversion rates, and consistency of growth among a group of animals.

As will be understood from the foregoing, as used herein the term ""Bacillus subtilis HF1 agent" or similar will be understood to mean any agent based on or derived from the deposited Bacillus subtilis HF1 including, but not limited to: · The deposited Bacillus subtilis HF1 strain

• A derived mutant strain therefrom

• Spores of either Bacillus subtilis HF1 strain

• A supernatant derived from either Bacillus subtilis HF1 strain

• A partially purified or concentrated supernatant derived from either Bacillus subtilis HF1 strain

• An active agent derived from any of these which has one or more of the activities described herein e.g. in respect of soil-borne filamentous phytopathogenic fungi or improving weight or animal health in broiler chickens.

As explained above, the invention provides compositions comprising one or more Bacillus subtilis HF1 agents as described above. Such compositions preferably further comprise other materials or carriers appropriate to their purpose.

Further aspects of the present invention include methods of use of the Bacillus subtilis HF1 agents for the control of plant disease or improving animal health

Preferred methods of the invention

Thus, in one aspect, the invention provides a method for treating or controlling disease in a plant, the method comprising contacting the plant with a Bacillus subtilis HF1 agent of the invention.

"Controlling" in this context should not be taken to imply complete eradication of the disease or pathogen from the plant to be protected, and includes also suppressing or otherwise reducing or minimizing the effects of a pathogen, or increasing yields (or reducing death rates) in plants affected by the pathogen.

The plant is typically a crop plant. Non-limiting Examples of plants which can be treated according to the invention are described in more detail below.

The disease is optionally a soil borne or air borne disease.

Preferably the disease is one caused by a phytopathogenic fungus. An example of a fungal target is powdery mildew.

The agent may be used to inhibit hyphal growth or fungal spore germination.

Thus the invention provides a method for controlling at least one phytopathogenic fungus, the method comprising contacting the fungus with a Bacillus subtilis HF1 agent of the invention. The method may be used to kill the pathogen.

The invention provides a method for soil remediation, the method comprising contacting the soil with a Bacillus subtilis HF1 agent of the invention. The methods of the invention may be used to improve crop production.

The invention provides a method for preservation of an agricultural product (for example a harvested crop), the method comprising contacting the crop with a Bacillus subtilis HF1 agent of the invention.

Optionally materials derived from Bacillus subtilis HF1 or mutants thereof are used in conjunction with other active agents e.g. pesticides or other disease control agents.

Use with plants

In another aspect, the invention provides a composition comprising at least one Bacillus subtilis HF1 agent of the invention, and an agriculturally acceptable carrier.

The Bacillus subtilis HF1 agent(s) of the invention are present in the composition in an amount effective to control the disease or pathogen of interest. The effective

concentration may vary depending on the form the Bacillus subtilis HF1 agent is used in, the environment to which the composition is to be applied, the type, concentration and degree of disease or pathogen infestation; temperature; season; humidity; stage in plant growing season; age of plant; method, rate and frequency of application; number and type of conventional fungicides, pesticides and the like being applied, and plant treatments (for example pruning, grazing, and irrigation). All factors may be taken into account in formulating the composition.

The compositions of the invention may be made by mixing one or more Bacillus subtilis HF1 agents with a desired agricultural carrier.

The compositions of the invention may include humectants, spreaders, stickers, stabilisers, penetrants, emulsifiers, dispersants, surfactants, buffers, binders, and other components typically employed in known art insecticidal or control compositions.

The composition of the invention may be in liquid or solid form, liquid compositions typically include water, saline or oils such as vegetable or mineral oils. Examples of vegetable oils useful in the invention are soy bean oil and coconut oil.

The compositions may be in the form of sprays, suspensions, concentrates, foams, drenches, slurries, injectables, gels, dips, pastes and the like.

Liquid compositions may be prepared by mixing the liquid agriculturally acceptable carrier with the Bacillus subtilis HF1 agent. Conventional formulation techniques may be used to produce liquid compositions.

