WO2019080638A1 - 一株杀菌固氮荧光假单胞菌及其发酵方法与应用 - Google Patents

一株杀菌固氮荧光假单胞菌及其发酵方法与应用

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WO2019080638A1
WO2019080638A1 PCT/CN2018/103224 CN2018103224W WO2019080638A1 WO 2019080638 A1 WO2019080638 A1 WO 2019080638A1 CN 2018103224 W CN2018103224 W CN 2018103224W WO 2019080638 A1 WO2019080638 A1 WO 2019080638A1
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cha0
nif
δrets
strain
nitrogen
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PCT/CN2018/103224
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English (en)
French (fr)
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张友明
涂强
于芳楠
荆晓姝
卞小莹
陈汉娜
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山东大学
德州迈科生物技术有限公司
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Priority to US16/759,077 priority Critical patent/US20210153507A1/en
Publication of WO2019080638A1 publication Critical patent/WO2019080638A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • 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
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the invention belongs to the field of biotechnology. Specifically, the present invention relates to a bactericidal nitrogen-fixing Pseudomonas fluorescens and its fermentation method and application, and particularly relates to its application in biological control.
  • Pseudomonas protegens (Pseudomonas protegens) is a plant biocontrol agent that secretes a variety of active substances and has a certain effect on bacteria, fungi, and larvae of soil-dwelling pests. Therefore, it has a great effect in the prevention and control of plant diseases. Prospects for development, with the potential to replace chemical pesticides.
  • Pseudomonas protegens CHA0 is isolated from tobacco roots, and the secondary metabolite 2,4-diacetylphloroglu-cinol (2,4-DAPG) is effectively produced.
  • the retS gene is a secondary metabolite 2,4-DAPG secreted by CHA0 and a negative regulator of related red pigment synthesis.
  • nitrogen in fertilizer is an indispensable nutrient for plants, and the most important way for nitrogen input in nature is biological nitrogen fixation. Studies have shown that nitrogen-fixing microorganisms can effectively provide nitrogen nutrition to plants for their absorption and utilization and promote their growth.
  • An object of the present invention is to provide a mutant strain of Pseudomonas fluorescens having bactericidal and nitrogen fixation ability in order to overcome the deficiencies of the prior art described above.
  • Bio-engineering method was used to knock out the wild-type Pseudomonas protegens CHA0 and the nitrogen-fixing gene cluster NiF.
  • the bactericidal nitrogen-fixing engineering strain CHA0- ⁇ retS-NiF was obtained.
  • the present invention provides a Pseudomonas protegens CHA0 mutant strain CHA0- ⁇ retS-NiF, which has the accession number: CGMCC No. 14476.
  • the present invention also provides a composition characterized in that the active ingredient is a Pseudomonas fluorescens mutant strain CHA0- ⁇ retS-NiF.
  • the composition may be a microbial agent.
  • the invention also relates to the use of the Pseudomonas fluorescens mutant strain CHA0- ⁇ retS-NiF for sterilizing plants, fixing nitrogen, promoting plant growth, increasing plant yield and/or controlling plant diseases.
  • the invention provides a method of producing a Pseudomonas fluorescens mutant strain CHA0- ⁇ retS-NiF9 comprising the steps of:
  • the invention further relates to a method of sterilizing plants, fixing nitrogen, promoting plant growth, increasing plant yield and/or controlling plant diseases, comprising administering to a plant or a seed thereof a Pseudomonas fluorescens mutant strain CHA0- ⁇ retS-NiF or comprising the strain Composition or microbial agent.
  • the plants to which the present invention relates may be monocotyledonous or dicotyledonous plants, such as cruciferae, grasses, liliaceae, and the like.
  • the mutant strain of Pseudomonas protegens CHA0 is CHA0- ⁇ retS-NiF, which is negative for Gram staining.
  • the cells are rod-shaped, the colonies are pale yellow, the edges of the colonies are nicked, and aerobic respiration is performed.
  • the growth state of KB medium is the best, and 28 °C is its optimum growth temperature.
  • the fermentation broth is khaki or light brick red with a large amount of foam; and wild type CHA0
  • the retS gene on the mutant strain CHA0- ⁇ retS-NiF was knocked out, and a NiF gene island with biological nitrogen fixation function was inserted.
  • the deposit number is CGMCC No.14476 (the depository: General Microbiology Center of China Microbial Culture Collection Management Committee, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, Date of Deposit: July 2017 31st).
  • the fermentation culture method of the Pseudomonas protegens CHA0 mutant strain CHA0- ⁇ retS-NiF comprises the following steps:
  • Seed activation The glycerol tube containing CHA0- ⁇ retS-NiF strain was taken out from the ultra-low temperature freezer storage box at -80 °C. After thawing, a small amount of bacterial liquid was taken and streaked on LB+genta 20 plate, and the temperature was adjusted at 30 °C. Incubate in the incubator for 20 h, randomly select 5 single colonies from the plate for colony PCR verification to ensure the correct target strain is obtained;
  • Fermentation tank culture The seed liquid is inoculated into a fermenter containing KB medium, and the inoculum amount is 5 to 10% (5 to 10 ml of seed liquid is added per 100 ml of KB medium), and the aeration amount is performed after the inoculation, and the dissolved oxygen is dissolved. , temperature, rotation speed, pH setting, cell density was measured every 6h, and the fermentation cycle was 96h.
  • the formula of the KB medium in the steps (2) and (3) is: 10 mL of glycerin per 1000 mL of water, 20 g of peptone, 1.5 g of K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O 1.5 g.
  • the culture condition of the shake flask seed in the step (2) is 30 ° C, 200 rpm.
  • the fermenter culture condition in the step (3) is: a temperature of 26 to 32 ° C, a pH of 6 to 7.5, a rotation speed of 300 to 600 rpm, and an aeration amount of 0.8 to 4.0 L/min. Oxygen 0.8-1.0L/min, dissolved oxygen is not connected with the rotation speed. After 12 to 24 hours of culture, 25 to 100 mL of 50% glucose aqueous solution is added, and then 25 to 100 mL is added every 2 to 6 hours. % glucose aqueous solution until the end of the fermentation, during which the volume ratio is 20% phosphoric acid, the ammonia water maintains the pH stability, and the volume ratio is 50% defoaming agent for defoaming.
  • the fermentation culture conditions in the step (3) are: a temperature of 28 ° C, a pH of 7, and a rotation speed of 600 rpm.
  • the present invention also provides a microbial agent comprising Pseudomonas protegens CHA0 mutant strain CHA0- ⁇ retS-NiF as an active ingredient.
  • the microbial agent is prepared by the step (3), wherein the cells cultured in the fermenter enter a stable period, and when the cell density reaches a maximum value, centrifugation is performed, and the cells are collected after lyophilization. That is.
  • the invention provides a fluorescent pseudomonas mutant strain CHA0- ⁇ retS-NiF and a microbial agent thereof as an active ingredient, and the potting test at room temperature proves that the strain can effectively promote plant growth. Mainly due to the knockout of retS gene, the mutant strain secretes the secondary metabolite 2,4-DAPG, and the bactericidal ability is enhanced. At the same time, the nitrogen-fixing gene cluster NiF is integrated to make the mutant strain have nitrogen-fixing ability, providing nitrogen source for plants and satisfying plants. The need for nitrogen sources during the growth process.
  • Figure 1 is a flow diagram of knocking out the rets gene on the CHA0 bacterial chromosome.
  • Figure 2 is a diagram showing the colony PCR verification of the fluorescent pseudomonas mutant strain CHA0- ⁇ retS of the present invention.
  • Figure 3 is a diagram showing the restriction endonuclease digestion of the expression plasmid pBeloBAC11-oriT-TnpA-genta-NiF by restriction endonuclease Kpn I constructed by the Red/ET direct cloning method of the present invention.
  • Fig. 4 is a flow chart showing the construction of a mutant strain of Pseudomonas fluorescens CHA0- ⁇ retS-NiF in the present invention.
  • Fig. 5 is a colony PCR verification diagram of a nitrogen-fixing mutant Pseudomonas fluorescens strain CHA0- ⁇ retS-NiF of the present invention.
  • Fig. 6 shows the results of bacteriostatic experiments against Pseudomonas fluorescens 1CHA0 (Pseudomonas protegens CHA0) and its mutant strains 2CHA0-NiF, 3CHA0- ⁇ retS and 4CHA0- ⁇ retS-NiF against Bacillus subtilis in the present invention.
  • Figure 7 is a chromatogram of the synthesis of antibiotic 2,4-DAPG by wild type strain CHA0 and its mutant strain.
