WO2013165607A1 - Souche bac03 inédite de bacillus amyloliquefaciens et ses méthodes d'utilisation - Google Patents

Souche bac03 inédite de bacillus amyloliquefaciens et ses méthodes d'utilisation Download PDF

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WO2013165607A1
WO2013165607A1 PCT/US2013/032246 US2013032246W WO2013165607A1 WO 2013165607 A1 WO2013165607 A1 WO 2013165607A1 US 2013032246 W US2013032246 W US 2013032246W WO 2013165607 A1 WO2013165607 A1 WO 2013165607A1
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plant
strain
bac03
composition
streptomyces
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Jianjun Hao
Qingxiao MENG
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Board Of Trustees Of Michigan State University
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • Streptomyces species like Streptomyces scabies, cause scabby diseases in potatoes, radishes, and other crops. Most of the current plant disease control methods have proven ineffective. Moreover, many synthetic fungicides are not permitted especially for organic production, and chemical fungicides do not prevent all diseases and often release toxic chemicals into the soil or food chain. The use of many agrochemicals are also banned or restricted due to environmental and health risks.
  • a novel strain of Bacillus amyloliquefaciens strain BAC03 is isolated and described.
  • the strain is one which has the identifying characteristics of the B. amyloliquefaciens BAC03 strain a representative sample of which has been deposited with the American Type Culture Collection (ATCC) as Deposit No. PTA- 12890 (hereinafter "ATCC No. PTA-12890).
  • ATCC American Type Culture Collection
  • PTA- 12890 accession No. PTA-12890
  • antimicrobial protein produced by this strain has 99% homology to reported LCI DNA sequences.
  • the bacterial strain promotes plant growth and enhances disease resistance.
  • the strain and mutants thereof are useful as a biological control agent. Use in reducing adverse impact of a pathogen on a plant or plant part is provided. Plants, plant parts or soils are treated by applying an effective amount of BAC03, a supernatant of the strain or cell suspension or other forms of BAC03.
  • FIGS. 1A-B Effect of Bacillus amyloliquefaciens BAC03 on disease severity and plant growth in potato and radish.
  • Panels Al and A2 Potato was planted in potting mix treated with 200 ml of S. scabies at final concentrations 10 6 CFU/cm 3 (treatment S), BAC03 atlO 5 CFU/cm 3 (treatment Bl), BAC03 at 10 6 CFU/cm 3 (treatment B2), and tryptic soy broth as a control (CK).
  • Panels B l and B2 Radish was planted in potting mix treated with 30 ml of BAC03 at final concentration of 10 5 CFU/cm 3 each time in the following treatments: bO (non- treated), bl [one time of application after planting (TAP)], b2 (2 TAPs), b3 (3 TAPs), and b4 (1 time before and 3 TAPs), with weekly intervals between applications. All pots were infested with Streptomyces scabies at 10 7 CFU/cm 3 potting mix. Fresh plant weight was measured in the end of experiment.
  • FIGS. 3A-B (A) Antimicrobial activity of Bacillus amyloliquefaciens BAC03 ammonium sulfate precipitation fraction (AS) against Streptomyces scabies; and (B) Test for bactericidal versus bacteriostatic activity of BAC03 AS. Streptomyces scabies was recovered after incubation with AS for 0, 1, 3, and 5 days, followed by rinsing off AS through a 0.22 ⁇ membrane.
  • FIG. 4 Scanning electron microscopy (SEM) of mycelial morphology of
  • Streptomyces scabies grown on yeast malt extract medium plates Ten microliters of sodium phosphate buffer (untreated), or AS (treated) was directly dropped onto a S. scabies 2-day-old colony. Five days later, the untreated colonies produced white spores, but the treated culture did not show any sporulation.
  • the culture specimen were processed following the method of Fan et al. (2011) and coated with metallic osmium in preparation for SEM.
  • FIG. 5 Test for active fraction of ammonium sulfate precipitate (AS) from Bacillus amyloliquefaciens BAC03 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation on SDS-PAGE, the gel was overlaid with yeast malt extract medium containing Streptomyces scabies (10 7 CFU/ml), and the inhibition zone was observed after 2 days of incubation at 28°C. Growth was detected by staining with thiazolyl blue tetrazolium bromide (MTT). The atomic mass of the active compound(s) was estimated by comparison to the position of the markers (in kDa).
  • AS ammonium sulfate precipitate
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • FIGS. 6A and 6B Effect of BAC03 to induce systemic resistance in potato against common scab.
  • Figure 6 A shows disease severity of four treatment groups, when exposed to BAC03, with or without exposure to S. scabies, and in control plants.