In one embodiment the compositions is in solid form. The composition may be produced by drying the liquid composition of the invention. Alternatively, a solid composition useful in the invention may be prepared by mixing Bacillus subtilis HF1 agents of the invention with a variety of inorganic or biological materials. For example, solid inorganic agricultural carriers may include carbonates, sulphates, phosphates or silicates, pumice, lime, bentonite, or mixtures thereof. Solid biological materials may include powdered palm husks, corncob hulls, and nut shells. The composition may be formulated as dusts, granules, seed coatings, wettable powders or the like. The compositions may be formulated before application to provide liquid compositions.

The compositions of the invention may be in the form of controlled release, or sustained release formulations.

In one embodiment the Bacillus subtilis HF1 agent is applied to a plant or to agricultural produce.

As explained above, the Bacillus subtilis HF1 agent may be applied to the environment of the pathogen, typically around the plants to be protected, or the soil in which they have been or will be planted. Spraying, dusting, soil soaking, seed coating, foliar spraying, misting, aerosolizing and fumigation are all possible application techniques.

Application rates may for example be 10 10 spores/hectare to 10 14 spores, preferably 10 12 to 10 13 spores per hectare.

Typical application rates may be 50 g/hectare to 10,000 g/hectare. Commonly from 100 g/hectare to 5,000 g/hectare, or 500 to 1500 g/hectare.

Alternatively, the agents of the invention may be applied to seeds as a seed soak. This may be followed by drying.

Alternatively, the agents of the invention are applied to a plant as a seedling

Applications may be once only or repeated as required. Application at different times in plant life cycles, are also contemplated. For example, at or after harvest to prevent or minimize post-harvest disease.

A wide range of plants may be treated using the compositions of the invention. Such plants include cereal, vegetable and arable crops, grasses, lawns, pastures, fruit trees and ornamental trees and plants. Preferred plants for treatment are flowering plants and medical plants.

One example plant is Pseudostellaria heterophylla.

Arable crops which may particularly benefit from use of the compositions and strain(s) of the invention include crucifers and brassicas. For example, cabbage, broccoli, cauliflower, brussel sprouts and bok choy.

Use with animals

Thus, in one aspect, the invention provides a method for promoting the growth or health of an animal, the method comprising administering to the animal a Bacillus subtilis HF1 agent of the invention.

The methods typically include oral administration of the agent (e.g. as a "probiotic formulation" or animal feed) to an animal. Oral administration includes, but is not limited to, delivery in feed (which includes in drinking water). Alternatives include by oral gavage or aerosol spray.

The gastrointestinal health of the animal may be improved by the method. Promoting growth will typically lead to increased rate in a treated animal compared to a control animal not receiving the Bacillus subtilis HF1 agent of the invention - for example a weight increase of at least 5%, 10%, 15% or 20% - for example between 5 and 25%..

Suitably the animal is a poultry, more suitably a chicken or turkey. If supplied in an animal feed, the feed may comprise between 10 4 and 10 9 cfu bacteria/gm of finished feed. Suitably the feed comprises between 10 5 and 5 x 10 7 cfu bacteria/gm feed. The Bacillus subtilis HF1 agents may be added to the feed during production, after production by the supplier or by the person feeding the animals, just prior to providing the food to the animals. The Bacillus subtilis HF1 strains used in the methods and compositions described herein are particularly suitable because they are capable of surviving (as spores) the heat and pressure conditions of the process of producing a dry pelleted feed product.

In other embodiments the animal is a human animal.

Methods for improving gastrointestinal tract health in such animals are provided.

In other embodiments the animal is a farmed aquatic animal such as a fish, a crustaceans (e.g. shrimp, prawn, crab), or a mollusc.

Methods of combating fungal infection in animals are also provided.

Thus in one aspect the invention provides an animal feed comprising a Bacillus subtilis HF1 agent disclosed herein. Where the agent is a strain or spores, the feed may (for example) comprise between about 10 4 and 10 9 cfu/gm finished feed.

The methods of increasing weight herein may be non-therapeutic.