  • Figure 8 shows the results of nitrogenase activity determination of the experimental strain Pseudomonas protegens CHA0 and its mutant strains CHA0-NiF, CHA0- ⁇ retS and CHA0- ⁇ retS-NiF.
  • Figure 9 shows the results of quantitative quantitative PCR detection of the expression levels of key genes NifD, NifK, NifN, NifM, NifQ, NifS, NifT in the engineering strains CHA0- ⁇ retS-NiF and Pseudomonas aeruginosa DSM4166.
  • Figure 10 shows the optimal pH growth of the mutant strain CHA0- ⁇ retS-NiF.
  • Figure 11 shows the optimal temperature growth of the mutant strain CHA0- ⁇ retS-NiF.
  • Figure 12 shows the optimal rotational speed of the mutant strain CHA0- ⁇ retS-NiF.
  • Figure 13 shows the growth of the mutant strain CHA0- ⁇ retS-NiF in a 5L fermentor.
  • Figure 14 shows the results of measurement of nitrogenase activity of different transformants of mutant strain CHA0- ⁇ retS-NiF.
  • Fig. 15A is a diagram showing the potted effect of Arabidopsis thaliana applying different microbial agents after transplanting into a pot for 4 weeks.
  • Fig. 15B is a schematic view showing the length of the diameter of the rosette after 4 weeks of transplanting different Arabidopsis thaliana into the pot.
  • Figure 16 shows the biological traits in each test treatment of the late growth stage of garlic.
  • the left side is the leaf of Process 1
  • the right side is the leaf of Process 4.
  • the mutant strain of Pseudomonas protegens CHA0 is CHA0- ⁇ retS-NiF, and its preservation number is CGMCC No.14476 (the depository: General Microbiology Center of China Microbial Culture Collection Management Committee, Address: Beichenxi, Chaoyang District, Beijing) No. 3, No. 3, Institute of Microbiology, Chinese Academy of Sciences, date of deposit: July 31, 2017).
  • the plasmid pBBR1-Rha-TEGpsy-kan (the plasmid can express a recombinase in Pseudomonas) is introduced into the wild type Pseudomonas protegens CHA0 by electroporation, and the electroporation is carried out.
  • the bacteria were applied to LB medium (LB medium components: tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 1 g/L, pH 7.0) + kanamycin (km, 30 ⁇ g/mL) On the plate, 12 single colony extraction plasmids were randomly selected for restriction enzyme digestion, and the correct transformants CHA0::pBBR1-Rha-TEGpsy-kan were screened;
  • step 16 electroporation into the CHA0::pBBR1-Rha-TEGpsy-kan obtained in step 1), using the Red/ET homologous recombination method, under the action of the recombinase, gentamicin resistance
  • the gene (genta) will replace the retS gene on the genome of Pseudomonas protegens CHA0, and the recombinant bacteria will be plated on LB medium + genta 15 ⁇ g/mL plate, and multiple single colonies will be randomly selected.
  • Colony PCR validation (the primers used in the validation were check-5'TGCTTCTACCGCAAGGACATC/check-3'GCTGATGAAGCACGAGAGCAC, as shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively), and the correct transformants CHA0:: ⁇ retS- were screened.
  • genta-loxM the primers used in the validation were check-5'TGCTTCTACCGCAAGGACATC/check-3'GC
  • the instructions indicate that the genta resistance gene in the recombinant has not been eliminated; if the LB plate colony grows and does not grow on the LB+genta 15 ⁇ g/mL plate, This indicates that the gena resistance gene in the recombinant has been eliminated.
  • the recombinants whose Genta resistance gene has been eliminated are picked for colony PCR verification and sequencing.
  • the primers are:
  • Figure 1 is a flow diagram of knocking out the rets gene on the CHA0 bacterial chromosome.
  • Figure 2 is a verification diagram of colony PCR reaction of CHA0- ⁇ retS. As shown in the figure, M is a Marker of DL 5,000 DNA, sample No. 1 is a wild type of CHA0 as a control, and sample No.
  • CHA0- ⁇ retS is a final transformant CHA0- ⁇ retS, under the action of Cre recombinase induced by IPTG, Mediating specific recombination between two loxM loci (sequences), the genta resistance gene sequence between loxM sites was deleted, thereby eliminating the exogenous resistance gene for Pseudomonas protegens CHA0 (Pseudomonas protegens CHA0)
  • the effects of growth, reproduction and colonization can be used with greater confidence.
  • Primer 1 AGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGCTTAAGTACGGCTACCTGGAGCTCGCGCCAGTG, as shown in SEQ ID NO.
  • Primer 2 TACGGCTACCTGGAGCTCGCGCCAGTGCTTGCCGACATCGAATCACGGCCGCTGCTGCAGCACGTGGTGGTCACCGGCCGGGATCCGTTTAAACACAAATGGCAAGGGCTAATG, as shown in SEQ ID NO. 2;
  • Primer 3 ATTGATGTTTTCCTTGGCCAGCGCCTCGAACATCCGGCTGGCGACGCCTGCGTGCGAACGCATACCGACACCGACGATAGGGATCCGTTTAAACGGTGTGGTAGCTCGCGTATT, as shown in SEQ ID NO.
  • Primer 4 GCGACACTATAGAATACTCAAGCTTGGCATGAATGCAGGTCGACTCTAGAGAATATTGATGTTTTCCTTGGCCAGCGCCTCGAAC, as shown in SEQ ID NO.
  • the expression plasmid pBeloBAC11-oriT-TnpA-genta-NiF (Fig. 3) was constructed and identified by restriction endonuclease Kpn I, and then the correct plasmid was electrotransferred into E. coli ET12567;
  • junction transfer was as follows: single colonies were picked from the plate, and Pseudomonas fluorescens CHA0- ⁇ retS (LB medium, 30 ° C) was cultured with Escherichia coli ET12567 (LB + genta 2 ⁇ g / mL + cm 10 ⁇ g / mL + km 1 ⁇ g / mL Base, 37 ° C) were separately cultured overnight; two overnight bacterial solutions were centrifuged at 7000 rpm for 1 minute. The Pseudomonas fluorescens CHA0- ⁇ retS and E.
  • coli ET12567 were washed twice with fresh LB medium, resuspended in 300 ⁇ L of LB medium, and each 50 ⁇ L suspension was mixed and mixed in a small area in the middle of the LB plate. , dry. After incubating for 4 h at 37 ° C, the plate was inverted and cultured in an incubator at 30 ° C overnight; the bacteria on the plate were scraped off with an inoculating loop, and suspended in 1 mL of sterile water, and 100 ⁇ L of the bacterial liquid was zigzag-lined.
  • NiF-check-2 CGATTCCAGCGTCGAATGAT
  • NiF-check-4 CAGCGGCACCTCGAGGAGT
  • the first four pairs of primers were used to verify whether the NiF nitrogen-fixing gene was integrated into the genome of Pseudomonas fluorescens CHA0- ⁇ retS, and the amplified PCR fragments were 1000 bp, 970 bp, 830 bp, and 1080 bp, respectively.
  • the fifth pair of primers was used to verify that the strain introducing NiF nitrogen-fixing gene was Pseudomonas fluorescens CHA0 instead of E. coli ET12567, and the PCR amplification result was a retS gene with a DNA fragment size of 3200 bp;
  • Fig. 5 shows that Marker is a marker of DL 5,000 DNA
  • ck1 is a mutant Pseudomonas fluorescens CHA0- ⁇ retS which has been knocked out of the retS gene
  • ck2 is Escherichia coli ET12567, which serves as a control group.
  • Samples 1, 2, and 3 were three CHA0- ⁇ retS-NiF transformants randomly picked from the plates, and each of the five pairs of primers described above was subjected to five colony PCR assays, which were obtained after repeated careful comparison.
  • the filter paper method was used to detect the inhibitory effect of Pseudomonas protegens CHA0 and its mutant strains CHA0-NiF, CHA0- ⁇ retS and CHA0- ⁇ retS-NiF on Bacillus subtilis.
  • the specific steps are as follows: :
  • Bacillus subtilis was centrifuged at 9000 rpm for 1 minute, and 100 ⁇ L of the bacterial solution was uniformly applied to the LB solid medium (15 g/L agar was added to the liquid LB medium).
  • LB solid medium 15 g/L agar was added to the liquid LB medium.
  • several 6 mm diameter double-layer filter paper sheets were placed on the plate, and 5 ⁇ L of the experimental strain Pseudomonas protegens CHA0 and its mutant strain CHA0-NiF, CHA0- ⁇ retS were cultured overnight.
  • the bacterial liquid with CHA0- ⁇ retS-NiF was dropped onto the filter paper sheets, respectively.
