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WO2024227302A1 - 一株贝莱斯芽孢杆菌及其应用 - Google Patents

一株贝莱斯芽孢杆菌及其应用 Download PDF

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WO2024227302A1
WO2024227302A1 PCT/CN2023/093264 CN2023093264W WO2024227302A1 WO 2024227302 A1 WO2024227302 A1 WO 2024227302A1 CN 2023093264 W CN2023093264 W CN 2023093264W WO 2024227302 A1 WO2024227302 A1 WO 2024227302A1
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colletotrichum
bacillus
strain
dactylonectria
neopestalotiopsis
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PCT/CN2023/093264
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English (en)
French (fr)
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张玮
燕继晔
刘梅
李兴红
李永华
王慧
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北京市农林科学院
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Publication of WO2024227302A1 publication Critical patent/WO2024227302A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present invention belongs to the field of microbial application, and specifically relates to a strain of Bacillus velezensis and its application in preventing and controlling plant diseases such as tomato gray mold.
  • Botrytis cinerea has a wide range of hosts and can infect a variety of fruits and vegetables such as grapes, strawberries, cherries, tomatoes, and watermelons, causing gray mold in crops. It occurs worldwide, and generally causes a yield loss of 10% to 20%, and in severe cases up to 60% or more. Chemical control is the main measure for preventing and controlling gray mold in crops in actual production (Kang Lijuan, Zhang Xiaofeng, Wang Wenqiao, et al. Resistance and fitness determination of gray mold. Journal of Pesticide Science, 2003, 2: 39-42.).
  • Botrytis cinerea develops resistance to chemical pesticides quickly, resulting in a reduction in the control effect of the pesticide (Hahn M. The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study. Journal of Chemical Biology, 2014, 7 (4): 133-141.).
  • Biopesticides are low-toxic, green and safe, and are in line with the concept of sustainable development (Kaur S, Samota M K, Choudhary M, et al.
  • Bacillus spp. is widely distributed in nature. The dormant spores produced under adverse conditions not only have extremely low water content, but also have the characteristics of acid resistance, salt resistance, high temperature resistance, and extrusion resistance. Its metabolism can also produce a variety of enzymes and more than a dozen antibacterial substances such as bacitracin, cyclolipids, and phage-like particles. Compared with other beneficial microorganisms, Bacillus spp.
  • Bacilus velezensis has attracted the attention of researchers due to its good antibacterial effect.
  • studies have shown that Bacilus velezensis plays a major role in preventing and controlling lettuce root rot caused by Rhizoctonia solani and tomato bacterial wilt caused by Ralstonia (Cao Y, Pi HL, Chandrangsu P, et al.
  • Bacillus velezensis BS87 and RK1 on strawberry wilt is equivalent to that of copper hydroxide (Nam MH, Park MS, Kim HG, et al. Biological control of strawberry Fusarium wilt caused by Fusarium oxysporumf.spfragariae using Bacilus velezensis BS87and RK1 formulation. Journal of Microbiology and Biotechnology, 2009, 19(5): 520-524.); The Bacillus velezensis 83 isolated from mango leaves by Balderas et al.
  • mango anthracnose has a control effect on mango anthracnose that is comparable to chemical treatment (Balderas-Ruiz KA, Bustos P, Santamaria RI, et al. Bacilus velezensis 83a bacterial strain from mango phyllosphere, useful for biological Control and plant growth promotion. AMB Express, 2020, 101: 7-10.).
  • the present disclosure provides a strain of Bacillus Velezii and applications thereof.
  • Bacillus velezensis of the present invention is classified and named Bacillus velezensis, collected from Huailai County, Zhangjiakou City, Hebei province, separated from diseased grape branch tissues of grape branch diseases, and named Bacillus velezensis BJ-1. It was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on December 16, 2021, and the deposit number is CGMCC No. 24113.
  • the Velez Bacillus of the present invention does not produce pigment on LB culture medium, and the colonies are milky white and opaque, round or nearly round, with rough edges and obvious wrinkles on the surface.
  • the physiological and biochemical characteristics of the Velez Bacillus BJ-1 are Gram-positive, can produce cellulase, and do not produce protease and amylase.
  • the Velez bacillus of the invention has antibacterial activity against plant pathogenic fungi and has a wide antibacterial spectrum.
  • the plant pathogenic fungi are Lasiodiplodia theobromae, Botryosphaeria dothidea, Neofusicoccum parvum, Diaporthe sojae), Diaporthe eres, Diaporthe honkonggensis, Botrytis cinerea, Colletotrichum viniferum, Colletotrichum sojae, Colletotrichum aenigma, Colletotrichum gloeosporioides, Colletotrichum fructicola, Colletotrichum siamense, Colletotrichum acutatum, Neopestalotiopsis sp., Neopestalotiopsis rosae, Coniella vitis, Dactylonectria alcacerensis, Dactylonectria macrodidyma, Lasiodiplodiapseudotheobromae, Dactylonectria torresensis, Fusarium oxysporum), binucleate
  • the present invention also provides a biological pesticide for plant diseases, wherein the active ingredient of the biological pesticide is the above-mentioned Bacillus Velezii.
  • the biopesticide also includes adjuvants and/or additives acceptable to pesticide formulations.
  • the biopesticide is a water dispersible granule containing 1.0 ⁇ 10 8 CFU/g or more of Bacillus Velez subtilis BJ-1; wherein, Bacillus Velez subtilis BJ-1 is the Bacillus Velez subtilis described in claim 1;
  • the water dispersible granules consist of the following components:
  • the final concentration of Bacillus velez BJ-1 is 1.0 ⁇ 10 8 -1.0 ⁇ 10 11 , preferably 2.0 ⁇ 10 10 ; polyvinyl alcohol 6.0%; sodium lauryl sulfate 4.0%; ammonium sulfate 4%; polyethylene glycol 4%; talc is supplemented to 100%, and the percentages are by mass.
  • the Velez Bacillus strain of the present invention shows good control effect on tomato gray mold caused by Botrytis cinerea both indoors and in the field.
  • the results of the indoor detached leaf inoculation test show that the Velez Bacillus BJ-1 fermentation liquid has good protection and treatment effects on tomato gray mold, and the relative control effects of 1 ⁇ 10 7 CFU/mL BJ-1 fermentation liquid on tomato gray mold can reach 68.95% and 88.28%, respectively.
  • the field test results show that the Velez Bacillus BJ-1 water dispersible granules have good control effects on tomato gray mold, and the relative control effect of 2.0 ⁇ 10 10 CFU/g Velez Bacillus BJ-1 water dispersible granules (450 g/mu) can reach 80.48%.
  • the Velez Bacillus of the present invention has biocontrol potential and can be used to prepare biocontrol agents and microbial fertilizers for plant pathogenic fungi.
  • the Bacillus Velezii provided in the present invention has good inhibitory effects on various plant pathogens, and shows good prevention effect on tomato gray mold caused by Botrytis cinerea in the field, so it has good application prospects for preventing and controlling gray mold of fruits and vegetables caused by Botrytis cinerea.
  • the strain has simple culture conditions, rapid growth and easy preservation, and is suitable for industrial production.
