WO1999059412A1 - Biocontrol agent and pesticide - Google Patents
Biocontrol agent and pesticide Download PDFInfo
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- WO1999059412A1 WO1999059412A1 PCT/CA1999/000426 CA9900426W WO9959412A1 WO 1999059412 A1 WO1999059412 A1 WO 1999059412A1 CA 9900426 W CA9900426 W CA 9900426W WO 9959412 A1 WO9959412 A1 WO 9959412A1
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- strain
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- antibiotic
- pkbl
- maculans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/25—Paenibacillus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/832—Bacillus
- Y10S435/838—Bacillus polymyxa
Definitions
- the present invention is directed toward a biocontrol agent and pesticide and, in particular, biocontrol agent and pesticide for inhibition of disease-causing fungi.
- Blackleg is a fungal disease of canola that is responsible for losses in crop yield and seed quality. The disease has spread throughout most of the Canadian prairies despite the use of fungicides and blackleg resistant canola cultivars. No successful inhibitor to the spread of blackleg exists, therefore another method of disease control is sought.
- Canola is an economically important crop in Canada and considerable losses in seed quality and yield are seen every year due to fungal diseases such as blackleg.
- the canola cultivars presently grown show varying degrees of susceptibility to blackleg, and. to date, there are also no varieties resistant to Sclerotinia white stem rot.
- Canola is an important agricultural product in Canada, with a cash value of over $300 million per year in Alberta alone.
- 1994 agronomists reported that the global consumption of vegetable oils is increasing by about 4% every year.
- To meet this global demand for canola oil Canada would need to grow 15% more than the 1994 acreage, and since that time there has been no abatement in the demand for canola oil.
- One of the major blocks to increasing the production of canola is the loss of crops to fungal diseases such as blackleg, Sclerotinia, Alternaria and Rhizoctonia.
- Much work has been done on developing blackleg disease tolerant canola cultivars. These new varieties help to improve the crop yield, however the canola cultivars are still not resistant to the fungal diseases.
- Fungicidal seed treatments are used for chemical control of disease-causing fungus.
- single applications at the time of seeding do not provide sustained protection for the plants, and considerable losses can still occur before the crop reaches a stage of growth where some natural resistance has developed.
- Multiple fungicide applications are undesirable from both an economic and environmental perspective, and fungicides are not effective against the fungal spores that can persist in infected canola stubble from year to year.
- Constant use of fungicides can select for fungicide-resistant fungi, so improved methods of controlling fungal diseases are needed to protect plants in the vulnerable seedling stage and throughout the growing season.
- Biological control of blackleg and other fungal diseases may offer an environmentally sound method for plant disease control.
- the soil bacterium has been isolated from canola roots in a canola production plot near Sedgewick, Alberta Canada.
- the soil bacterium is a new strain of Paenibacillus polymyxa (formerly defined as Bacillus polymyxa) and has been called PKB 1.
- a sample of the bacterium was deposited on May 18, 1998 with the American Type Culture Collection (ATCC). bearing ATCC Accession Number 202127.
- the soil bacterium produces an antibiotic after sporulation of the vegetative cells.
- the antibiotic is primarily spore associated.
- the antibiotic exhibits pesticidal activity against some bacteria and fungi. In particular, the P.
- PKB1 and the peptide antibiotic from PKB1 offer antifungal activity against Leptosphaeria maculans, the fungus that causes blackleg disease in canola, as well as other economically important disease-causing fungi including Sclerotinia sclerotiorum, Marasmius oreades, Pythium pythioides, Rhizoctonia solani,
- PKB1 of P. polymyxa can be used as a biocontrol agent against blackleg and other fungal diseases of canola.
- the present invention relates to a novel strain of bacteria, referred to herein as PKBl, which has an inhibitory affect on fungi such as L. maculans and S. sclerotiorum.
- the present invention also relates to the antibiotic isolated from the bacterial strain PKBl, referred to herein as the PKBl antibiotic, and the peptides of the antibiotic, referred to herein as the PKBl peptides, which provide the inhibitory affect against fungi.
- the bacteria, antibiotic and peptides of the present invention can be used as pesticides and biocontrol agents against disease-causing fungi, for example, in crop plants.
- One aspect of the invention pertains to an isolated Paenibacillus polymyxa strain PKB 1 that acts as an inhibitory agent against Leptosphaeria maculans and other disease- causing fungi such as for example, Sclerotinia sclerotiorum. Marasmius oreades. Pythium pythioides, Rhizoctonia solani, Fusarium avenaceum and Alternaria brassicae.
- An "isolated” or “purified” bacterial strain is substantially free of materials from its natural environment including soil and biological matter including other bacterium or plant matter.
- the language “substantially free of materials from its natural environment” includes preparations or cultures of the bacterium in which the bacterium is separated from components of the environment in which it is naturally found. In one embodiment, the language “substantially free of materials from its natural environment” includes cultures having less than about 20% (by count) of non- PKB 1 bacteria (also referred to herein as contaminating bacteria, contaminating bacteria does not include bioactive mutants or modified forms of strain PKB 1 ), more preferably less than
- non-PKBl bacteria 10% (by count) of non-PKBl bacteria and most preferably less than about 5% non-PKBl bacteria.
- the invention also pertains to bioactive mutants or modified forms of strain PKB 1 which retain their inhibitory affect against L. maculans.
- bioactive mutants or modified forms of strain PKB 1 is intended to include bacterium which have naturally mutated or by manipulations such as, for example, chemical or UV mutation or genetic modification or transformation been modified to have other characteristics such as, for example, antibiotic resistance.
- inhibition is a reduction in the growth or development of the fungi, for example, against control systems.
- Standard assays such as those described herein, can be used to determine the ability of the strain or bioactive mutants or modified forms thereof to act against the fungi of interest. The standard assays can be conducted in vitro or in the field.
- the strain or bioactive mutants or modified forms thereof can be in vegetative or spore state. They can be in culture, cell suspension, dried, dead or viable or in any other form such that they are capable of inhibiting L. maculans and preferably other disease-causing fungi.
- Another aspect of the invention pertains to methods for detecting the presence of the bacterial strain of the present invention in a biological sample.
- the methods involve contacting a biological sample (e.g. a soil sample) with a compound or an agent capable of detecting PKBl cells or nucleic acids such that the presence of PKBl is detected in the biological sample.
- a biological sample e.g. a soil sample
- the compound or agent can be, for example, a labeled or labelable nucleic acid probe capable of hybridizing to PKBl bacterium nucleic acids.
- the isolated antibiotic contains at least one of the spore-associated peptides of Paenibacillus polymyxa strain PKBl or contains at least one peptide which is sufficiently homologous to the amino acid sequence of one of the spore-associated peptides of Paenibacillus polymyxa strain PKB 1 and maintains the ability to inhibit the development of L. maculans.
- the antibiotic of the present invention comprises at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain and that have inhibitory affect against L. maculans, or at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 or, preferably, at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 or between 896 to 897.
- the invention also provides an isolated preparation of the PKBl antibiotic.
- the antibiotic preparation comprises at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 or, preferably, at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 or between 896 to 897 and has a inhibitory affect against L. maculans.
- the antibiotic includes two primary peptides.
- the peptides are each comprised of eight amino acids in a branched cyclic sequence and are nearly identical in form.
- One peptide has a molecular weight (MW) of about 897 to 898 and the second form has a molecular weight of about 883 to 884.
- Another aspect of the invention pertains to an isolated peptide of the present invention or a fragment, or portion, e.g. a bioactive fragment or portion, thereof.
- the isolated peptide or bioactive fragment thereof can inhibit the development of L. maculans.
- the isolated peptide or bioactive fragment thereof is sufficiently homologous to the amino acid sequence of one of the spore-associated peptides of Paenibacillus polymyxa strain PKBl and maintains the ability to inhibit the development of L. maculans.
- the peptide or bioactive fragment of the present invention comprise the amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or the amino acid sequence of a spore-associated peptides of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 and preferably those spore- associated peptides of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 and/or between 896 to 897 and have inhibitory affect against L. maculans.
- the peptide or bioactive fragment of the present invention comprises one valine, one alanine, one threonine.
- the peptide or bioactive fragment of the present invention comprises the amino acid sequence according to Figure 6B.
- the invention also provides an isolated preparation of the peptide according to the present invention.
- the peptide of the preparation comprise the amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or the amino acid sequence of a spore-associated peptides of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 and preferably those spore-associated peptides of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 and/or between 896 to 897 and have inhibitory affect against L. maculans.