  • the plate was placed at 30 ° C overnight;
  • Figure 6 shows that the inhibition zone of the Pseudomonas protegens CHA0 mutant strains 3CHA0- ⁇ retS and 4CHA0- ⁇ retS-NiF, which have been knocked out of the retS gene, is more than the non-knockout res gene of Pseudomonas fluorescens 1CHA0 and The inhibition zone of 2CHA0-NiF was significantly larger (the inhibition zone diameter of the 3 and 4 samples was 2.7 cm, and the inhibition zone diameter of the 1 and 2 samples was 2.3 cm), indicating that it inhibited Bacillus subtilis. The ability to be enhanced after the ⁇ retS gene knockout.
  • the experimental strain Pseudomonas protegens CHA0 and its mutant strains CHA0-NiF, CHA0- ⁇ retS and CHA0- ⁇ retS-NiF were respectively cultured in KB medium at 150 rpm for 24 h at 30 ° C, then 1 mL was added. Resin, the bacteria solution and the resin were shaken for 24 hours, centrifuged at 8000 rpm for 10 min, and the resin was collected and added with an equal volume of ethyl acetate overnight. The supernatant was again centrifuged and steamed to obtain an extract, which was reconstituted with 1 mL of methanol for HPLC detection.
  • HPLC detection conditions reverse phase column Thermo Scientific Acclaim TM C18 (2.1 mm ⁇ 100 mm, 2.2 ⁇ m), column temperature 30 ° C; mobile phase: composed of 0.1% aqueous acetic acid (solvent A) and acetonitrile (solvent B); chromatographic procedure: 0-5 min, 5% solvent B; 5-20 min, 5%-95% solvent B; 20-25 min, 95% solvent B; flow rate 0.5 mL/min.
  • Ultraviolet (UV) light (2,4-DAPG, ⁇ 270 nm) was monitored at 250 nm, 270 nm, 290 nm, and 310 nm, respectively.
  • MS measurements were performed on an amaZon velocity mass spectrometer and ultra high resolution Qq-Time-Of-Flight using a standard ESI (electrospray ionization) source.
  • Figure 7a shows that 1, WT CHA0; 2, CHA0::Nif; 3, CHA0- ⁇ retS; 4, CHA0- ⁇ retS-Nif, the results show that the production of 2,4-DAPG by engineering bacteria knocking out the res gene is much higher.
  • the yield of the antibiotic 2,4-DAPG was increased by about 100-fold in strains that did not knock out retS.
  • Figure 7b shows the UV absorption peak and mass spectrum of the antibiotic 2,4-DAPG.
  • Nitrogenase activity detected ethylene peak area x (anaerobic bottle volume - sample volume) / (standard ethylene gas peak area x reaction time x protein concentration).
  • Fig. 8 show that the Pseudomonas fluorescens CHA0 mutant strain with nitrogen-fixing gene island (NiF) has nitrogenase activity, but the activity expression is different. Among them, the mutant strain of retS-Nif1 has the highest nitrogenase activity. The strain was frozen and used for subsequent fermentation experiment field trials.
  • Real-time PCR was used to detect the expression levels of key genes in the nitrogen-fixing gene island (NiF) in CHA0- ⁇ retS-NiF and Pseudomonas aeruginosa DSM4166.
  • the engineering strains CHA0- ⁇ retS-NiF and Pseudomonas aeruginosa DSM4166 were cultured in KB medium for 24 h, then transferred to a nitrogen-free medium for 6 h, and the total RNA of the target strain was extracted using the RNAPure kit, followed by inversion.
  • the kit purchased from Takara, Japan
  • cDNA complementary DNA strand
  • RR820A The expression levels of seven key genes NifD, NifK, NifN, NifM, NifQ, NifS, NifT in the nitrogen-fixing gene island (Nif).
  • Figure 9 shows that these seven key genes have a certain relative expression in the engineering strain CHA0- ⁇ retS-NiF, and are greater than their expression level in Pseudomonas aeruginosa DSM4166, thus demonstrating the fluorescent A nitrogenase is produced in the bacterium CHA0 (Pseudomonas protegens CHA0).
  • Seed activation Remove the strained glycerol tube from the ultra-low temperature freezer storage box at -80 °C. After thawing, use 1 ul of inoculating loop to draw a small amount of bacterial liquid and scribe it on LB+genta 20 plate, and place it in biochemical incubator. Incubate at 30 ° C, 20 h, and randomly select 5 single colonies from the plate for colony PCR verification to ensure the correct target strain was obtained;
  • Shake flask seed culture Use 1 uL inoculation loop to scrape a small amount of bacteria into the KB medium on the LB+genta 20 plate with activated seeds.
  • the flask volume is 500 mL bottle body liquid 100 mL KB culture.
  • Base placed in a full temperature shaking incubator at 30 ° C, 200 rpm, 20h culture;
  • KB medium is formulated: 10mL of glycerol per 1000mL of water, peptone 20g, K 2 HPO 4 1.5g, MgSO 4 ⁇ 7H 2 O 1.5g;
  • the pH values in the above step (3) were set to 6, 6.5, 7 and 7.5, respectively, and the remaining conditions and steps were unchanged, and the CHA0- ⁇ retS-NiF strain was cultured, and the CHA0- ⁇ retS-NiF strain under different pH culture conditions was cultured. The growth was compared and the results are shown in Figure 10.
  • the temperature in the above step (3) was set to 26 ° C, 30 ° C and 32 ° C, and the remaining conditions and steps were unchanged, and the CHA0- ⁇ retS-NiF strain was cultured, and the CHA0- ⁇ retS-NiF strain under different temperature culture conditions was cultured. The growth was compared and the results are shown in Figure 11.
  • 28 °C is the optimum temperature for the growth of the engineering strain CHA0- ⁇ retS-NiF, and the target strain under the 32 °C culture condition shows a poor growth state from the beginning, and after 36 hours, 28 At °C, 30 °C, 26 °C, the cell growth rate began to change, and the growth advantage of the cells at 28 °C was relatively obvious. This may be related to the temperature-regulating microbial metabolic pathways that affect the rate of enzyme reaction.
  • the rotation speeds of the above step (3) were set to 300rmp, 400rmp and 500rmp respectively, and the remaining conditions and steps were unchanged.
  • the strain of CHA0- ⁇ retS-NiF was cultured, and the growth of CHA0- ⁇ retS-NiF strain under different rotation conditions was carried out. In contrast, the results are shown in Figure 12.
  • 600 rpm is the optimum growth speed of the bacteria, but the growth advantage between 600 rpm and 500 rpm is not very obvious, which indicates that the oxygen demand during the growth of the cells can be basically satisfied under the condition of 500 rpm.
  • Oxygen solubility can affect the metabolic pathway of microorganisms, product yield, enzyme activity, proper oxygen concentration is conducive to the growth of cells, but too high oxygen concentration will accelerate the oxidation of cells, so that the bacteria enter the decline phase early.
  • Seed activation Remove the strained glycerol tube from the ultra-low temperature freezer storage box at -80 °C. After thawing, use 1 ul of inoculating loop to draw a small amount of bacterial liquid and scribe it on LB+genta 20 plate, and place it in biochemical incubator. Incubate at 30 ° C for 20 h;
  • Shake flask seed culture Use 1 uL inoculation loop to scrape a small amount of bacteria into the KB medium on the LB+genta 20 plate with activated seeds.
  • the flask volume is 500 mL bottle body liquid 100 mL KB culture.
  • Base placed in a full temperature shaking incubator at 30 ° C, 200 rpm, 20h culture;
  • KB medium is formulated: 10mL of glycerol per 1000mL of water, peptone 20g, K 2 HPO 4 1.5g, MgSO 4 ⁇ 7H 2 O 1.5g;
  • Fermentation tank culture entangle the cotton soaked in alcohol near the inoculating mouth of the 5L tank, ignite, and after the flame surrounds the inoculation port, use the tweezers to unscrew the screw of the inoculating port. At this time, pour the liquid in the shake flask quickly. In the tank, quickly screw the screw back to the inoculating port before the flame is extinguished.
  • the inoculum is 10% of the total volume of the liquid KB medium in the fermenter.
  • the fermentation product was centrifuged, and the centrifuged cells were freeze-dried to have a dry weight of 15.9 g (DCW/L).
  • the nitrogenase activity of the mutant transformants CHA0- ⁇ retS-NiF was determined by acetylene reduction method.
