WO2025038470A2 - Compositions et procédés de lutte biologique - Google Patents

Compositions et procédés de lutte biologique Download PDF

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Publication number
WO2025038470A2
WO2025038470A2 PCT/US2024/041770 US2024041770W WO2025038470A2 WO 2025038470 A2 WO2025038470 A2 WO 2025038470A2 US 2024041770 W US2024041770 W US 2024041770W WO 2025038470 A2 WO2025038470 A2 WO 2025038470A2
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WIPO (PCT)
Prior art keywords
plant
bacillus amyloliquefaciens
disease
formulation
contacting
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US2024/041770
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English (en)
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WO2025038470A9 (fr
WO2025038470A3 (fr
Inventor
Robert Mcbride
Karen Hunt
Jamie Bacher
Veronica Garcia
Safira SUTTON
James Pearce
Jia Jie LI
Kelly TRINIDAD
Adam FREEDMAN
William ESTELL
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Kureha America Inc
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Kureha America Inc
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Publication of WO2025038470A2 publication Critical patent/WO2025038470A2/fr
Publication of WO2025038470A3 publication Critical patent/WO2025038470A3/fr
Publication of WO2025038470A9 publication Critical patent/WO2025038470A9/fr
Priority to MX2026001677A priority Critical patent/MX2026001677A/es
Priority to CONC2026/0001515A priority patent/CO2026001515A2/es
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof

Definitions

  • pathogens on plants may be a problem. For example, they can cause diseases or other issues with the plants themselves, or with produce. Accordingly, there exists a need for improved methods and compositions to combat plant pathogens.
  • An aspect of the present disclosure is a method comprising contacting a plant, a seed, or soil comprising a plant, with a formulation including (i) a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1, and/or (ii) with one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • the formulation may have a pH in a range from about 6 to about 11.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, or the soil comprising the plant, wherein the one or more pathogenic organisms is selected from the group consisting of a fungus, a bacterium, and a nematode.
  • a further aspect of the present disclosure is a formulation comprising a Bacillus amyloliquefaciens spore comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1, wherein the formulation has a pH in a range from about 6 to about 11 and comprises viable Bacillus amyloliquefaciens after storage at a temperature of from about 18°C to about 25 °C or at a temperature of up to about 50°C for at least 14 days.
  • An additional aspect of the present disclosure is a method for treating a disease in a plant, a seed, or soil comprising a plant.
  • the method comprises steps of: obtaining a plant, a seed, or soil comprising a plant having a disease; and contacting the plant, the seed, or soil comprising the plant, with a formulation that includes (i) Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1, and/or (ii) with one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • the formulation may have a pH in a range from about 6 to about 11.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, or the soil comprising the plant and responsible for the disease; and the disease is Asian soybean rust, rice blast, brown spot, narrow brown spot, crown rot, sour rot (citrus, post-harvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), citrus variegated chlorosis, Black rot, soybean cysts, or root knots.
  • the disease is Asian soybean rust, rice blast, brown spot, narrow brown spot, crown rot, sour rot (citrus, post-harvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), citrus variegated chlorosis, Black rot, soybean cysts, or root knots.
  • Another aspect of the present disclosure is a method for preventing or reducing severity of a disease in a plant, a seed, or soil comprising a plant.
  • the method comprises steps of: obtaining a plant, a seed, or soil comprising a plant at risk for contracting a disease and contacting the plant, the seed, or soil comprising the plant, with a formulation including (i) a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1, and/or (ii) with one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • the formulation may have a pH in a range from about 6 to about 11.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, or the soil comprising the plant and responsible for the disease; and the disease is Asian soybean rust, rice blast, brown spot, narrow brown spot, crown rot, sour rot (citrus, post-harvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), citrus variegated chlorosis, Black rot, soybean cysts, or root knots.
  • the disease is Asian soybean rust, rice blast, brown spot, narrow brown spot, crown rot, sour rot (citrus, post-harvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), citrus variegated chlorosis, Black rot, soybean cysts, or root knots.
  • FIG. 1 shows the sequence alignments results of gene clusters in the BC17B genome as compared to known Bacillus genome sequences in accordance with one or more embodiments.
  • FIG. 2 shows digital images of the inhibition of Xanthomonas campestris on R2A medium by BC17B as compared to control cells.
  • FIG. 3A and FIG. 3B show the tabulated average Xanthomonas campestris-xelated disease severity scores for disc 1 and disc 2, respectively.
  • FIG. 4 A shows the degree of blackspot control for the three experimental conditions at 25 days post-treatment.
  • FIG. 4B show digital images comparing the appearance of PBS-treated lettuce
  • FIG. 5 is a graph showing the degree of inhibition of wound size and weight achieved by BC17B in peaches with wounds infected by Botrytis cinerea.
  • FIG. 6 is a graph showing the incidence of blossom blight in blueberries treated with BC17B.
  • FIG. 7A is a graph showing the incidence of Botrytis on grape clusters treated with BC17B.
  • FIG. 7B is a graph showing the severity of Botrytis on grape clusters treated with BC17B.
  • FIG. 7C is a graph showing the disease index of Botrytis for grape clusters treated with BC17B.
  • FIG. 8A is a graph showing the incidence of Downey Mildew on grape leaves treated with BC17B.
  • FIG. 8B is a graph showing the severity of Downey Mildew on grape leaves treated with BC17B.
  • FIG. 8C is a graph showing the disease index of Downey Mildew for grape leaves treated with BC17B.
  • FIG. 9A is a graph showing the incidence of Powdery Mildew on grape clusters treated with BC17B.
  • FIG. 9B is a graph showing the severity of Powdery Mildew on grape clusters treated with BC17B.
  • FIG. 9C is a graph showing the disease index of Powdery Mildew for grape clusters treated with BC17B.
  • FIG. 10A is a graph showing the incidence of Powdery Mildew on grape leaves treated with BC17B.
  • FIG. 10B is a graph showing the severity of Powdery Mildew on grape leaves treated with BC17B.
  • FIG. 10C is a graph showing the disease index of Powdery Mildew for grape leaves treated with BC17B.
  • FIG. 11 is a graph showing percent (%) disease reduction for cucumbers inoculated with downey mildew spores and treated with BC17B.
  • FIG. 12 show digital images of the inhibition of Monilinia fructicola by BC17B.
  • FIG. 13 A is a graph showing inhibition of Panama Wilt psuedostem disease caused by Fusarium oxysporum for Cavendish bananas treated with Bacillus amyloliquefaciens strain BC17B or Propiconazole chemical fungicide.
  • FIG. 13B includes digital images of whole plants and pseudostems of Cavendish bananas treated with Bacillus amyloliquefaciens strain BC17B and grown in soil infested with Fusarium oxysporum.
  • FIG. 14A includes graphical data showing BC17B colonization in banana plants.
  • FIG. 14B includes digital images of banana root, stem, and leaves, and plates from which data were generated.
  • FIG. 15 is a graph showing Bacillus amyloliquefaciens strain BC17B protects blueberry plants against the endophytic bacterial pathogen Xylella fastidiosa, where disease was rated based on percent of symptomatic and asymptomatic leaves per plant.
  • FIG. 16 shows weekly disease severity for Pierce’s Disease in Grenache grape vines treated with Bacillus amyloliquefacens BC17B.
  • FIG. 17A is a graph showing incidence of Leaf Scorch in almond tree leaves, Xylella fastidiosa (Xfa): infected (with Breakthru® adjuvant, Xfa, Brk); infected and treated (Xfa, BC17B, Brk), uninfected but treated with adjuvant as a control (Brk) or uninfected, untreated.
  • BC17B and Breakthru® were applied by foliar spray.
  • FIG. 17B is a graph showing incidence of Leaf Scorch in almond tree leaves, Xylella fastidiosa subsp. multiplex (Xfa mpx): infected (with Breakthru® adjuvant, Xfa mpx, Brk); infected and treated (Xfa mpx, BC17B, Brk), uninfected but treated with adjuvant as a control (Brk) or uninfected, untreated. BC17B and Breakthru® were applied by foliar spray.
  • FIG. 17C is a graph showing severity of Leaf Scorch in almond tree leaves, Xylella fastidiosa (Xfa): infected (with Breakthru®adjuvant, Xfa, Brk); infected and treated (Xfa, BC17B, Brk), uninfected but treated with adjuvant as a control (Brk) or uninfected, untreated.
  • BC17B and Breakthru® were applied by foliar spray.
  • FIG. 17D is a graph showing severity of Leaf Scorch in almond tree leaves, Xylella fastidiosa subsp. multiplex (Xfa mpx): infected (with Breakthru® adjuvant, Xfa mpx, Brk); infected and treated (Xfa mpx, BC17B, Brk), uninfected but treated with adjuvant as a control (Brk) or uninfected, untreated. BC17B and Breakthru® were applied by foliar spray.
  • FIG. 17E is a graph showing incidence of Leaf Scorch in almond tree leaves, Xylella fastidiosa (Xfa): infected (Xfa); infected and treated (Xfa, BC17B), or uninfected, untreated. BC17B was applied by injection.
  • Xfa Xylella fastidiosa
  • FIG. 17F is a graph showing incidence of Leaf Scorch in almond tree leaves, Xylella fastidiosa subsp. multiplex (Xfa mpx): infected (Xfa mpx); infected and treated (Xfa mpx, BC17B), or uninfected, untreated. BC17B was applied by injection.
  • FIG. 17G is a graph showing severity of Leaf Scorch in almond tree leaves, Xylella fastidiosa (Xfa): infected (Xfa); infected and treated (Xfa, BC17B), or uninfected, untreated. BC17B was applied by injection.
  • Xfa Xylella fastidiosa
  • FIG. 17H is a graph showing severity of Leaf Scorch in almond tree leaves, Xylella fastidiosa subsp. multiplex (Xfa mpx): infected (Xfa mpx); infected and treated (Xfa mpx, BC17B), or uninfected, untreated. BC17B was applied by injection.
  • FIG. 18A is a graph showing the number of colony forming unit per gram (CFU/g) of Bacillus amyloliquefaciens BC17B that persisted epiphytically on the stem tissue of almond trees three months after contact.
  • FIG. 18B is a graph showing the number of CFU/g of Bacillus amyloliquefaciens
  • FIG. 19 is a graph showing reduction of nematode load in soil treated with Bacillus amyloliquefaciens strain BC17B.
  • FIG. 20 is a graph showing long lasting protection of roots from root knot nematodes in cucumber plants treated with Bacillus amyloliquefaciens strain BC17B.
  • FIG. 21 is a graph showing the disease index of rice blast for rice treated with Bacillus amyloliquefaciens strain BC17B.
  • FIG. 22 is a graph showing the disease index of narrow brown spot for rice treated with Bacillus amyloliquefaciens strain BC17B.