- the peptide or bioactive fragment of the present invention comprises one valine, one alanine, one threonine, two aspartic acids, one proline, one acetyl-modified leucine and one glutamic acid and being capable of inhibiting L. maculans.
- the peptide or bioactive fragment of the present invention comprises the amino acid sequence according to Figure 6B.
- the peptide is at least about 50-60%, preferably at least about 65-70%, more preferably at least about 75-80%, and even more preferably at least 85. 90, 95% or more homologous to the entire amino acid sequence of Figure 6B.
- the isolated peptide comprises an amino acid sequence which is at least about 60-70% or more homologous to the amino acid sequence of Figure 6B and has an one or more of the following activities: 1) it can inhibit development of L. maculans and 2) it can inhibit development of S. sclerotiorum.
- the peptide or a bioactive fragment thereof, as defined above, can be operatively linked to another peptide to form a fusion protein.
- the PKBl peptide or a bioactive fragment thereof can be incorporated into a pesticide composition comprising the peptide and an acceptable carrier or enhancer.
- the P. polymyxa antibiotic producing strain PKBl according to the present invention offers promise for use as a biocontrol agent because it is active against many fungal diseases. Biocontrol agents are relatively inexpensive to mass produce because they are grown in large volume fermenters and require little or no downstream processing other than, in some embodiments, drying.
- the bacterium of the present invention is a preferred organism for use as a biocontrol agent because it forms dormant spores which are extremely resistant to heat, drying, chemical treatment, radiation and ultraviolet light exposure. This makes it possible to store dried cells of Paenibacillus polymyxa, PKBl for long periods with little loss of viability. The same-spore forming ability occurs in the soil, making it possible for the introduced P. polymyxa strain to survive long periods of dryness or harsh conditions.
- Figure 1 A is a Euclidian chart from the fatty acid methyl ester analysis (FAME analysis) of
- Figure IB is a phylogenetic tree diagram showing the relatedness of P. polymyxa PKBl to other gram positive bacteria with known 16S rRNA sequences. Relatedness was determined using RDP and the tree diagram was made using a software program called TreeviewTM.
- Figure 2 is graph showing the growth curve and the antibiotic production of PKBl at 10°C.
- Figure 3 is a reverse phase high performance liquid chromatography trace of the antifungal antibiotic. The active fraction is found at peaks 17.0 and 17.7. A linear mobile phase gradient was used of (A) acetonitrile, 60% B to 100% B. The flow rate was 1.0 ml/min, detection at 214nm, fractions collected every 60 seconds.
- Figure 4 is a photo of the 16.5% acrylamide SDS-PAGE. Peptide was stained with coomassie blue. Bands seen correspond to the molecular weight standard of 2.3kDa.
- Figure 5 is a bioautograph of the 16.5%> acrylamide SDS-PAGE using L. maculans as the indicator organism. Zones of inhibition that correspond to the bands in the SDS-PAGE of Figure 4 can be seen.
- Figure 6A is a chart of the fast atom bombardment mass spectrum of pure antifungal antibiotic. Two components are seen to determine an active antibiotic, one at 883.5 molecular weight and the other at 897.5 molecular weight. The sample was ionized using a glycerine/thioglycerol matrix.
- Figure 6B is a proposed structural formula for antibiotic peptide 883.5 MW.
- Figure 7 shows scanning electron micrographs of germination of pycnidiospores of Leptosphaeria maculans (a) without bacterial treatment and (b) treated with Paenibacillus polymyxa PKB 1 on a cellophane membrane after two days incubation at room temperature; (c) without bacterial treatment and (d) treated with Paenibacillus polymyxa PKBl on cellophane membrane after three days incubation at room temperature; (e) without bacterial treatment and (f) treated with bacterium on the canola leaf surface after three days incubation in a greenhouse.
- Figure 8 shows DNA patterns on 23 bacterial strains of Paenibacillus polymyxa and Bacillus spp. amplified with primers OPA07 (A), OPA08 (B), OPA13 (C), and OPA14 (D).
- Figure 9 shows autoradiographs of Southern blot of 22 bacterial strains of Paenibacillus polymyxa and Bacillus spp. DNA digested with Hindlll (A,B), Pstl(C) and EcoRl (D,E, and F), and hybridized with Pl-7 (A,C, and D), Pl-8 (E), Pl-14 (B,F).
- Figure 10 shows dot-blotting of 23 bacterial strains of Paenibacillus polymyxa and Bacillus spp. with probes 1-7 (A) and 1-8(B), and 52 unknown bacteria strains from compost and canola stubble probed with PI -7 (C) and PI -8(D).
- Figure 11 shows the DNA sequence of probe Pl-8 (SEQ ID NO:6).
- Figure 12 shows the PCR products of 23 bacterial strains of Paenibacillus polymyxa and Bacillus spp. amplified with primers (A) Jl and JY1, and (B) Jl and JY2.
- Figure 13 shows photos of (A) a plate showing the inhibitory effect of strain 97-003 inoculated compost and (B) a control plate.
- the present invention relates to a novel strain of bacteria, referred to herein as PKB 1 , which has an inhibitory affect on fungi such as L. maculans and S. sclerotiorum.
- PKB 1 novel strain of bacteria
- the present invention also relates to the antibiotic isolated from the bacterial strain PKB 1 , referred to herein as the PKBl antibiotic, and the peptides of the antibiotic, referred to herein as the PKBl peptides, which provide the inhibitory affect against fungi.
- the bacteria, antibiotic and peptides of the present invention can be used as pesticides and biocontrol agents against disease-causing fungi, for example, in crop plants.
- the bacterial strain PKBl which is a. Paenibacillus polymyxa, has been deposited with the American Type Culture Collection (ATCC), Manassas, Virginia, on May 18, 1998 and assigned Accession Number 202127.
- the antibiotic extracted from the bacteria is a peptide antibiotic including primarily two cyclic peptides of eight amino acids each. The sequence of one of the peptides is shown in Figure 6B.
- the peptides or bioactive fragments thereof according to the invention have inhibitory affect against disease-causing fungi.
- One aspect of the invention pertains to an isolated Paenibacillus polymyxa strain PKBl that acts as an inhibitory agent against Leptosphaeria maculans and other disease- causing fungi such as for example.
- An "isolated” or “purified” bacterial strain is substantially free of materials from its natural environment including soil and biological matter including other bacterium or plant matter.
- the language “substantially free of materials from its natural environment” includes preparations or cultures of the bacterium in which the bacterium is separated from components of the environment in which it is naturally found.
- the language “substantially free of materials from its natural environment” includes cultures having less than about 20%> (by count) of non- PKBl bacteria (also referred to herein as contaminating bacteria, contaminating bacteria does not include bioactive mutants or modified forms of strain PKBl), more preferably less than 10%) (by count) of non-PKBl bacteria and most preferably less than about 5% non-PKBl bacteria.
- the invention also pertains to bioactive mutants or modified forms of strain PKB 1 which retain their inhibitory affect against L. maculans.
- bioactive mutants or modified forms of strain PKB 1 is intended to include bacterium which have naturally mutated or by manipulations such as, for example, chemical or UV mutation or genetic modification or transformation been modified to have other characteristics such as, for example, antibiotic resistance.
- inhibition is a reduction in the growth or development of the fungi, for example, against control systems.
- Standard assays such as those described herein, can be used to determine the ability of the strain or bioactive mutants or modified forms thereof to act against the fungi of interest.
- the standard assays can be conducted in vitro or in the field.
- the strain or bioactive mutants or modified forms thereof can be in vegetative or spore state. They can be in culture, dried, dead or viable or in any other form such that they are capable of inhibiting L. maculans and preferably other disease-causing fungi.
- Another aspect of the invention pertains to isolated PKBl antibiotic and peptides or bioactive fragments or portions thereof.
- An “isolated” or “purified” antibiotic or peptide or bioactive fragments thereof is substantially free of cellular material when produced by extraction from a bacterial system, or chemical precursors or other chemicals when chemically synthesized.
- the language “substantially free of cellular material” includes preparations of PKB 1 peptide in which the antibiotic or peptide is separated from cellular components of the bacteria, or in particular, the bacterial spores on which it is produced.
- the language "substantially free of cellular material” includes preparations having less than about 30%> (by dry weight) of non-PKBl peptides or protein (also referred to herein as contaminating protein), more preferably less than 20%> (by dry weight) of non-PKBl peptides or protein, still more preferably less than about 10%> (by dry weight) of non-PKBl peptides or protein and most preferably less than about 5%(by dry weight) of non-PKBl peptides or protein.