  • Figure 6B shows biomass measurements.
  • FIGS. 7 A and 7B Effect of biomaterials on potato common scab in the field in location 1.
  • Figure 7 A shows disease severity and
  • Figure 7B shows plant weight.
  • FIGS. 8 A and B Effect of biomaterials on potato common scab in the field in location 2.
  • Figure 8A shows disease severity and
  • Figure 8B shows plant weight.
  • Bacillus amyloliquefaciens is an aerobic bacterium that is a gram-positive motile, rod-shaped bacterium. See, for example Kang et al., US20110274673.
  • strain BAC03 Bacillus amyloliquefaciens
  • An embodiment is to a strain having all the identifying, biological and genetic characteristics of strain BAC03.
  • the bacterial strain is an alternative to chemical control strategies since they are environmentally friendly and safe to humans.
  • the bacteria produce antimicrobial substances that serve as antibiotics used to inhibit other microorganisms, such as plant pathogens.
  • This strain has been isolated from a disease-suppressive soil and sequenced.
  • the bacteria have been shown to have antimicrobial activity.
  • the antimicrobial portion which is able to kill or inhibit a range of microorganisms, especially soilborne plant pathogens, was isolated and identified as having 99% homology to reported nucleotide sequences encoding an LCI protein, an antimicrobial substance, and disclosed here as SEQ ID NO: 1.
  • the term "the bacteria”, “the strain”, “the bacterial strain”, depending on the context may be used interchangeably to refer to the BAC03 strain, the antimicrobial portion of BAC03 (viz., LCI) or both.
  • the bacterial strain also stimulates plant growth by increasing biomass and inducing the plant to defend against soil borne pathogens.
  • the strain has been demonstrated to have antimicrobial activity on plant pathogens including bacterial and fungal pathogens. Examples include, without limitation, agronomically important crop, vegetable and nut pathogens such as Streptomyces scabies (potato common scab), Agrobacterium tumefaciens, Clavibacter michiganensi, Phytophthora cambivora, Phytophthora capsici, Rhizactonia solani and Sclerotinia sclerotiorum. Still further examples of pathogens include those listed in Table 2 below.
  • the strain In some cases of pathogen inhibition, the strain possesses a greater antimicrobial effect (inhibition zone) than a known biocontrol product containing Bacillus subtilis sold under the trade name SERENADETM and produced by AgraQuest. Furthermore, BAC03 promotes plant growth (e.g. increasing plant biomass) in plants such as potato, radish, tomato, turnip, beet, cucumber, pepper and carrot.
  • a plant is used broadly herein to include any plant at any stage of development, or to part of a plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed, and a plantlet.
  • a plant cell is the structural and physiological unit of the plant, having a protoplast and a cell wall.
  • a plant cell can be in the form of an isolated single cell or aggregate of cells such as a friable callus, a cultured cell, or can be part of a higher organized unit, for example, a plant tissue, plant organ, or plant.
  • a plant cell can be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant.
  • a seed which includes multiple plant cells and is capable of regenerating into a whole plant, is considered a plant cell for purposes of this disclosure.
  • a plant tissue or plant organ can be a seed, protoplast, callus, or any other groups of plant cells that is organized into a structural or functional unit.
  • Particularly useful parts of a plant include harvestable parts and parts useful for propagation of progeny plants.
  • a harvestable part of a plant can be any useful part of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots, and the like.
  • a part of a plant useful for propagation includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks, and the like.
  • Tissue culture is capable of regenerating plants.
  • the regenerable cells in such tissue cultures will be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, stalks or the like.
  • plants may be regenerated from the tissue cultures.
  • the disclosed bacterial strain may be used with any plant species, whether monocotyledonous or dicotyledonous. Examples of monocotyledonous plants include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats.
  • dicotyledonous plants include, but are not limited to tomato,potato tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli, cauliflower, Brussels sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals.
  • Brassica oleracea e.g., cabbage, broccoli, cauliflower, Brussels sprouts
  • radish, carrot, beets eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals.
  • a culture of the microbial strain has been deposited with the ATCC, 10801 University Boulevard., Manassas, Va. 20110-2209 USA on May 10, 2012 and, as noted above, has been designated as ATCC No. PTA- 12890.
  • the strain or the antimicrobial protein when referred to as biologically pure or isolated refers to the strain or protein separated from materials with which it is normally associated in nature.
  • a representative sample of a biologically pure or isolated culture of BAC03 strain refers to the strain deposited as ATCC No. PTA- 12890.
  • a monoculture of a strain, or a strain associated with other strains, or with compounds or materials that it is not normally found in nature, are examples of a biologically pure strain.