  • FIG. 23 is a graph showing the disease index of brown spot for rice treated with Bacillus amyloliquefaciens strain BC17B.
  • FIG. 24 is a graph showing the disease index of sheath blight for rice treated with Bacillus amyloliquefaciens strain BC17B.
  • FIG. 25 A includes graphs showing the mycelial mass diameter and Mycosphaerella spore count in banana plants treated with Bacillus amyloliquefaciens strain BC17B, or a PBS control.
  • FIG. 25B is a graph showing percent Mycosphaerella in BC17B-treated plants relative to a PBS -treated control.
  • FIG. 26A shows the efficacy of different treatments: BC17B; Serenade®, a biological control product; and Tilt®, a chemical control product, at controlling Black Sigatoka on bananas in greenhouses.
  • FIG. 26B shows the standard area under the disease progression curve for different treatments: BC17B; Serenade®, a biological control product; and Tilt®, a chemical control product, at controlling Black Sigatoka on bananas in greenhouses.
  • FIG. 27 A is a graph showing disease severity in Xylellafastidiosa infection in olive trees treated with BC17B and Breakthru® surfactant.
  • FIG. 27B is a graph showing disease incidence in Xylella fastidiosa infection in olive trees treated with Bacillus amyloliquefaciens BC17B and Breakthru® surfactant.
  • FIG. 28A shows the average severity scores for evaluation of internal symptoms of Panama Wilt caused by Fusarium oxysporum.
  • FIG. 28B shows representative images of cross-sections of banana plants treated with Bacillus amyloliquefaciens BC 17B (left) or water (right).
  • FIG. 29 shows digital images of the inhibition of Penicillium digitatum, the causative agent of clear rot in citrus, by Bacillus amyloliquefaciens BC17B.
  • FIG. 30 is a graph showing the disease rating of soybean plants infected with Asian Soybean Rust, untreated and treated with Proline or BC17B.
  • FIG. 31 A is a graph showing the stability of BC17B spores over two-week incubation period at 54°C.
  • FIG. 3 IB is a digital image of the retained efficacy of BC17B at inhibiting Botrytis cinerea in a standard in vitro confrontation assay after two weeks incubation at 54°C.
  • compositions and methods for reducing growth of or killing pathogenic organisms are capable of reducing growth or killing pathogenic organism(s) that effect plants of agricultural importance.
  • Compositions and methods disclosed herein can be formulated or adjusted to be used at various points in the production process.
  • compositions can be formulated for use prior to harvest, for example by incorporating the composition into an irrigation line or administration in combination with a fertilizer.
  • Compositions can also be formulated for use post-harvest and/or during processing, packaging, transportation, storage, and commercial display of the produce.
  • Compositions for post-harvest may be sprayed on the harvested produce or used by applying the composition to a packaging material that is used to store or ship the produce.
  • these compositions can show improved efficacy when compared to commercial biocontrol compositions.
  • embodiments disclosed herein relate to a biocontrol microbe (or “microbe”) capable of treating, reducing, or killing one or more plant pathogens.
  • a microbe of the disclosure for example, a Bacillus amyloliquefaciens BC17B, may be an engineered or recombinant microbe.
  • the microbe may comprise additional nucleic acids sequences, mutations, or other sequences deviations from a microbe found in nature.
  • the microbe may include a Bacillus amyloliquefaciens BC17B strain having a genome in accordance with Deposit Number PTA-127137 in the ATCC® Patent Depository.
  • the microbe of one or more embodiments may include a Bacillus amyloliquefaciens microbe.
  • the Bacillus amyloliquefaciens may be a Bacillus amyloliquefaciens BC17B strain (also referred to herein as “BC17”).
  • the Bacillus amyloliquefaciens BC17B strain includes a 16S ribosomal RNA (rRNA) sequence of SEQ ID NO: 1.
  • the Bacillus amyloliquefaciens microbe may include a 16S rRNA that includes a sequence in accordance with SEQ ID NO: 1.
  • the Bacillus amyloliquefaciens may include a 16S rRNA sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to SEQ ID NO: 1.
  • the Bacillus amyloliquefaciens may include a 16S rRNA sequence at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, identical to SEQ ID NO: 1.
  • the Bacillus amyloliquefaciens includes a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1. In some embodiments, the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • a Bacillus amyloliquefaciens BC17B strain of the present disclosure comprises a nucleic acid sequence having a percent sequence identity to SEQ ID NO: 1 and/or SEQ ID NO 2 in a range having a lower limit of any one of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.99%, and 100% and an upper limit of any one of 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, and 100% and an upper limit of any one
  • the microbe has one or more gene clusters that are related to the biosynthesis of one or more compounds.
  • the gene clusters are responsible for biosynthesis of known antibiotic compounds and other activities were identified.
  • the microbe may have a plurality of percent gene cluster similarities to known Bacillus genome sequences as described in FIG. 1, which shows example compounds and gene clusters related to the synthesis of these compound found in the microbes of the disclosure.
  • the microbe may include gene clusters having within a certain percentage known to Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of fengycin having a gene cluster similarity in a range having a lower limit of any one of 75%, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, and 86% similarity to known Bacillus genome sequences and an upper limit of any one of 86%, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of fengycin having a similarity of at least 75%, at least 80%, or at least 85% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of plipastatin having a gene cluster similarity in a range having a lower limit of any one of 15%, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26% similarity to known Bacillus genome sequences and an upper limit of any one of 26%, 27, 28, 29, 30, 31, 32, 33, 34, and 35% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of plipastatin having a similarity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of butirosin A and/or butirosin B having a gene cluster similarity in a range having a lower limit of any one of 0%, 1 , 2, 3, 4, 5, 6, and 7% similarity to known Bacillus genome sequences and an upper limit of any one of 7%, 7.5, 8, 9, 10, 12, 15, and 20% or greater similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of butirosin A and/or butirosin B having a similarity of at least 1%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, and at least 25% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of rhizocticin A having a gene cluster similarity in a range having a lower limit of any one of 0%, 1, 2, 3, 4, 5, 5.5, and 6% similarity to known Bacillus genome sequences and an upper limit of any one of 6.5%, 7, 7.5, 8, 9, 10, 12, 15, and 20% or greater similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of rhizocticin A having a similarity of at least 1%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, and at least 25% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of Macrolactin H having a gene cluster similarity in a range having a lower limit of any one of 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90% similarity to known Bacillus genome sequences and an upper limit of any one of 90%, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of Macrolactin H having a similarity of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of difficidin having a gene cluster similarity in a range having a lower limit of any one of 35%, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 55% similarity to known Bacillus genome sequences and an upper limit of any one of 45%, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of difficidin having a similarity of at least 35%, at least 40%, at least 45%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of bacilysin having a gene cluster similarity in a range having a lower limit of any one of 35%, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 55% similarity to known Bacillus genome sequences and an upper limit of any one of 45%, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of bacilysin having a similarity of at least 35%, at least 40%, at least 45%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of bacillibactin having a gene cluster similarity in a range having a lower limit of any one of 35%, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 55% similarity to known Bacillus genome sequences and an upper limit of any one of 45%, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of bacillibactin having a similarity of at least 35%, at least 40%, at least 45%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of surfactin having a gene cluster similarity in a range having a lower limit of any one of 35%, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 55% similarity to known Bacillus genome sequences and an upper limit of any one of 45%, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of surfactinhaving a similarity of at least 35%, at least 40%, at least 45%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to known Bacillus genome sequences.
  • the microbe of one or more embodiments may have a gene cluster for the production of bacillaene having a gene cluster similarity in a range having a lower limit of any one of 35%, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 55% similarity to known Bacillus genome sequences and an upper limit of any one of 45%, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% similarity to known Bacillus genome sequences, where any lower limit can be paired with any mathematically compatible upper limit.
  • the microbe includes a gene cluster for the production of bacillaene having a similarity of at least 35%, at least 40%, at least 45%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to known Bacillus genome sequences.
  • a microbe or a formulation including a microbe of one or more embodiments, for example, a Bacillus amyloliquefaciens BC17B strain may be capable of reducing disease by protecting a plant against a fungus, bacteria, nematodes, or insects.
  • a microbe of the disclosure e.g., a Bacillus amyloliquefaciens BC17B strain
  • a microbe of one or more embodiments, for example, a Bacillus amyloliquefaciens BC17B strain may reduce disease by broadly protecting a plant against a fungus, bacteria, nematodes, or insects.
  • a microbe of one or more embodiments, for example, a Bacillus amyloliquefaciens BC17B strain may reduce disease and act as broad-spectrum fungicide, bactericide, nematicide, or insecticide.
  • a microbe or a formulation including a microbe of one or more embodiments, for example, a Bacillus amyloliquefaciens BC17B strain may reduce disease by protecting a plant against one or more pathogenic organisms belonging to the genus of Phakopsora, Magnaporthe, Cochliobolus, Sphaerulina, Geotrichum, Microsphaera, Blumeria, Podosphaera, Peronospora, Pseudoperonospora, Xanthomonas, Xylella, Heterodera, Colletotrichum, or Meloidogyne.
  • the one or more pathogenic organisms may be Phakopsora pachyrhizi, Magnaporthe grisea, Cochliobolus miyabeanus, Sphaerulina oryzina, Geotrichum spp, Microsphaera diffusa, Blumeria graminis, Podosphaera leucotricha, Peronospora manshurica, Peronospora effusa, Peronospora belbahrii, Colletotrichim musae, Pseudoperonospora cubensis, Xylella fastidiosa, Xanthomonas campestris, Heterodera glycines, Meloidogyne hapla, Meloidogyne incognita, or Meloidogyne enterolobii.
  • a microbe or a formulation including a microbe in accordance with the present disclosure may be capable of reducing Asian soybean rust, rice blast, brown spot, narrow brown spot, sour rot (citrus, post-harvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), Black rot, soybean cysts, or root knots.
  • a microbe or a formulation including a microbe of the disclosure may reduce Asian soybean rust, rice blast, brown spot, narrow brown spot, sour rot (citrus, post-harvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), Black rot, soybean cysts, or root knots by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing Asian soybean rust disease caused by a Phakopsora e.g., Phakopsora pachyrhizi, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing rice blast disease caused by a Magnaporthe e.g., Magnaporthe grisea, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing a brown spot disease caused by a Cochliobolus e.g., Cochliobolus miyabeanus, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Cochliobolus e.g., Cochliobolus miyabeanus
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing narrow brown spot disease caused by a Sphaerulina e.g., Sphaerulina oryzina, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefacien .
  • a Sphaerulina e.g., Sphaerulina oryzina
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing a sour rot (citrus, post-harvest) disease caused by a Geotrichum e.g., Geotrichum spp., by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Geotrichum e.g., Geotrichum spp.