- the language “substantially free of chemical precursors or other chemicals” includes preparations of PKBl peptides in which the peptides are separated from chemical precursors or other chemicals which are involved in the synthesis of the peptides.
- the language "substantially free of chemical precursors or other chemicals” includes preparations having less than about 30%> (by dry weight) of chemical precursors or non-PKB 1 chemicals, more preferably less than 20% (by dry weight) of chemical precursors or non-PKB 1 chemicals, still more preferably less than about 10%> (by dry weight) of chemical precursors or non-PKBl chemicals and most preferably less than about 5%(by dry weight) of chemical precursors or non-PKBl chemicals.
- PKB 1 antibiotic or peptides or bioactive fragments thereof are free of contaminating proteins from the same bacteria from which the antibiotic or peptides are derived. Typically, such antibiotic and peptides are produced by extraction from the bacteria which produces them. Isolated PKB 1 antibiotic and peptides or bioactive fragments or portions thereof according tot he present invention act against L. maculans.
- the antibiotic of the present invention comprises at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain and that have inhibitory affect against L.
- the invention also provides an isolated preparation of the PKBl antibiotic.
- the antibiotic preparation comprises at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 or, preferably, at least one peptide having an amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 or between 896 to 897 and has a inhibitory affect against L. maculans.
- the antibiotic includes two primary peptides.
- the peptides are each comprised of eight amino acids in a branched cyclic sequence and are nearly identical in form.
- One peptide has a molecular weight (MW) of about 897 to 898 and the second form has a molecular weight of about 883 to 884.
- Another aspect of the invention pertains to an isolated peptide of the present invention or a fragment, or portion, e.g. a bioactive fragment or portion, thereof.
- the isolated peptide or bioactive fragment thereof can inhibit the development of L. maculans.
- the isolated peptide or bioactive fragment thereof is sufficiently homologous to the amino acid sequence of one of the spore-associated peptides of Paenibacillus polymyxa strain PKBl and maintains the ability to inhibit the development of L. maculans.
- the peptide or bioactive fragment of the present invention comprise the amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or the amino acid sequence of a spore-associated peptides of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 and preferably those spore- associated peptides of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 and/or between 896 to 897 and have inhibitory affect against L. maculans.
- the peptide or bioactive fragment of the present invention comprises one valine, one alanine, one threonine, two aspartic acids, one proline, one acetyl-modified leucine and one glutamic acid and being capable of inhibiting L. maculans.
- the peptide or bioactive fragment of the present invention comprises the amino acid sequence according to Figure 6B.
- the invention also provides an isolated preparation of the peptide according to the present invention.
- the peptide of the preparation comprise the amino acid sequence of a spore-associated peptide of Paenibacillus polymyxa strain, or the amino acid sequence of a spore-associated peptides of Paenibacillus polymyxa strain deposited with ATCC as Accession Number 202127 and preferably those spore-associated peptides of Paenibacillus polymyxa strain PKBl having molecular weights of between 883 to 884 and/or between 896 to 897 and have inhibitory affect against L. maculans.
- the peptide or bioactive fragment of the present invention comprises one valine, one alanine, one threonine, two aspartic acids, one proline, one acetyl-modified leucine and one glutamic acid and being capable of inhibiting L. maculans.
- the peptide or bioactive fragment of the present invention comprises the amino acid sequence according to Figure 6B.
- the peptide is at least about 65-70%), more preferably at least about 75-80%>, and even more preferably at least 85, 90, 95%) or more homologous to the entire amino acid sequence of Figure 6B.
- the isolated peptide comprises an amino acid sequence which is at least about 60-70%) or more homologous to the amino acid sequence of Figure 6B and has an one or more of the following activities: 1) it can inhibit development of L. maculans and 2) it can inhibit development of S. sclerotiorum.
- the language "sufficiently homologous" refers to the peptides or fragments thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g. an amino acid residue which has a similar side chain as an amino acid residue in the peptides of the present invention) amino acid residues to an amino acid sequence of a peptide of the PKB 1 antibiotic while retaining its inhibitory affect against L. maculans.
- the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of one sequence for optimal alignment with he other sequence).
- the amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position.
- Bioactive fragments or portions of the PKBl peptides include amino acid sequences derived from the amino acid sequence of the (insert) and exhibit the inhibitory affect against
- PKB 1 antibiotic and peptides are preferably produced by extraction from bacterial strain PKBl.
- the extraction procedure includes treating the spores of the bacterium with a solvent of medium polarity of, for example, between 5.5 to 7.5 using the nine point scale of N. Godfrey (Solvent selection via miscibility number, Chemtech pp 359-363, 1972).
- the extraction is preferably carried out with methanol or acetic acid.
- the peptides of the present invention can be produced recombinantly or by chemical synthesis.
- the invention also provides PKBl chimeric or fusion proteins.
- chimeric or “fusion” proteins comprise a PKBl peptide operatively linked to a non-PKBl peptide or protein.
- a PKB 1 peptide is a peptide or bioactive fragment or portion thereof as defined hereinbefore
- a non-PKB 1 peptide or protein refers to a peptide or protein having an amino acid sequence corresponding to a protein which is not substantially homologous to any one of the PKBl peptides, e.g. a protein that is different from the PKBl peptides and which is derived from the same or a different organism.
- the term "operatively linked" is intended to indicate that the PKBl peptide and the non-PKB 1 peptide or protein are fused in-frame to each other.
- the non-PKB 1 peptide or protein can be fused to the PKB 1 peptide in any suitable way provided that it does not eliminate the antibiotic function of the peptide.
- Such fusion or chimeric proteins can be selected to enhance, for example, delivery, handling, purification or effect of the PKBl peptide from which it is formed. Fusion or chimeric proteins can be produced by any desired means.
- the bacterial strain, antibiotic and peptides of the present invention can be used as a pesticide against some bacteria and fungi.
- the bacterial strain or bioactive mutants or modified forms thereof of the present invention can be used as a pesticide and, in particular, a biocontrol agent against some bacteria and fungi including Leptosphaeria maculans, Sclerotinia sclerotiorum, Pythium pythioides, Marasmius oreades, Rhizoctonia solani, Fusarium avenaceum and Alternaria brassicae.
- the bacteria is preferably applied to a crop to act against the fungal diseases therein.
- the bacteria can be applied in any suitable way including in a live or dead, vegetative state, in a spore state.
- the bacteria can be dried or hydrated.
- the bacteria is applied in a viable form which permits it to sustain itself in the soil to provide a biocontrol effect over a long period of time. Bioassay methods can be used to determine the presence of the bacteria in the environment.
- PKBl antibiotic or PKBl peptides or bioactive fragments or portions thereof can be used as a pesticide against some bacteria and fungi including Leptosphaeria maculans, Sclerotinia sclerotiorum, Marasmius oreades,
- PKB 1 antibiotic or PKB 1 peptides or bioactive fragments or portions thereof can be applied to a crop at any desired stage of crop growth to act against the fungal diseases therein. Fusion proteins of the PKBl peptides can also be used as pesticides.
- the bacterial strain, antibiotic and peptides of the present invention can be applied as a pesticide in any desired way.
- the bacterial strain, antibiotic and peptides are applied in a carrier to facilitate application and to reduce crop maintenance time.
- the PKBl bacterial strain is cultured in compost and applied to a crop with the compost.
- biochemical tests were used to analyze the bacterium. These tests characterized the bacterium as Bacillus polymyxa. Since the time of the early work, the genus Bacillus has undergone a redefinition based on 16S rRNA sequence homology. It was desirable to characterize this isolate on the basis of molecular tests as well. Using 16S rRNA homology the isolate was determined to be Paenibacillus polymyxa.
- Biochemical tests designed to determine the identity of unknown bacteria was used to analyze the bacterium. These tests can specifically differentiate between different species of the genus Paenibacillus. Two different Bacillus spp. were obtained from the American Type Culture Collection (ATCC) and used as standards in these tests; B. macerans ATCC accession no. 8244 and B. circulans ATCC accession no. 4513. B. polymyxa NCIB 4868 was used as the B. polymyxa type culture. Preliminary taxonomic studies on strain PKB 1 indicated that the organism was most closely related to Bacillus polymyxa. Biochemical tests confirmed the isolate as B. polymyxa after comparison of the test results with the test results of three Bacillus type cultures. B.