  • the strain encompasses its various forms, including by way of example without limitation, cell suspensions, spores, vegetative cells or whole culture of the strain which may be used to provide the antimicrobial properties of BAC03.
  • the whole culture obtained may be prepared into whole culture powder, for example, by freeze-drying the whole culture as it is.
  • the vegetative cells may be prepared as a cell precipitate, for example, by centrifuging whole culture after the cultivation to remove contaminants, further centrifuging the resulting supernatant, and then washing the cells precipitated.
  • the spores may be prepared as freeze-dried spore powder, for example, by suspending the cell precipitate obtained above in distilled water and freeze-drying the resulting suspension.
  • the strain may be a mutant (also referred to as a variant) of BAC03, a representative sample of which has been deposited as ATCC PTA- 12890 that has all the identifying characteristics of the strain and retains antimicrobial properties to the pathogens described.
  • Such mutants may be those having a genome that hybridizes under conditions of high stringency to the genome of the strain a representative sample of which has been deposited as ATCC PTA- 12890.
  • the mutants may be those having recA, recN, cheA and gyrA sequences which hybridize under stringent conditions to the recA (SEQ ID NO. 2), recN (SEQ ID NO. 3), cheA (SEQ ID NO. 4) and gyrA sequences (SEQ ID NO. 5) of BAC03, a representative sample of which has been deposited as ATCC PTA- 12890.
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (e.g., genomic or cDNA libraries) from a chosen organism.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the DNA sequences disclosed.
  • Methods for probe preparation for hybridization and for cDNA or genomic libraries construction are generally known in the art and are disclosed (see e.g.
  • the nucleic acid sequences disclosed herein, or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding sequences.
  • probes include sequences that are unique among the sequences to be screened and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • sequences may alternatively be used to amplify corresponding sequences from a chosen plant by polymerase chain reaction (PCR). This technique may be used to isolate sequences from a desired plant or as a diagnostic assay to determine the presence of sequences in a plant.
  • Hybridization techniques include hybridization screening of DNA libraries plated as either plaques or colonies (Sambrook et al., 1989). Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent
  • hybridization condition is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization or washing conditions or both, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g. , 10 to 50 nucleotides) and at least about 60°C for long probes (e.g. , greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 50°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 0.1% SDS at 37°C, and a wash in 0.1X SSC at about 60 to 65°C.
  • sequences that correspond to and which hybridize to the nucleotide sequence disclosed herein will be at least about 50% to about 99% homologous; about 70% homologous, and even about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous or more with the disclosed sequence. That is, the sequence similarity between probe and target may range, sharing at least about 50%, about 70%, and even about 85% or more sequence similarity.
  • T m can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem. , 138:267-284
  • T m 81.5°C + 16.6 (log M) + 0.41 (%GC) -0.61 (% form) - 500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe.
  • T m is reduced by about 1°C for each 1 % of mismatching; thus, T m , hybridization or wash conditions or both can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • a mutant may also be a strain having a genomic sequence having at least about 85% to about 95% identity to the genome of BAC03 as represented in ATCC No. PTA-12890.
  • the mutant may be a strain having a recA, recN, cheA and gyrA sequences which have at least about 85% to about 95% identity to the recA (SEQ ID NO. 2), recN (SEQ ID NO. 3), cheA (SEQ ID NO. 4) and gyrA (SEQ ID NO. 5) sequences of BAC03, a representative sample of which has been deposited as ATCC No. PTA-12890. Any such mutant is one which retains the antimicrobial properties described herein.
  • a mutant may be one which produces the antimicrobial protein, an LCI protein.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length promoter sequence, or the complete promoter sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (e.g., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least about 20 to about 100 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Optimal alignment of sequences for comparison can use any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed "PILEUP" (Morrison, (1997) Mol. Biol. Evol. 14:428-441 , as an example of the use of PILEUP); by the local homology algorithm of Smith & Waterman ⁇ Adv. Appl. Math. 2: 482 (1981)); by the homology alignment algorithm of Needleman & Wunsch (/. Mol. Biol. 48:443-453 (1970)); by the search for similarity method of Pearson ⁇ Proc. Natl. Acad. Sci.
  • BLAST algorithm Another example of algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et al, (1990) /. Mol. Biol. 215: 403- 410.
  • the BLAST programs (Basic Local Alignment Search Tool) of Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410) searches under default parameters for identity to sequences contained in the BLAST "GENEMBL" database.
  • a sequence can be analyzed for identity to all publicly available DNA sequences contained in the GENEMBL database using the BLASTN algorithm under the default parameters.