  • Fig. 1 Colony morphology and scanning electron microscopy photograph of strain BJ-1.
  • top Escherichia coli JM109
  • bottom left Bacillus subtilis IPEP6
  • bottom right strain BJ-1.
  • top Escherichia coli JM109
  • bottom left Bacillus subtilis IPEP6
  • bottom right strain BJ-1.
  • top Escherichia coli JM109
  • bottom left Bacillus subtilis IPEP6
  • bottom right strain BJ-1.
  • Fig. 5 is the detection result of lipase of strain BJ-1
  • top Escherichia coli JM109
  • bottom left Bacillus subtilis IPEP6
  • bottom right strain BJ-1.
  • Fig. 6 is the test result of glucose fermentation of strain BJ-1
  • left Escherichia coli JM109
  • middle strain BJ-1
  • right Bacillus subtilis IPEP6.
  • Fig. 7 is the test result of sucrose fermentation of strain BJ-1;
  • left Escherichia coli JM109
  • middle strain BJ-1
  • right Bacillus subtilis IPEP6.
  • Fig. 8 is the detection result of lactose fermentation of strain BJ-1;
  • left Escherichia coli JM109
  • middle strain BJ-1
  • right Bacillus subtilis IPEP6.
  • FIG9 is the detection result of mannitol fermentation of strain BJ-1
  • left Escherichia coli JM109
  • middle strain BJ-1
  • right Bacillus subtilis IPEP6.
  • FIG10 is the detection result of inositol fermentation of strain BJ-1
  • left Escherichia coli JM109
  • middle strain BJ-1
  • right Bacillus subtilis IPEP6.
  • Fig. 12 Phylogenetic tree based on 16S rDNA.
  • the pathogen was isolated from diseased grape branches and trunks in Haidian District, Beijing, and the tissue separation method was used to isolate the pathogen.
  • the method was as follows: (1) observe and record the symptoms of grape branch and trunk diseases; (2) cut 2 cm of branches and trunks at intervals of 10 cm from the parts with different disease severity, remove the bark of the grape branch and trunk diseases, cut out 5 mm2 tissue blocks from the diseased and healthy junctions of the branch and trunk tissues, disinfect them with 2% sodium hypochlorite for 2 minutes, 70% ethanol for 30 seconds, and wash them with sterile water for 3 times. Put sterilized filter paper on the tissue blocks and dry them.
  • strain BJ-1 a strain with antagonistic effect on pathogenic fungi was obtained and named BJ-1.
  • the colony morphology of strain BJ-1 is shown in Figure 1. The colonies are round or nearly round, milky white, with rough edges and inconspicuous wrinkles on the surface.
  • Detection medium amylase selection medium (LB medium + 1.0% soluble starch): peptone 10g, yeast extract 5g, NaCl 5g, soluble starch 10g, agar 20g, distilled water 1000mL, pH7.8.
  • Detection method Take 10 ⁇ L of the test strains (target strains and control strains (Escherichia coli and Bacillus subtilis) and shake them. coli, a control strain that does not have starch hydrolase activity, was added dropwise to the test plate, dried and cultured at 28°C for 24-48 hours, then covered and stained with dilute iodine solution, rinsed with clean water, and the size of the transparent circle was observed.
  • Test results As shown in Figure 2, it can be seen that a transparent circle is produced around the target strain BJ-1, indicating that the strain can produce amylase and is positive for starch hydrolysis.
  • the control strain Escherichia coli has no transparent circle around it and does not have starch hydrolysis enzyme activity.
  • Detection plate Contains 10% skim milk water agar medium (agar powder, 1g/100mL water). Use triple skim milk, heat it in a microwave oven first, remove any fat film, and then use a pipette to draw 10mL and add it to 100mL of sterilized, cooled water agar medium.
  • Detection method shake culture the strain to be tested in NA medium overnight, take 10 ⁇ L and drop it onto the test plate, dry it and culture it at 28°C, and observe the protein degradation zone after 12 hours.
  • Test results As shown in Figure 3, it can be seen that there is an obvious transparent circle around the target strain BJ-1, indicating that the strain can produce protease and protease hydrolysis is positive.
  • the control strain Escherichia coli has no transparent circle around it and has no protease activity.
  • Detection plate half LB medium (the amount of carbon source and nitrogen source is halved), plus 0.2% sodium carboxymethyl cellulose.
  • Detection method shake the test strain and the target strain in NA medium overnight, take 10 ⁇ L and drop it on the test plate, dry it and culture it at 28°C for 24h, wash off the colonies, add 1g/L Congo red solution to stain for 1h, then soak and wash twice with 1MNaCl solution for 30min each time to observe whether there is cellulose hydrolysis zone around the colonies.
  • Test results As shown in Figure 4, it can be seen that a white transparent circle can be produced around the target strain BJ-1, indicating that the strain can produce cellulase and cellulase hydrolysis is positive.
  • the control strain Escherichia coli has no transparent circle around it and does not have cellulase hydrolysis activity.
  • Detection culture medium Oil culture medium: peptone 10g, beef extract 5g, peanut oil or sesame oil 10g, 1.6% neutral red aqueous solution 1mL, agar 15g, distilled water 1000mL, pH7.2.
  • Detection method Prepare the oil culture medium and sterilize it for later use. Before pouring the plate, shake the oil culture medium thoroughly to evenly distribute the oil, and then pour it into the culture dish. After the plate solidifies, drop the strain to be tested onto the plate, dry it, and culture it at 28°C for 24 hours. Observe the colonies at the bottom of the plate. If red spots appear, it means that the fatty acid is hydrolyzed, which is a positive reaction.
  • Detection medium Sugar fermentation basic medium: peptone 10g, NaCl 5g, distilled water 1000mL, adjust pH to 7.6, add 1.6% bromocresol purple ethanol solution.
  • Glucose (1%), lactose (0.75%), sucrose (0.75%), mannitol (0.75%), and inositol (0.75%) were added to the sugar fermentation basal medium, and each tube was placed in a Dulbecco's fermenter.
  • the strains to be tested were inoculated into the sugar-containing medium, and the uninoculated culture medium was used as a control.
  • the culture was cultured at 30°C for 7 days, and the color changes of each test tube and the presence of bubbles in the Dulbecco's fermenter were observed.
  • the indicator bromocresol purple has an indication range of pH 5.2 (yellow) to 6.8 (purple).
  • the results of the glucose fermentation experiment are shown in FIG6 .
  • the color of the test tube culture medium of the test strain BJ-1 changed from purple to yellow, and no bubbles were generated in the Dulbecco tube, indicating that the test strain can decompose glucose and produce acid but not gas.
  • the results of the sucrose fermentation experiment are shown in FIG7 .
  • the test tube culture medium of the tested strain BJ-1 strain changed from purple to yellow, and no bubbles were generated in the Dulbecco tube, indicating that the tested strains can decompose sucrose and produce acid but not gas.
  • the results of the mannitol fermentation experiment are shown in FIG9 .
  • the color of the test tube culture medium of the test strain BJ-1 changed from purple to yellow, and no bubbles were generated in the Duroc tube, indicating that the test strain can decompose mannitol and produce acid but not gas.