- polymyxa strain PKBl differs from the B. polymyxa type culture in several ways.
- the environmental isolate grows more vigorously on most culture media and produces more extracellular polysaccharides.
- the type culture B. polymyxa is not able to inhibit the growth of L. maculans.
- Fatty acid methyl ester analysis was performed on the bacterial isolate according to the method of Stockman et al (Stockman, L., Roberts, G.D. and Smith, D.H., "Identification of mycobacteria by cell wall composition with the HP microbial identification system" Abstracts of the 87th meeting of American Society of Microbiology, 1987) . Comparison of fatty acid profiles of bacterial cell walls is a common method to determine the relatedness of bacterial strains.
- the data from the FAME is entered into a cluster program such as the dendrogram program that produces unweighted pair matchings and form a diagram such as a tree that displays the relatedness of the organisms tested, measured in
- Euclidean distances A distance of 25 or less means the organisms are related at the genus level and 10 or less means they are related at the species level.
- the isolate was within 3 Euclidean distances of the type culture. Therefore, the PKB 1 is the same genus and species as the type culture B. polymyxa.
- the forward primer, 8F encompassed the forward amplification primer sequence 16F27 from Hauben et al. (Hauben L., L. Vauterin. J. Swings and E.R.B. Moore. 1997. Comparison of 16S ribosomal DNA sequences of all Xanthomonas species. International Journal of Systematic Bacteriology. 47, pp 328-335) and a sequence that contained sites for the restriction enzymes Sacl, EcoRl and Xba.
- the reverse primer sequence (1403R) encompassed the reverse amplification primer sequence 1387R from Marchesi et al. (Marchesi J.R., Sato T., Weightman A.J., Martin T.A., Fry J.C., Hiom S.J., and Wade W.G. 1998. Design and evaluation of useful bacterium specific PCR primers that amplify genes coding for bacterial 16S rRNA. Applied and
- the restriction enzyme sites were added to the PCR primers so that if it were necessary to clone the PCR fragment into a plasmid for further genetic analyses, there would be an easy means to prepare it.
- the primer sequences were compared with those for the E. coli 16S rRNA sequence (Carbon P., Ehresmann C, Ehresmann B. and Ebel J-P. 1979. The complete nucleotide sequence of the ribosomal 16S RNA from Escherichia coli. European Journal of
- the primers described above extend from nucleotides 8 to 27 in a forward direction and from nucleotides 1403 to 1386 as a reverse complement to the reported sequence, which is a total of 1541 nucleotides in length.
- the amplification primers described above were used as sequencing primers to obtain the end portions of the P. polymyxa PKBl 16S rRNA sequence.
- Other sequencing primers were designed based on a series of 10 primers described by Hauben et al. (1997). Modifications to the suggested sequences were made based on the sequence for P. polymyxa which was obtained using the amplification primers. A summary of these primers is shown in Table 0A.
- primer 8F is listed as SEQ ID NO: 1
- primer 704F is listed as SEQ ID NO:2
- primer 1176F is listed as SEQ ID NO:3
- primer 358R is listed as SEQ ID NO:4
- primer 1106R is listed as SEQ ID NO:5
- primer 1403R is listed as SEQ ID NO:7.
- the temperature program was designed based on those of Hauben et al. (1997) and Marchesi et al. (1998), with modifications developed experimentally to suit P. polymyxa gene amplification, and is shown in Table OB.
- PCR generation of the 8F-1403R PCR fragment for sequencing was carried out in a set of 16 identical reaction tubes. Reactions contained: 1.2 mL 25 ng/mL genomic DNA, 0.67 mL each of 30 pmol/mL primer 8F and 30 pmol/mL primer 1403R, 0.4 mL 25mM dNTP mix (25 mM each of dATP, dCTP, dGTP and dTTP, Boeringher Mannheim), 5 ml 1 OX PCR buffer, 0.75 mL 0.1 M MgC12, 28.25 mL sterile deionized, distilled water and 1 mL 5 units/mL taq DNA polymerase (synthesized by A.
- PCR buffer contained 0.5 g KC1, 8.48 g tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 8.8), 1 mL 1% Triton X- 100 per 100 mL. in deionized, distilled water. After preparation, a small amount of sterile mineral oil was put in each tube The taq DNA polymerase was not added to the reaction mix until the PCR program had run for approximately 2 min; at this time the PCR tubes were opened, enzyme was added beneath the mineral oil layer, and the program was allowed to continue. This 'hot start' procedure was used in order to decrease possible nonspecific product formation.
- a DNA fragment was detected at approximately 1.4 kilobases (kb); this was excised with a razor and removed from the agarose using the GenecleanTM kit (BIO 101, Vista, CA). For this procedure, agarose gel bands approximately 1.5 x 0.5 x 0.5 cm were placed in sterile 1.5-mL Eppendorf tubes and dissolved in 900 mL Geneclean Nal solution at 55°C for 10 min. DNA was extracted onto glass beads by adding 10 mL Geneclean glass milk solution, mixing by inversion, incubating at room temperature for 10 min, centrifuging at maximum speed for
- the glass bead and DNA pellet was washed three times by resuspending in 300 mL Geneclean New Wash buffer, centrifuging 5 s and discarding the supernatant. After the final wash step, the pellet was air dried for 10 min and resuspended in 20 mL sterile deionized, distilled water by adding a lOmL portions of water, resuspending the glass beads, warming to 55°C for 10 min, then centrifuging 30 s and collecting the water two times.
- the DNA concentration was measured by fluorometry; if the sample was not concentrated enough for sequencing (200 mg/mL) then the remaining DNA preparation was precipitated by adding 60 mL 95%> ethanol, freezing at -20°C for at least 30 min, centrifuging 10 min, discarding the supernatant and air drying the DNA pellet, which was resuspended in the appropriate amount of sterile deionized, distilled water to provide a sample for sequencing. Samples were checked by electrophoresis on a 0.8 agarose gel as well as measuring their DNA concentration by fluorometry.
- Sequencing of the 16S rRNA gene fragment The 1400 bp DNA fragment generated by PCR of P. polymyxa genomic DNA using primers 8F and 1403R was sequenced. Samples were submitted for sequencing in the form of PCR product (template) as 5 mL of a 200 mg/mL solution in sterile deionized, distilled water and primers (each separately at a concentration of 3 pmol/mL in sterile deionized, distilled water).
- BLAST search protocol The P. polymyxa PKBl 16S rRNA sequence was submitted to the basic local alignment search tool (BLAST) sequence alignment system. This search tool was developed by the National Center for Biotechnology Information (USA). The BLAST search tool provides a sequence database for DNA, RNA and proteins (Peruski, L.F. Jr. and Peruski, A.H. 1997. The Internet and the New Biology: Tools for Genomic & Molecular Research, American Society for Microbiology, Washington, D.C. pp. 52-59). The web site for this is http://www.ncbi.nlm.nih.gov, and the reference for its use is Altschul et al. (Altshul. S.F.,
- Ribosomal database project search protocol The P. polymyxa PKBl 16S rRNA sequence was submitted to the Ribosomal Database Project (RDP) for sequence alignment and for the generation of a specific phylogenetic tree (Maidak B.L., G.J. Olsen. N. Larson, R. Overbeek, M.J. McCaughey and C.R. Woese. 1997.
- the RDP ribosomal database project). Nucleic Acids Research. 25, 109-110.).
- the web site for the RDP was formerly http://rdpwww.life.uiuc.edu ; the new site is http:www.cme.msu.edu/RDP. Both were used for this project.
- a phylogenetic tree showing the results of the study is shown in Figure IB.
- the P. polymyxa strain of the present invention was originally isolated and observed to inhibit L. maculans when grown on potato dextrose agar plates (PDA). This medium has a pH of 5. Most bacteria grow better in less acidic conditions, so a search for a medium that allowed optimum growth and antibiotic production was undertaken. Many different kinds of both complex and defined media were used as growth substrates for the antifungal antibiotic producing P. polymyxa. Potato dextrose broth with added nutrients was also tested. Temperature and aeration were varied in order to determine the optimal growth conditions. Inoculum size and pH of the medium were also tested for their effect on antibiotic production.
- Scale-up cultures for use in 15 litre fermenters were tested to determine whether the organism could produce antibiotic in large quantities.
- Growth of the bacterium in broth culture was measured by determining the optical density (OD) of the culture at 600nm with a spectrophotometer at regular time intervals during the growth cycle.
- the life cycle of the bacterium was recorded at the same time by making wet mount slides from the broth culture and viewing at 400X magnification under a microscope.