  • HSPs high scoring sequence pairs
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • BLAST refers to the BLAST algorithm which performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin (1993), Proc. Natl. Acad. Sci. USA 90:5873-5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • GAP Global Alignment Program
  • GAP uses the algorithm of Needleman and Wunsch (/. Mol. Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • Default gap creation penalty values and gap extension penalty values in the commonly used Version 10 of the Wisconsin PackageTM (Accelrys, Inc., San Diego, CA) for protein sequences are 8 and 2, respectively.
  • the default gap creation penalty is 50 while the default gap extension penalty is 3.
  • Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
  • a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • a general purpose scoring system is the BLOSUM62 matrix (Henikoff and Henikoff (1993), Proteins 17: 49-61), which is currently the default choice for BLAST programs. BLOSUM62 uses a combination of three matrices to cover all contingencies. Altschul, J. Mol. Biol. 36: 290-300 (1993), herein incorporated by reference in its entirety and is the scoring matrix used in Version 10 of the Wisconsin PackageTM (Accelrys, Inc., San Diego, CA) (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).
  • sequence identity or “identity” in the context of two nucleic acid sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (e,g., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • Identity to the sequences disclosed would mean a polynucleotide sequence having at least about 65% to about 99% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% identity, at least 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • “application” of the disclosed strain is intended to refer to exposing the plant, plant part, or soil with or without the plant or plant part to the strain in any convenient manner, such that adverse impact of a pathogen is reduced.
  • the adverse impact of a pathogen on a plant or plant part may be measured by any of a variety of options available to one skilled in the art, including by way of example without limitation, determination of improved plant growth, growth rate, biomass, increased survival rates, plant length, weight, leaf area, flower fertility, pollen fertility, seed weight or yield, seed germination, tuber weight or any combination of these, as compared to a plant exposed to the pathogen but which has not had the disclosed strain applied.
  • the strain may be applied to plants and soil in any convenient manner.
  • the strain may be applied as a stand-alone living organism, since its spores can tolerate extreme environmental conditions. It may be provided as a suspension in whole broth culture (liquid culture containing cells and media) or supernatant of whole broth culture (liquid broth with cells removed by centrifugation, filtration or other of the many available methods) or precipitants having the antimicrobial LCI protein or an extraction of the protein or granular formulas.
  • the application method is not limited by manner of producing or combining the strain with other components and one skilled in the art appreciates many variables exist.
  • the strain or supernatant or cell suspension or the like may also be combined with other compositions of interest, and provided in a convenient form, such as a formula, liquid, granule, suspension, microencapsulation or powder, for example.
  • liquid compositions may include water, a saline solution or other aqueous solution.
  • inactive additives, plant health enhancing compositions, stabilizers or other components desired may be added.
  • the strain or any compositions combined with the strain may be applied in any advantageous manner, and may, for example, be applied prior to pathogen infection, at the time of infection, after infection, or as a seed treatment, and in multiple treatments.
  • Application can also include, for example, applying to the soil in contact with the plant or plant part.
  • the concentration of the strain may need to be determined such that it does not affect seedling germination.
  • the strain may be applied in a convenient strength to the seedling or in the soil.
  • An effective amount of the strain is an amount sufficient to reduce adverse impact of a pathogen compared to a plant to which the strain has not been applied.
  • the concentration of the strain may be dictated by economical considerations. For example, concentrations of 10 7 CFU cm "3 would be effective, yet costs may indicate a range of up to 10 6 CFU cm " .
  • economic considerations may also impact a concentration.
  • an inhibition rate of about 15% was observed when the concentration of purified antimicrobial substance was 15 ⁇ g/ml and an inhibition of almost 100% was observed with a concentration of 75 ⁇ g/ml.
  • kits for the control, prevention or treatment of plant disease or inducing or promoting plant growth where the kit includes the bacterial strain or protein composition and instructional material for applying the bacterial composition to control, prevent or treat a plant pathogen infection, or induce or promote plant growth as described herein.
  • the instructions for applying the disclosed bacterial composition can be any form of instruction means. Such instructions include, but are not limited to, written instruction material (e.g., a label, a booklet, a brochure, a pamphlet), oral instructional material (such as on an audio cassette or CD) or video instructions (such as on a video tape or DVD).
  • a biologically pure or isolated bacterial culture having a gene sequence possessing at least 85% sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
  • An isolated polynucleotide having SEQ ID NO: 1 and having antimicrobial or plant growth promoting activity having SEQ ID NO: 1 and having antimicrobial or plant growth promoting activity.
  • a composition comprising any of the embodiments 1-12.
  • a composition of embodiment 13 further comprising one or more other compositions.