  • Staining fluid and reagents crystal violet, Lugol's iodine solution, 95% alcohol, safranin.
  • Staining method The strain to be tested and the target strain are cultured on the LB plate for 48 hours. Take a clean glass slide, add a drop of distilled water on the left and right of the glass slide, take a bacterial smear according to the aseptic operation method, apply the test bacteria on the left and the target strain on the right to make a concentrated bacterial suspension. Take another clean glass slide and apply 1-2 loops of the test bacterial suspension just made on the left to make a thin smear, and take 1-2 loops of the concentrated bacterial solution of the target strain on the right to make a thin coating. Let the smear dry naturally, hold one end of the glass slide with the bacterial film facing up, and fix it by passing through a flame 2-3 times.
  • Microscopic examination First use low power, then high power, and finally use oil objective to observe and determine the Gram staining reactivity of the bacteria.
  • the results of Gram staining are shown in FIG11 .
  • the bacteria of strain BJ-1 are all short rod-shaped with blunt ends.
  • the stained cells are purple, indicating that they are Gram-positive bacteria.
  • the preserved bacterial liquid was spread on a plate and cultured at 25°C for 3 days.
  • the bacteria were scraped off with an inoculation loop and washed out with LB liquid culture medium.
  • the bacterial genomic DNA was extracted using the Bacterial Genomic DNA Rapid Extraction Kit from Beijing Bomade Gene Technology Co., Ltd.
  • the genomic DNA of BJ-1 was extracted and used as a template for PCR amplification using the bacterial 16S rDNA universal primers 27f and 1492r.
  • the reaction system used for PCR amplification was as follows: 25 ⁇ L of 2 ⁇ Taq PCR Mix, 2 ⁇ L of DNA template (100 ng/ ⁇ L), 1 ⁇ L of each of upstream and downstream primers (10 ⁇ mol/L), and sterile ddH 2 O was used to make up to 50 ⁇ L of the system.
  • the PCR amplification product was detected by electrophoresis, and the fragment length was about 1500 bp (sequence 1 in the sequence list).
  • the PCR product was verified by 1% agarose gel electrophoresis and sent to Beijing Bomed Gene Technology Co., Ltd. for sequencing.
  • the sequencing results of the 16S rDNA PCR amplified fragments of the BJ-1 strain are shown in Sequence 1 in the sequence table.
  • the obtained sequences were compared with the sequences in the GenBank database of the NCBI website to obtain the strain species with the highest similarity to the sequenced 16S rDNA sequence.
  • the results showed that the 16S rDNA sequence similarity of the BJ-1 strain to that of Bacillus velezensis BCRC 17467 T was 99.85%. It was preliminarily identified as a strain of a closely related population of Bacillus velezensis.
  • strain BJ-1 was aggregated with Bacillus velezensis strain AB78 (GenBank accession number: MN100588.1). Based on this, strain BJ-1 was identified as Bacillus velezensis.
  • Strain BJ-1 was deposited in the China General Microbiological Culture Collection (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing), with a deposit date of December 16, 2021, and a deposit number of CGMCC No. 24113.
  • the confrontation culture method was used to determine the antagonistic activity of strain BJ-1 with 59 strains of 26 plant pathogenic fungi (the information of plant pathogenic fungi strains is shown in Table 1, which were identified by the Plant Disease Prevention Laboratory of Beijing Academy of Agricultural and Forestry Sciences according to morphological and molecular identification methods and Koch's postulates) as target bacteria.
  • BJ-1 was cultured on LB solid medium at 25°C for 2-3 days, and then a single colony was picked and placed in 1mL LB liquid medium, and cultured at 37°C and 200rpm/min for 24h for use.
  • 59 pathogenic bacteria strains were cultured on PDA plates for 3-5 days, and a hole was punched in the edge area of the colony to make a pathogen cake with a diameter of 5mm, which was placed on the PDA plate.
  • 4 ⁇ L of the cultured bacterial suspension was dripped on a filter paper with a diameter of 6mm, which was placed about 1cm away from the edge of the plate.
  • Four filter papers were placed on one plate, and the same amount of clean water was dripped on the filter paper as a control.
  • Inhibition rate (%) (control colony radius - treated colony radius) / control colony radius ⁇ 100%
  • strain BJ-1 The antibacterial activity of strain BJ-1 against 59 strains of 26 plant pathogenic fungi was measured by confrontation culture method. The results are shown in Table 1. The experimental results show that strain BJ-1 has obvious inhibitory effect on different plant pathogenic fungi tested. The inhibitory effect on mycelium growth was between 53.76% and 98.45%, and the inhibitory effect on mycelium growth of Diaporthesojae OSS1, which causes grape vine blight, was the best, with an inhibition rate of up to 98.45%. The results showed that BJ-1 has a broad spectrum of inhibitory effects on common fruit and vegetable plant pathogens.
  • the strain of tomato gray mold (Botrytis cinerea) was preserved by the Plant Disease Research Laboratory of the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences and identified by conventional methods.
  • the preserved strain was transferred to a PDA plate and cultured in a 25°C incubator.
  • the original powder of biocontrol bacteria BJ-1 was prepared with sterile water as solvent into four concentration gradients of 1 ⁇ 10 5 spores/mL, 1 ⁇ 10 6 spores/mL, 5 ⁇ 10 6 spores/mL and 1 ⁇ 10 7 spores/mL for use.
  • the treatment with the agent adopts the spray method.
  • the nozzle of the throat sprayer evenly sprays the bacterial solution on the back of the tomato leaves, and the water just overflows from the leaf surface.
  • inoculate with tomato gray mold cake (4mm in diameter) so that the side with hyphae faces down and fits the inoculation point of the leaf.
  • the determination of the therapeutic effect is to first inoculate the pathogen, and then treat the inoculated tomato leaves with different bacterial solutions and sterile water 24 hours after inoculation.
  • the methods and materials used are the same as those for the protective effect determination.
  • the cross method was used to measure the diameter of the lesions on the fruits of each treatment, and the relative protection effect was calculated according to the formula.
  • the calculation formula is as follows:
  • strain BJ-1 The control effect of strain BJ-1 on tomato gray mold was measured by the in vitro leaf inoculation method, and the results are shown in Table 2. The results showed that the four concentrations of strain BJ-1 suspension had a certain protective effect on tomato gray mold. With the increase of concentration, the protection and treatment effects gradually improved. Among them, the BJ-1 fermentation liquid with a concentration of 1 ⁇ 10 7 CFU/mL had the best protection and treatment effects, which were 68.95% and 88.28% respectively.
  • the control agent was 10 billion CFU/g Bacillus subtilis wettable powder (Baiwo Co., Ltd., USA) Co., Ltd., pesticide registration number: PD20160669) had a protective and therapeutic effect of 49.19% and 20.08% against tomato gray mold, which was significantly lower than that of strain BJ-1. Therefore, the control effect of strain BJ-1 against tomato gray mold was significantly better than that of the control agent 10 billion CFU/g Bacillus subtilis wettable powder.