Nevertheless, in other aspects, the invention provides:

(i) a Bacillus subtilis HF1 agent disclosed herein for use in the methods of treatment or therapy described herein;

(ii) use of a Bacillus subtilis HF1 agent disclosed herein in the preparation of a

composition (medicament or veterinary product) for the in the methods of treatment or therapy described herein.

Definitions

"Pathogen" refers to microorganisms (fungi or bacteria) that cause disease or damage to plants or animals. For example the damage may relate to plant growth, yield,

reproduction or viability, and may be cosmetic damage. Preferably the damage is of commercial significance. In a preferred embodiment the term "pathogen" is a filamental fungal organism that causes disease in plants. Preferably the plants are cultivated plants.

The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises", and the terms "including", "include" and "includes" are to be interpreted in the same manner. The term "consisting essentially of" when used in this specification refers to the features stated and allows for the presence of other features that do not materially alter the basic characteristics of the features specified.

The term "effective amount" as used herein means an amount effective to control or eradicate pests, particularly insect pests.

The term "biologically pure culture" or "biologically pure isolate" as used herein refers to a culture of the relevant strain of the invention comprising at least 90%, preferably 95%, preferably 99% and more preferably at least 99.5% cells of the relevant strain.

The term "animal" includes mammals such as humans and farmed or companion or wild animals (e.g. a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orang utang, gibbon)) and also fishes, birds, reptiles, a crustaceans, molluscs. Preferred animals are farmed or aquafarmed animals.

FIGURES

Figure 1 - The monoclonal morphology of HF1 cultivated on LB (Lysogeny Broth, 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCI) agar medium was irregular with lobate margins and folding surface.

Figure 2 - The growth curve of HF1 in different media. Note: LB, the LB liquid medium; 1 % and 2.8% Endo, the glucose concentration of medium was 1 % and 2.8% respectively. Figure 3A - Suppression effects of HF1 to important phytopathogens.

Figure 3B - Suppression effects of HF1 to fungi: A = rice blast strain strain 1-2-5; B= Magnaporthe grisea appressorium strain 3-2; C= rice blast strain 5-2-2.

Figure 4 - Suppression of HF1 broth without live cells to the hyphal growth of

Colletot chum gloeosporiodes. Note: A, HF1 broth was sterilized under 121 °C for 10 minuntes; B, HF1 broth was filtrated through a biofilter.

Figure 5 - The suppression of HF1 broth to the spore germination of Alternaria alternata. Note: A, HF1 broth was sterilized under 121 °C for 10 minuntes; B, HF1 broth was filtrated througth a biofilter. The broth was diluted by (A) 100 and (B) 500 x.

Figure 6 - Biocontrol to the soil-borne diseases and promoting growth effects of HF1 on Pseudostellaria heterophylla.

Figure 7 - Suppression effects of the HF1 LB broth on the powdery mildew. Note: the target crop of A and B was cucumber and pea respectively. HF1 LB Broth contained living cells of HF1 were diluted by 300 x. Concentration of Azoxystrobin and ether phenolic were diluted by 1000 χ according to the operating manual.

Figure 8 - Bio-preservation effects of HF1 to strawberries. Note: A, Control; B, 0.2% Chitosan; C, HF1 with 0.2% Chitosan. Figure 9 - Growth promoting effects of HF1 on broiler chickens.

Figure 10 - The phylogenetic tree of HF1 based on the 16S rDNAs. Note: the 16S rDNA sequnces from different species were downloaded from the NCBI database.

B.atrophaeus JCM 9070, NR_024689.1 ; B.carboniphilus JCM9731 , NR_024690.1 ;

SJ/exus lF015715, NR_024691 .1 ; B.halodurans ATCC 27557, NR_1 12056.1 ; B.lentus NCIMB8773 , NR_040792.1 ; B.marinus, AB021 190.1 ; B.mojavensis IF015718,

NR_024693.1 ; B.mycoides 273 , NR_036880.1 ; B.niacini IF015566, NR_024695.1 ;

B.psychrosaccharolyticus ATCC 23296, NR_040793.1 ; B.vallismortis DSM,

NR_024696.1 ; B.weihenstephanensis DSM, NR_024697.1 ; B.amyloliquefaciens BG1 1 , KF699870.1 . The fellowing 16S rDNA sequences were from the genome sequences. B.anthracis Ames, CP010792.1 ; B.cereus HN002, GQ478254.1 ; B.licheniformis ATCC 14580, CP000002.3; B.megaterium DSM 319, CP009920.1 ; B.pumilus SAFR-032, CP000813.1 ; B.subtilis 168, CP010052.1 ; B.thuringiensis 97-27, AE017355.1 .