  • a composition comprising any of the embodiments of 1-14 in a concentration up to 10 7 CFU.
  • composition comprising spores, cells, whole culture or cell suspension of any of the embodiments 1-15.
  • a composition comprising an ammonium sulfate precipitated fraction of a supernatant of a whole cell culture of Bacillus amyloliquefaciens BAC03.
  • kits for increasing plant growth comprising a bacterial culture of any of
  • kits for reducing disease severity of a plant pathogen comprising a bacterial culture of any of embodiments 1-17, and instructions for use of the culture for reducing disease severity of a plant pathogen.
  • a method of reducing adverse impact of a plant pathogen on a plant or plant part comprising applying to the plant, plant part or a soil an effective amount of any of embodiments 1-17.
  • a method of reducing adverse impact of a plant pathogen on a plant or plant part comprising applying to the plant, plant part or a soil an effective amount of a composition comprising:
  • a a polynucleotide having SEQ ID NO: 1 ;
  • a method of producing a composition for reducing adverse impact of a soil plant pathogen on a plant or plant part comprising producing a composition comprising biologically pure or isolated culture of a Bacillus amyloliquefaciens strain BAC03 deposited as ATCC No. PTA- 12890.
  • Streptomyces scabies selected from Streptomyces scabies, Streptomyces acidiscabies, Streptomyces stelliscabiei, Streptomyces aureofaciens, Streptomyces sp., Agrobacterium tumefaciens, Pseudomonas syringae, Clavibacter michiganensi, Cladosporium cucumerinum, Fusarium solani, Cryphonectria parasitica, Phytophthora capsici, Penicillium citrinum, Phytophthora cambivora, Phytophthora cinnamomi, Botrytis cinerea, Rhizactonia solani and Sclerotinia sclerotiorum.
  • a method for promoting plant growth comprising:
  • a plant growth promoting bacteria comprising a biologically pure or isolated culture of Bacillus amyloliquefaciens BAC03 deposited as ATCC NO. PTA- 12890 and mutants or variants thereof.
  • BAC03 A bacterial strain, designated BAC03, was isolated from a Michigan potato field that is suppressive to potato common scab.
  • Bacillus subtilis QST713 was obtained from the commercial biocontrol product SERENADETM (AgraQuest Inc. Davis, CA, USA) as a positive control.
  • Bacillus amyloliquefaciens FZB42 was obtained from the Bacillus Genetic Stock Center (Columbus, OH, USA). All other microorganisms used in this study are listed in Table 1.
  • BAC03 was identified based on sequence analysis of selected genes. BAC03 cultured in 200 ⁇ tryptic soy broth (TSB, EMB Chemical Inc., Gibbstown, NJ, USA) for 24 hours was used for DNA extraction. The FastDNA Spin Kit (MP Biomedicals, Solon, OH, USA) was used following the manufacturer's instructions. Polymerase chain reaction (PCR) was conducted to amplify fragments of the 16S rDNA, recA and recN (DNA repair and recombination proteins), cheA (a histidine kinase), and gyrA (gyrase subunit A) genes with primers listed in Table 1.
  • TAB tryptic soy broth
  • recA and recN DNA repair and recombination proteins
  • cheA a histidine kinase
  • gyrA gyrase subunit A
  • Primers for gene cheA and gyrA were designed based on the consensus sequence of these two genes retrieved from several B. amyloliquefaciens strains in National Center for Biotechnology Information (NCBI) database, and validated with DNAMAN software (Lynnon Corporation, Quebec, Canada).
  • Each PCR reaction had a total volume of 25 ⁇ , containing 5 U Taq DNA polymerase, IX Taq polymerase PCR buffer (Promega, Madison, WI, USA), 200 mM dNTP mixture, 0.2 mM of each primer, and 1 ⁇ (2 to 25 ng) of template DNA.
  • Thermocycler (Bio-Rad Scientific Inc, Hercules, CA, USA) settings were as follows: an initial denaturation for 5 min at 94°C, followed by 36 cycles of 40 s at 94°C, 40 s at 58°C, and 1.5 min at 72°C; and extension for 7 min at 72°C.
  • the PCR products were purified with a PCR purification kit (Denville Scientific Inc, Metuchen, NJ, USA), and sequenced at the Michigan State University Genomic Technology Support Facility (East Lansing, MI, USA). The sequences were analyzed using the BLAST algorithm against the GenBank database.
  • subtilis QST713 which were cultured for 24 hours in TSB, were placed as a drop on a sterile filter paper disk (5 mm diam., Whatman #1, Piscataway, NJ, USA), which was placed on the agar medium with 2 disks per plate, at 2 cm distance from each other. After the plates were incubated at 28°C in darkness for 3 days, the diameter of the inhibition zone (if any) was measured with a ruler. Each isolate was replicated four times. The experiment was conducted twice.