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing olive quick decline syndrome, Pierce’s disease (grapes), citrus variegated chlorosis, or leaf scorch (almonds, coffee) caused by a Xylella e.g., Xylella fastidiosa, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing black rot disease caused by a Xanthomonas e.g., Xanthomonas campestris, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Xanthomonas e.g., Xanthomonas campestris
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing soybean cyst disease caused by a Heterodera e.g., Heterodera glycines, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Heterodera e.g., Heterodera glycines
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing root knot disease caused by a Meloidogyne, e.g., Meloidogyne hapla, Meloidogyne incognita, or Meloidogyne enterolobii, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Meloidogyne e.g., Meloidogy
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing powdery mildew disease caused by a Microsphaera, e.g., Microsphaera diffusa, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Microsphaera e.g., Microsphaera diffusa
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing powdery mildew disease caused by a Blumeria, e.g., Blumeria graminis, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the formulation or the Bacillus amyloliquefacien .
  • a Blumeria e.g., Blumeria graminis
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing powdery mildew disease caused by a Podosphaera, e.g., Podosphaera leucotricha, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Podosphaera e.g., Podosphaera leucotricha
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing downy mildew disease caused by a Peronospora, e.g., Podosphaera leucotricha, Peronospora manshurica, Peronospora effusa, or Peronospora belbahrii, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Peronospora e.g.,
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing downy mildew disease caused by a Pseudoperonospora, e.g., Pseudoperonospora cubensis, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Pseudoperonospora e.g., Pseudoperonospora cuben
  • a Bacillus amyloliquefaciens BC17B strain or a formulation including the BC17B strain of the present disclosure may be capable of reducing crown rot caused by a Colletotrichum, e.g., Colletotrichim musae, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, relative to a control plant having a disease and not contacted with the Bacillus amyloliquefaciens.
  • a Colletotrichum e.g., Colletotrichim musae
  • biocontrol microbe may be as described above.
  • the microbe of one or more embodiments can be grown in a culture.
  • the microbe can be isolated and purified from the culture.
  • the microbe purified from the culture may include a vegetative cell, a spore of the microbe, or any combinations thereof.
  • the culture used to grow the microbe may include a solid medium or a semi-solid medium.
  • the culture used to grow the microbe may include a liquid medium.
  • the culture can include a bioreactor. Any suitable bioreactor can be used to grow the microbe. Examples of bioreactors include, but are not limited to a flask, continuously stirred tank bioreactor (CSTR), a bubbleless bioreactor, an airlift reactor, and a membrane bioreactor.
  • CSTR continuously stirred tank bioreactor
  • a supernatant of the culture can be collected.
  • the supernatant may include one or more molecules synthesized by the microbe.
  • the one or more molecules synthesized by the microbe can be isolated or purified from the supernatant, or otherwise enriched to generate a solution including the one or more molecules.
  • the supernatant, the isolated one or more molecules, the purified one or more molecules, the enriched solution including one or more molecules, or any combination thereof may be added to a formulation to produce a biocontrol composition in accordance with one or more embodiments.
  • the one or more molecules may be isolated and/or purified from the supernatant and the purified and/or isolated molecules may be applied to a plant, e.g., as a fraction isolated or purified from the liquid and including the one or more molecules.
  • the one or more molecules may be enriched, purified, or isolated such that the concentration of the one or more molecules may be increased.
  • the one or more molecules may be enriched, purified, or isolated such that the number of different molecules in a solution is reduced.
  • the supernatant can be applied as the formulation as described elsewhere herein.
  • the one or more molecules may include a molecule selected from the group consisting of lipopeptides, polyketides, peptides, dipeptides, polypeptides, cyclic polypeptides, polyenes, aminoglycosides, surfactants, siderophores, and combinations thereof.
  • the lipopeptides may include one or more selected from the group consisting of fengycin, plipastatin, and surfacticin.
  • the polyketides may include one or more selected from macrolactin H, and difficidin.
  • the peptide compounds may include one or more selected from bacilysin, and rhizocticin A.
  • the aminoglycoside compounds may include one or more selected from butirosin A, and butirosin B.
  • the surfactant compound includes surfactin.
  • the siderophore may be a bacillibactin compound.
  • the one or more molecules (e.g., one or more small molecules) synthesized by the microbe may have antibiotic, anti-fungal, anti-nematodal, or anti-insect properties.
  • the one or more molecules may include a molecule with previously identified antibacterial properties.
  • compositions disclosed herein relate to a biocontrol composition (or “formulation”).
  • the composition of one or more embodiments may include a Bacillus amyloliquefaciens microbe, one or more molecules synthesized by the Bacillus amyloliquefaciens microbe, or any combination thereof.
  • the Bacillus amyloliquefaciens microbe and the one or more molecules synthesized by the Bacillus amyloliquefaciens may be as previously described.
  • An additional embodiment of the present disclosure is a formulation including a Bacillus amyloliquefaciens spore that includes a nucleic acid comprising a sequence that is a certain percentage (for example, at least 99.8%) identical to SEQ ID NO: 1.
  • the formulation may include viable Bacillus amyloliquefaciens after storage at a temperature in a particular range and a particular number of days, such as from about 18°C to about 25°C or at a temperature of up to about 50°C for at least 14 days, the stable formulation comprises viable Bacillus amyloliquefaciens after storage at a temperature of up to about 50°C for 14 days.
  • the microbe is present as part of a formulation.
  • the microbe may be included in a biocontrol composition that is a stable formulation, such as a liquid formulation or a dry formulation.
  • the liquid formulation may be a flowable or aqueous suspension.
  • the liquid formulation may include the microbe, a secondary metabolite thereof, or both suspended in water, oil, or a combination thereof (e.g., in an emulsion).
  • a dry formulation may include a wettable powder, a dry flake, a dust, or a granule.
  • a wettable powder can be applied to the plant, the seed, the flower, or the produce thereof as a suspension.
  • a dust can be applied to the plant, the seed, or the produce thereof dry, such as to seeds or foliage.
  • a granule can be applied dry or can be mixed with an aqueous fluid (e.g., water) to create a suspension or dissolved in the aqueous fluid to make a solution.
  • the microbe, a secondary metabolite thereof, or any combination thereof can be formulated as a microencapsulation.
  • the microbe, a secondary metabolite thereof, or any combination thereof has a protective inert layer.
  • the protective inert layer can comprise any suitable polymer.
  • the formulation includes spores.
  • the formulation may include Bacillus amyloliquefaciens spores.
  • Spore-containing compositions can be applied to treat a plant pathogen by one or more methods described herein. Spore-containing compositions according to one or more embodiments herein may extend the shelf life of the formulation.
  • the formulation includes vegetative cells.
  • the formulation may include Bacillus amyloliquefaciens vegetative cells.
  • Vegetative cellcontaining compositions can be applied to treat a plant pathogen by methods described herein.
  • Vegetative cells may proliferate and increase the efficacy of the biocontrol composition in accordance with one or more embodiments.
  • vegetative cells in the formulation may be capable of proliferating after treatment application, increasing the surface area of the plant that is exposed to the formulation.
  • vegetative cells in the formulation may be capable of proliferating after application, thereby increasing the amount of the survival time of the formulation, and thus, extending the time the formulation has efficacy.
  • the vegetative cells may proliferate and compete for nutrients with a pathogen.
  • the vegetative cells of one or more embodiments may be capable of actively producing one or more secondary metabolites with anti-pathogen properties.
  • vegetative cells in a formulation may become spores, allowing them any advantages of spores.
  • the formulation may include an additional compound.
  • the additional compound may include one or more selected from the group consisting of a buffer salt, carrier, a surfactant, a wetting agent, a penetrant, an emulsifier, a spreader, a sticker, a stabilizer, a nutrient, a binder, a desiccant, a thickener, a dispersant, a UV protectant, and combinations thereof.
  • the carrier may be a liquid carrier, a mineral carrier, or an organic carrier.
  • the additional compound may include a surfactant that is an agricultural adjuvant.
  • the surfactant may include an organo-modified siloxane.
  • the surfactant can include non-ionic surfactants.
  • the surfactant may be commercially available, such as a Breakthru® surfactant available from Evonik.
  • the surfactant is included in the formulation in an amount in a range having a lower limit of any one of a non-zero amount, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.075 wt%, 0.1 wt%, 0.15 wt%, 0.18 wt%, and 0.2 wt% and an upper limit of any one of 0.2 wt%, 0.22 wt%, 0.25 wt%, 0.27 wt%, 0.3 wt%, 0.5 wt%, 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, and 25 wt%, where any lower limit can be paired with any mathematically compatible upper limit.
  • the formulation may be formulated as or included in a solution.
  • the formulation may be dissolved in water.
  • the composition may include an aqueous solution including one or more of at least one buffer salt, a Bacillus amyloliquefaciens microbe, and one or more molecules synthesized by the Bacillus amyloliquefaciens microbe.
  • the formulation may be buffered to maintain a pH or osmolarity.
  • the formulation may have a pH or osmolarity suitable for uptake via a plant.
  • the formulation may comprise a pH or osmolarity suitable for uptake via a plant without changing the pH or osmolarity of cells in the plant.
  • the formulation may be at a pH of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.
  • the formulation may be at a pH of no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or less.
  • the formulation has a pH in a range having a lower limit of any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and an upper limit of any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, where any lower limit can be paired with any mathematically compatible upper limit.
  • the formulation may be at a pH from about 3 to about 8.
  • the formulation may be at a pH from about 5 to about 8.
  • the formulation may be at a pH from about 3 to about 8.
  • the formulation may be at a pH from about 6 to about 9.
  • the formulation may be at a pH from about 3 to about 8.
  • the formulation may be at a pH from about 6 to about 11.
  • the formulation may be at a pH from about 6 to about 8.
  • the formulation may be at a biological pH of about 7.4, such as in a range from a pH of about 7 to about 7.5.
  • the formulation may be a stable formulation having an osmotic concentration in a range from 0.05 mM to 1.5 M solute.
  • the formulation may have an osmotic concentration in a range having a lower limit of any one of 0.05 mM, 0.075 mM, 0.8 mM, 0.9 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM 5.0 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 250 mM, and 500 mM, and an upper limit of any one of 1.0 mM, 2.5 mM, 5.0 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 250 mM, 500 mM, IM, and 1.5 M solute, where any lower limit can be paired with any mathematically compatible upper limit.
  • the formulation may be stable such that the formulation is capable of maintaining efficacy or viability after storage.
  • the formulation may be stable after a period of time having a range from 1 day to 12 years or more. In some embodiments, the formulation is stable after a period of time having a range with a lower limit of any one of 1 day, 2 days,
  • the formulation may be stable after storage after 1 day.
  • the formulation may be stable after storage after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22, days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more.
  • the formulation may be stable after storage after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12, months, or more.
  • the formulation may be stable after storage after 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12, years, or more.
  • the formulation may be stable after storage for the period of time as described above and under a storage temperature.