Figure 1 1 - The relationship between the biomass (OD) and inhibition zone size

In the shaker, the HF1 was cultured in ENDO medium. The OD was measured at different culture time and at same time the fungus growth inhibition was tested using the sterilized broth by cylinder plate method. The fungus used is Alternaria alternata

EXAMPLES

Example 1 - Isolation and molecular identification of HF1

The bacteria strain HF1 was isolated from the cucumber (Cucumis sativus) rhizosphere soil.

According to the genome annotation of HF1 , the contig15 (1627 bp) was the 16S rDNA sequence. This has the following sequence:

1 tttatcggag agtttgatcc tggctcagga cgaacgctgg cggcgtgcct aatacatgca

61 agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac

121 gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg

181 ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt acagatggac

241 ccgcggcgca ttagctagtt ggtgaggtaa tggctcacca aggcaacgat gcgtagccga

301 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca

361 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg

421 aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt cgaatagggc

481 ggtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta

541 atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc

601 ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact

661 tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga

721 ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt

781 ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg

841 ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt

901 acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg

961 tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct

1021 agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc

1081 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag

1141 cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac

1201 gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa

1261 gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag

1321 tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt

1381 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg

1441 aagtcggtga ggtaaccttt taggagccag ccgccgaagg tgggacagat gattggggtg

1501 aagtcgtaac aaggtagccg tatcggaagg tgcggctgga tcacctcctt tctaaggata

1561 ttatacggaa tataagacct tgggtcttat aaacagaacg ttccctgtct tgtttagttt

1621 tgaagga

Based on the alignment of 16S rDNA sequence by Basic Local Alignment Search Tool (BLAST), the 16S rDNA sequence of HF1 has the highest similar to that of Bacillus subtilis sp., for example Bacillus subtilis subsp. subtilis str. 168 (100%), which is the Bacillus subtilis standard stain. And then the phylogenetic tree was reconstructed through MAGE6.0 with Neighbor-joining method (see Figure 10) and show HF1 belonged to Bacillus subtilis spp.. At last, the strain HF1 was named as Bacillus subtilis HF1 , abbreviated to HF1 in the Examples hereafter.

Example 2 - Characteristics of HF1

HF1 is a gram positive, aerobic, sporeforming bacteria, and its colony morphology cultivated on LB (Lysogeny Broth, 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCI) agar medium was irregular with lobate margins (Figurel ).

HF1 is very sensitive to some antibiotics, such as Chloromycetin, Kanamycin,

Gentamicin, Spectinomycin, Rifampicin, Ampicillin, Tetracycline, Streptomycin, Penicillin and Carbenicillin.

The initial OD 6 oo value of HF1 was 0.267 ± 0.0015 in 500 mL flasks with 200 mL LB liquid medium at 170 rpm and 28 °C. The OD 6 oo of HF1 after inoculation for 24 hours reached 5.76 ± 0.03 (Figure 2).

Example 3. Preferred medium for HF1 large scale production

An preferable medium for HF1 was found, and named "Endo", which contained glucose10-28 g/L, KN0 3 6 g/L, KH 2 P0 4 1.2 g/L, MgS0 4 -7H 2 01 .2 g/L, NasCeHsCv 0.2 g/L, CaCI 2 « 2H 2 0 105 mg/L, FeS0 4 « 7H 2 0 16 mg/L, EDTA 2.1 mg/L, H 3 B0 3 2.86 mg/L, ZnS0 4 « 7H 2 0 0.222 mg/L, MnCI 2 « 4H 2 0 1 .81 mg/L, Na 2 Mo0 4 0.021 mg/L and

CuS0 4 « 5H 2 0 0.07 mg/L. Compared with LB medium, the growth status of HF1 was similar in the two types of Endo media, which contained 10 or 28 g/L glucose, but their production of HF1 were twice as much as that of LB medium under the same condition (Figure 2). The OD 6 oo of HF1 at 24 hours after inoculation was 1 1.22 ± 0.04 or 1 1.46 ± 0.1 1 in Endo containing 10 or 28 g/L glucose.