  • BAC03 Bacillus subtilis
  • a dual culture method was used. Briefly, one mycelial plug (5 mm diam.) were cut from freshly growing cultures using a sterile cork borer (5 mm diam), and transferred to approximately the center of a potato dextrose agar (PDA, EMB Chemical Inc., Gibbstown, NJ, USA) plate. Two paper discs (5 mm diam.) were placed on either side of the plug at 2 cm distance from the plug. An aliquot of 15 ⁇ of BAC03 or QST713 was added to each filter paper disc using a pipette. The plates were incubated at 22°C for 3 to 4 days. The inhibition zone was measured as described above.
  • PDA potato dextrose agar
  • the potato seedlings were transferred to a 3.78 L plastic pot with potting mix, infested with S. scabies by pouring the inoculum to the top of the potting mix at a final concentration of 10 6 colony forming unit (CFU)/cm 3 .
  • Liquid culture of B AC03 was added to the potting mix as a drench to give a final concentration of 10 5 (treatment B l) and 10 6 (treatment B2) CFU/cm 3 .
  • Treatment with BAC03 was done two times; once when seedlings were transplanted and a second 20 days later.
  • a positive control using S. scabies inoculum only (S), and a negative control of non-infested potting mix mixed with tryptic soy broth at a level similar to the amount added with B2 (CK) were used.
  • S. scabies inoculum only (S) S. scabies inoculum only
  • CK non-infested potting mix mixed with tryptic soy broth at a level similar to the amount added with B2
  • Radish cv. "Cherry Belle”, Burpee Inc. Warminster, PA, USA
  • seeds were pre- germinated in a Petri dish (VWR International, LLC, Radnor, PA, USA) with moist filter paper overnight at 25 °C. After germination, seedlings were sown in a 1 L pot containing autoclaved potting mix infested with S. scabies (10 7 CFU/cm 3 ), in a growth chamber (23°C and 14 h light).
  • BAC03 was applied (10 5 CFU/cm 3 for each time) as the following treatments: bO: non-treated; bl : 1 time after planting (TAP); b2: 2 TAPs; b3: 3 TAPs; and b4: 1 time before planting and 3 TAP.
  • TAP time after planting
  • b2 2 TAPs
  • b3 3 TAPs
  • b4 1 time before planting and 3 TAP.
  • the intervals between BAC03 applications were one week.
  • Reactions were prepared in triplicate in 96-well optical plates using the following master mix: 10 ⁇ of ABI SYBR Green PCR master mix (2 X) (Applied Biosystems, Carlsbad, CA, USA), 2 ⁇ of 1/10 diluted potting mix DNA, 0.5 ⁇ of each primer (10 ⁇ ), and 7 ⁇ of sterile ddH 2 0. External standards for quantification consisted of six
  • the potting mix in the pots was covered with a plastic film to avoid contamination from foliar spray.
  • S. scabies (10 6 CFU/cm 3 potting mix) was slowly poured onto the surface of potting mix evenly 5 days after transplanting, and repeated two weeks later. A negative control of inoculation with S. scabies only, and 5 replicates were used for each treatment. Potato tubers were harvested after 3 months and the lesion severity was rated as above.
  • BAC03 was grown in 200 ml of TSB at 28°C on a shaker incubator at 180 rpm for 48 h. The culture was centrifuged at 15,000 g for 20 min at 4°C and the supernatant was collected for all extractions.
  • BAC03 culture supernatant was extracted using ammonium sulfate precipitation as described by Sutyak et al. (2008) with a little modification as follows. Eighty percent ammonium sulfate was added to the supernatant, and incubated at 4°C overnight. The mixture was centrifuged at 15,000 g for 30 min at 4°C, and the supernatant was discarded. The precipitate was dissolved in 10 mM sodium phosphate buffer (pH 6.0), and applied to a Sephadex G-50 column (Pure Biotech LLC, Middlesex, NJ, USA) and centrifuged at 2000 g for 3 min.
  • the eluted fluid was passed through a 0.22 ⁇ filter membrane (Millipore, Billerica, MA, USA), and the antimicrobial activity of this substance against S. scabies was tested by the agar diffusion assay as described above.
  • This extract was designated as ammonium sulfate precipitated material (AS) for the rest of the study.
  • AS ammonium sulfate precipitated material
  • the material from the ammonium sulfate precipitation produced larger inhibition zones (> 2 cm) and thus further study concentrated on this material.