  • Example 5 Field test of the efficacy of 2.0 ⁇ 10 10 CFU/Kbeles Bacillus BJ-1 water dispersible granules against tomato gray mold
  • the test agent was 2.0 ⁇ 10 10 CFU/g Bacillus velutipes BJ-1 water dispersible granules (the final concentration of BJ-1 mother powder was 2.0 ⁇ 10 10 CFU/g; polyvinyl alcohol 6.0%; sodium dodecyl sulfate 4.0%; ammonium sulfate 4%; polyethylene glycol 4%; talc powder was supplemented to 100%, wherein the percentages are by mass); the control agent was 100 billion spores/g Bacillus subtilis wettable powder (Jiangxi Zhengbang Crop Protection Co., Ltd., pesticide registration number: PD20151587).
  • the experimental crop is tomato, variety "Jingcai 8", and the growth period is the fruiting period.
  • the information of pesticide treatment is shown in Table 3.
  • the area of each plot is 20m 2 , and each treatment is repeated 4 times.
  • the first application was on December 29, 2022, and the pesticide was applied once every 7 days for a total of 3 times.
  • the disease control efficacy survey was conducted on January 20, 2023.
  • the survey standard was based on the agricultural industry standard of the People's Republic of China, "Guidelines for Field Efficacy Tests of Pesticides (I): Fungicides for Control of Vegetable Gray Mold" (GB/T 17980.28-2000).
  • test agent 2.0 ⁇ 10 10 CFU/g Bacillus subtilis BJ-1 water dispersible granules had a good control effect on tomato gray mold, with an average control effect of 72.38% to 80.48%.
  • the average control effects of the test agent 2.0 ⁇ 10 10 CFU/g Bacillus subtilis BJ-1 water dispersible granules in treatment 1 (112.5 g/mu), treatment 2 (225 g/mu), treatment 3 (450 g/mu) and the control agent 100 billion spores/g Bacillus subtilis wettable powder (70 g/mu) were 72.38%, 76.05%, 80.48%, and 66.48%, respectively.
  • BJ-1 water dispersible granules treatment 1 112.5 g/mu
  • treatment 2 225 g/mu
  • treatment 3 450 g/mu
  • the control agent 100 billion spores/g Bacillus subtilis wettable powder (70 g/mu). Therefore, 2.0 ⁇ 10 10 CFU/g of Bacillus sp.
  • BJ-1 water dispersible granules can be used as a pesticide for controlling tomato gray mold. It can be sprayed before or at the early stage of tomato gray mold onset. The recommended dosage of the preparation is 225-450 g/mu.

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Abstract

提供了一株贝莱斯芽孢杆菌BJ-1菌株及其应用。该菌株的保藏编号为CGMCC No.24113。该菌株具有对植物病原菌的抑菌活性且具有较宽的抑菌谱。该菌株发酵液对番茄灰霉病具有较好的保护和治疗作用。该菌株具有生防潜力，可用于制备针对植物病原真菌的生防菌剂和微生物菌肥。

Description

一株贝莱斯芽孢杆菌及其应用 技术领域
本发明属于微生物应用领域，具体涉及一株贝莱斯芽孢杆菌(Bacillus velezensis)及其在防治番茄灰霉病等植物病害上的应用。
背景技术
灰葡萄孢(Botrytis cinerea)寄主广泛，可侵染葡萄、草莓、樱桃、番茄、西瓜等多种果蔬，引起作物灰霉病，在世界范围内均有发生，一般情况下造成的产量损失为10％～20％，严重时高达60％及以上。化学防治是当前实际生产中防控作物灰霉病的主要措施(康立娟,张小风,王文桥,等.灰霉菌的抗药性与适合度测定.农药学学报,2003,2:39-42.)。然而由于灰葡萄孢具有繁殖快速、遗传变异大和适合度高等特点，对化学农药的抗性产生快，导致药剂的防治效果降低(Hahn M.The rising threat of fungicide resistance in plant pathogenic fungi:Botrytis as a case study.Journal of Chemical Biology,2014,7(4):133-141.)。此外，长期、反复和大量使用化学农药可引起土壤、水体和大气的污染，破坏了生态平衡。生物农药具有低毒、绿色和安全等特点，符合可持续发展理念(Kaur S,Samota M K,Choudhary M,et al.How do plants defend themselves against pathogens-Biochemical mechanisms and genetic interventions.Physiology and Molecular Biology of Plants,2022,28(2):485-504.)，因此开发应用于作物灰霉病的生物农药迫在眉睫。芽孢杆菌(Bacillus spp.)