Example 4. HF1 has a wide antimicrobial spectrum, showing HF1 is a great potential biocontrol agent to suppress plant diseases

Different fungous phytopathogens were separately inoculated on solid PDA medium (Potato Dextrose Agar, 200 g/L potato, 20 g/L dextrose, 10 g/L agar) and cultured at 28 °C for about 4 days. Different agar disks with mycelium of 0.5 cm in diameters made by a punch from the margins of the colony with the same growth ability, were inoculated on the middle of the Petri dish with PDA medium. And then 1 μί HF1 LB culture liquid was inoculated at about 2 cm away from agar disks. They were cultured for about 5 days and suppression effects of HF1 were investigated.

According to culture results, the strain HF1 can suppress the hyphal growth of some important soil-borne filamentous phytopathogen fungi (Figure 3), such as Fusarium spp., Colletot chum spp., Rhizoctonia spp., Phytophthora parasitica., Alternaria spp.

Gaeumannomyces graminis, Verticillium dahliae, Calonectria nivale, Ceratobasidium cornigerum, Botryosphaeria berengeriana, Stemphylium solani, Coniothyrium spp, Phoma crystallifera, Myrmecridium schulzeri, Corynespora cassiicola and Magnaporthe grisea.

Under the conditions used in the assay, the growth of Phytophthora nicotianae and Aspergillus flavus was too fast to show anti-fungicidal effect of the broth, although it is believed that the HF1 broth may show activity against these strains at higher

concentrations

In a separate experiment, under the conditions used, the broth did not suppress the growth of E. coli.

Results revealed that HF1 can be used as a biocontrol or soil remediation agent to suppress the soil borne disease.

Example 5. The broth of HF1 had inhibition activities to the hyphal growth

To evaluate the suppression effect of HF1 broth from the 2-day LB liquid media culture on the fungus growth, Oxford-cup tests were carried out. The trials of confront culture were prepared according to Example 4.

HF1 was cultured for about 2 days in LB at 170 rpm and 28 °C. After centrifugation, the supernatant was collected, and then a part of supernatant was sterilized under 121 °C for 10 minutes and the other was filtered by a biofilter (diameters 0.22 μηη).

The Oxford cups were put on the medium and each cup was added about 100 μί of the above broths. After about 4 days, the results can be recorded (Figure 4). The results showed that the LB culture broth of HF1 sterilized by high temperature and/or biofiltration has the significant effect on the suppression to the hyphal growth of fungus. Example 6. The broth of HF1 can inhibit the fungus spore germination

The fungus, Alternaria alternata, was cultured on PDA solid medium for about 5 days. About 5 ml. sterile water was added into Petri dish (9 cm), and the mycelium were scraped off and washed by water, and then spores were gained through filtration and the density was about 1000 spores/mL.

1 ml_ of HF1 LB broth was prepared as in Example 5 in LB medium and added into 100 mL or 500 mL PDA medium. And then HF1 broth was diluted by 100 and 500 x, respectively. About 0.2 mL spore suspension liquid was spread onto the PDA solid medium plates.

According to the results (Figure 5), diluted 100 χ HF1 broth can completely inhibit the spore germination of Alternaria alternate. However the inhibitive rate (computed as the following Formula 1 ) of the diluted 500 χ HF1 broth sterilized by high temperature was 80.25±3.57% and higher than that of the diluted 500 x HF1 filtrated broth (about

20.45±2.43%).

Formulal Inhibitive rate (%) = 2£rX 100% Note: CK, the number of colonies in controls; T, the number of colonies in treatments.