  • the lipopeptide fraction showed low activity ( ⁇ 0.5 cm) and was not examined further.
  • the concentration of protein in the AS was determined using a Bradford Protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) with a
  • Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) based on a standard curve constructed for colorimetric absorbance at 595nm.
  • Both BAC03 liquid culture and its derived AS fraction were examined for their reaction to various factors, including temperatures, pH levels, enzymes, and chemicals.
  • temperature the antimicrobial activity was assessed after being incubated at temperatures of 40, 60, 80, and 100°C for 30 min, or 121°C (autoclaving) for 15 min.
  • pH was adjusted from 1.0 to 14.0 by whole pH units using sterile 1 N HC1 or 1 N NaOH. Material was incubated at these pH levels overnight at 4°C. Prior to assessing activity, the pH was readjusted to pH 7.0.
  • Enzymes were tested for their effect on activity by incubating the BAC03 culture or the AS fraction with 10 mg/ml of the following enzymes for 2 h at the optimal temperature for each enzyme according to the manufacturer's instruction. Enzymes were trypsin (MP Biomedicals, Solon, OH, USA, 25°C, pH 7.6); proteinase K (Sigma-Aldrich, Inc. St Louis, MO, 37°C, pH 7.5); pepsin (Sigma- Aldrich, Inc., 37°C, pH 2.0); a-chymotrypsin (Sigma-Aldrich, Inc., 25°C, pH 7.8); and catalase (MP Biomedicals, 25°C, pH 7.0). Effect of various chemical solvents (Table 3) was tested by incubating 5 h at 25°C with 10% organic solvents. Antimicrobial activities of the treated culture and the AS fraction were tested against S. scabies using the disc diffusion assay as described above.
  • the AS fraction was obtained by dodecyl sulfate-polyacrylamide gel
  • SDS PAGE electrophoresis
  • the gel was vertically cut into two parts. One part of the gel, containing the sample and molecular weight standards, was stained with Coomassie Blue protein stain. The other part, containing the same sample, was tested for antimicrobial activity using the method of Naclerio et al. (1993). Briefly, the gel was treated with 20% isopropanol-10 mM Tris-HCl (pH 7.5) for 3 h, rinsed for 1.5 h in 10 mM Tris-HCl (pH 7.5), and washed twice with distilled water for a total of 1 h.
  • the gel was placed in a Petri dish with 1% water agar, and overlaid with 0.8% YME mixed with a bacterial suspension of S. scabies (10 7 CFU/ml).
  • the plate was incubated at 28°C for 48 h, then dyed with thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) to stain living organisms, and observed for any unstained zone(s).
  • MTT thiazolyl blue tetrazolium bromide
  • LC/MS/MS chromatography-mass spectrometry
  • the 16s rDNA sequence of BAC03 had a similarity higher than 99% with both B. subtilis and B. amyloliquefaciens. However, based on fragment sequences of recA (SEQ ID NO. 2), recN (SEQ ID NO. 3), cheA (SEQ ID NO. 4) and gyrA sequences (SEQ ID NO. 5), the results showed the highest similarity between strain BAC03 and B. amyloliquefaciens. Antimicrobial activity of BAC03
  • z LB Lysogeny broth medium
  • PDA potato dextrose agar
  • 523 bacterial screening medium (SigmaAldrich, Inc. St Louis, MO, USA).
  • Table 2 shows that BAC03 displays antagonistic activity against a broad spectrum of bacteria, fungi, and oomycetes. BAC03 showed significantly greater inhibition zones (P ⁇ 0.05) than QST713 on 13 out of 21 test organisms.
  • Foliar spray with BAC03 did not affect potato growth or disease severity; sprayed plants had disease severity average ratings of 0.76 ⁇ 0.16, and tuber weight of 48.04 + 2.09 g/pot; non-treated plants had disease severity of 0.75 + 0.17 and tuber weight of 47.82 + 1.55 g/pot.
  • BAC03 Effects of BAC03 on Plant Emergence [0073] Emergence of potato and radish reacted differently to BAC03 treatment. BAC03 did not affect the amount of emergence of potato seedlings, regardless of concentrations. However for radish, only 20 + 5.8 % plants emerged during the course of experiment when treated with BAC03 culture at 10 4 CFU/cm 3 potting mix, compared to 100% radish emerged for the control. No radish emergence was visible following treatments with BAC03 at 10 5 and 10 6 CFU/cm 3 potting mix.
  • the AS fraction was picked for further study.
  • the AS fraction significantly reduced the S. scabies population when evaluated by the co- culturing method, and the antimicrobial effect was positively correlated with the AS concentration (Fig. 3 A).