  • the storage temperature may be a temperature in a range having a lower limit of any one of at least 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C,
  • the formulation may be stable after storage at a temperature of at least 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C,
  • the formulation may be stable after storage of at least 1 day, 2, days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or more, at a storage temperature as described above.
  • the formulation is stable after storage of at least 1 day, 2, days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12, days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or more, at a temperature of at least 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11 °C, 12°C, 13°C, 14°C, 15°C,
  • the formulation may be stable after storage after 1 month, 2 months,
  • the formulation may be stable after storage after 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12, years, or more, at least 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C,
  • the formulation after storage may include viable Bacillus amyloliquefaciens.
  • the formulation after storage may comprise a reduction of number of viable Bacillus amyloliquefaciens of no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less.
  • the formulation after storage include a reduction of number of viable Bacillus amyloliquefaciens in a range from 0% to 90%.
  • the formulation after storage may include a reduction of number of viable Bacillus amyloliquefaciens in a range having a lower limit of any one of 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 60% and 70% and an upper limit of any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, and 90%, where any lower limit can be paired with any mathematically compatible upper limit.
  • the reduction of viability may be determined via comparison or metric of viability, for example, colony forming units per volume, or optical density of a solution.
  • reduction of viability may comprise comparing the CFU/ml of a freshly made formulation versus the CFU/ml of a formulation that was stored for a longer period of time.
  • the storage of the formulation may maintain at least a portion of the efficacy of the formulation prior to storage.
  • the formulation after storage may comprise a reduction of efficacy against a fungus, bacteria, nematode, or insect of no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less.
  • the formulation after storage may include a reduction of efficacy against a fungus, bacteria, nematode, or insect of in a range from 0% to 90%.
  • the formulation after storage includes a reduction of efficacy against a fungus, bacteria, nematode, or insect of in a range having a lower limit of any one of 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 60% and 70% and an upper limit of any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, and 90%, where any lower limit can be paired with any mathematically compatible upper limit.
  • the reduction of efficacy may be determined via comparison or calculation of disease severity index, disease severity, average disease severity, percent average disease severity, disease index, average disease index, percent disease index, or other metric of agricultural disease.
  • reduction of efficacy determination may include comparing the reduction of disease severity of a plant that was applied a freshly made formulation versus the reduction of disease severity of a plant that was applied a formulation that was stored for a longer period of time.
  • the formulation can be formulated such that the microbes can replicate once they are applied or delivered to the target habitat.
  • the formulation may be capable of replicating once applied to one or more of soil proximate to the plant, the plant, a seed, and a plant product (i.e., produce) growing from or harvested from the plant.
  • the formulation may have anti-pathogen activity, such as prevention of growth of a pathogen or reduction of growth of a pathogen on a plant, a seed, or a produce thereof.
  • the formulation may be capable of preventing growth of a pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, or at least 5 days, or longer.
  • the formulation may be capable of preventing growth of a pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days or longer.
  • the formulation may be capable of preventing growth of a pathogen on the plant, seed, or produce thereof for over 10 days.
  • the formulation may be capable of reducing growth of the pathogen on the plant, seed, or produce thereof relative to growth of the pathogen on a control that is a plant, a seed, flower, or a produce thereof not exposed to the formulation.
  • the control can be a plant, a seed, or a produce thereof to which no anti-pathogen agent has been applied or can be a plant, a seed, flower, or produce thereof to which a commercially available antipathogen agent has been applied.
  • the commercially available control may include a Serenade® product (e.g., Serenade® Aso and/or Serenade® Opti) obtainable from Bayer Crop Sciences (US), which includes an active ingredient of a QST713 strain of Bacillus subtilis.
  • the formulation can reduce growth of a pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, or at least 5 days.
  • the formulation may be capable of reducing growth of a pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
  • the formulation may be capable of reducing growth of a pathogen on the plant, seed, or produce thereof for over 10 days.
  • the formulation may be capable of reducing growth of the pathogen of at least 25% relative to growth of the pathogen on a control.
  • the formulation may be capable of reducing growth of the pathogen of at least 60% relative to growth of the pathogen on the control.
  • the formulation may be capable of reducing growth of the pathogen of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60 % 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more relative to growth of the pathogen on the control.
  • biocontrol composition may be as described previously.
  • the biocontrol composition may be Bacillus amyloliquefaciens microbe including a nucleic acid a sequence that is at least 99.8% identical to SEQ ID NO: 1, and/or one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • the biocontrol composition includes a formulation containing Bacillus amyloliquefaciens includes a nucleic acid including a sequence that is at least 99.8% identical to SEQ ID NO: 1, and/or one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • Bacillus amyloliquefaciens includes a nucleic acid including a sequence that is at least 99.8% identical to SEQ ID NO: 1, and/or one or more molecules synthesized by said Bacillus amyloliquefaciens may be provided in a stable formulation as described herein.
  • the methods for using the biocontrol composition may include a method of treating a plant, seed, soil including the plant, or any combination thereof with a biocontrol composition of one or more embodiments.
  • a method for using a biocontrol composition is directed to a method of plant and/or produce treatment.
  • An additional aspect of the present disclosure is a method for treating a disease in a plant, a seed, or soil comprising a plant.
  • the method of one or more embodiments may include obtaining a plant, a seed, and/or soil that includes a plant having a disease.
  • the method may include contacting the plant, the seed, or soil comprising the plant, (i) with a Bacillus amyloliquefaciens that includes a nucleic acid comprising a sequence that is a certain percentage (e.g., at least 99.8%) identical to SEQ ID NO: 1, and/or (ii) with one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • the Bacillus amyloliquefaciens may be formulated in a biocontrol composition in accordance with one or more embodiments.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, and/or the soil including the plant, where the pathogenic organism is responsible for the disease.
  • Another aspect of the present disclosure is a method for preventing or reducing severity of a disease in a plant, a seed, or soil comprising a plant.
  • the method comprises steps of: obtaining a plant, a seed, or soil comprising a plant at risk for contracting a disease and contacting the plant, the seed, or soil comprising the plant, (i) with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is a certain percentage (e.g., at least 99.8%) identical to SEQ ID NO: 1, and/or (ii) with one or more molecules synthesized by said Bacillus amyloliquefaciens.
  • the Bacillus amyloliquefaciens may be formulated in a biocontrol composition in accordance with one or more embodiments.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, and/or the soil comprising the plant, where the pathogenic organism is responsible for the disease.
  • the method of plant treatment may be used to treat one or more plant pathogens and/or one or more plant diseases.
  • the one or more plant pathogens may include at least one plant pathogen described herein.
  • the disease may include, but is not limited to, Asian soybean rust, rice blast, brown spot, narrow brown spot, crown rot, sour rot (citrus, postharvest), olive quick decline syndrome, Pierce’s disease (grapes), leaf scorch (almonds, coffee), citrus variegated chlorosis, Black rot, soybean cysts, or root knots.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, and/or the soil including the plant, where the pathogenic organism is responsible for the disease.
  • one or more molecules are secreted by the Bacillus amyloliquefaciens into a liquid including the Bacillus amyloliquefaciens such that the contacting includes contacting the liquid with one or more of the plant, the seed, or the soil including the plant and/or contacting a fraction isolated or purified from the liquid and the one or more molecules with one or more of the plant, the seed, or the soil including the plant.
  • the one or more molecules secreted from Bacillus amyloliquefaciens is purified prior to the contacting step.
  • One or more methods of the present disclosure is a method that includes contacting one or more of a plant, a seed, or a soil including a plant with a biocontrol composition (e.g., a biocontrol microbe and/or a formulation in accordance with one or more embodiments).
  • the step of contacting may include dusting, dipping, rolling, injecting, rubbing, spraying, and/or brushing one or more of a plant, seed, or soil including the plant.
  • the contacting includes contacting a leaf, root, fruit, seed, flower, or stem of the plant with the biocontrol microbe and/or biocontrol composition.
  • the contacting reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, the soil comprising the plant, or any combination thereof.
  • the contacting may kill, reduce growth, reduce reproduction, or any combination thereof of an infectious fungus or a diseasecausing endophyte.
  • the contacting may prevent a disease or reduces the incidence and/or severity of a disease of the plant.
  • the contacting may prevent a disease or reduces the incidence and/or severity of a disease of the plant.
  • one or more molecules synthesized by said Bacillus amyloliquefaciens kills, damages, slows the growth of, reduces the reproduction of the fungus, bacterium, or nematode, or any combination thereof by any of the following modes of action: damage to the cell membrane; disintegration of the cell membrane or osmotic pressure imbalance; changes to cell membrane permeability; formation of pores in the cell membrane; formation of conglobation structures; reducing concentrations or availability of polysaccharide or other components of biofilm formation, or by downregulation for genes required for formation of such compounds; induction of plant immune responses; downregulation of genes required for cell wall synthesis, protein production, or DNA replication; inhibition of glucosamine synthase; inhibition of protein synthesis; iron chelation, or chelation of other key chemicals that prevents access by the pathogen; inhibition of biofilm formation or swarming motility.
  • the lipopeptides may damage the cell membrane; disintegrate the cell membrane or create osmotic pressure imbalance, change cell membrane permeability, form pores in the cell membrane; form conglobation structures, reduce concentrations or availability of polysaccharide or other components of biofilm formation or downregulate genes required for formation of such compounds, and/or induce plant immune responses.
  • lipopeptides include fengycin, plipastatin, surfacticin, or related compounds
  • the fengycin may damage the cell membrane;
  • the plipastatin may change cell membrane permeability, forms pores in the cell membrane, and/or forms conglobation structures; and
  • the surfacticin may disintegrate the cell membrane or create osmotic pressure imbalance, reduce concentrations or availability of polysaccharide or other components of biofilm formation or downregulates genes required for formation of such compounds, and/or induces plant immune responses.
  • the related compound of a compound herein may include a raw material, a precursor or a metabolite of such compound.
  • the related compound of a compound includes a catalyst, an enzyme, a co-enzyme or other component of an enzyme that is incorporated in a chemical reaction for generating such compound or a precursor thereof.
  • the polyketides downregulate genes required for cell wall synthesis, protein production, or DNA replication or inhibit biofilm formation or swarming motility.
  • Difficidin or related compounds may downregulate genes required for cell wall synthesis, protein production, or DNA replication or inhibits biofilm formation or swarming motility.
  • the peptides e.g., bacilysin, rhizocticin A, or related compounds
  • the aminoglycosides e.g., butirosin A, or butirosin B or related compounds
  • the siderophores modify iron chelation, or chelation of other key chemicals which prevents access by the pathogen.
  • the one or more pathogenic organisms may be selected from the group consisting of a fungus, a bacterium, and a nematode.
  • the contact with the biocontrol composition reduces growth of or kills, one or more pathogenic organisms present on or inside the plant, the seed, or the soil comprising the plant.
  • the pathogenic organism may be selected from the group consisting of a fungus, a bacterium, and a nematode.