Example 7. HF1 can be used as a soil remediation agent for suppression of some soil- borne diseases and improving the crop production

To investigate effects of HF1 as a soil remediation agent, the target medical plant,

Pseudostellaria heterophylla was selected. The soil-borne diseases of P. heterophylla, such as root rot caused by Fusarium oxysporum and Rhizoctonia solani and damping off caused by R. solani, can be very serious in the field because of the practice of successive planting.

A field with serious soil-borne diseases was used as the trial field. The field was grouped into 10 trial blocks. One group of 5 blocks was treated with the HF1 broth with living cells, and the others were used as the control. 300 seedlings were planted in every block (about 2.0 m long and 1.5 m wide). The death rate (as the following Formula 2) was recorded at the harvest time.

The seeds were soaked by diluted 100 χ HF1 broth with living cells for about 2 minutes and dried in the air in October. The seeds were then planted in the soil. After the emergence of seedlings, each of planting seedlings was watered with about 20 mL 300 χ HF1 broth with living cells. When the next spring came, seedlings came back to growth, and were watered with about 20 mL 300 χ HF1 broth with living cells for each plant. For the control, seedlings were watered with 20 mL water for each plant.

Formula 2 Death rate (%) = « %

Note: CK, the number of plants in controls; T, the number of plants in treatments.

In the field trial, the death rates of treatments and controls were 28.29±6.39% and 63.45±8.56% respectively. The storage roots of the treatment were bigger than those of the control (Figure 6), and average fresh weights of treatments and controls were 958.05±89.35 kg/ha and 343.45±88.35 kg/ha respectively. The results revealed HF1 can be a potential soil remediation suppressing the soil-borne diseases and improving the crop production. Example 8. HF1 LB broth can suppress powdery mildew

HF1 was cultured in LB liquid medium. The broth with living cells was called "HF1 LB broth".

HF1 LB broth with living cells was diluted by 300x and applied as a foliar spray

Diluted (1000x) azoxystrobin or ether phenolic was used as a positive control

Water was used as a negative control (CK).

100 plants were used for treatment, and sprayed once every 5-7 days for about 3 times. The disease grading as the following: 0, no spots; 1 , the ratio of the infected area and the whole leaf area was less than 25%; 3, the rate of the infected area and the whole leaf area was between 25% and 50%; 5, the rate of the infected area and the whole leaf area was between 50% and 75%;7, the rate of the infected area and the whole leaf area was more than 75% 0 The disease indexes (%) were∑(the disease gradingxnumber of the corresponding leaves)/(The total number of the investigated leavesxthe highest disease grading)]x100%. Control effects (%) were [1 - (disease index of the treatment/disease index of the control)] *100%.

In the greenhouse, the control effects of HF1 LB broth on the cucumber powdery mildew {Sphaerotheca fuliginea (Schlecht) Poll) were about 89.46±3.65% and that of the positive control were about 91 .26±4.1 1 %. For peas, the control effects of HF1 LB with living cells on the powdery mildew (Erysiphe pi ' si ' DC) were about 81.23±2.31 % and that of the positive control were about 84.98±3.71 %. The results revealed that the HF1 LB with living cells can suppress the powdery mildew.

Example 9. HF1 LB broth can be used as a bio-preservative

To investigate the bio-preservation effects of HF1 , the HF1 broth was heated at 100 °C for about 10 minutes and centrifuged at 12,000 rpm. The supernatant was collected and diluted by 100 x. Perishable strawberry was the target fruit. The strawberries were soaked in sterile water (the negative control) and 0.2% chitosan or 100 χ supernatant of HF1 LB broth with 0.2% chitosan for about 20-30 seconds and air dried. The treated fruit was put into fresh-keeping boxes at room temperature. Ten strawberries were used for each treatment and every treatment was replicated for three times.

Five days after treatment, 100%, 82.35±4.32% and 13.45±2.45% strawberries of the negative controls, 0.2% chitosan and HF1 LB broth with 0.2% chitosan respectively were mouldy and rotten (Figure 7).

Thus HF1 LB broth can be used as a bio-preservative.

Example 10. HF1 broth can be used as the feed additive to promote the growth of target animals.