  • the mutant strain CHA0- ⁇ retS-NiF with the highest nitrogenase activity was selected from different transformants for subsequent pot experiment:
  • Nitrogenase activity difference in detected ethylene peak area ⁇ (gas phase volume/injection amount in a flask) / (standard ethylene gas peak area ⁇ reaction time ⁇ protein concentration).
  • Protein concentration determination was determined according to the method of Coomassie Brilliant Blue:
  • Wild-type Arabidopsis Col-0 was used as the test object.
  • the test conditions were as follows: temperature was 20 ° C, light intensity was 80 ⁇ mol ⁇ m -2 ⁇ s -1 , photoperiod: 16 hours light, 8 hours dark; test was divided into 4 Groups: the original strain strain DSM4166 containing the nitrogen-fixing gene cluster was applied as a positive control with no nitrogen fertilizer applied and wild type Pseudomonas fluorescens strain CHA0 as a negative control, and the preferred nitrogen-fixing enzyme activity of step 1 above was applied.
  • the mutant strain CHA02-3 was used as the test group, and the potting effect was as shown in Fig. 15A.
  • the diameter of the rosette diameter of Arabidopsis thaliana was measured to compare the growth state of Arabidopsis thaliana, and the diameter measurement result of the rosette was as shown in 6B.
  • the lotus leaf diameter of Arabidopsis thaliana was superior to the negative control in the test group in which the nitrogen fertilizer was not applied, but the original strain DSM4166 containing the nitrogen-fixing gene cluster was applied, and the test group of the P. fluorescens strain CHA0- ⁇ retS-NiF was applied. . Furthermore, the Arabidopsis rosette leaf diameter to which the Nigrobacterium fluorescens strain CHA0- ⁇ retS-NiF was applied was superior to the Arabidopsis rosette leaf diameter to which the original strain DSM4166 containing the nitrogen-fixing gene cluster was applied (Fig. 15B).
  • Treatment 1 farmers used to fertilize, including N 45kg/hm 2 , P 2 O 5 22.5kg/hm 2 , K 2 O 22.5kg/hm 2 , organic fertilizer 40kg/mu, high-nitrogen high-potassium compound fertilizer for topdressing;
  • Treatment 2 Optimized fertilization, formula fertilizer N 30kg/hm 2 , P 2 O 5 16kg/hm 2 , K 2 O 24kg/hm 2 , bio-organic fertilizer 200kg/hm 2 ; formula fertilizer for topdressing (18-5-17 Humic acid type) 20kg/mu, mixed with garlic, and used in the spring when topdressing, according to the actual situation, use carbendazim, methyl thiophanate and tonic before fertilization;
  • Treatment 3 Microbial Inoculant - Pseudomonas fluorescens - Cloud (purchased from the market, from Shandong Tylenol Pharmaceutical Co., Ltd.)
  • Fertilization is consistent with optimized fertilization, seed dressing with Pseudomonas fluorescens, fluorescein Pseudomonas liquid is applied with water at the time of sowing, before winter, during regreening, and spring, and when the fertilizer is applied;
  • Treatment 4 Microbial Inoculant - Pseudomonas fluorescens CHA0- ⁇ retS-NiF
  • the fertilization is consistent with the optimization treatment, and the seedlings are mixed with Pseudomonas fluorescens, and the Pseudomonas fluorescens liquid is applied with water at the time of sowing, before winter, during the regreening period and in the spring, and when the fertilizer is applied;
  • the application rate of nitrogen fertilizer is 2/3 of optimized fertilization, and the phosphorus and potassium are consistent.
  • the other fertilization is consistent with the optimized treatment.
  • the seedlings are mixed with Pseudomonas fluorescens, and the fluorescent fakes are applied with water during sowing, before winter, during regreening, and in spring. Cytobacterial liquid, when applying fertilizer;
  • the dosage form of the microbial agent is liquid, and the effective viable cell count is ⁇ 1 billion/ml, and the dosage is 2 kg/mu.
  • the data of the above two tables are the growth indexes of garlic measured at the seedling stage and the winter return period. The measured data are consistent with the results of the field test observation.
  • the three treatments of Pseudomonas fluorescens are in the early stage (Miao The leaf width and root enzyme activity of garlic were significantly promoted, and the most obvious promotion was treatment 4, which reached 38.5%. That is because the fluorescent Pseudomonas agent replaces the seed dressing of Tonin, and the inhibition of the growth of the bacteria also causes damage to the beneficial bacteria around the garlic body, which indirectly hinders the growth of the garlic in the early stage of growth, and the Pseudomonas fluorescens agent The garlic seedlings are growing vigorously.
  • the higher yield of garlic was the three treatments of the experimental group with the addition of Pseudomonas fluorescens.
  • the yield increase was 9.21%, 19.25% and 15.52%, respectively, compared with the control group 1 farmers.
  • the highest yield of garlic treatment was the treatment of Pseudomonas fluorescens CHA0- ⁇ retS-NiF, which was 2050.54 kg/mu: the yield was followed by Pseudomonas fluorescens, which was treated with Pseudomonas fluorescens CHA0- ⁇ retS-NiF.
  • Pseudomonas fluorescens which was treated with Pseudomonas fluorescens CHA0- ⁇ retS-NiF.
  • Pseudomonas fluorescens-cloud treatment yield was 1877.16 kg/mu with an increase rate of 9.21%.
  • the promotion of Pseudomonas fluorescens-cloud on garlic yield was not as good as that of Pseudomonas fluorescens CHA0- ⁇ retS-NiF.
  • the three processed garlic stalks added with Pseudomonas fluorescens had higher yields of 347.73 kg/mu, 375.74 kg/mu and 365.89 kg/mu, respectively, compared with the farmers' customary treatment group of treatment 1. They increased by 9.06%, 17.85% and 14.76% respectively.
  • the use of Pseudomonas fluorescens has a significant effect on the garlic yield of garlic.
  • the most promoting effect was the treatment of Pseudomonas fluorescens CHA0- ⁇ retS-NiF.
  • the yield of garlic stalk increased by 17.85% compared with the control.
  • the yield of garlic stalks in the nitrogen-reduced group of 5 P. fluorescens CHA0- ⁇ retS-NiF engineering bacteria was treated. It is slightly less than treatment 4, but overall remains at a relatively average level.
  • the main diseases of garlic are leaf blight and root rot.
  • Garlic leaf blight is one of the common diseases on garlic.
  • Each vegetable area has different degrees of occurrence, mainly affecting the garlic cultivated in open fields.
  • the number of precipitation is high, and the year when the rainfall is large is serious.
  • the disease is serious, it often causes the dead leaves of the disease, the premature aging of the plants, the reduction of the garlic, the spoilage of the garlic, and directly affects the yield.
  • the disease index is calculated by counting the number of garlic diseases in the plot.