在自然界分布广泛，且逆境下产生的休眠体芽孢不仅含水量极低，且具有耐酸、耐盐、耐高温、抗挤压等特点，其代谢还可产生多种酶类以及杆菌肽、环脂、类噬菌体颗粒等十几种抗菌物质，相比其他有益微生物，芽孢杆菌存活率高、繁殖能力强、环境适应能力与抗逆能力强，是目前生物农药产品中使用最广泛的一类生防菌(IvicaI,Janakiev T,M,et al.Plant-associated Bacilus and Pseudomonas antimicrobial activities in plant disease suppression via biological control mechanisms-A review,Physiological and Molecular Plant Pathology,2022,117:101754)。芽孢杆菌的种类多，在生物农药应用方面较多的种类包括枯草芽孢杆菌(Bacilus subtilis)、解淀粉芽孢杆菌(Bacilus amlyoliquefaciens)、多 粘类芽孢杆菌(Paenibaciluspolmyxa)、蜡样芽孢杆菌(Bacilus cereus)、地衣芽孢杆菌(Bacilus licheniformis)等。贝莱斯芽孢杆菌(Bacilus velezensis)近年来由于具有较好的抑菌效果得到了研究人员的关注，如有研究表明，贝莱斯芽孢杆菌对防治立枯丝核菌引起的生菜根腐病和雷尔氏菌引起的番茄青枯病起主要作用(Cao Y,Pi HL,ChandrangsuP,et al.Antagonism of two plant-growth promoting Bacilus velezensis isolates against Ralstonia solanacearum and Fusarium oxysporum.Scientific Reports,2018,8(1):4360.)；贝莱斯芽孢杆菌BS87和RK1对草莓枯萎病的田间防治效果与氢氧化铜相当(Nam MH,Park MS,Kim HG,et al.Biological control of strawberry Fusarium wilt caused by Fusarium oxysporumf.spfragariae using Bacilus velezensis BS87and RK1formulation.Journal of Microbiology and Biotechnology,2009,19(5):520-524.)；Balderas等人从芒果树叶中分离得到的贝莱斯芽孢杆菌83对芒果炭疽病的防治效果与化学处理相当(Balderas-Ruiz KA,Bustos P,Santamaria RI,et al.Bacilus velezensis 83a bacterial strain from mango phyllosphere,useful for biological Control and plant growth promotion.AMB Express,2020,101:7-10.)。
发明内容
为了解决上述问题，本公开提供了一株贝莱斯芽孢杆菌及其应用。
本发明的贝莱斯芽孢杆菌，分类命名为贝莱斯芽孢杆菌Bacillus velezensis，采集自河北省张家口怀来县，分离自葡萄枝干病害的葡萄病枝干组织，命名为贝莱斯芽孢杆菌(Bacillus velezensis)BJ-1，已于2021年12月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号)，保藏编号为CGMCC No.24113。
本发明的贝莱斯芽孢杆菌在LB培养基上不产色素，菌落为乳白色不透明，圆形或近圆形，边缘不光滑，表面有明显褶皱。所述贝莱斯芽孢杆菌BJ-1的生理生化特征为革兰氏染色阳性，可产纤维素酶，不产蛋白酶、淀粉酶。
本发明的贝莱斯芽孢杆菌具有对植物病原真菌的抑菌活性并且具有较宽的抑菌谱。
所述植物病原真菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe  sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、Colletotrichum viniferum、Colletotrichum sojae、隐秘炭疽菌(Colletotrichum aenigma)、胶孢炭疽菌(Colletotrichum gloeosporioides)、果生炭疽菌(Colletotrichum fructicola)、暹罗炭疽菌(Colletotrichum siamense)、尖孢炭疽菌(Colletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniella vitis、Dactylonectria alcacerensis、Dactylonectria macrodidyma、Lasiodiplodiapseudotheobromae、Dactylonectria torresensis、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、枝状枝孢菌(Cladosporium cladosporioides)、核盘菌(Sclerotinia sclerotiorum)。对上述26种植物病原真菌59个菌株的菌丝生长抑制率在53.76％-98.45％之间。该菌株在植物真菌病害生物制剂开发方面具有非常好的应用前景。
本发明还提供一种针对植物病害的生物农药，所述生物农药的活性成分为上述的贝莱斯芽孢杆菌。
所述生物农药还包括农药制剂可接受的辅料和/或助剂。
在本发明一个优选的实施方式中，所述生物农药为包含1.0×108CFU/克以上的贝莱斯芽孢杆菌BJ-1的水分散粒剂；其中，贝莱斯芽孢杆菌BJ-1为权利要求1所述的贝莱斯芽孢杆菌；
优选的，所述水分散粒剂由下述组分组成：
贝莱斯芽孢杆菌BJ-1终浓度为1.0×108-1.0×1011，优选为2.0×1010；聚乙烯醇6.0％；十二烷基硫酸钠4.0％；硫酸铵4％；聚乙二醇4％；滑石粉补足至100％，所述百分含量为质量百分含量。
同时，本发明的贝莱斯芽孢杆菌菌株对由灰葡萄孢引起的番茄灰霉病在室内和田间均表现较好的防效。室内离体叶片接种试验结果表明，贝莱斯芽孢杆菌BJ-1发酵液对番茄灰霉病具有较好的保护和治疗作用，1×107CFU/mLBJ-1发酵液对番茄灰霉病的保护和治疗作用相对防效分别可达68.95％和88.28％。田间试验结果表明，贝莱斯芽孢杆菌BJ-1水分散粒剂对番茄灰霉病具有较好的防治效果，2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂处理(450克/亩)的相对防效可达80.48％。本发明的贝莱斯芽孢杆菌具有生防潜力，可用于制备针对植物病原真菌的生防菌剂和微生物菌肥。
本公开的本发明中提供的贝莱斯芽孢杆菌对多种植物病原菌具有较好的抑制效果，对灰葡萄孢引起的番茄灰霉病在田间表现较好的防效，因此其对于防治灰葡萄孢引起的果蔬灰霉病较好的应用前景。该菌株培养条件简单、生长迅速且容易保存，适合用于工业生产。
附图说明
图1菌株BJ-1的菌落形态和扫描电镜照片。
图2菌株BJ-1的淀粉酶检测结果；
其中，上：大肠杆菌Escherichia coliJM109，左下：枯草芽孢杆菌Bacillus subtilisIPEP6，右下：菌株BJ-1。
图3菌株BJ-1的蛋白酶检测结果；
其中，上：大肠杆菌Escherichia coliJM109，左下：枯草芽孢杆菌Bacillus subtilisIPEP6，右下：菌株BJ-1。
图4菌株BJ-1的纤维素酶检测结果；
其中，上：大肠杆菌Escherichia coliJM109，左下：枯草芽孢杆菌Bacillus subtilisIPEP6，右下：菌株BJ-1。
图5菌株BJ-1的脂肪酶的检测结果；
其中，上：大肠杆菌Escherichia coliJM109，左下：枯草芽孢杆菌Bacillus subtilisIPEP6，右下：菌株BJ-1。
图6菌株BJ-1的葡萄糖发酵的检测结果；
其中，左：大肠杆菌Escherichia coliJM109，中：菌株BJ-1，右：枯草芽孢杆菌Bacillus subtilisIPEP6。
图7菌株BJ-1的蔗糖发酵的检测结果；