  • the inhibition was almost 100% when the AS concentration was 75 ⁇ g/ml.
  • a reduction in the number of S. scabies colonies following the removal of the AS fraction was observed after co-incubation for different times, and the antimicrobial effect was enhanced with increasing exposure time to the AS fraction (Fig. 3B).
  • the culture broth and ammonium sulfate precipitate (AS) were exposed to various treatments, including:chemical solvent (10%) for 5 h at 25°C temperatures of 40, 60, 80, and 100°C for 30 min, and 121°C (autoclaving) for 15 minpH levels from 1.0 to 14.0 (every single unit) using sterile 1 N HQ or 1 N NaOH, and was readjusted to pH 7.0 after incubation overnight at 4°C.
  • a subset of levels is shown as pH 1, 3, 5, 10, 12, 14 in the table enzymes (10 mg/ml) for 2 h, including trypsin (25°C, pH 7.6), proteinase K (37°C, pH 7.5), pepsin (37°C, pH 2.0), oc-chymotrypsin (25°C, pH 7.8), and catalase (25°C, pH 7.0).
  • Culture broth strain BAC03 was cultured in TSB for 24 hours (10 6 CFU/ml) in a shaker with 180 rpm.
  • AS crude antimicrobial substance, which was extracted by ammonium sulfate precipitation from the culture broth supernatant, and filtered through Sephadex G-50 column and 0.22 ⁇ filter membrane.
  • the AS fraction affected S. scabies sporulation.
  • the S. scabies colonies showed no color change throughout the 7 day incubation period, compared to non-treated colonies, which changed to white or whitish grey.
  • the morphology of the mycelia observed by SEM displayed spiral spore chain formation on non- treated plates but no such structure formed on AS-treated cultures during the time period examined (Fig. 4).
  • the sequence similarity of the amplified putative LCI was 99% between strains BAC03 and B. amyloliquefaciens FZB42 (accession number: CP000560.1) or C-31 (accession number: FJ904931.1).
  • the DNA sequence and putative translated protein sequence were compared to those of reported B. amyloliquefaciens strains. All of the strains had a putative protein of 94 amino acids.
  • the LCI protein sequence for BAC03 was 100% identical with strain C31 (Hu et al. 2010), but had one amino acid different from FZB42. [0078]
  • a reduction in scab severity was observed in both potato and radish, where the population of S. scabies was significantly suppressed by BAC03, and suppression was increased as the time of application was increased.
  • the experiment is repeated with measurement including shoot length, fresh and dry shoot weight, fresh and dry root weight and compared to other strains of biocontrol product.
  • Table 4 Effect of BAC on growth of seven plant types. Two applications of BAC03 (10 s CFU/cm "3 ) were drenched into the soil after plant germination and two weeks later. The same volume of media was used for control.
  • BAC03 The ability of BAC03 to induce systemic resistance is determined by a split root system.
  • potato roots of a plant were split into two parts, A and B, and each part placed in a separate pot when the root system was well developed.
  • Four groups were created including: Group I, roots inoculated with BAC3 and S. scabies in root part A and with S. scabies in root part B; Group II, root inoculated with BAC03 in root part A and S. scabies in root part B; Group III, roots inoculated with S. scabies in root part A and non- treated in root part B; and Group IV, roots not treated in both root parts A and B.
  • Example 3 The experiment of Example 3 may be repeated using radish, pepper or tomato. Briefly, the plant roots may be split and placed into two separate pots infested with a pathogen. To one of the two pots may be applied the Bacillus solution and disease suppression may be evaluated. Assessing of disease suppression may be done by evaluating plant defense-related enzymes such as peroxidase (POX), polyphenol oxidase (PPO) or phenylalanine ammonia lyase (PAL). The plant growth promoting effect of the bacteria may also be evaluated by assaying for the production of indoleacetic acid (IAA), NH 3 , or HCN, and presence of phosphate solubilization and ACC-deaminase. These secreted substances or reactions are correlated with the plant growth promotion.
  • POX peroxidase
  • PPO polyphenol oxidase
  • PAL phenylalanine ammonia lyase
  • IAA indoleacetic acid

Abstract

Une souche BAC03 inédite de bacillus amyloliquefaciens est isolée et caractérisée. Ladite souche BAC03 protège les plantes contre les organismes pathogènes des plantes présents dans le sol et favorise la croissance des plantes. L'invention concerne également des méthodes d'utilisation de ladite souche.
PCT/US2013/032246 2012-04-30 2013-03-15 Souche bac03 inédite de bacillus amyloliquefaciens et ses méthodes d'utilisation WO2013165607A1 (fr)

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