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Abstract

提供了一株杀菌固氮荧光假单胞菌(Pseudomonas protegens)CHA0-ΔretS-NiF及其发酵方法与应用。所述菌株的保藏编号为 CGMCC No.14476，最优培养条件为pH值为7，温度为28℃，转速为600rpm。还提供了一种以荧光假单胞菌CHA0-ΔretS-NiF为活性成分的微生物菌剂。所述荧光假单胞菌CHA0-ΔretS-NiF具有较强的固氮和杀菌能力，可用于防治植物病害及促进植物生长。

Description

一株杀菌固氮荧光假单胞菌及其发酵方法与应用 技术领域
本发明属于生物技术领域。具体地，本发明涉及一株杀菌固氮荧光假单胞菌及其发酵方法与应用，尤其涉及其在生物防治中的应用。
背景技术
荧光假单胞菌(Pseudomonas protegens)是一种植物生防菌，能分泌多种活性物质，在抗细菌、真菌、土栖害虫幼虫等方面都具有一定效果，因此在植物病害防治中具有很大的开发前景，具有替代化学农药的潜质。
荧光假单胞菌CHA0(Pseudomonas protegens CHA0)分离自烟草根系，产生的次生代谢产物2，4-二乙酰基藤黄酚(2，4-diacetylphloroglu-cinol，2，4-DAPG)能够有效地防治Gaeu-mannomyces graminis var.tritici引起的小麦全蚀病，Thielaviopsis basicola引起的烟草黑根腐病和Ralstonia solanacearum引起的番茄细菌性青枯病。retS基因是CHA0分泌的次生代谢产物2，4-DAPG及相关红色素合成负调控因子。
近年来，由于化肥的过量使用使土壤机制发生变化，土壤连作障碍、次生盐渍化、板结和酸化情况严重等的现状，极大地阻碍农作物的产量提高。其中化肥中的氮是植物不可缺少的营养元素，而大自然中氮输入的最主要途径为生物固氮。研究表明固氮微生物可以有效地为植物提供氮营养，供其吸收与利用，促进其生长。
发明内容
本发明的目的是为克服上述现有技术的不足，提供具有杀菌和固氮能力的荧光假单胞菌突变菌株。使用生物工程手段对野生型Pseudomonas protegens CHA0进行了retS基因的敲除及固氮基因簇NiF的整入，最终获得杀菌固氮工程菌CHA0-ΔretS-NiF。
具体地，本发明提供了荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株CHA0-ΔretS-NiF，其保藏编号为：CGMCC No.14476。
本发明还提供了一种组合物，其特征在于，其活性成分为荧光假 单胞菌突变菌株CHA0-ΔretS-NiF。所述组合物可以为微生物菌剂。
本发明还涉及荧光假单胞菌突变菌株CHA0-ΔretS-NiF在对植物杀菌、固氮、促进植物生长、增加植物产量和/或防治植物病害方面的应用。
另一方面，本发明提供了生产荧光假单胞菌突变菌株CHA0-ΔretS-NiF9的方法，其包括以下步骤：
a)敲除荧光假单胞菌CHA0基因组中的retS基因；和
b)将固氮斯氏假单胞菌DSM4166基因组中的NiF固氮基因岛整体克隆到步骤a)得到的菌株中，使之异源表达。
本发明另外涉及对植物杀菌、固氮、促进植物生长、增加植物产量和/或防治植物病害的方法，包括给植物或其种子施用荧光假单胞菌突变菌株CHA0-ΔretS-NiF或包含所述菌株的组合物或微生物菌剂。
本发明涉及的植物可以是单子叶或双子叶植物，例如十字花科、禾本科、百合科植物，等等。
荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株为CHA0-ΔretS-NiF，对其革兰氏染色为阴性菌株，细胞为杆状，菌落呈淡土黄色，菌落边缘缺刻，进行有氧呼吸，在KB培养基中生长状态最佳，28℃为其最适生长温度，通常，经过一段时间的KB培养后其发酵液为土黄色或者浅砖红色，且伴有大量泡沫产生；和野生型的CHA0细菌相比，突变菌株CHA0-ΔretS-NiF染色体上retS基因被敲除，同时插入了具有生物固氮功能的NiF基因岛。
其保藏编号为CGMCC No.14476(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号中国科学院微生物研究所，保藏日期：2017年7月31日)。
荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株CHA0-ΔretS-NiF的发酵培养方法，包括以下步骤：
(1)种子活化：从-80℃超低温冷冻存储箱中取出含有CHA0-ΔretS-NiF菌种的甘油管，待解冻后取少量菌液划线于LB+genta 20平板上，于30℃恒温生化培养箱中倒置培养20h，从平板上随机挑选5个单菌落进行菌落PCR验证以确保获得正确的目的菌株；
(2)摇瓶种子培养：将活化好的CHA0-ΔretS-NiF菌种接入KB培养基中，放置在全温振荡培养箱中培养20h，得到种子液；
(3)发酵罐培养：将种子液接种到装有KB培养基发酵罐中，接种量为5～10％(每100mlKB培养基加入5～10ml种子液)，接种完毕后进行通气量，溶氧，温度，转速，pH设置，每隔6h取菌液测定细胞密度，发酵周期为96h。
作为优选的技术方案之一，步骤(2)与步骤(3)所述KB培养基的配方为：每1000mL水中含甘油10mL，蛋白胨20g，K 2HPO 41.5g，MgSO 4·7H 2O 1.5g。
作为优选的技术方案之一，步骤(2)中摇瓶种子的培养条件为30℃，200rpm。
作为优选的技术方案之一，步骤(3)所述发酵罐培养条件为：温度为26～32℃，pH为6～7.5，转速为300～600rpm，通气量为0.8～4.0L/min，溶氧0.8～1.0L/min，溶氧不与转速串联，培养12～24小时后流加25～100mL的质量分数为50％葡萄糖水溶液，此后每间隔2～6小时流加一次25～100mL的50％葡萄糖水溶液，直至发酵结束，期间用体积比为20％磷酸，氨水维持pH的稳定，体积比为50％消泡剂进行消泡。
作为进一步优选的技术方案之一，步骤(3)所述发酵培养条件为：温度为28℃，pH为7，转速为600rpm。
本发明还提供了一种以荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株CHA0-ΔretS-NiF为活性成分的微生物菌剂。
作为优选的技术方案之一，所述微生物菌剂的制备方法为：步骤(3)所述发酵罐培养的菌体进入稳定期，细胞密度达到最大值时进行离心，冷冻干燥后收集菌体，即得。
本发明的有益效果：
本发明提供一株荧光假单胞菌突变菌株CHA0-ΔretS-NiF的发酵方法，其最适生长条件为pH＝7、温度28℃、转速600rpm。通过现有的文献可知，对于荧光假单胞菌发酵方法的报道鲜少，因此突变菌株CHA0-ΔretS-NiF的最适生长条件的确定既有利于对其扩大培养，得到大量菌体，增加了产业化生产的可能性，同时也为同属的其他荧光假单胞菌发酵培养提供借鉴。
本发明提供了一株荧光假单胞突变菌株CHA0-ΔretS-NiF及以其为活性成分的菌剂，室温盆栽试验证明该菌株能有效地促进植物生长。主要由于retS基因的敲除增加突变菌株分泌次生代谢产物2，4-DAPG 产量，杀菌能力得到增强，同时固氮基因簇NiF的整入使得突变菌株具有固氮能力，为植物提供氮源，满足植物生长过程中对氮源的需求。
附图说明
图1为在CHA0细菌染色体上敲除retS基因的流程图。
图2为本发明中荧光假单胞突变菌株CHA0-ΔretS的菌落PCR验证图。
图3为本发明中通过Red/ET直接克隆方法构建的表达质粒pBeloBAC11-oriT-TnpA-genta-NiF经过限制性内切酶Kpn I的酶切鉴定验证图。
图4为本发明中荧光假单胞菌突变菌株CHA0-ΔretS-NiF的构建实验流程图。
图5为本发明中固氮突变的荧光假单胞菌株CHA0-ΔretS-NiF的菌落PCR验证图。
图6显示了本发明中荧光假单胞菌①CHA0(Pseudomonas protegens CHA0)及其突变菌株②CHA0-NiF，③CHA0-ΔretS和④CHA0-ΔretS-NiF对枯草芽孢杆菌(Bacillus subtilis)的抑菌实验的结果。
图7为野生型菌株CHA0及其突变菌株合成抗生素2，4-DAPG产量的色谱图。
图8显示了实验菌株荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF的固氮酶活性测定结果。
图9显示了荧光定量PCR检测工程菌株CHA0-ΔretS-NiF和固氮斯氏假单胞菌DSM4166基因组中关键基因NifD、NifK、NifN、NifM、NifQ、NifS、NifT表达量的结果。
图10显示突变菌株CHA0-ΔretS-NiF生长最优pH值探究。
图11显示突变菌株CHA0-ΔretS-NiF生长最优温度探究。
图12显示突变菌株CHA0-ΔretS-NiF生长最优转速探究。