其中，左：大肠杆菌Escherichia coliJM109，中：菌株BJ-1，右：枯草芽孢杆菌Bacillus subtilisIPEP6。
图8菌株BJ-1的乳糖发酵的检测结果；
其中，左：大肠杆菌Escherichia coliJM109，中：菌株BJ-1，右：枯草芽孢杆菌Bacillus subtilisIPEP6。
图9菌株BJ-1的甘露醇发酵的检测结果；
其中，左：大肠杆菌Escherichia coliJM109，中：菌株BJ-1，右：枯草芽孢杆菌Bacillus subtilisIPEP6。
图10菌株BJ-1的肌醇发酵的检测结果；
其中，左：大肠杆菌Escherichia coliJM109，中：菌株BJ-1，右：枯草芽孢杆菌Bacillus subtilisIPEP6。
图11菌株BJ-1的革兰氏染色结果。
图12基于16SrDNA系统进化树。
生物材料保藏
名称：贝莱斯芽孢杆菌(Bacillus velezensis)BJ-1；
分类命名：贝莱斯芽孢杆菌Bacillus velezensis；
保藏日：2021年12月16日；
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号)；
保藏编号：CGMCC No.24113。
具体实施方式
实施例1.贝莱斯芽孢杆菌(Bacillus velezensis)BJ-1的获得和鉴定
采集自北京市海淀区，分离自表现葡萄枝干病害症状的葡萄病枝干组织，采用组织分离法进行病原菌分离，方法如下：(1)观察并记录葡萄枝干病样的症状；(2)对不同发病程度的部分每间隔10cm各切取2cm的枝干，去掉葡萄枝干病样的树皮，从枝干组织的病健交界处切出5mm2的组织块，用2％的次氯酸钠消毒2分钟，70％的乙醇消毒30s，无菌水洗3次，把组织块上放灭菌的滤纸晾干，待水分吸干之后把组织块放在PDA平板上，每个PDA平板放置4～5个组织块，封口膜封口；(3)于25℃，黑暗条件下培养3d，3d后观察、统计菌落生长情况并计算相应的分离比率；(4)挑取菌落边缘少量菌丝于新的PDA或其他易诱导产孢的培养基中，待产生分生孢子后进行单孢分离。在分离病原真菌的过程中，发现一株细菌对病原真菌具有很好的拮抗作用，将其在LB培养基平板划线纯化，获得细菌的纯培养物，经鉴定，获得了一株对病原菌具有拮抗作用的菌株，将其命名为BJ-1，菌株BJ-1的菌落形态如图1所示，菌落为圆形或近圆形，乳白色，边缘不光滑，表面有不明显褶皱。
1菌株BJ-1的生理生化特征
1.菌株的生理生化鉴定
1.1淀粉酶的检测
检测培养基：淀粉酶选择培养基(LB培养基+1.0％可溶性淀粉)：蛋白胨10g，酵母膏5g，NaCl 5g，可溶性淀粉10g，琼脂20g，蒸馏水1000mL，pH7.8。
检测方法：取10μL摇培的待测菌株(目标菌株和对照菌株(大肠杆菌和枯草芽孢杆 菌)，对照菌株大肠杆菌不具有淀粉水解酶活性)滴加到检测平板上，晾干后28℃恒温培养24-48h，然后用稀碘液覆盖染色，清水冲洗，观察透明圈大小。
检测结果：如图2所示，可以看出，在目标菌株BJ-1的周围有透明圈产生，说明该菌株能产生淀粉酶，淀粉水解阳性。对照菌株大肠杆菌的周围无透明圈，不具有淀粉水解酶活性。
1.2蛋白酶检测
检测平板：含10％的脱脂牛奶水琼脂培养基(琼脂粉，1g/100mL水)，选用三元脱脂牛奶，先微波炉加热，如果有脂肪膜则去掉，再用移液枪吸取10mL加到100mL已灭菌冷凉的水琼脂培养基内。
检测方法：将待测菌株在NA培养基中摇培过夜，取10μL滴加到检测平板，晾干后28℃恒温培养，12h后观察蛋白降解圈。
检测结果：如图3所示，从图中可以看出，在目标菌株BJ-1的周围有较明显的透明圈产生，说明该菌株能产生蛋白酶，蛋白酶水解阳性。对照菌株大肠杆菌的周围无透明圈，不具有蛋白酶活性。
1.3纤维素酶检测
检测平板：半LB培养基(碳源、氮源的量减半)，加0.2％的羧甲基纤维素钠。
检测方法：将待测菌株和目标菌株在NA培养基中摇培过夜，取10μL滴加到检测平板上，晾干后于28℃恒温培养24h，洗掉菌落，加入1g/L刚果红溶液染色1h，然后用1MNaCl溶液浸泡洗涤2次进行脱色，每次浸泡30min，观察菌落周围有无纤维素水解圈。
检测结果：如图4所示，可以看出，在目标菌株BJ-1的周围能产生白色透明圈，说明该菌株能产生纤维素酶，纤维素酶水解阳性。对照菌株大肠杆菌的周围无透明圈，不具有纤维素水解酶活性。
1.4脂肪酶检测
检测培养基：油脂培养基：蛋白胨10g，牛肉膏5g，花生油或香油10g，1.6％中性红水溶液1mL，琼脂15g，蒸馏水1000mL，pH7.2。
检测方法：配置油脂培养基，灭菌后备用。倒平板前，将油脂培养基充分震荡使油脂均匀分布，再倒入培养皿中。平板凝固后将待测菌株滴加到平板上，晾干后28℃恒温培养24h。观察平板底层的菌落，如果出现红色斑点，即说明脂肪酸被水解，为阳性反应。
检测结果：如图5所示，可以看出，所有待测菌株的平板底层的菌落周围无红色斑点出现，说明它不能产生脂肪酶，脂肪酶水解阴性。
1.5糖醇发酵实验
检测培养基：糖发酵基础培养基：蛋白胨10g、NaCl5g、蒸馏水1000mL，调PH7.6，加入1.6％溴甲酚紫乙醇溶液。
检测方法：糖发酵基础培养基按照分别加入葡萄糖(1％)、乳糖(0.75％)、蔗糖(0.75％)、甘露醇(0.75％)、肌醇(0.75％)，分装试管后，每管放入一杜氏发酵罐，将待测菌株分别接入含糖培养基中，以不接种的培养液作为对照，30℃培养7d，观察各试管颜色的变化及杜氏发酵罐中有无气泡产生。指示剂溴甲酚紫的指示范围为pH5.2(黄色)～6.8(紫色)。
检测结果：
葡萄糖发酵实验结果如图6所示，从图中可以看出，待测菌株BJ-1的试管培养基颜色均由紫色变为黄色，杜氏管中无气泡产生，说明待测菌株能分解葡萄糖，产酸不产气。
蔗糖发酵实验结果如图7所示，从图中可以看出，待测菌株BJ-1菌株的试管培养基颜色均有紫色变为黄色，杜氏管中无气泡产生，说明待测菌株均能分解蔗糖，产酸不产气。
乳糖发酵实验结果如图8所示，从图中可以看出，待测菌株BJ-1的试管培养基颜色都没有变化，杜氏管中无气泡产生，说明待测菌株不能分解乳糖。
甘露醇发酵实验结果如图9所示，从图中可以看出，待测菌株BJ-1的试管培养基颜色均由紫色变为黄色，杜氏管中无气泡产生，说明待测菌株能分解甘露醇，产酸不产气。
肌醇发酵实验结果如图10所示，从图中可以看出，待测菌株BJ-1的试管培养基颜色没有变化，杜氏管中无气泡产生，说明待测菌株不能分解肌醇。
2菌株的革兰氏染色
2.1材料与方法
染色液和试剂：结晶紫、卢戈氏碘液、95％酒精、番红。
染色方法：待测菌株和目标菌株在LB平板上培养48h，取干净载玻片一块，在载玻片的左、右各加一滴蒸馏水，按无菌操作法取菌涂片，左边涂待测菌，右边涂目标菌株，制成浓菌悬液。再取干净载玻片一块将刚制成的待测菌菌悬液挑1-2环涂在左边制成薄的涂片，将目标菌株的浓菌液取1-2环涂在右边制成薄涂面。让涂片自然晾干，手持玻片一端，让菌膜朝上，通过火焰2-3次固定。加适量(以盖满细菌涂面)的结晶紫染色液染色1min，倾去染色液，用水小心的冲洗。滴加卢戈氏碘液，媒染1min。用水洗去碘液，将玻片倾斜，连续点滴95％乙醇通过涂面涂色20-25s至流出液无色，立即水洗。滴加番红复染3-5min。用水洗去涂片上的番红染色液。将染色的涂片放空气中晾干。
镜检：先用低倍，再用高倍，最后用油镜观察，并判断菌体的革兰氏染色反应性。
2.2实验结果
革兰氏染色的结果如图11所示，菌株BJ-1的菌体均为短杆状，两端钝圆，经染色细胞为紫色，说明是革兰氏阳性菌。
3菌株BJ-1的分子鉴定
3.1基因组DNA的提取：
将保存的菌液涂平皿后，25℃培养3d后，用接种环刮下菌体后使用LB液体培养基洗出，采用北京博迈德基因技术有限公司细菌基因组DNA快速提取试剂盒提取细菌的基因组DNA。
3.2 16S rDNA的PCR扩增：
对BJ-1进行基因组DNA的提取，并以这株细菌基因组DNA为模板，选用细菌16S rDNA通用引物27f和1492r进行PCR扩增。