  • the pathogenic organism may be as described herein.
  • the one or more pathogenic organisms may infect the leaves, fruits, flowers, stems, seeds, or roots of the plant.
  • the one or more pathogenic organisms may cause an infection inside the plant.
  • the one or more pathogenic organisms causes an infection inside the plant via the xylem, the phloem, or an internal or external structural element.
  • the structural element may include a stem, trunk, corm, pseudostem, or bulb.
  • the one or more pathogenic organisms causes an infection to the plant by entering the roots of the plant or leaf stomata of the plant.
  • the one or more pathogenic organisms may include an infectious fungus or a disease-causing endophyte.
  • the method includes preparing and/or forming the biocontrol composition.
  • the preparing and/or forming the biocontrol composition may include one or more of growing, synthesizing, or culturing one or more components of the biocontrol composition.
  • the preparing and/or forming the biocontrol composition may include adding a Bacillus amyloliquefaciens microbe and/or one or more molecules synthesized by said Bacillus amyloliquefaciens microbe to an aqueous solution.
  • the method may include adding an amount of the Bacillus amyloliquefaciens microbe and/or one or more molecules synthesized by the Bacillus amyloliquefaciens microbe to an aqueous solution in an amount in a range from IxlO 2 CFU/mL (colony forming units per milliliter) to IxlO 20 CFU/mL or more.
  • the Bacillus amyloliquefaciens microbe may be added to an aqueous solution in an amount in a range from IxlO 2 CFU/mL, 2.5xl0 2 CFU/mL, 5xl0 2 CFU/mL, IxlO 3 CFU/mL, 2.5xl0 3 CFU/mL, 5xl0 3 CFU/mL, IxlO 4 CFU/mL, 2.5xl0 4 CFU/mL, 5xl0 4 CFU/mL, IxlO 5 CFU/mL, 2.5xl0 5 CFU/mL, 5xl0 5 CFU/mL, IxlO 6 CFU/mL, 2.5xl0 6 CFU/mL, 5xl0 6 CFU/mL, IxlO 7 CFU/mL, 2.5xl0 7 CFU/mL, 5xl0 7 CFU/mL, IxlO 8 CFU/mL, 2.5xl0 8 CFU/mL, 2.5
  • the amount of (i) the Bacillus amyloliquefacien , and/or (ii) the one or more molecules synthesized by the Bacillus amyloliquefaciens in the formulation is IxlO 2 CFU/mL or greater, IxlO 5 CFU/mL or greater, or IxlO 10 CFU/mL or greater.
  • the method may include adding one or more selected from the group consisting of sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, and combinations thereof to the aqueous solution.
  • the method includes adding at least one buffer salt to the aqueous solution in a non-zero amount to about 25 wt% (weight percent).
  • At least one buffer salt may be added to the aqueous solution in a range from a non-zero amount, 0.05 wt%, 0.1 wt%, 1 wt%, 5 wt%, 7.5 wt%, 10 wt%, 12.5 wt%, and 15 wt% and an upper limit of any one of 12.5 wt%, 15 wt%, 17.5 wt%, 20 wt%, and 25 wt%, where any lower limit can be paired with any mathematically compatible upper limit.
  • the method may include adjusting the pH of the solution to a value in a range having a lower limit of any one of 3, 4, 5, 6, 6.25, 6.5, 6.75, 7.0, 7.2, and 7.4 and an upper limit of any one of 7.4, 7.5, 7.7, 7.9, 8, 8.5, 9, 9.5, 10, 10.5, and 11, where any lower limit can be paired with any mathematically compatible upper limit.
  • Any herein-disclosed aspect or embodiment may include harvesting a crop from the plant, and contacting the harvested crop with the Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is a certain percentage (e.g., at least 99.8%) identical to SEQ ID NO: 1.
  • the harvested crop is a fruit, nut, leaf, vegetable, and/or root.
  • the method of one or more embodiments may include obtaining one or more of a plant, a seed, or soil including the plant having a disease, obtaining a plant, a seed, or soil including the plant at risk for contracting a disease, or combinations thereof.
  • the plant treatment may include inoculation of any one of a plant, seed, or soil comprising the plant with a composition including a Bacillus amyloliquefaciens microbe such that the composition contacts one or more portions of the plant, seed, soil including the plant, or any combination thereof.
  • a method of plant treatment may include contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens microbe.
  • the Bacillus amyloliquefaciens includes a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1.
  • inoculating one or more of the plant, seed, or soil including the plant reduces growth of a Fusarium fungus, a Mycosphaerella fungas, or both on the plant.
  • the inoculation reduces the of Panama disease, leaf spot disease, or both on the plant.
  • the Panama disease includes a pseudostem disease. The method of one or more embodiments may reduce pseudostem disease reduced by at least 1%, at least 5%, at least 7%, at least 10%, at least 25%, or at least 50%.
  • the method reduces pseudostem disease is reduced by an amount in a range from 15% to at least 85%, relative to a control plant having pseudostem disease and not contacted with the Bacillus amyloliquefaciens.
  • the method reduces pseudostem disease in a range having a lower limit of any one of 15%, 20%, 25%, and 30%, and an upper limit of any one of about 70%, 75% 80%, and 85% or more, relative to a control plant having pseudostem disease and not contacted with the Bacillus amyloliquefaciens.
  • the Fusarium fungus treated by a method of one or more embodiments may include Fusarium oxysporum.
  • the Mycosphaerella fungus comprises Mycosphaerella fijiensis or Mycosphaerella musicola.
  • the plant may be a fruit plant, such as a banana tree.
  • the leaf spot disease may include Black sigatoka.
  • disruption of photosynthesis can reduce fruit yield by up to 50%. Infection with black Sigatoka can interrupt ripening, causing fruit to ripen prematurely and unevenly, and as a result become unsuitable for export.
  • a treatment that reduces black sigatoka may be beneficial in plants.
  • a method of plant treatment includes inoculating a plant, seed, or soil comprising the plant with a Bacillus amyloliquefaciens to treat a Xanthomonas pathogen.
  • the inoculation includes contacting the microbe with one or more of the plant seed, or soil including the plant with Bacillus amyloliquefacien .
  • the contact reduces growth of the Xanthomonas pathogen including, but not limited to, Xanthomonas campestris on the plant.
  • the contact reduces blackspot development on the plant.
  • the blackspot development is reduced by about 50-85%, relative to a control plant having blackspot and not contacted with the Bacillus amyloliquefaciens. In some embodiments, the blackspot development is reduced by at least 10%.
  • a treatment method may include contacting a nematode with a Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens.
  • the step of contacting the nematode may include inoculating a plant that includes the nematode with the Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens.
  • the method or the step of contacting the nematode may include inhibiting a nematode-mediated plant disease.
  • the contacting can kill the nematode, reduce growth of the nematode, inhibits or reduces the reproduction of the nematode, or any combination thereof.
  • the nematode comprises a root-knot nematode or a soybean cyst nematode.
  • a treatment method of one or more embodiments may be directed to a method of treatment to reduce the growth of a Xylella bacterium (e.g., Xylella fasticliosa), Phakopsora pachyrhizi, or reduce the development of soybean rust on the plant on a plant.
  • the method may include contacting a plant, seed, or soil that includes the plant with a Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens.
  • the contacting may include, for example, inoculation.
  • the contact reduces growth of a Xylella bacterium on the plant, reduces development of vascular disease in the plant (e.g., leaf scorching), or both.
  • the plant includes a nut tree, such as an almond tree.
  • the plant includes a fruit plant, such as an olive tree, a berry plant (e.g., a blueberry plant), a grape vine, or any combination thereof.
  • the contacting step includes contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens .
  • the contacting may include spraying.
  • a treatment method is directed to reducing the development of a rice disease, such as in a rice plant.
  • the method may include contacting, such as inoculating, a plant, seed, a soil including the plant, or any combination thereof with a Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens.
  • the contact includes contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens.
  • the rice disease may include rice blast, narrow brown spot, brown spot, or sheath blight.
  • the rice disease in the plant is reduced by at least 10%, relative to the rice disease in a plant without the contact.
  • a treatment method is directed to a method of post-harvest protection.
  • the method for post-harvest production may include a contacting step (e.g., an inoculation step) of a harvested crop with a biocontrol composition in accordance with one or more embodiments.
  • the contact prevents a disease, reduces the incidence and/or severity of a disease, or combinations thereof.
  • the harvested from may include a fruit, including, but not limited to a citrus fruit (e.g., an orange).
  • the is infected or under threat of infection from a plant pathogen, such as at least one of Fusarium spp., Botrytis spp., Penicillium spp., Geotrichum spp. and Rhizoctonia spp.
  • a plant pathogen such as at least one of Fusarium spp., Botrytis spp., Penicillium spp., Geotrichum spp. and Rhizoctonia spp.
  • the orange is infected or under threat of infection from Penicillium digitatum.
  • infection is prevented or disease severity is reduced.
  • the method may include contacting a plant, seed or produce thereof, or soil comprising the plant with a microbe, such as a Bacillus amyloliquefaciens.
  • the Bacillus amyloliquefaciens may include a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1.
  • the contact may kill, prevent growth, or reduce growth of a pathogen such as a fungus, bacterium, nematode, or insect on or in the plant, seed or produce thereof, or soil.
  • the contact may prevent, reverse, or reduce development of a disease related to the pathogen on the plant.
  • Applying the microbe or formulation to the plant can comprise dusting, dipping, rolling, injecting, rubbing, spraying, or brushing the plant with the microbe or formulation.
  • the biocontrol composition can be applied to the produce immediately prior to harvest or immediately after harvesting or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of harvesting. In some cases, the biocontrol composition is applied by the entity doing the harvesting, in a process treating the produce immediately prior to harvest or postharvest, by the entity packaging the produce, by the entity transporting the produce, or by the entity commercially displaying the produce for sale, or a consumer.
  • the formulation can reduce growth of the pathogen on the plant, seed, or produce thereof relative to growth of the pathogen on a control that is a plant, a seed, flower, or a produce thereof not exposed to the formulation.
  • the control can be a plant, a seed, or a produce thereof to which no anti-pathogen agent has been applied or can be a plant, a seed, flower, or produce thereof to which a commercially available anti-pathogen agent has been applied.
  • the commercially available control may include a Serenade® product (such as Serenade® Opti and/or Serenade® Aso obtainable from Bayer Crop Science US), which includes an active ingredient of a QST 713 strain of Bacillus subtilis.
  • the formulation can reduce growth of a pathogen on the plant, seed, or produce thereof for at least 1 , at least 2, at least 3, at least 4, or at least 5 days.
  • the formulation can reduce growth of a pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
  • the formulation can reduce growth of a pathogen on the plant, seed, or produce thereof for over 10 days.
  • the formulation can reduce growth of the pathogen of at least 25% relative to growth of the pathogen on the control.