- Antibiotic production by the culture was measured using a plate bioassay with L. maculans as the indicator organism.
- the antifungal antibiotic was extracted from culture pellets with 100% methanol and placed into wells scored into the agar of the bioassay plate. After 48 h, a zone of inhibition in the lawn of fungus was seen indicating that the sample contained antifungal antibiotic.
- Culture conditions were varied to determine an optimal medium and optimal growth conditions for the production of the antifungal antibiotic.
- the optimization of conditions was based on the assumption that the amount of and the stage of growth and antibiotic production were directly related. Rich media that can support much bacterial growth like nutrient broth, beef extract broth, and Landry's medium were tested and the resulting growth and antibiotic production was compared to potato dextrose broth (PDB).
- PDB potato dextrose broth
- Bacterial growth increased on rich media but antibiotic production decreased; the amount of antibiotic produced could not be increased by simply increasing bacterial growth.
- PDB potato dextrose broth
- the type and quantity of carbon source needed for optimal antibiotic production was tested by adding these compounds to PDB; glucose, sucrose, dextrose, starch, soy meal and different amino acids.
- the type and quantity of trace vitamins and minerals needed for optimal antibiotic production were also tested by adding these to PDB; biotin, ascorbic acid, sodium nitrate, potassium phosphate, sodium sulphate and magnesium sulphate.
- Some of these nutrient combinations increased the antibiotic production of the P. polymyxa strain PKB 1 , but not in a reproducible manner.
- the amount of antibiotic production of P. polymyxa in PDB and in potato dextrose agar (PDA) was consistently high so these media was selected for growth of the bacterium.
- the rate of aeration, pH of the medium and growth temperature were also tested to determine optimal growth conditions.
- antibiotic production was optimized at 200 ml PDB in a
- Bacillus spp. are well known for their production of peptide antibiotics.
- P. polymyxa in particular makes a family of closely related small peptide antibiotics called polymyxins.
- the purification protocol developed took into consideration that this antibiotic might be a small protein, between 2 to 20 amino acids, like the polymyxins. Commercially obtained polymyxins were ineffective against L. maculans. The PKB 1 bacterium does not appear to produce polymyxins since it could not inhibit any of the bacteria that polymyxin producing P. polymyxa strains can. Therefore, although the antibiotic peptide according to the present invention may be similar in structure, it was determined not to be one of the known polymyxins. Preliminary, purification experiments showed that the antibiotic was associated with the cell or spores rather than with culture broth.
- Antibiotic was extracted from either fresh cultures or from freeze dried spore stocks. Antibiotic was initially extracted from the Paenibacillus strain into 100%) methanol. Successful extraction of the antibiotic was also achieved using 0.1 % acetic acid. However most of the work was done using methanol. A small scale purification protocol was designed because pure antibiotic was only required for characterization which typically involves small amounts of sample. The purification steps were also regulated by the need for techniques that were compatible with organic solvents. The primary methanol extract was condensed to 2 ml by rotary evaporation and centrifuged.
- the white precipitate was resuspended in acetonitrile: water (1 :3) containing 0.1% NaH 2 P0 4 and loaded onto a ⁇ Bondapak C-18 (Millipore- Waters) reverse phase HPLC column.
- the components were eluted using a gradient mobile phase of (A) acetonitrile: water (1: 3) containing 0. 1% NaH 2 PO 4 and (B) acetonitrile.
- P. polymyxa PKBl was grown in a 10 litre fermenter in PDB. Cells were harvested after sporulation and freeze dried. Antibiotic samples were extracted from 1 gram of freeze dried spores. The spores were rinsed with water, resuspended in 60 ml of 100%) HPLC grade methanol and left to sit for 60 minutes. The spores were then pelleted by centrifugation and the methanol supernatant saved. This crude antibiotic extract was condensed to 3 ml by rotoevaporation and the extract was periodically filtered using a 0.45 ⁇ m star filter from Costar (Fisher Scientific. Ontario, Canada).
- the condensed crude extract was loaded onto a Sephadex LH-20 gel filtration column, using the same method as noted above and a mobile phase of 100% methanol at a flow rate of 0.2 ml/min. Fractions were collected every 10 minutes and the absorbance of the fractions at
- the active fractions from the Sephadex column were condensed to 5 ml and loaded onto a reverse phase Sep-Pak C18 column in 50%> methanol.
- the antibiotic was eluted from this column using 100% methanol at a flow rate of 1.2 ml./min.
- the active fractions were dried under nitrogen.
- the dried sample was resuspended in 200 ⁇ l of acetonitrile:water (1 :3) plus 0.1% NaH 2 PO 4 and loaded onto the reverse phase C18 HPLC.
- the mobile phase was a gradient of acetonitrile: water (1 :3) plus 0.1 %> sodium dihydrogen orthophosphate and acetonitrile.
- the acetonitrile was ramped in a 0% to 60% linear gradient over 2.5 minutes, held for 10 minutes, followed by a further linear gradient from 60%> to 100% over 37.5 minutes, held for 10 minutes, then ramped down to 0%> by 65 minutes.
- the active fractions were eluted from 9 to 15 minutes.
- the gel was washed in water and then placed on the surface of a bioassay plate.
- Soft agar inoculated with the indicator organism was poured over the gel and allowed to solidify. If there is any antibiotic in the gel it diffuses into the agar of the bioassay plate. In so doing, it inhibits the indicator organism and a zone of inhibition results.
- the antibiotic was characterized by amino acid composition analysis and mass spectroscopy.
- the mass spectrum was measured by two methods, one using glycerine/thioglycerol matrix fast atom bombardment spectroscopy and the other by metastable analysis which is a type of double mass spectroscopy called MS-MS.
- the peptide was further characterized by measuring its stability at a range of temperatures over time, its reaction to proteases, ester hydrolysis, coloration reactions with ninhydrin and other dyes and stains, solubility in a variety of solvents and the inhibitory spectrum towards a variety of fungi and a limited number of bacteria.
- the first step in the purification process was chromatography of the methanol extract on a Sephadex LH-20 gel filtration column, to separate components on the basis of size and their affinity toward methanol as the mobile phase. Extract components were monitored at the time of elution by measuring the effluent absorbance at 280nm. This wavelength can detect certain amino acids such as tyrosine and tryptophan. The absorbance profile indicated that the fractions with antifungal activity still contained contaminating components because it contained two broad peaks. Active sample was then passed through a Sep-Pak cartridge, a small scale reverse phase column that separates components on the basis of polarity such that more polar components elute first.
- the antibiotic is one of the first components to elute from the column, suggesting that it is relatively polar.
- the antibiotic containing fractions were then run on a reverse phase HPLC column and the resulting effluent monitored for absorbance at 214 nm to detect materials containing peptide bonds (Fig. 3).
- the fractions were tested for activity using the well bioassay method and the activity profile was compared to the HPLC absorbance trace.
- the presence of the antifungal peptide was monitored during the purification procedure by a well bioassay and by a bicinchoninic acid assay (BCA) that measures the protein concentration in the sample (Table 1A).
- Table 1A The antifungal activity and amount of protein in the antibiotic extract throughout the purification procedure.
- the hydrolyzed sample showed ninhydrin positive spots, indicating the presence of amino acids, whereas the material which had not been treated by hydrolysis gave no ninhydrin reaction. This suggests that the active fraction is made up of amino acids but that the unhydrolyzed form of the antibiotic is either a cyclic or an N-terminally blocked peptide.
- the peptide could not be successfully sequenced by the Edman degradation method.
- the N-terminal amino acid is derivatized by a chemical and the peptide bond between it and the next amino acid is cleaved. This frees the derivatized amino acid from the rest of the peptide so it can be characterized by chromatography after comparison to amino acid standards. In this way, the sequence of amino acids can be determined since they are cleaved from the peptide one by one. That the Edman degradation was unsuccessful shows that the N-terminal is either blocked by a chemical modification or it is involved in a peptide bond, making the peptide cyclic.
- the antibiotic was incubated with carboxypeptidase A over a 3 hour time period and the activity measured by a well bioassay.
- Carboxypeptidase A cleaves the peptide bond joining the last amino acid at the carboxy terminus to the rest of the peptide. It will continue cleavage of the carboxy terminal amino acid until there is no more substrate available. If there was a carboxyl terminal amino acid available for the protease to use as a substrate on the antibiotic then the antifungal activity of the sample would disappear. The sample remained active after carboxypeptidase A protease treatment, suggesting that there is no C- terminal amino acid available, probably due to the peptide being cyclic.