PCR扩增所用引物：
27f：5’-AGAGTTTGATCCTGGCTCAG-3’
1492r：5’-TACCTTGTTACGACTT-3’
PCR扩增所用反应体系：2×Taq PCR Mix 25μL，DNA模板(100ng/μL)2μL，上下游引物(10μmol/L)各1μL，用无菌ddH2O补足50μL体系。
PCR反应程序：94℃3min；94℃30s，54℃30s，72℃90s，35个循环；72℃10min；保温：4℃。
PCR扩增产物通过电泳检测，片段长度约为1500bp(序列表中序列1)。
3.3序列分析与分子鉴定
PCR产物经1％琼脂糖凝胶电泳验证后，送至北京博迈德基因技术有限公司测序。BJ-1菌株的16S rDNA PCR扩增片段测序结果分别如序列表中序列1所示。所获得序列与NCBI网站GenBank数据库中的序列进行比对，获得与测序16S rDNA序列相似度最高的菌株种类。结果显示，BJ-1菌株的与贝莱斯芽孢杆菌Bacillus velezensis BCRC 17467T的16S rDNA序列相似度为99.85％。将其初步鉴定为贝莱斯芽孢杆菌近缘种群菌株。
根据基因片段序列，利用MEGA软件，基于16S rDNA序列利用ML方法构建系统发育树，如图12所示，结果表明菌株BJ-1与贝莱斯芽孢杆菌菌株AB78(GenBank登录号：MN100588.1)聚合到一起，据此，将菌株BJ-1鉴定为贝莱斯芽孢杆菌Bacillus velezensis。将菌株BJ-1保藏于中国普通微生物菌种保藏管理中心(地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)，保藏日期2021年12月16日，保藏编号为CGMCC No.24113。
实施例3.贝莱斯芽孢杆菌(Bacillus velezensis)BJ-1的抑菌活性测定
采用对峙培养法，以26种植物病原真菌的59个菌株(植物病原真菌菌株信息见表1，由北京市农林科学院植病综防研究室按照形态学及分子鉴定方法和柯赫氏法则进行鉴定)为靶标菌，测定菌株BJ-1的拮抗活性。
将BJ-1在LB固体培养基上25℃培养2～3d，后挑取单菌落于1mL的LB液体培养基内，37℃200rpm/min摇培24h，备用。将59个病原菌菌株在PDA平板上培养3～5d，用打孔器在菌落边缘区域打孔制成直径为5mm的病原菌菌饼，放置到PDA平板，同时吸取4μL培养好的菌悬液滴加在直径为6mm的滤纸片上，放置距平板边缘约1cm处，1个板放4个滤纸片，并以滤纸片上滴加同样剂量的清水作为对照，重复3次，28℃培养箱中培养5d，观察抑菌带的产生，测量相对内生拮抗细菌BJ-1方向病原菌菌丝生长的半径(D)和抑菌带宽度(d)，计算抑菌率。
抑制率(％)＝(对照菌落半径-处理菌落半径)/对照菌落半径×100％
表1.菌株BJ-1对59株植物病原真菌的抑菌活性


通过对峙培养法，测得菌株BJ-1对26种植物病原真菌59个菌株的抑菌活性，测定结果如表1所示。由实验结果可知，菌株BJ-1对供试的不同植物病原真菌都具有明显的抑制 作用，对菌丝生长的抑制率在53.76％-98.45％之间，对引起葡萄蔓枯病的大豆间座壳(Diaporthesojae)OSS①菌丝生长抑制效果最佳，抑制率可达98.45％。研究结果表明，BJ-1对常见果蔬植物病原菌的抑制作用有广谱性。
实施例4.离体叶片接种法测定菌株BJ-1对番茄灰霉病的防治作用
1实验方法
1.1病原菌制备
番茄灰霉病菌(Botrytis cinerea)菌株由北京市农林科学院植保所植病研究室保存，经过常规方法鉴定。挑取保存菌种转接到PDA平板上，25℃培养箱培养。
1.2药剂配制
生防菌BJ-1原粉以无菌水为溶剂配制成浓度分别为1×105个孢子/mL、1×106个孢子/mL、5×106个孢子/mL和1×107个孢子/mL四个浓度梯度备用。
1.3试验处理
1.3.1菌株BJ-1对番茄灰霉病的保护作用的测定
药剂处理采用喷雾法，将喉头喷雾器的喷头将菌液均匀喷施到番茄叶背面，以叶面刚好溢水为准。24h后用番茄灰霉菌菌饼(直径4mm)接种，使有菌丝的一面向下和叶片接种点贴合，叶柄处用湿润脱脂棉包裹，然后放入底部铺有双层滤纸的保鲜盒保湿，25℃培养3-5d后检查结果。以上每重复6-8个叶片，每处理3次重复，以清水和对照药剂处理(100亿CFU/克枯草芽孢杆菌可湿性粉剂)为对照。
1.3.2菌株BJ-1对番茄灰霉病的治疗作用的测定
治疗作用的测定是先接种病菌，接种后24h再用不同的菌液及无菌水处理接种的番茄叶片，其所用方法及材料与保护作用测定相同。
1.4数据统计分析
采用十字交叉法测量各处理果实的病斑直径，根据公式计算相对防效。计算公式如下：
2实验结果
通过离体叶片接种方法，测得菌株BJ-1对番茄灰霉病的防治效果，结果如表2所示。结果表明，四个浓度的菌株BJ-1菌悬液对番茄灰霉病都具有一定的保护作用，随着浓度的增加，保护和治疗效果逐步提升，其中浓度为1×107CFU/mL的BJ-1发酵液保护和治疗效果最佳，分别为68.95％和88.28％。对照药剂100亿CFU/克枯草芽孢杆菌可湿性粉剂(美国拜沃股份 有限公司，农药登记证号：PD20160669)对番茄灰霉病的保护和治疗效果分别为49.19％和20.08％，显著低于菌株BJ-1的保护和治疗效果。因此菌株BJ-1对番茄灰霉病的防治效果显著优于对照药剂100亿CFU/克枯草芽孢杆菌可湿性粉剂。
表2菌株BJ-1对番茄灰霉病的防治效果(室内离体叶片接种)
实施例5. 2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂对番茄灰霉病的田间防效测定
1.材料与方法
1.1试验药剂
供试药剂为2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂(BJ-1母粉终浓度为2.0×1010CFU/克；聚乙烯醇6.0％；十二烷基硫酸钠4.0％；硫酸铵4％；聚乙二醇4％；滑石粉补足至100％，其中百分含量为质量百分含量)；对照药剂为1000亿孢子/克枯草芽孢杆菌可湿性粉剂(江西正邦作物保护股份有限公司，农药登记证号：PD20151587)。
1.2供试植物
试验作物为番茄，品种“京彩8”，生长期为结果期。
1.3试验方法
药剂处理信息如表3，每个小区面积为20m2，每个处理重复4次。在番茄灰霉发病初期进行喷雾施药。2022年12月29日第一次施药，每7天施药1次，共施药3次，于2023年1月20日进行病害防效调查。调查标准参照中华人民共和国农业行业标准，《农药田间药效试验准则(一)：杀菌剂防治蔬菜灰霉病》(GB/T 17980.28-2000)进行。
表3试验药剂试验设计
病情指数和防治效果计算方法如下述公式：

1.4数据统计与分析
利用SPSS 25.0软件对试验数据进行统计分析，运用邓肯氏新复极差法(DMRT)法对试验结果数据进行差异显著性检验。
2.结果
由田间调查结果可知，供试药剂2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂对番茄灰霉病有很好的防治效果，平均防治效果72.38％～80.48％。供试药剂2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂处理1(112.5克/亩)、处理2(225克/亩)、处理3(450克/亩)与对照药剂1000亿孢子/克枯草芽孢杆菌可湿性粉剂(70克/亩)药后平均防治效果分别为72.38％、76.05％、80.48％、66.48％。
方差分析结果表明：在0.05水平上，供试药剂2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂处理1(112.5克/亩)、处理2(225克/亩)、处理3(450克/亩)与对照药剂1000亿孢子/克枯草芽孢杆菌可湿性粉剂(70克/亩)之间防效存在显著性差异。在0.01水平上，供试药剂2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂处理1(112.5克/亩)、处理2(225克/亩)、处理3(450克/亩)与对照药剂1000亿孢子/克枯草芽孢杆菌可湿性粉剂(70克/亩)防效之间存在极显著性差异。因此，2.0×1010CFU/克贝莱斯芽孢杆菌BJ-1水分散粒剂可以作为一种防治番茄灰霉病的药剂使用，于番茄灰霉病发病前或发病初期进行喷雾施药，制剂推荐用量为225～450克/亩。