图13显示突变菌株CHA0-ΔretS-NiF在5L发酵罐生长情况。
图14显示突变菌株CHA0-ΔretS-NiF不同转化子固氮酶活性测定结果。
图15A为施加不同菌剂的拟南芥移栽入盆4周后的盆栽效果图。
图15B为施加不同菌剂拟南芥移栽入盆4周后莲座直径长度示意图。
图16显示大蒜的生长后期各试验处理中的生物学性状。其中左边的为处理1的叶子，右边的为处理4的叶子。
保藏信息
分类名称：荧光假单胞菌(Pseudomonas protegens)突变菌株CHA0-ΔretS-NiF
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心
保藏单位地址：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：2017年7月31日
保藏号：CGMCC No.14476
具体实施方式
下面结合附图和实施例对本发明进行进一步的阐述，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。
荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株为CHA0-ΔretS-NiF，其保藏编号为CGMCC No.14476(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号中国科学院微生物研究所，保藏日期：2017年7月31日)。
实施例1
荧光假单胞菌突变菌株CHA0-ΔretS的筛选方法，具体操作步骤如下：
(1)把质粒pBBR1-Rha-TEGpsy-kan(该质粒可以在假单胞菌中表达重组酶)通过电转的方式导入到野生型的荧光假单胞菌CHA0(Pseudomonas protegens CHA0)中，将电转后的细菌涂布于LB培养基(LB培养基成分：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠1g/L，pH值7.0)+卡那霉素(km，30μg/mL)的平板上，随机挑选12个单菌落提取质粒进行酶切鉴定，筛选到正确的转化子CHA0：：pBBR1-Rha-TEGpsy-kan；
(2)敲除荧光假单胞菌CHA0(Pseudomonas protegens CHA0) 基因组上的retS基因。将线性DNA片段loxM-genta(此片段通过PCR的方法得到，所用的一对引物为RetS-Genta-loxM-5’GCACACGCCCTTGCCGTGCGGTCATTACGCCGCGCATAGTTATAATCAGGCATCAACCAACGAAGGGATTTCGCCAGCTGAATTACATTCCCAACCG/RetS-Genta-loxM-3’TGGAGCATGGTGGGAGCTCACGACTAAAGGAGGGCGAGCGAGAGTTTAACAGGCGCCGCAGAGCCTGTCGGCTCACAACTTAAATGTGAAAGTGGGTC，分别如SEQ ID NO.15和SEQ ID NO.16所示)电转到步骤1)得到的CHA0：：pBBR1-Rha-TEGpsy-kan中，利用Red/ET同源重组的方法，在重组酶的作用下，庆大霉素抗性基因(genta)将替换荧光假单胞菌CHA0(Pseudomonas protegens CHA0)基因组上的retS基因，将重组后的细菌涂布于LB培养基+genta 15μg/mL的平板上，随机挑选多个单菌落进行菌落PCR验证(验证时用的引物为check-5’TGCTTCTACCGCAAGGACATC/check-3’GCTGATGAAGCACGAGAGCAC，分别如SEQ ID NO.13和SEQ ID NO.14所示)，筛选到正确的转化子CHA0：：ΔretS-genta-loxM；
(3)消除CHA0：：ΔretS-genta-loxM中的genta抗性基因。把能够表达Cre重组酶的PCM157质粒电转导入CHA0：：ΔretS-genta-loxM中，涂布于LB培养基+四环素(tet 25μg/mL)的平板上筛选。将得到的重组子接种到1mL LB+tet 25μg/mL液体培养基中，900rpm，30℃培养过夜。将50μL过夜培养的菌液转接到新鲜的1mL LB+tet 25μg/mL液体培养基中，900rpm，30℃培养3小时后，加入1mM的异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导，继续培养2小时后用蓝色接种环将菌液Z字形划线于LB平板上，待长出单菌落后将分别双划线于LB和LB+genta 15μg/mL两种平板上30℃过夜培养。如果在两种平板上都长出了单菌落，说明说明该重组子内的genta抗性基因没有被消除；如果在LB平板菌落生长出来，而在LB+genta 15μg/mL的平板上不生长，则说明该重组子内的genta抗性基因已被消除。挑取此类genta抗性基因已被消除的重组子进行菌落PCR验证和测序，引物为：
check-5’TGCTTCTACCGCAAGGACATC/
check-3’GCTGATGAAGCACGAGAGCAC；分别如SEQ ID NO.13和SEQ ID NO.14所示。
(4)将PCR验证和测序后正确的转化子CHA0-ΔretS分装冻存，用于后续的抑菌、室温盆栽和田间试验。
图1为在CHA0细菌染色体上敲除retS基因的流程图。图2为CHA0-ΔretS的菌落PCR反应验证图。从图中显示，M为DL 5,000DNA的Marker，1号样品为CHA0野生型作为对照，2-10号样品为最终的转化子CHA0-ΔretS，在经过IPTG诱导产生的Cre重组酶的作用下，介导两个loxM位点(序列)之间的特异性重组，使loxM位点间的genta抗性基因序列被删除，从而消除了外源抗性基因对于荧光假单胞菌CHA0(Pseudomonas protegens CHA0)生长、繁殖和定殖等方面的影响，可以更加放心的使用。
实施例2
荧光假单胞菌突变菌株CHA0-ΔretS-NiF的筛选方法，具体操作步骤如下：
(1)利用Red/ET直接克隆的方法，先使用限制性内切酶Afl II和Ssp I对固氮斯氏假单胞菌DSM4166(Pseudomonas stutzeri DSM4166)的基因组DNA进行酶切，获得的49kb大小NiF固氮基因岛的DNA片段凝胶电泳验证正确后，再连接到相应的表达载体上，所使用的引物为：
Primer 1：AGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGCTTAAGTACGGCTACCTGGAGCTCGCGCCAGTG，如SEQ ID NO.1所示；
Primer 2：TACGGCTACCTGGAGCTCGCGCCAGTGCTTGCCGACATCGAATCACGGCCGCTGCTGCAGCACGTGGTGGTCACCGGCCGGGATCCGTTTAAACACAAATGGCAAGGGCTAATG，如SEQ ID NO.2所示；
Primer 3：ATTGATGTTTTCCTTGGCCAGCGCCTCGAACATCCGGCTGGCGACGCCTGCGTGCGAACGCATACCGACACCGACGATAGGGATCCGTTTAAACGGTGTGGTAGCTCGCGTATT，如SEQ ID NO.3所示；
Primer 4：GCGACACTATAGAATACTCAAGCTTGGCATGAATGCAGGTCGACTCTAGAGAATATTGATGTTTTCCTTGGCCAGCGCCTCGAAC，如SEQ ID NO.4所示；
构建了表达质粒pBeloBAC11-oriT-TnpA-genta-NiF(图3)，通过限制性内切酶Kpn I进行酶切鉴定，然后将结果正确的质粒电转入到大肠杆菌ET12567中；
(2)通过接合转移把来自大肠杆菌ET12567中的质粒pBeloBAC11-oriT-TnpA-genta-NiF导入荧光假单胞菌CHA0-ΔretS中，然后通过转座的方式，NiF基因可随机插入到CHA0的基因组DNA中(图4)。接合转移详细操作为：平板挑取单菌落，把荧光假单胞菌CHA0-ΔretS(LB培养基，30℃)与大肠杆菌ET12567(LB+genta 2μg/mL+cm 10μg/mL+km1μg/mL培养基，37℃)分别过夜培养；将两种过夜的菌液7000rpm，离心1分钟。用新鲜的LB培养基分别将荧光假单胞菌CHA0-ΔretS和大肠杆菌ET12567洗涤两遍后，用300μL LB培养基重悬，分别各取50μL悬浮液，混匀，小范围涂在LB平板中间，晾干。在37℃条件下孵育4h后，将平板在30℃的培养箱中倒置培养过夜；用接种环将平板上的菌刮下来，用1mL无菌水悬浮混匀，取100μL菌液Z字形划线涂布于PMM培养基(磷酸氢二钾8g/L，磷酸二氢钾5g/L，硫酸铵1g/L，琥珀酸钠6.6g/L，调节pH至7.0，灭菌后加入1M硫酸镁1.2mL/L)+genta 25μg/mL平板上，30℃倒置培养2天，直至单菌落出现；两天后长出菌落，挑单菌落接种于1mL LB+genta 25μg/mL过夜培养，之后用以下的5对引物进行菌落PCR验证，引物为：