The low doses of antibiotics can promote the animal body weight, but can seriously affect the animal, and ultimately human, health, and also damage the environment.

Safer less harmful feed additives to replace antibiotics in animal feeding are needed.

In China, Bacillus subtilis had been approved as an additive by the ministry of agriculture for use in feeding white feather broiler. Compared with negative controls, the final weight increase is about 3.5% by using regular Bacillus subtilis strain, which is not significantly different to antibiotic feeding.

Based on our experiments described herein, we found small doses of (0.1 %-1 % in drinking water) HF1 could significantly promote the white feather broiler body weight increase.

In a first experiment, HF1 was cultured in liquid LB and Endo medium, respectively. The broth was heated at 100°C for about 10 minutes after 10,000rpm centrifugation.

The LB broth was diluted by water to 1 %, 0.5% and 0.25%, and the Endo broth was also diluted by water to 1 %.

The diluted broth was used as the daily drinking water for the broiler chicken (Gallus gallus domesticus Baiyu) fed on basal feed from 1 day to 42 days. The broiler chickens fed on the basal feed and water without any additive were used as the negative control (CK); and those fed on the basal feed with 15 mg/kg virginiamycin as additive were used as the positive control.

Every treatment contained six groups and every group had 10 chickens. Every 7 days, the chicken weight was recorded at about 8 o'clock before feeding.

The average weight of chickens drinking 1 %, 0.5% and 0.25% LB broth and 1 % Endo broth was 2771.90±19.06 g, 2698.38±13.92 g, 2651.55±6.28 g, 2775.23±39.69 g respectively when trials were finished on day 42.

The average weight of CK and the positive control were 2309.90±26.39 g and

2331 .95±16.60 g.

Compared with CK, the weight increasing ratio of chickens drinking 1 %, 0.5% and 0.25% LB broth and 1 % Endo broth were 20.01 ±1.85%, 16.82±1 .79%, 14.80±1.73%,

20.15±1.80%, respectively.

Compared with positive control, the weight increasing ratio of chickens drinking 1 %, 0.5% and 0.25% LB broth and 1 % Endo broth were 13.73±1.33%, 10.71 ±1.24%, 8.79±1.16%, 13.86±1.26%.

According to the anatomy results, we did not find abnormal symptoms in the livers, hearts, spleens, lungs, kidneys, duodenum and jejunums of the chickens compared with the control or antibiotic treatments. We also did not find anything abnormal in the blood biochemistry.

However, the villi present in the duodenum of the treated animals were thinner and longer, and more splenic corpuscles could be found. Without wishing to be bound by theory, these changes may lead to a health benefit to the chicken health and hence the weight promoting benefits.

In other experiments, at 42 days of age, was HF1 group weight is significant higher than those of the negative control and positive controls: compared with negative control, the weight was increased by 28%; compared with a commercial Bacillus subtilis probiotic supplement and different antibiotics (virginiamycin, amoxicillin), the weight was increased by 16% and 25%. Additionally the beneficial activity was demonstrated when chickens were fed with HF1 culture dried on a solid, either starch or a rice husk / starch mixture (results not shown).

Example 1 1. The stain HF1 was safe to animals tested by the acute toxicity trial.

CD-1 mice with 20±2 g were selected as the target animals to investigate the acute toxicity of HF1. The mice were divided into 5 groups. Each group had six mice with 3 males and 3 females. The mice were not fed one day before the trial. Each group was separately given with 0.4 ml 0.8% NaCI, 10 9 cfu/mL, 10 11 cfu/mL, 10 12 cfu/mL and HF1 LB broth without dilution, once by gavage. And then mice were reared normally for about 15 days. All the mice were killed 15 days later. The clinic performances of the mice were observed.

All tested mice were alive at the end of experiments. The clinic performances of the tested mice were not different from those of the control (the group 0.8% NaCI). The clinic performances include daily feeding, drinking, coat color, faeces, weight, death rate, and general anatomical results. According to the anatomical results, there were no visually abnormal tissues in any of the mice. Acute toxicity tests showed that Bacillus strain HF1 should be safe to mammals.

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