  • the formulation can reduce growth of the pathogen of at least 60% relative to growth of the pathogen on the control.
  • the formulation can reduce growth of the pathogen of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60 % 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more relative to growth of the pathogen on the control.
  • the plant may include a flower, seed, and/or produce thereof.
  • the plant, flower, seed, or produce thereof may include an almond, apricot, apple, artichoke, banana, barley, beet, blackberry, blueberry, broccoli, Brussels sprout, cabbage, cannabis, capsicum, carrot, celery, chard, cherry, citrus, corn, cucurbit, cucumber, date, fig, garlic, grape, herb, spice, kale, lettuce, mandarin, oil palm, olive, onion, orange, pea, pear, peach, peanut, papaya, parsnip, pecan, persimmon, plum, pomegranate, potato, quince, radish, raspberry, rose, rice, sloe, sorghum, soybean, spinach, strawberry, sweet potato, tobacco, tomato, turnip greens, walnut, or wheat.
  • the plant, seed, flower, and/or produce thereof may belong to a genus of Prunus, Malus, Cynara, Musa, Hordeum, Beta, Rubus, Vaccinium, Brassica, Cannabis, Capsicum, Daucus, Apium, Citrus, Zea, Cucumis, Curcubita, Phoenix, Ficus, Allium, Vitis, Elaeis, Olea, Pisum, Pyrus, Arachis, Carica, Carya, Pastinaca, Diospyros, Punica, Solanum, Cydonia, Raphanus, Rubus, Rosa, Oryza, Sorghum, Glycine, Spinacia, Fragaria, Ipomoea, Nicotiana, Juglans, or Triticum.
  • the plant, seed, flower, or produce thereof may include a plant or produce thereof can be from the family Rosaceae.
  • the plant, flower, seed, or produce thereof from the family Rosaceae can be from the genus Rubus, such as a raspberry or blackberry, Fragaria, such as a strawberry, Pyrus such as a pear, Cydonia such as a quince, Prunus, such as an almond, peach, plum, apricot, cherry or sloe, Rosa, such as a rose, or Malus, such as an apple.
  • the plant, seed, flower, or produce thereof can be a plant or produce thereof from the family Ericaceae.
  • the plant, seed, flower, or produce thereof from the family Ericaceae can be from the genus Vaccinium, such as a blueberry.
  • the plant, seed, flower, or produce thereof can be a plant or produce thereof from the family Ericaceae.
  • the plant, seed, flower, or produce thereof from the family Ericaceae can be from the genus Vaccinium, such as a blueberry.
  • the plant, seed, flower, or produce thereof can be a plant or produce thereof from the family Vitaceae.
  • the plant, seed, flower, or produce thereof from the family Vitaceae can be from the genus Vitis, such as a grape.
  • the plant is an almond tree, apricot tree, apple tree, artichoke plant, banana tree, barley, beet, blackberry plant, blueberry bush, broccoli plant, brassica plant, Brussels sprout plant, cabbage plant, cannabis plant, capsicum plant, carrot plant, celery plant, chard plant, cherry tree, citrus tree, corn stalk, cucurbit plant, cucumber plant, date palm, fig tree, garlic plant, grape vine, herb plant, spice plant, kale plant, lemon tree, lettuce plant, lime tree, mandarin tree, oil palm, olive tree, onion plant, orange tree, pea shoot, pear tree, peach tree, peanut plant, papaya tree, parsnip plant, pecan tree, persimmon tree, plum tree, pomegranate plant, potato plant, quince plant, radish plant, raspberry plant, rose plant, rice plant, sloe plant, sorghum plant, soybean plant, spinach plant, strawberry plant, sweet potato plant, tobacco plant, tomato vine, turnip greens
  • the microbe may colonize, or grow, on a portion of the plant. For example, inoculating the plant may produce an endophyte.
  • the microbe may colonize a rhizome of the plant.
  • the microbe may colonize a sucker of the plant.
  • the microbe may be associated with the plant throughout a harvest of portions of the plant. For example, the microbe may colonize a sucker of a subsequent year’s crop of the plant. For example, the microbe may colonize a rhizome of the plant between harvests.
  • the microbe may maintain its properties between harvests or growths of new portions of the plant.
  • embodiments herein relate to a method for producing an endophyte (e.g., an endosymbiont).
  • the method for producing an endophyte includes one or more steps for using a biocontrol composition as described herein.
  • the method for producing an endophyte may include contacting one or more of a leaf, root, or stem of a plant with Bacillus amyloliquefaciens or a formulation including the Bacillus amyloliquefaciens.
  • the contacting produces an endophyte inside the plant or seed.
  • the produced endophyte may protect the plant or seed against a pathogen.
  • the contacting occurs before the one or more pathogenic organisms are present on or inside the plant, the seed, or the soil comprising the plant.
  • the Bacillus amyloliquefaciens is capable of colonizing a rhizome of the plant between harvests, a sucker of a plant crop of a subsequent year, or both.
  • the Bacillus amyloliquefaciens may colonize a rhizome of the plant between harvests.
  • the Bacillus amyloliquefaciens may colonize a sucker of a subsequent year’s crop of the plant.
  • the endophyte is transmitted to the progeny of the plant and protects said progeny of the plant against a fungus, bacterium, or nematode.
  • the endophyte may be transmitted via seed germination or via cuttings or transplanting.
  • the method for producing an endophyte may include inoculating a plant with a Bacillus amyloliquefaciens microbe or a formulation including the Bacillus amyloliquefaciens microbe.
  • inoculating the plant with a Bacillus amyloliquefaciens microbe or a formulation including the Bacillus amyloliquefaciens microbe allows for the production of an endophyte comprising the Bacillus amyloliquefaciens.
  • the plant includes a banana plant, a Brassica, a lettuce, a blueberry plant, an almond tree, an olive tree, a grape vine, or any combination thereof.
  • the microbe may be vertically transmitted from a plant to a plant’s progeny.
  • the plant’s progeny may be conferred the benefits of the microbe.
  • the endophyte may be transmitted to the progeny of the plant and may protects the progeny of the plant against a fungus, bacterium, nematode, or insect.
  • the transmission to a progeny, or generation of progeny may comprise seed germination, growth from cuttings, or via transplanting.
  • the method of producing an endophyte in a plant enables protection of the plant against a pathogen, such as a fungus, bacterium, or nematode.
  • a pathogen such as a fungus, bacterium, or nematode.
  • the fungus includes Fusarium, such as Fusarium oxysporum.
  • the bacterium includes a Xanthomonas, such as Xanthomonas campestris, a Xylella (e.g., Xylella fasticliosa), or combinations thereof.
  • the nematode includes a root-knot nematode or a soybean cyst nematode.
  • disease severity index generally refers to a score representing the degree of disease symptoms visible on the plant.
  • a given disease severity index may have a particular number (or range of numbers) of spots on the leaves indicative of a disease.
  • a plant that has more symptoms of the disease has a higher disease severity index than a plant that has a lower disease severity index.
  • Different species of plants may have a different disease severity index associated with it.
  • the term “disease severity” or “average disease severity” or “percent average disease severity” generally refers to the degree of disease symptoms which is visible on a plant or population of plants. The disease severity may be calculated by the percentage of the plant that is covered by disease symptoms. The percent average disease severity may be calculated for a population using by assessing the disease severity of each plant and averaging the disease severity of each plant.
  • the term “disease index”, “average disease index” or “percent average disease index” generally refers to a score for a population of plants representing the degree of disease symptoms visible in a population of plants.
  • the disease index may be calculated as the disease incidence multiplied by the disease severity.
  • the average disease index may be calculated based on a disease severity index or score for an individual plant, number of plants with that disease severity index, the total number of plants, the maximal disease index, and the percent disease incidence in order to create a weighted average representing the average disease severity.
  • a general calculation of the percent average disease index may be done as a [sum(number of plants in a given score multiplied by the score)]/ [(total number of plants multiplied by the maximal score)] multiplied by 100.
  • Embodiment 1 A method of treatment, comprising:
  • Embodiment 2 The method of embodiment 1 , wherein the plant comprises a fruit plant.
  • Embodiment 3 The method of embodiment 1, wherein the fruit plant comprises a banana tree.
  • Embodiment 4 The method of embodiment 1, wherein the Panama disease comprises pseudostem disease.
  • Embodiment 5 The method of embodiment 4, wherein the pseudostem disease is reduced by at least 10%.
  • Embodiment 6 The method of embodiment 4, wherein the pseudostem disease is reduced by about 25-75%, relative to a control plant having pseudostem disease and not contacted with the Bacillus amyloliquefaciens.
  • Embodiment 7 The method of embodiment 1, wherein the contact comprises contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens.
  • Embodiment 8 The method of embodiment 1, wherein the Fusarium fungus comprises Fusarium oxysporum.
  • Embodiment 9 The method of embodiment 1, wherein nucleic acid comprises a sequence that is 100% identical to SEQ ID NO: 1.
  • Embodiment 10 A method of producing an endophyte, comprising:
  • Embodiment 11 The method of embodiment 10, wherein the Bacillus amyloliquefaciens comprises a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1.
  • Embodiment 12 The method of embodiment 11, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 13 The method of embodiment 10, wherein the contact comprises contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens.
  • Embodiment 14 The method of embodiment 10, wherein the Bacillus amyloliquefaciens colonizes a rhizome of the plant between harvests.
  • Embodiment 15 The method of embodiment 10, wherein the Bacillus amyloliquefaciens colonizes a sucker of a subsequent year’s crop of the plant.
  • Embodiment 16 The method of embodiment 10, wherein the endophyte protects the plant against a pathogen.
  • Embodiment 17 The method of embodiment 16, wherein the pathogen comprises a fungus, bacterium, or nematode.
  • Embodiment 18 The method of embodiment 17, wherein the fungus comprises a Fusarium.
  • Embodiment 19 The method of embodiment 18, wherein the Fusarium comprises Fusarium oxysporum.
  • Embodiment 20 The method of embodiment 17, wherein the bacterium comprises a Xanthomonas .
  • Embodiment 21 The method of embodiment 20, wherein the Xanthomonas comprises Xanthomonas campestris.
  • Embodiment 22 The method of embodiment 17, wherein the bacterium comprises a Xylella.
  • Embodiment 23 The method of embodiment 22, wherein the Xylella comprises Xylella fastidiosa.
  • Embodiment 24 The method of embodiment 17, wherein the nematode comprises a root-knot nematode or a soybean cyst nematode.
  • Embodiment 25 A method of treatment, comprising:
  • Embodiment 26 The method of embodiment 25, wherein the Bacillus amyloliquefaciens comprises a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1.
  • Embodiment 27 The method of embodiment 25, wherein the plant comprises a Brassica or lettuce.