NiF-check-1 GGTCTACCAGCTCGACCT/
NiF-check-2 CGATTCCAGCGTCGAATGAT；
NiF-check-3 GCTGACCTCCTTGAGGTGCT/
NiF-check-4 CAGCGGCACCTCGAGGAGT；
NiF-check-5 GATAGAGCAGGTCCTCGAT/
NiF-check-6 GGTGCTCTACGTCAGCCATT；
NiF-check-7 CGACAGATCCTGATTACCGT/
NiF-check-8 TACCCTCGACCAGCTTGAGCA；
check-5’TGCTTCTACCGCAAGGACATC/
check-3’GCTGATGAAGCACGAGAGCAC；分别如SEQ ID NO.5～SEQ ID NO.14所示；
前4对引物用于验证NiF固氮基因是否已经整体整合到荧光假单 胞菌CHA0-ΔretS的基因组当中，扩增出的PCR片段的分别为1000bp、970bp、830bp、1080bp。第5对引物是为了验证导入NiF固氮基因的菌株是荧光假单胞菌CHA0而不是大肠杆菌ET12567，其PCR扩增结果是DNA片段大小为3200bp的retS基因；
(3)将菌落PCR验证后正确的转化子CHA0-ΔretS-NiF送去测序，结果正确的分装冻存，用于后续的抑菌、室温盆栽和田间试验。
图5显示，M为DL 5,000DNA的Marker，ck1为已经敲除了retS基因的突变荧光假单胞菌CHA0-ΔretS，ck2为大肠杆菌ET12567，二者作为对照组。1，2，3号样品为从平板上随机挑取的3个CHA0-ΔretS-NiF转化子，各用了上述的5对引物做了5次菌落PCR验证，经过反复仔细比对后发现获得的数据与预期结果吻合，从而证明固氮斯氏假单胞菌DSM4166(Pseudomonas stutzeri DSM4166)中的NiF固氮基因已经整体整合到已经敲除了retS基因的突变荧光假单胞菌CHA0-ΔretS的基因组当中，从而获得了正确的转化子CHA0-ΔretS-NiF。
本发明得到的几种荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF的相关信息，如表1所示。
表1.各个菌株的相关信息
Figure PCTCN2018103224-appb-000001
Figure PCTCN2018103224-appb-000002
实施例3
采用滤纸片法，检测荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF对枯草芽孢杆菌(Bacillus subtilis)的抑制作用，具体操作步骤如下：
(1)将枯草芽孢杆菌(Bacillus subtilis)和实验菌株荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF分别接种到1mL LB液体培养基，900rpm，30℃过夜培养；
(2)第二天把枯草芽孢杆菌(Bacillus subtilis)9000rpm，离心1分钟后，留100μL菌液均匀涂布在LB固体培养基(在液体LB培养基的基础上加入15g/L的琼脂)平板上，待晾干后在该平板上放置若干个直径6mm的双层滤纸片，取5μL过夜培养的实验菌株荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF的菌液分别滴加到滤纸片上。将此平板放置在30℃条件下培养过夜；
(3)第三天观察平板上各个小滤纸片周边的抑菌圈大小。
图6显示，已经敲除了retS基因的荧光假单胞菌CHA0(Pseudomonas protegens CHA0)突变菌株③CHA0-ΔretS和④CHA0-ΔretS-NiF的抑菌圈比未敲除retS基因的荧光假单胞菌①CHA0和②CHA0-NiF的抑菌圈明显要大(③和④样品的抑菌圈直径为2.7cm，而①和②样品的抑菌圈直径为2.3cm)，从而说明其抑制枯草芽孢杆菌(Bacillus subtilis)的能力，在ΔretS基因敲除后得到了增强。
实施例4
retS基因敲除后导致抗生素2，4-DAPG产量提升的高效液相色谱分析
将实验菌株荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF分别于KB培养基中经过150rpm，30℃的条件培养24h后，加入1mL树脂，待菌液与树脂共震荡24h后8000rpm离心10min收集树脂并加入等体积乙酸乙酯过夜萃取，再次离心收集上清进行旋蒸得到浸膏，加入1 mL甲醇复溶进行HPLC检测。HPLC检测条件：反相柱Thermo Scientific Acclaim TM C18(2.1mm×100mm，2.2μm)，柱温30℃；流动相：由0.1％乙酸水溶液(溶剂A)和乙腈(溶剂B)组成；色谱程序：0-5min，5％溶剂B；5-20min，5％-95％溶剂B；20-25min，95％溶剂B；流速为0.5mL/min。分别在250nm，270nm，290nm和310nm处监测紫外(UV)光(2，4-DAPG，λ＝270nm)。MS测量在amaZon速度质谱仪和超高分辨率Qq-Time-Of-Flight上进行，使用标准ESI(电喷雾电离)源。
图7a显示，1，WT CHA0；2，CHA0：：Nif；3，CHA0-ΔretS；4，CHA0-ΔretS-Nif，结果表明敲除retS基因的工程菌合成2，4-DAPG的产量远远高于未敲除retS的菌株，抗生素2，4-DAPG的产量提升了约100倍。图7b为抗生素2，4-DAPG紫外吸收峰及质谱图。
实施例5
乙炔还原法测定固氮酶活性
(1)将实验菌株荧光假单胞菌CHA0(Pseudomonas protegens CHA0)及其突变菌株CHA0-NiF，CHA0-ΔretS和CHA0-ΔretS-NiF分别接种于LB培养基中，30℃培养8h。
(2)在4℃下，5000rpm离心10min后收集菌液，用0.85％的生理盐水洗涤三遍。用无氮培养基(葡萄糖10g/L，磷酸二氢钾0.2g/L，七水合硫酸镁0.2g/L，氯化钠0.2g/L，二水合硫酸钙0.2g/L，碳酸钙5g/L，调至pH 7.0-7.2，113℃灭菌30min)重悬至OD＝1.0。
(3)在100mL的厌氧培养瓶中加入18mL的无氮培养基以及2mL上述菌液，排除空气并置换高纯氩气，使厌氧瓶成密封状态。加入1％的氧气，30℃，250rpm，培养6h后，抽取10％的混合气体并注入10％的乙炔气体，30℃，250rpm，培养4h后开始取样测定。取样用无菌注射器从瓶中取100μL混合气体，注入气相色谱仪进行乙烯含量测定。以不接菌注入乙炔的厌氧瓶为对照。
(4)固氮酶活性＝检测到的乙烯峰面积×(厌氧瓶体积-样品体积)/(标准乙烯气体峰面积×反应时间×蛋白浓度)。
图8结果显示，整入了固氮基因岛(NiF)的荧光假单胞菌CHA0突变菌株都具有固氮酶活性，但活性表达量不同，其中retS-Nif1这一突变菌株的固氮酶活性最高，分装冻存该菌株，用于后续的发酵实验 田间试验。
实施例6
荧光定量PCR检测固氮基因岛(NiF)中的相关关键基因在CHA0-ΔretS-NiF和固氮斯氏假单胞菌DSM4166中的表达量。
将工程菌株CHA0-ΔretS-NiF和固氮斯氏假单胞菌DSM4166分别于KB培养基培养24h后，移至无氮培养基继续培养6h，使用RNAPure试剂盒提取目的菌株总RNA，随后通过反转录试剂盒(购自日本Takara公司)将RNA中的基因组DNA去除并且合成互补DNA链(cDNA)。以GAPDH基因为内参基因，荧光定量分析(
Figure PCTCN2018103224-appb-000003
Premix Ex Taq TM II(Tli RNaseH Plus)Code No.RR820A)固氮基因岛(Nif)中7个关键基因NifD、NifK、NifN、NifM、NifQ、NifS、NifT的表达量。图9显示，这7个关键基因在工程菌株CHA0-ΔretS-NiF中都具有一定的相对表达量，且均大于其在固氮斯氏假单胞菌DSM4166的表达水平，从而证明了在荧光假单胞菌CHA0(Pseudomonas protegens CHA0)中产生了固氮酶。
实施例7
荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株CHA0-ΔretS-NiF发酵方法
(1)种子活化：从-80℃超低温冷冻存储箱中取出菌种甘油管，待解冻后使用1ul接种环沾取少量菌液划线于LB+genta 20平板上，倒置于生化培养箱中，30℃，20h恒温培养，从平板上随机挑选5个单菌落进行菌落PCR验证以确保获得正确的目的菌株；