- the temperature stability of the antibiotic was measured at -70°C, 4°C, 25 °C and 100°C.
- the antibiotic retained its activity after 30 min at 100°C. 3 weeks at 25°C. 4 months at 4°C and indefinitely at -70 °C.
- the antibiotic is soluble in methanol, ethanol, acetonitrile, propanol and acetic acid and slightly soluble in water.
- the antifungal activity of the antibiotic had been tested against fungi other than L. maculans and against some bacteria.
- the fungi that were inhibited by this antibiotic include:
- the antibacterial spectrum of this antibiotic is limited because not many bacteria have been tested, however some bacteria have been found to be sensitive to this antibiotic, including: Micrococcus luteus, Streptomyces clavuligerus and Escherichia coli ESS.
- Table IB The inhibition profile, solubility and stability of the antifungal antibiotic.
- the mass spectrum of the peptide antibiotic was first taken using a gentle ionization method so as to not fragment the sample.
- This spectrum showed that the active material consisted of two components, one with a molecular weight (MW) of 883.5 and the other at 897.5 (Fig. 6A).
- the two components could not be two fragments of a larger component since the gentle ionization procedure was followed.
- These two components were investigated further by taking a mass spectrum of the sample using the metastable technique and a mass spectroscopy method that employs two mass spectroscopy instruments linked in tandem. It allows for a sample containing two components to be separated by holding one of the components at the first MS and fractionating the other component through the second MS.
- Post source decay (PSD) mass spectroscopy was used to determine the amino acid composition of each of the two components.
- the composition for the component having a molecular weight of 883.5 is listed in Table 2.
- Table 2 The amino acid composition of the 883.5 MW antifungal antibiotic.
- One postulated structural formula is shown in Figure 6B.
- the PSD data for the 897.5 MW component indicated that it also contained eight amino acids corresponding to the composition of the 883.5 MW peptide, except that it contains isoleucine instead of the valine.
- the PSD data also indicated that there were other similarly sized peptides which were extracted from the spores of the Paenibacillus polymyxa, strain PKBl including peptides having molecular weights of 912, 914, 920, 928, 942.
- the antibiotic components do not appear to be ribosomally produced but rather produced by peptide synthetase activity.
- strain PKB 1 could be used as an agent for biocontrol of blackleg on canola crops.
- Paenibacillus polymyxa has two properties that make it an attractive biocontrol agent. First, it is a native soil bacterium and, second, it can form dormant spores. Effect of strain PKB 1 on L. maculans pycnidiospore germination and viability was studied. A loopful of P. polymyxa from an actively growing culture was transferred to 5 ml of nutrient broth and incubated at room temperature for three days. A bacterial suspension was then prepared in sterile distilled water and adjusted to approximately 1 x 10 cells ml "1 . Fresh pycnidiospore suspension of a virulent strain of P.
- maculans was prepared using a 10 day old fungal culture grown on V-8 juice-Rose Bengal agar amended with streptomycin sulfate (200 ppm). The suspension was adjusted to 1 x 10 6 pycnidiospores ml "1 .
- a small piece of cellophane was cut from each plate every 24 h for three days, mounted on a microscope slide, stained with lactophenol cotton blue and observed with a compound microscope. The percentage of germinated pycnidiospores and length of germ tubes were recorded. The experiment was repeated once.
- Pycnidiospores mixed with the bacterial suspension fluoresced dull yellow when treated with FungoLight, indicating reduced viability; pycnidiospores not treated with bacteria fluoresced bright yellow with a red cylindrical object inside each cell.
- Micro Antibiotic Extraction To monitor the antibiotic production during a growth assay, 1.5ml sample was taken from the culture, the cells were harvested by centrifugation at 13,000xg for 5 min and the supernatant was saved for measurement of the culture pH. The cell pellet was resuspended into 200ul of methanol for 20 min and then the cells were pelleted again and the methanol extract was assayed for antibiotic activity in a well bioassay.
- Macro Antibiotic Extraction The cells in 200ml of culture broth were pelleted at lO.OOOxg for 20 min and the supernatant was discarded; alternatively I gram of freeze dried cells was used. The cells were washed once with distilled water and then resuspended into 60ml of methanol and held for 1 hour. The resuspended cells were pelleted by centrifugation and the methanol extract (referred to as primary methanol extract) was used in the antifungal antibiotic purification protocol.
- Fermentation and Freeze-drying Fermenters were used to grow 10, 30 and 50 litre batches of P. polymyxa. PDB was used as the growth medium and 5 and 2.5% inocula from TSB starter cultures were used to initiate growth. Aeration was regulated at 15 L/min. temperature at
- Well Bioassay 100ml of molten potato dextrose agar (PDA) was poured into 15 x 15cm bioassay plate. After solidification 800ul of P. maculans pycnidiospores in 20%> glycerol were spread on the surface as the indicator organism. 10mm diameter wells were cut from the plate with a sterile cork borer and 75 ul amounts of sample were placed in the wells. The diameters of the zones of inhibition of the fungus around the well are measured in millimetres after 24 h incubation at room temperature.
- BCA Assay An assay kit from Pierce Chemicals was used as the source of reagents and protein standards. The standard protocol methods designed by this company are used. A standard curve was generated using bovine serum albumin and the unknown samples were compared to this for identification of the protein concentration.
- Sep-Pak Chromatography The pooled active -fractions from the Sephadex chromatography were condensed by evaporation under nitrogen to 5ml from 14ml. The fraction was diluted to 50%) methanol and loaded onto a Sep-Pak CA 8 cartridge (Millipore Waters). This is a small reverse phase C 18 column. The active fraction was eluted with methanol at a flow rate of
- Reverse Phase HPLC The pooled active fractions from the Sep-Pak were condensed to dryness under nitrogen and redissolved into 200, 400 or 600 ul of mobile phase liquid. The sample was loaded onto a uBondapak C-18 RP-HPLC column (Millipore Waters) and eluted with one of two different mobile phase gradients at a flow rate of 1.Oml/min collecting one min fractions.
- One gradient was (A) acetonitrile: water (1 :3) containing 0.1%> NaH : PO 4 and (B) acetonitrile with the gradient varying from 0 to 100%> (B) over 90 min.
- Bioautograph Following SDS-PAGE, the gel was washed in distilled water and then placed onto solidified PDA in a bioassay plate. Soft PDA was inoculated with 500ul of P. maculans pycnidiospores and then poured over the gel and allowed to solidify. Antibiotic will elute from the gel and enter the PDA where it can inhibit the fungus and cause a zone of inhibition that correlates to where the antibiotic migrated to in the gel during electrophoresis.
- Genomic DNA was extracted generally according to the procedure described by Sambrook et. al. (Sambrook, J.E. et.al. Molecular cloning: A Laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, NY 1989).
- PCR reactions were carried out in a thermal cycler (Thermolyne, Temp. Tronic, Bamstead/Thermolyne Corporation, Dubuque, IA, U.S.A.). The amplified products were run electrophoreses in 1 % agarose gels, stained with ethidium bromide and photographed under UN light. One kb D ⁇ A ladder (Gibco-BRL) was used as a D ⁇ A size marker. All reactions were performed twice.
- DNA of PKBl was amplified with primers OPA07, OPA08, OPA13 and OPA14.
- Four DNA fragments of 0.61, 0.71, 0.27 and 0.62 kb were excised from agarose gel of PCR products amplified with each primer respectively and purified with DNA Purification Kit (Boehringer Mannheim Corp.).
- the purified DNA fragments were separately labeled with alkali labile Dig-11-dUTP in a simultaneous amplification in a thermocycler by using the same PCR profile as described previously.
- Estimation of the yield of Dig-labeled probe was done according to the method of Genius System User's Guide for Membrane Hybridization, Version 3.0 (Boehringer Mannheim Corp.). The probes were stored at -20°C for later use.
- the genomic DNA of 23 Bacillus and Paenibacillus strains was digested with restriction endonucleases, Eco RI, Pst I, and Hind III (Gibco-BRL) separately at 37°C for 1 h.
- the DNA digests were electrophoresed on 1%> agarose gel. A 21 kb dig-labeled marker (Boehringer Mannheim Corp.) was used.
- Probed DNA was detected with CSDP (disodium 3-(4-methoxyspiro [1,2-dioxetane- 3,2' ⁇ 5'-chloro ⁇ tricyclo (3.31.F '7 ) decan[-4-yl) phenyl phosphate) using a DIG Luminescence Detection Kit (Genius 7, Boehringer Mannheim Corp.).