以上所述实施例仅展示了本发明的几种实施方式，其描述较为具体，但对本发明而言只是说明性的，而非限制性的。对于本领域普通技术人员来说，在不脱离所附权利要求所限定的精神和范围的情况下，可做出许多修改、变化和改进，这些都属于本发明的保护范围。
项目名称:WHPI230126
状态:已生成
创建日期:2023-05-02
最近一次修改:2023-05-10
基本信息
当前申请
申请号:before
知识产权局:CN
申请号:
申请日:
申请人档案名:WHPI230126
最早优先权申请
知识产权局:CN
申请号:2023104827243
申请日:2023-05-02
优先权标识
发明名称
申请人和发明人:
发明人姓名:张玮
语言:zh
拉丁名称:Zhang Wei
居住地址:
通信地址:
申请人姓名或名称:北京市农林科学院
语言:zh
拉丁名称:Beijing Academy of Agriculture and Forestry Sciences
居住地址:
通信地址:
序列
序列1:"Seq_1"
特征
残基

Claims (11)

  1. 一株贝莱斯芽孢杆菌，名称为贝莱斯芽孢杆菌(Bacillus velezensis)BJ-1，其特征在于，所述贝莱斯芽孢杆菌已保存于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为为CGMCC No.24113。
  2. 权利要求1所述的贝莱斯芽孢杆菌在制备抑制植物病原真菌的生防菌剂或微生物菌肥或生物农药中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述植物病原菌为可可毛色二孢(Lasiodiplodiatheobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthesojae)、甜樱间座壳(Diaportheeres)、Diaporthehonkonggensis、灰葡萄孢(Botrytis cinerea)、Colletotrichum viniferum、Colletotrichum sojae、隐秘炭疽菌(Colletotrichum aenigma)、胶孢炭疽菌(Colletotrichum gloeosporioides)、果生炭疽菌(Colletotrichum fructicola)、暹罗炭疽菌(Colletotrichum siamense)、尖孢炭疽菌(Colletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsisrosae、Coniellavitis、Dactylonectriaalcacerensis、Dactylonectriamacrodidyma、Lasiodiplodiapseudotheobromae、Dactylonectriatorresensis、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、枝状枝孢菌(Cladosporium cladosporioides)、核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  4. 一种针对植物病原菌的生防菌剂，其特征在于，所述生防菌剂的活性成分为权利要求1所述的贝莱斯芽孢杆菌。
  5. 根据权利要求4所述的生防菌剂，其特征在于，所述植物病原菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、Colletotrichum viniferum、Colletotrichum sojae、隐秘炭疽菌(Colletotrichum aenigma)、胶孢炭疽菌(Colletotrichum gloeosporioides)、果生炭疽菌(Colletotrichum fructicola)、暹罗炭疽菌(Colletotrichum siamense)、尖孢炭疽菌(Colletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniella vitis、Dactylonectria alcacerensis、Dactylonectria macrodidyma、Lasiodiplodiapseudotheobromae、Dactylonectria torresensis、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、枝状枝孢菌(Cladosporium cladosporioides)、核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  6. 一种针对植物病害的微生物菌肥，其特征在于，所述微生物菌肥的活性成分为权利要求1所述的贝莱斯芽孢杆菌。
  7. 根据权利要求6所述的微生物菌肥，其特征在于，导致所述植物病害的植物病原菌为可可毛色二孢(Lasiodiplodia theobromae)、葡萄座腔菌(Botryosphaeria dothidea)、小新壳梭孢(Neofusicoccum parvum)、大豆间座壳(Diaporthe sojae)、甜樱间座壳(Diaporthe eres)、Diaporthe honkonggensis、灰葡萄孢(Botrytis cinerea)、Colletotrichum viniferum、Colletotrichum sojae、隐秘炭疽菌(Colletotrichum aenigma)、胶孢炭疽菌(Colletotrichum gloeosporioides)、果生炭疽菌(Colletotrichum fructicola)、暹罗炭疽菌(Colletotrichum siamense)、尖孢炭疽菌(Colletotrichum acutatum)、Neopestalotiopsis sp.、Neopestalotiopsis rosae、Coniella vitis、Dactylonectria alcacerensis、Dactylonectria macrodidyma、Lasiodiplodiapseudotheobromae、Dactylonectria torresensis、尖孢镰刀菌(Fusarium oxysporum)、双核丝核菌融合群A、双核丝核菌融合群G、链格孢(Alternaria alternata)、枝状枝孢菌(Cladosporium cladosporioides)、核盘菌(Sclerotinia sclerotiorum)中的一种或多种。
  8. 一种针对植物病害的生物农药，其特征在于，所述生物农药的活性成分为权利要求1所述的贝莱斯芽孢杆菌。
  9. 根据权利要求8所述的生物农药，其特征在于，所述生物农药还包括农药制剂可接受的辅料和/或助剂。
  10. 根据权利要求8所述的生物农药，其特征在于，所述生物农药为包含1.0×108CFU/克以上的贝莱斯芽孢杆菌BJ-1的水分散粒剂；其中，贝莱斯芽孢杆菌BJ-1为权利要求1所述的贝莱斯芽孢杆菌；
  11. 根据权利要求10所述的生物农药，其特征在于，所述水分散粒剂由下述组分组成：
    贝莱斯芽孢杆菌BJ-1终浓度为1.0×108-1.0×1011；聚乙烯醇6.0％；十二烷基硫酸钠4.0％；硫酸铵4％；聚乙二醇4％；滑石粉补足至100％，所述百分含量为质量百分含量。
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