  • Embodiment 28 The method of embodiment 25, wherein the blackspot development is reduced by about 50-85%, relative to a control plant having blackspot and not contacted with the Bacillus amyloliquefaciens.
  • Embodiment 29 The method of embodiment 26, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 30 A method of treatment, comprising:
  • Embodiment 31 The method of embodiment 30, wherein the Xanthomonas bacterium comprises Xanthomonas campestris.
  • Embodiment 32 The method of embodiment 30, wherein the plant comprises a lettuce or Brassica.
  • Embodiment 33 The method of embodiment 30, wherein the blackspot development is reduced by at least 10%.
  • Embodiment 34 The method of embodiment 30, wherein the blackspot development is reduced by about 50-85%, relative to a control plant having blackspot and not contacted with the Bacillus amyloliquefaciens.
  • Embodiment 35 The method of embodiment 30, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 36 A method, comprising: contacting a nematode with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1.
  • Embodiment 37 The method of embodiment 36, wherein the contacting kills, or reduces growth, or reproduction of the nematode.
  • Embodiment 38 The method of embodiment 36, wherein the nematode comprises a root-knot nematode or a soybean cyst nematode.
  • Embodiment 39 The method of embodiment 36, wherein contacting the nematode with the Bacillus amyloliquefaciens comprises inoculating a plant comprising the nematode with the Bacillus amyloliquefaciens.
  • Embodiment 40 The method of embodiment 36, wherein the contacting inhibits a nematode-mediated plant disease.
  • Embodiment 41 The method of embodiment 36, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 42 A method of treatment, comprising: inoculating a plant, seed, or soil comprising the plant with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1 ; wherein the contact reduces growth of a Xylella bacterium on the plant, or reduces development of vascular disease in the plant.
  • Embodiment 43 The method of embodiment 42, wherein the plant comprises a nut tree.
  • Embodiment 44 The method of embodiment 42, wherein the nut tree comprises an almond tree.
  • Embodiment 45 The method of embodiment 42, wherein the plant comprises a fruit plant.
  • Embodiment 46 The method of embodiment 42, wherein the fruit plant comprises an olive tree.
  • Embodiment 47 The method of embodiment 42, wherein the fruit plant comprises a berry plant.
  • Embodiment 48 The method of embodiment 42, wherein the berry plant comprises a blueberry plant.
  • Embodiment 49 The method of embodiment 42, wherein the fruit plant comprises a grape vine.
  • Embodiment 50 The method of embodiment 42, wherein the contact comprises contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefacien .
  • Embodiment 51 The method of embodiment 50, wherein contacting comprises spraying.
  • Embodiment 52 The method of embodiment 42, wherein the Xylella bacterium comprises Xylella fastidiosa.
  • Embodiment 53 The method of embodiment 42, wherein the vascular disease comprises leaf scorching.
  • Embodiment 54 The method of embodiment 42, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 55 A method of treatment, comprising: inoculating a plant, seed, or soil comprising the plant with a Bacillus amyloliquefaciens, wherein the plant comprises an almond tree or blueberry plant; and wherein the contact reduces growth of a Xylella bacterium on the plant, or reduces development of a vascular disease in the plant.
  • Embodiment 56 The method of embodiment 55, wherein the contact comprises contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens.
  • Embodiment 57 The method of embodiment 56, wherein contacting comprises spraying.
  • Embodiment 58 The method of embodiment 55, wherein the Xylella bacterium comprises Xylella fastidiosa.
  • Embodiment 59 The method of embodiment 55, wherein the vascular disease comprises leaf scorch.
  • Embodiment 60 The method of embodiment 55, wherein the Bacillus amyloliquefaciens comprises a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1
  • Embodiment 61 The method of embodiment 60, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 62 A method of treatment, comprising: inoculating a plant, seed, or soil comprising the plant with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1 ; wherein the contact reduces growth of a Mycosphaerella fungus on the plant, or reduces development of a leaf spot disease on the plant.
  • Embodiment 63 The method of embodiment 62, wherein the plant comprises a fruit plant.
  • Embodiment 64 The method of embodiment 63, wherein the fruit plant comprises a banana tree.
  • Embodiment 65 The method of embodiment 62, wherein the contact comprises contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefaciens.
  • Embodiment 66 The method of embodiment 62, wherein the Mycosphaerella fungus comprises Mycosphaerella fijiensis or Mycosphaerella musicola.
  • Embodiment 67 The method of embodiment 62, wherein the leaf spot disease comprises Black sigatoka.
  • Embodiment 68 The method of embodiment 62, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 69 A method of treatment, comprising: inoculating a plant, seed, or soil comprising the plant with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1 ; wherein the plant comprises rice, and the contact reduces development of a rice disease.
  • Embodiment 70 The method of embodiment 69, wherein the contact comprises contacting a leaf, root, or stem of the plant with the Bacillus amyloliquefacien .
  • Embodiment 71 The method of embodiment 69, wherein the rice disease comprises rice blast, narrow brown spot, brown spot, or sheath blight.
  • Embodiment 72 The method of embodiment 69, wherein the rice disease in the plant is reduced by at least 10%, relative to the rice disease in a plant without the contact.
  • Embodiment 73 The method of embodiment 69, wherein the sequence of the nucleic acid is 100% identical to SEQ ID NO: 1.
  • Embodiment 74 A method of post-harvest protection, comprising: inoculating a harvested crop with a composition comprising the harvested crop with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1, wherein the contact prevents a disease or reduces the incidence and/or severity of a disease.
  • Embodiment 75 The method of embodiment 74, wherein the harvested crop is a fruit.
  • Embodiment 76 The method of embodiment 75, wherein the fruit is a citrus fruit.
  • Embodiment 77 The method of embodiment 76, wherein the citrus fruit is an orange.
  • Embodiment 78 The method of embodiment 74, wherein the harvested crop is infected or under threat of infection from at least one of Fusarium spp., Botrytis spp., Penicillium spp., Geotrichum spp. and Rhizoctonia spp.
  • Embodiment 79 The method of embodiment 77, wherein the orange is infected or under threat of infection from Penicillium digitatum.
  • Embodiment 80 The method of embodiment 79, wherein infection is prevented or disease severity is reduced.
  • Embodiment 81 A method of treatment, comprising: inoculating a plant, seed, or soil comprising the plant with a Bacillus amyloliquefaciens comprising a nucleic acid comprising a sequence that is at least 99.8% identical to SEQ ID NO: 1 ; wherein the contact reduces growth of a Phakopsora pachyrhizi on the plant, or reduces development of soybean rust on the plant.
  • Example 1 Inhibition of Xanthomonas campestris growth in vitro by Bacillus amyloliquefaciens BC17B
  • Example 2 Inhibition of Xanthomonas campestris growth on detached lettuce leaves by Bacillus amyloliquefaciens BC17B
  • Bacterial leaf spot control was investigated for BC17B compared to Serenade® Opti (includes 26.2t% of QST713 strain of Bacillus subtil is) and a control treatment of phosphate buffered saline (PBS). Treatments were applied to Xanthomonas campestris- inoculated lettuce leaf discs. BC17B was applied as a solution in PBS at 8mL (milliliters) of 2.5xl0 7 colony forming unit (CFU)/mL on each leaf. Ratings for severity were taken 1, 4, 7, 11, 14, and 18 days after treatment. Experiments were performed in duplicate (disc 1 and disc 2) with 15 replicates per duplicate.
  • PBS phosphate buffered saline
  • FIG. 3 A and FIG. 3B show average disease severity scores for disc 1 and disc 2, respectively. Letters in FIGs. 3A and 3B indicate whether the scores are significantly different for each time point (different letters) or not significantly different (same letter).
  • Example 3 Bacillus amyloliquefaciens BC17B inhibits lettuce blackspot disease caused by Xanthomonas campestris
  • FIG. 4 A shows the degree of blackspot control for the three experimental conditions at 25 days post-planting.
  • BC17B in PBS elicited the highest degree of disease control at 72.5%, which was superior to a control that was treated with Serenade® ASO fungicide (having 1.34% of QST713 strain of Bacillus subtilis) having 49.7%.
  • FIG. 4B compares the appearance of PBS-treated lettuce and BC17B in PBS- treated lettuce, which shows more dense and fuller plant structure with the BC17B solution treatment as compared to only PBS.
  • Example 4 Bacillus amyloliquefaciens BC17B inhibits Botrytis cinerea on wounded peaches
  • the ability of a microbe to protect peaches from Botrytis cinerea wound infection was evaluated.
  • the candidate microbe evaluated was Bacillus amyloliquefaciens strain BC17B.
  • BC17B is a strain of Bacillus amyloliquefaciens that comprises a 16S rRNA sequence provided in SEQ ID NO: 1.
  • Four wounds were created on the surface of each peach of 2mm (millimeter) diameter and 1 mm depth. 10 pL of BC17B at 2xl0 8 cells/mL was applied into each wound and the treatment allowed to dry for 4 hours. 10 pL of Botrytis spores were applied into the wounds (excluding the negative (-) controls) and allowed to dry for 1 hour.
  • the peaches were stored in a plastic container with high humidity at room temperature. At day 3, the following properties were assessed: 1) Number of infected wounds; 2) lesion diameters for all the wounds; and 3) weight of disease tissue around wound.
  • the peaches having BC17B treatment showed clear protection from Botrytis infection, as shown in FIG. 5, which includes graphical data for the mean lesion diameter (in millimeters, mm) and the main disease weight (grams, g) for BC17B treated samples, negative controls, and positive controls.
  • the graphs of FIG. 5 show combined means of three experiments.
  • Example 5 Bacillus amyloliquefaciens BC17B reduces Blossom Blight caused by Botrytis cinerea in blueberries
  • BC 17B in PBS was applied at a high, medium, and low rate.
  • the volume of product applied was 2 L liters) for the high rate, 1 L for the medium rate and 500 mL (milliliters) for the low rate.
  • Botrytis blossom blight pressure was high and all treatments significantly reduced incidence of disease as shown in FIG. 6.
  • Treatment with a solution of BC17B in PBS reduced disease in a dose-dependent manner, with the high rate reducing blossom blight by 57% compared to the untreated control (UTC).
  • An industry standard chemical treatment completely controlled the disease and resulted in 100% improvement as compared to the UTC.
  • blueberry bushes were monitored for symptoms of phytotoxicity throughout the season. No symptoms of phytotoxicity were observed.
  • Example 6 Bacillus amyloliquefaciens BC17B reduces Botrytis, Powdery Mildew, and Downey Mildew on Grape Leaves in the field
  • BC17B was determined to reduce Botrytis, Powdery and Downey Mildew on Grape Leaves in the field in a dose-dependent manner (e.g., as shown in FIGs. 7A-10C).
  • the industry standards as controls provided the most control across all three diseases.
  • the higher doses of BC17B demonstrated efficacies reached approximately the efficacies observed for the comparative standard experiments.