- CSDP disodium 3-(4-methoxyspiro [1,2-dioxetane- 3,2' ⁇ 5'-chloro ⁇ tricyclo (3.31.F '7 ) decan[-4-yl) phenyl phosphate
- DIG Luminescence Detection Kit Genius 7, Boehringer Mannheim Corp.
- the specificity of the probes was also determined by dot blotting method using 23 known P. polymyxa and Bacillus strains and 64 unknown bacterial strains.
- Isolates 96-1 and 97-1 are the two subcultures of the original isolate of Paenibacillus polymyxa (Strain PKBl). Strains 96-2 to 96-5 were isolated from canola stubble that were buried in soil after treatment with compost infested with the strain PKB 1. These strains seem to be the isolates of PKBl strain as these have identical DNA pattern.
- Isolates R-l and R-2 are the two antibiotic resistant mutants of PKBl that were developed in the laboratory. These are also similar to the patterns of other PKB 1 strains (96-1 to 96-5).
- 97-2 is Colistinus Koyama strain of Paenibacillus polymyxa available from ATCC.
- 97-3 is NCIB 4686 strain, a type strain of Paenibacillus polymyxa. DNA patterns of both 97-2 and 97-3 are different from PKBl (Strains 96-1 to 96-5 and R-l and R-2).
- DNA patterns are of Paenibacillus and other Bacillus species obtained from Dr. Richard Coleman, ARC, Vegreville, or other colleagues elsewhere.
- DNA of the antibiotic resistant bacterial mutants could also be probed (isolates 12 and 13 in Fig. 9C-F). DNA of the bacterial isolates from compost was tested in Southern blot hybridization but none of them could be probed (strains 12-22 in Fig. 9A and 9B).
- Probes P 1 -7 and P 1 -8 were used to detect the DNA of bacterial strains with dot- blotting method.
- PKB 1 and all related strains in the specific Paenibacillus sp. could be probed (Fig. 10A and 10B). This is confirmed that the probes are species specific. Once the specific probe is developed, dot-blot method is very simple and easy to be used in the detection of the bacterium.
- DNA of four probes were cloned and sequenced with the length of 609 bp (p 1 -7), 713 bp (Pl-8). 270 bp (Pl-13) and 617 bp (Pl-14). Two sequences could be translated into polypeptides with no interruption stop codons in some reading frames.
- One sequence has some identity with the protein required to initiate sporulation in a Bacillus sp. The other has some identity with an enzyme produced by a Paenibacillus sp. Genetic markers of this bacterial strain were determined by randomly amplified polymo ⁇ hic DNA-polymerase chain reaction (RAPD-PCR). Four DNA fragments, specific for PKBl, were selected for making probes.
- RAPD-PCR randomly amplified polymo ⁇ hic DNA-polymerase chain reaction
- the four probes were used to hybridize with Southern blots of Eco RI, Pst I, and Hind III restriction digests of 23 strains of Paenibacillus polymyxa and seven Bacillus sp.
- the probes were species specific and hybridized only with DNA of PKBl and related strains belonging to the same Paenibacillus sp. but not with that of other Bacillus spp.
- the four probes were cloned and sequenced to design specific primers for the biocontrol agent. Eight primers were designed, synthesized and tested with 23 strains mentioned above. The designed primers were strain specific and amplified only one band of PKBl . The specific primers will be used for the detection of this biological control agent in field studies to control diseases of canola.
- Paenibacillus polymyxa strain PKB 1 significantly reduced blackleg incidence and severity on the susceptible cultivar Westar but not on the resistant cultivar Quantum (Table 4).
- Strain PKB 1 had profound effect on the viability of the fungus in the growth chamber (Table 5). Under most conditions, bacterium treated stubble had significantly lower survival of the fungus than the untreated check on those treated with Tilt ® . This may be because strain
- Each sample was kept for 2 weeks at each of the five different temperatures, 20°C, 10°C, -5°C, 10°C, 20°C in a cycle.
- strain PKB 1 was found to have inhibitory effect on all the fungi tested.
- significant inhibitory effect of the bacterial filtrate was observed on mycelial growth of all the fungi tested (Table 6). The results suggest that PKBl has a potential of biological control not only against blackleg but also against other diseases of canola.
- the bacterium, Paenibacillus polymyxa strain PKBl. has significant inhibitory effect on the growth and development of Leptosphaeria maculans.
- the bacterium PKB 1 is capable of inhibiting growth of different isolates of several fungi which cause important diseases on canola such as root rot, Sclerotinia stem rot and black spot.
- Tilt ® significantly reduced number of pycnidia on stubble and bacterium significantly reduced survival of P. maculans under growth chamber condition.
- Bacterial strains within a Paenibacillus species are more genetically related and distinguishable among the Bacillus species based on the DNA patterns. It is possible to detect the biocontrol agent PKB 1 from other Paenibacillus species using DNA patterns by
- Pure P. polymyxa antibiotic is obtained according to example III and dried.
- An Eppendorf tube containing 721 ug of the dried, purified antibiotic is washed with 5 separate 1 ml aliquots of 70%> methanol then taken to a volume of 70 ml which gave a stock solution of 10 ppm.
- a pycnidiospore suspension of P. maculans is prepared by covering an actively sporulating plate of the fungus with sterile distilled water and scraping with a flame-sterilized glass hockey stick. A haemocytometer is used to determine a spore concentration of 5.5 x 10 7 spores/ml.
- Potato dextrose broth (PDB) is prepared according to package directions and autoclaved at 121 °C for 25 minutes. After cooling, the broth is divided into 90 ml portions. The first 90 ml aliquot received 10 ml of the 10 ppm stock antibiotic solution to yield a 1 ppm solution of the antibiotic in PDB.
- the PDB is then subdivided into small 60 x 15 mm Petri plates (10 ml per plate). Each concentration yielded 8 plates, 4 of which are inoculated with 10 ul of the pycnidiospore suspension of P. maculans while the remaining 4 received 100 ul of the spore suspension.
- Plates are placed on a rotary shaker (50 rpm) at room temperature until the development of mycelium occurred.
- Whatman No. 1 filter papers (100 mm) are oven dried in a metal tray for 24 hours at 60 °C prior to harvesting the mycelium. Papers are weighed before harvest and the weight recorded. Mycelium is collected on the filter papers using a Buchner funnel under vacuum pressure. Filter papers are removed onto several layers of paper towel to remove excess liquid then paper-clipped to envelopes and placed in the dryer at 60 °C for 24 hours. Papers are fastened to envelopes to prevent them from being blown around in the dryer. After 24 hours, the papers are re-weighed and the weight of the mycelium calculated.
- Rhizoctonia inoculum was prepared by scraping mycelium from the surface of an agar plate with a flame-sterilized scalpel. The mycelium was placed in a manual tissue macerator in 5 ml of sterile distilled water and ground until the solution was consistently cloudy and there were no large clumps of mycelium.
- Potato dextrose broth was prepared according to package directions and autoclaved at 121 °C for 25 minutes. After cooling, the broth was divided into 90 ml portions. The first 90 ml aliquot received 10 ml of the 10 ppm stock antibiotic solution to yield a 1 ppm solution of the antibiotic in PDB. This was shaken vigorously then 10 ml was removed and added to a second 90 ml aliquot of PDB to yield a 0.1 ppm solution of the antibiotic in PDB. This serial dilution was continued to give solutions of 0.01 ppm, 0.001 ppm and 0.0001 ppm of antibiotic in PDB. Controls consisted of plain PDB.
- the PDB was then subdivided into small 60 x 15 mm Petri plates (10 ml per plate).
- Whatman No. 1 filter papers (100 mm) were oven dried in a metal tray for 24 hours at 60 °C prior to harvesting the mycelium. Papers were weighed before harvest and the weight recorded. Mycelium was collected on the filter papers using a Buchner funnel under vacuum pressure. Filter papers were removed onto several layers of paper towel to remove excess liquid then paper-clipped to envelopes and placed in the dryer at 60 °C for 24 hours. Papers were fastened to envelopes to prevent them from being blown around in the dryer. After 24 hours, the papers were re-weighed and the weight of the mycelium calculated.
- Mycelium was harvested 16 days after the start of the experiment. An ANOVA test run on the data indicated a significant concentration effect.
- Table 10 contains the mean mycelial dry weights for each treatment as well as the Tukey separations.
- Table 10 Mean mycelial dry weights of Rhizoctonia grown in PDB amended with various concentrations of antibiotic.
- Weights represent the mean of 4 replicates, 1 plate per replicate. Means followed by the same letter are not significantly different at P ⁇ 0.05.
- primer Jl is listed as SEQ ID NO:9
- primer JY1 is listed as SEQ ID NO: 10
- primer JY2 is listed as SEQ ID NO:8.
- RFLP analysis of the PCR products amplified with primers Jl and JY1 from 10 bacterial strains was conducted with three restriction endonucleases, Rsa I, Mse I. and Sau3A I to check the polymo ⁇ hism on the restriction site of these 10 bacteria.
- the reaction mix contained 2 ⁇ l DNA of the PCR product, 1 ⁇ l (5 units) restriction endonuclease, 2 ⁇ l reaction buffer and 15 ⁇ l water.
- the reaction was carried out in a 37°C water bath for 1 h.
- the digests were separated on 1.5%) agarose gel at 100 V.
- the gel was stained with ethidium bromide and photographed under UV light.
- DNA of two bacterial strains (97-4, lane 11 and 96-9, lane 12 in Fig. 12) that had no inhibitory effect to P. maculans but were inhibitory to Rhizoctonia solani and other pathogens could also be amplified with these designed primers.
- One strain of Erwinia sp. that was also included in the PCR test did not yield any PCR product.
- a complete restriction map was generated for the DNA fragment of PKBl (#1, lane 1 in Fig. 12) and 97-4 (#11, lane 12 in Fig. 12), respectively, using DNA StriderTM1.2 computer software. These two DNA fragments differed in one restriction endonuclease site, Tthlll II site was absent in strains 96-9 and 97-4. Because this enzyme (Tthlll II) is not commercially available at the present time, the RFLP procedure could not be conducted. However, this enzyme should prove to be a useful marker for differentiating PKB 1 from other non-anti-blackleg P. polymyxa strains based on the RFLP pattern once it becomes commercially available.
- M. oreades isolates Two M. oreades isolates (one from gill and one from stipe) were obtained from a fungal mushroom collected from a fairy ring in the Vegreville, Alberta, Canada area. The fungal cultures were grown in Malt-potato dextrose agar (MPDA) and kept at a temperature of4°C.
- MPDA Malt-potato dextrose agar
- the bacterial mutant strain 97-006 significantly inhibited the mycelial growth of both isolates of M. oreades (Table 13). The colony diameter of the fungus co-inoculated with the bacterium was significantly reduced.
- the composts were prepared under the supervision of the Compost Technology Centre (CTC), Olds, Alberta, Canada. Mature and near mature composts (cattle manure and wood chips) were pasteurized by steaming 0.5 m 3 of each for 5 hours at 80°C in a soil steamer to destroy the resident microorganisms, thus enhancing the effectiveness and viability of the intended PKBl spore inoculum. After cooling to 45°C, 0.25 m 3 of each material was spread out in layers, and soybean meal and/or spores of the bacterium were added and well mixed with the compost. The spore suspension (1 x 10 9 cfu/ml) was prepared in distilled water (pH 7.4) from the freeze-dried spores. The suspension was further diluted to 3.7-4.2 x 10 6 cfu ml.
- the inoculated composts were incubated in 0.25 m 3 commercial composters to enhance the population of PKB 1.
- the two soybean meal supplemented portions were incubated in forced aeration composters and without aeration in passive aeration composters.
- the oxygen concentration reached ⁇ 18% indicating the start of the maturing phase, lm 3 of the material was steam pasteurized at 80° C for 5 hours and then left to cool to 40° C in the steam wagon.
- the control half of the pasteurized compost had only the 13.5kg soybean meal added.
- Two 0.25m 3 compost bins were filled with material from each treatment and incubated with forced aeration. Temperature, oxygen concentration, moisture and pH were monitored .
- the pH of the material rises to ⁇ 8 because of the added soybean meal.
- the material was harvested after 27 days of incubation and stored in jute bags. At weekly intervals during the composting period, samples were taken for spore enumeration and pathogen inhibiting assessment.
- the inhibitory effect of PKBl re-isolated from compost was confirmed by using two methods.
- a loop of the bacterium was placed at four places on a PDA plate, around an agar plug of P. maculans culture. Inhibition zones in the culture around the bacterial colonies were checked after 10 days incubation at 22°C.
- 0.1 ml of the diluted compost suspension and 0.9 ml of P. maculans spore suspension were mixed and then spread onto four PDA plates. Inhibition of fungal growth was observed after one week of incubation at room temperature.
- composts containing PKB 1 were mixed with greenhouse soil ( 1 : 1 ,v:v) and filled in 10-cm fiber pots.
- Five sclerotia were buried in the mixture and incubated in a growth chamber at a constant temperature of 20°C with 12 h light. The pots were kept moist for the duration of the experiment. Pots without compost-bacterium were used as controls. There were four replications for each treatment. Germinated sclerotia and the number of apothecia formed were counted three weeks after seeding; counting was repeated at 10-day interval for another 30 days.
- compost (C4) + bacterium significantly inhibited germination of sclerotia of S. sclerotiorum three weeks after inoculation compared to the control (Table 14), whereas the other compost treatments had no significant effect. Also, the number of apothecia in compost C4 was the least compared with other composts. Some of the batches of composts did not have significant effect. Total number of viable bacterial cells in these batches was found to be quite low. This suggests that the substrate of the compost may influence the success of establishment and overall performance of strain PKB 1. In experiment two, where compost was not mixed with any greenhouse soil or ProMix, compost or compost + bacterium completely inhibited the germination of sclerotia. In the control treatment (sclerotia buried in ProMix), 58%> of sclerotia germinated and produced apothecia.
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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EP99920487A EP1079692A1 (en) | 1998-05-20 | 1999-05-20 | Biocontrol agent and pesticide |
AU38057/99A AU758577B2 (en) | 1998-05-20 | 1999-05-20 | Biocontrol agent and pesticide |
US09/700,486 US6602500B1 (en) | 1998-05-20 | 1999-05-20 | Paenibacillus polymyxa strain ATCC 202127 for biocontrol of bacteria and fungi |
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CA2,238,289 | 1998-05-20 | ||
CA2238289A CA2238289C (en) | 1998-05-20 | 1998-05-20 | Biocontrol agent and fungicide for blackleg disease |
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WO1999059412A1 true WO1999059412A1 (en) | 1999-11-25 |
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PCT/CA1999/000426 WO1999059412A1 (en) | 1998-05-20 | 1999-05-20 | Biocontrol agent and pesticide |
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US (1) | US6602500B1 (en) |
EP (1) | EP1079692A1 (en) |
AU (1) | AU758577B2 (en) |
CA (1) | CA2238289C (en) |
WO (1) | WO1999059412A1 (en) |
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US11766045B2 (en) | 2016-12-01 | 2023-09-26 | Indigo Ag, Inc. | Modulated nutritional quality traits in seeds |
US11807586B2 (en) | 2016-12-23 | 2023-11-07 | The Texas A&M University System | Fungal endophytes for improved crop yields and protection from pests |
US11516989B2 (en) | 2017-03-01 | 2022-12-06 | Indigo Ag, Inc. | Endophyte compositions and methods for improvement of plant traits |
US11985931B2 (en) | 2017-03-01 | 2024-05-21 | Indigo Ag, Inc. | Endophyte compositions and the methods for improvement of plant traits |
EP3629742A4 (en) * | 2017-04-27 | 2022-01-05 | Flinders University Of South Australia | Bacterial inoculants |
US11882838B2 (en) | 2017-04-27 | 2024-01-30 | The Flinders University Of South Australia | Bacterial inoculants |
US10743535B2 (en) | 2017-08-18 | 2020-08-18 | H&K Solutions Llc | Insecticide for flight-capable pests |
WO2022023109A1 (en) | 2020-07-31 | 2022-02-03 | Basf Se | New agrochemical formulations for fusaricidin producing bacteria |
WO2022136003A1 (en) | 2020-12-23 | 2022-06-30 | Basf Se | Mixtures and compositions comprising fusaricidin a, fusaricidin b and fungicides |
Also Published As
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CA2238289C (en) | 2013-08-06 |
AU3805799A (en) | 1999-12-06 |
CA2238289A1 (en) | 1999-11-20 |
EP1079692A1 (en) | 2001-03-07 |
AU758577B2 (en) | 2003-03-27 |
US6602500B1 (en) | 2003-08-05 |
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