  • Example 7 Bacillus amyloliquefaciens BC17B inhibits downy mildew on cucumbers
  • Plants Two-week-old Straight Eight cucumber plants (1st true leaf stage) were used for the downy mildew bioassay. Six plants (replicates) were used for each treatment. Downy mildew spores were harvested from heavily infected leaf material. Spore suspension was diluted to 10 5 spores/mL in water before spraying.
  • FIG. 11 shows percent disease reduction from water controls for the various conditions, with higher bars indicative of better control.
  • BC17B showed dose-dependent efficacy at reducing downy mildew. No other side effects such as phytotoxicity or plant growth promotion were observed.
  • Example 8 Bacillus amyloliquefaciens BC17B inhibits Monilinia fructicola (brown rot) in vitro
  • BC17B spores effectively inhibited Monilinia fructicola (e.g., as shown in FIG. 12).
  • BC17B supernatant represents cell-free fermentate, following centrifugation. In the absence of cells minimal protection was observed.
  • Example 9 Bacillus amyloliquefaciens BC17B inhibits Panama Wilt caused by Fusarium oxysporum
  • Cavendish banana plantlets were removed from pots and placed in sample holders covering the root system, which were then filled with 200 mF of each treatment (Bacillus amyloliquefaciens BC17B solution, Propiconazole chemical fungicide, or no treatment). After 30 minutes, plants were removed from sample holders, allowed to dry, then planted in 500 g of potting soil previously infested with Fusarium oxysporum var. cubense (except for a non-infested negative control condition). 2-5 plantlets were used per condition.
  • Plants were placed in a greenhouse with a 16h photoperiod and diurnal temperature range of 79°-86°F. Entire plants were photographed with measuring sticks to assess health and height, and healthy banana leaves were counted and recorded. Pseudostems were first cut 3 inches above soil line and photographed, then removed from the pot, shaken free of soil, cut vertically, and photographed again to assess the remaining pseudostem/corm. Dissected pseudostem photographs were analyzed using software to assess percentages of infected corm.
  • Bacillus amyloliquefaciens BC17B treatment outperformed the chemical Propiconazole fungicide and significantly reduced Fusarium oxysporum symptom development in pseudostems as compared to the positive control (e.g., as shown in FIG. 13A and FIG. 13B).
  • Example 10 Root-inoculated Bacillus amyloliquefaciens BC17B becomes endophytic & epiphytic
  • FIG. 14A shows the relative abundance of BC17B detected on leaf, root, and stem after 45 days. Controls included plants not treated with BC17B. There was some background detection in control plants due to Bacillus amyloliquefaciens strains other than BC17 cross reacting with primers for BC17B.
  • FIG. 14B shows representative images of collected samples and agar plates. These results demonstrate that root-inoculated BC17B becomes endophytic and epiphytic and persists for at least 45 days.
  • Example 11 Bacillus amyloliquefaciens BC17B protects blueberry plants against the endophytic bacterial pathogen Xylella fastidiosa
  • Blueberry pants in single pots were inoculated via injection with 5 pl of Xylella fastidiosa (10 8 CFU/mL).
  • the plants were treated with 5 pl of Bacillus amyloliquefaciens BC17B (10 8 CFU/mL, with 0.2 wt% Breakthru® surfactant (obtainable from Evonik) via foliar spray, or 5 pl of PBS.
  • a randomized complete block trial design was used in a greenhouse with temperature ranging from 15°C-32°C for up to 16 weeks. Crop response and efficacy against Xylella infection severity was assessed weekly after disease incidence became apparent for controls (8-10 wks.). Disease was rated based on percent (%) of symptomatic and asymptomatic leaves per plant.
  • Plants inoculated with Xylella fastidiosa and treated with the BC17B solution showed significantly decreased disease severity and delayed onset when compared to plants inoculated with Xylella fastidiosa alone as shown in FIG. 16.
  • Example 13 Evaluating protection against Eeaf Scorch, as caused by the bacterial pathogen Xylella fastidiosa in almond trees
  • Xylella fastidiosa (Xfa) or Xylella fastidiosa sp. multiplex (Xfa mpx) was injected at three consecutive nodes on the basal part of stem two year old almond trees in 1 gallon pots.
  • Example 14 Bacillus amyloliquefaciens strain BC17B persists as an epiphyte and endophyte on almond tree stems
  • Bacillus amyloliquefaciens strain BC17B The ability of Bacillus amyloliquefaciens strain BC17B to persist as an epiphyte and endophyte on almond trees was evaluated. Bacillus amyloliquefaciens strain BC17B in combination with a surfactant was sprayed onto two year old almond trees in 1 gallon pot plants. Plant stem and leaf samples are collected three months post-treatment.
  • plant tissues were washed in 70% ethanol, followed by 8% sodium hypochlorite for 2 minutes, to kill or remove surface organisms. Samples were rinsed with sterile water four times. Plant tissues were crushed in PBS, and then processed for serial dilution plating on semi-selective antibiotic media and CFU calculation. The rinse solution was also plated. Plates were incubated at 37°C for 24 hours, and BC17 CFU enumerated.
  • Example 15 Bacillus amyloliquefaciens strain BC17B exhibits nematocidal activity in soil
  • strain A396 cells and spent fermentation media (94.46%); 8 qt/a; obtainable from Maronne Bio Innovations, Inc.), or Vydate® L (Chemical nematicide having 24% Oxamyl [Methyl NN -dimethyl-N-[(methyl carbamoyl)Oxy]-I-thiooxamimidate]; 4 pt/a; obtainable from DuPont).
  • Example 16 Bacillus amyloliquefaciens strain BC17B exhibits nematocidal activity on roots
  • strain A396 cells and spent fermentation media (94.46%); 8 qt/a; obtainable from Maronne Bio Innovations, Inc.), or Vydate® L (Chemical nematicide having 24% Oxamyl [Methyl NN -dimethyl-N-[(methyl carbamoyl)Oxy]-I-thiooxamimidate]; 4 pt/a; obtainable from DuPont), or a no treatment control.
  • Example 17 Efficacy of Bacillus amyloliquefaciens strain BC17B against rice blast, narrow brown spot, brown spot, and sheath blight
  • the BC17B solution exhibited very good control of the disease index of rice blast, including comparable or even more consistent control than chemical treatment as shown in FIG. 21.
  • the BC17B solution exhibited very good control of the disease index of narrow brown spot, including comparable control to chemical treatment and superior to B. subtilis as shown in FIG. 22.
  • the BC17B solution exhibited very good control of the disease index of brown spot, including comparable control to chemical treatment and superior to B. subtilis as shown in FIG. 23.
  • the BC17B solution exhibited very good control of the disease index of sheath blight, including comparable control to chemical treatment and superior to B. subtilis as shown in FIG. 24. Phytotoxicity was monitored throughout both seasons, and none was observed.
  • Example 18 Efficacy of Bacillus amyloliquefaciens strain BC17B against banana fungal disease Black Sigatoka
  • results from the in planta experiment are shown in FIGs. 26A-26B, where different letters indicate statistical differences. Also shown is the area under the curve for plants treated with BC17B that were not infected. In particular, the results show that the BC17B- PBS solution reduced Black Sigatoka to an extent statistically indistinguishable from Tilt®, while crops treated with Serenade® and untreated crops were statistically indistinguishable.
  • Example 19 Evaluating protection against bacterial pathogen Xylella fastidiosa in olive trees
  • Xylella fastidiosa was injected at three consecutive nodes on the basal part of stem two year old olive trees in 1 gallon pots.
  • the solution including Bacillus amyloliquefaciens strain BC17B was sprayed onto the plants in combination with a surfactant. The incidence and severity of disease was monitored for 28 days after application.
  • Treatment with Bacillus amyloliquefaciens strain BC17B in PBS showed significantly lower levels of disease severity (e.g., as shown in FIG. 27 A) and incidence (e.g., as shown in FIG. 27B) when compared to a treatment with surfactant alone.
  • Example 20 Bacillus amyloliquefaciens BC17B inhibits Panama Wilt caused by Fusarium oxysporum in field trials
  • Plantlets were removed from pots and substrate was removed from the roots, which were then covered with 100 mL per plant with the BC17B solution adjusted to 5 x 10 8 CFU/mL; or water, or Serenade®. Plants were then removed and allowed to dry for 30 minutes.
  • FIG. 28A shows the average severity scores for evaluation of internal symptoms of Panama Wilt caused by Fusarium oxysporum. Different letters denote statistically different severity scores.
  • FIG. 28B shows representative images of cross-sections of banana plants treated with Bacillus amyloliquefaciens BC 17B (left) or water (right). The Bacillus amyloliquefaciens BC17B solution treatment statistically significantly reduced Fusarium oxysporum symptom development in pseudostems compared with positive control.
  • Example 21 Bacillus amyloliquefaciens BC17B inhibits Penicillium digitatum on post-harvest citrus fruit
  • Example 22 Bacillus amyloliquefaciens BC17B inhibits Asian Soybean Rust in greenhouses
  • Soybean plants were treated, allowed to dry for 2 to 4 hours, then sprayed with a urediniospore suspension (10 5 spores / mL). After inoculation, plants were incubated in high humidity overnight, then placed on a greenhouse bench.
  • Results are shown in FIG. 30, where different letters indicate statistical differences. Disease ratings were measured as percent leaf area covered by lesions. An average of 10 plants were used for each treatment. It was observed that solutions including BC17B reduced Asian soybean rust to an extent comparable with Proline, a common chemical control agent.
  • Example 23 Bacillus amyloliquefaciens BC17B is stable when produced in spore form
  • BC17B was prepared as spores and stored in PBS at 54°C for two weeks. Viable CFU counts were determined at 0, 7, and 14 days. Antifungal activity was assessed in a confrontation assay with Botrytis cinerea. The spore solution of BC17B showed no loss of viability or efficacy after two weeks at 54°C (e.g., as shown in FIGs. 31 A and 3 IB).
  • ordinal numbers for example, first, second, third
  • an element that is, any noun in the application.
  • the use of ordinal numbers does not imply or create a particular ordering of the elements or limit any element to being only a single element unless expressly disclosed, such as by the use of the terms “before,” “after,” “single,” and other such terminology. Rather, the use of ordinal numbers is to distinguish between the elements.
  • a first element is distinct from a second element, and the first element may encompass more than one element and succeed (or precede) the second element in an ordering of elements.

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Abstract

La divulgation concerne des compositions et des procédés de lutte biologique. La composition de lutte biologique peut comprendre une souche de Bacillus amyloliquefaciens. La composition de lutte biologique peut comprendre une solution aqueuse ayant un pH dans une plage comprise entre environ 6 à environ 11. Le procédé peut comprendre la mise en contact d'une plante, d'une graine, d'un fruit, d'une feuille ou d'un sol contenant la plante avec la composition de lutte biologique.
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