(2)摇瓶种子培养：用1uL接种环在活化好种子的LB+genta 20平板上刮取少量菌体接入KB培养基中，摇瓶的装瓶量为500mL瓶体装液100mL KB培养基，放置于全温振荡培养箱30℃，200rpm，20h培养；KB培养基的配方为：每1000mL水中含有甘油10mL，蛋白胨20g，K 2HPO 41.5g，MgSO 4·7H 2O 1.5g；
(3)发酵罐培养：待摇瓶的种子液培养成功后，在超净工作台中将种子液吸入规格为60ml的注射器中，然后通过穿刺法将菌液从接种口注射到1L罐中，接种量为发酵罐中盛有液体KB培养基总体积的5％，接种完毕后对通气量，溶氧，温度，转速，pH进行智能设置：通气量0.8L/min，溶氧量0.8L/min，溶氧不与转速串联；每隔6h取菌液测定 细胞密度，发酵周期为96h，培养24h后开始流加25mL的50％葡萄糖水溶液，随后每间隔6h流加一次25mL的50％葡萄糖水溶液，直到发酵结束，期间用20％磷酸，氨水维持pH的稳定，50％消泡剂进行消泡。
将上述步骤(3)所述pH值分别设置为6，6.5，7和7.5，其余条件及步骤不变，培养CHA0-ΔretS-NiF菌株，将不同pH培养条件下的CHA0-ΔretS-NiF菌株的生长情况进行对比，结果见图10。
由图10可以看出在48h的培养期间，不同的pH生长环境对菌体的密度并没有产生较大影响，生长步伐几本保持一致，但48h过后，可能由于细胞密度达到了一定程度，分泌不利于菌体生长的代谢物质浓度达到了自身承受的最高能力从而阻碍了细胞的繁殖，最终导致在偏酸或偏碱的环境下菌体量不高。
将上述步骤(3)所述温度分别设置为26℃，30℃和32℃，其余条件及步骤不变，培养CHA0-ΔretS-NiF菌株，将不同温度培养条件下的CHA0-ΔretS-NiF菌株的生长情况进行对比，结果见图11。
由图11可以看出，28℃为工程菌株CHA0-ΔretS-NiF生长的最适温度，32℃培养条件下的目的菌株从一开始就表现出了不佳的生长状态，而到了36h后，28℃，30℃，26℃条件下各自的细胞增长量开始发生变化，最终28℃条件下菌体的生长优势相对明显。这可能与温度调控微生物的代谢途径，影响酶的反应速率有关。
将上述步骤(3)所述转速分别设置为300rmp，400rmp和500rmp，其余条件及步骤不变，培养CHA0-ΔretS-NiF菌株，将不同转速培养条件下的CHA0-ΔretS-NiF菌株的生长情况进行对比，结果见图12。
由图12可知600rpm为该菌的最适生长转速，但600rpm与500rpm之间的生长优势并不十分明显，这说明在500rpm的条件下也基本能满足菌体生长期间对氧的需求量。氧溶量可影响微生物的代谢途径，产物产量，酶的活性，适当的氧浓度有利于菌体生长，但过高的氧浓度会加速细胞的氧化，使菌体提早进入衰亡期。
由上所述，本发明工程菌株CHA0-ΔretS-NiF的最优环境生长条件为pH＝7，温度为28℃，转速为600rpm。
实施例8
采用CHA0-ΔretS-NiF最优环境生长条件对其进行5L罐的扩大培养及收集菌体
(1)种子活化：从-80℃超低温冷冻存储箱中取出菌种甘油管，待解冻后使用1ul接种环沾取少量菌液划线于LB+genta 20平板上，倒置于生化培养箱中，30℃，20h恒温培养；
(2)摇瓶种子培养：用1uL接种环在活化好种子的LB+genta 20平板上刮取少量菌体接入KB培养基中，摇瓶的装瓶量为500mL瓶体装液100mL KB培养基，放置于全温振荡培养箱30℃，200rpm，20h培养；KB培养基的配方为：每1000mL水中含有甘油10mL，蛋白胨20g，K 2HPO 41.5g，MgSO 4·7H 2O 1.5g；
(3)发酵罐培养：在5L罐接种口附近缠绕浸湿酒精的棉花，点燃，待火焰包围接种口后用镊子将接种口的螺丝旋开，此时快速将摇瓶中的菌液倒入罐中，在火焰熄灭以前迅速将螺丝拧回接种口，接种量为发酵罐中盛有液体KB培养基总体积的10％，接种完毕后通气量，溶氧，温度，转速，pH智能设置(温度28℃，pH＝7，转速600rpm，通气量4L/min，溶氧量1.0L/min，溶氧不与转速串联)；每隔6h取菌液测定细胞密度，发酵周期为96h，培养12h后开始流加100mL的50％葡萄糖，随后每间隔2h流加一次50mL的50％葡萄糖，直到发酵结束，期间用20％磷酸，氨水维持pH的稳定，50％消泡剂进行消泡。
(4)待菌体进入稳定期，细胞密度达最大值时进行离心，冷冻干燥后收集菌体。
从图13可以看出与1L发酵罐相比，菌体在5L发酵罐的生长对数期增长率明显增大，而且最终的菌体密度也有所增加，OD值为40.76，但对数期的生长趋势是大体相同的，即对数前期的生长率都优于对数阶段的中后期。主要原因为：5L罐的通风量与转速方面都优于1L罐，而且在培养过程中搅拌扇叶可以将菌体分散，避免结团，增加菌体与培养基的接触面积，利于氧的吸收。
发酵完成后，将发酵产物进行离心，对离心后的菌体进行冷冻干燥，菌体干重为15.9g(DCW/L)。
实施例9
荧光假单胞菌突变菌株CHA0-ΔretS-NiF在杀菌固氮、促进植物生长方面的应用
1、采用乙炔还原法对突变菌株CHA0-ΔretS-NiF不同的转化子进行固氮酶活性测定，从不同的转化子中挑选出固氮酶活性最高的突变 菌株CHA0-ΔretS-NiF进行后续盆栽试验：
(1)将-80℃保藏的指定实验菌株接种于LB液体培养基中，30℃培养8h；
(2)在4℃下，5000rpm离心10min后收集菌液，用0.85％的生理盐水洗涤三遍，用无氮培养基重悬至OD＝1.0；
(3)在100mL的厌氧培养瓶中加入18mL的无氮培养基以及2mL上述菌液，排除空气并置换高纯氩气，使厌氧瓶成密封状态；加入1％的氧气，30℃，250rpm，培养6h后，抽取10％的混合气体并注入10％的乙炔气体，30℃，250rpm，培养4h后开始取样测定；取样用无菌注射器从瓶中取100μL混合气体，注入气相色谱仪进行乙烯含量测定，以不接菌注入乙炔的厌氧瓶为对照。
(4)固氮酶活性＝检测到的乙烯峰面积之差×(三角瓶中气相体积/进样量)/(标准乙烯气体峰面积×反应时间×蛋白浓度)。
蛋白浓度测定按照考马斯亮蓝的方法测定：
①将20mL菌液4000rpm离心10min后，弃掉上清；
②用200μL 0.5M NaOH重悬。煮沸5min后，加入200μL 0.5M HCl，12000rpm离心10min；
③取100μL上清加入900μLG250，混匀，静止5min后，在OD595处测定蛋白含量。
突变菌株CHA0-ΔretS-NiF不同转化子固氮酶活性测定结果如图14所示，不同的转化子由于固氮基因岛NiF随机插入到CHA0细菌染色体上位置的不同，所表达的固氮酶活性大小也不相同，结果显示转化子CHA0 2-3表达的固氮酶含量最高，甚至高于携带NiF的野生型菌株DSM4166。
2、使用野生型拟南芥Col-0为试验对象，试验条件：温度为20℃，光照强度80μmol·m -2·s -1，光照周期：16小时光照，8小时黑暗；试验分为4个组：以未施加氮肥与施加野生型的荧光假单胞菌株CHA0菌液作为阴性对照，施加含有固氮基因簇的原始菌株DSM4166菌液作为阳性对照，施加上述步骤1优选的固氮酶活性最高的突变菌株CHA02-3菌液为试验组，盆栽效果如图15A所示。
3、测量拟南芥的莲座直径长度，以此比较拟南芥的生长状态，莲座直径长度测量结果如6B所示。
由图15A和图15B可以看出，在拟南芥盆栽试验中，以未施加氮肥与施加野生型的荧光假单胞菌株作为阴性对照(Control和CHA0)，施加含有固氮基因簇的原始菌株DSM4166作为阳性对照(DSM4166)，施加CHA0-ΔretS-NiF菌液为试验组(NIF)。结果表明，未施加氮肥与施加野生型荧光假单胞菌株的拟南芥长势均不好，叶小茎短(图15A)。在未施加氮肥，但施加了含有固氮基因簇的原始菌株DSM4166的阳性对照组以及施加固氮荧光假单胞菌株CHA0-ΔretS-NiF的试验组中，拟南芥的莲座叶直径均优于阴性对照。此外，施加了固氮荧光假单胞菌株CHA0-ΔretS-NiF的拟南芥莲座叶直径优于施加了含有固氮基因簇的原始菌株DSM4166的拟南芥莲座叶直径(图15B)。
实施例10
CHA0工程菌菌剂在大蒜上的田间试验报告
1试验时间：2017年10月-2018年6月
2试验地点：山东省济宁市鱼台县鱼城镇前蒋村
3试验作物：杂交蒜(白皮蒜)
4试验处理：本试验分为6个处理，分别为
处理1：农民习惯施肥，其中N 45kg/hm 2、P 2O 522.5kg/hm 2、K 2O 22.5kg/hm 2，有机肥40kg/亩，追肥时用高氮高钾复合肥；
处理2：优化施肥，配方肥N 30kg/hm 2、P 2O 516kg/hm 2、K 2O 24kg/hm 2，生物有机肥200kg/hm 2；追肥时用配方肥(18-5-17腐殖酸型)20kg/亩，用多宁拌蒜种，开春追肥时根据实际情况施肥前用恶霉灵、甲基托布津和多宁；
化控措施(根据实际再选择)：缩节胺和芸苔素内酯
处理3：微生物菌剂-荧光假单胞菌-云(从市场上购置，来自山东泰诺药业有限公司)
施肥与优化施肥一致，用荧光假单胞菌拌种，在播种时、入冬前、返青期、开春随水冲施荧光假单胞菌液，追肥时冲施菌液；
处理4：微生物菌剂-荧光假单胞菌CHA0-ΔretS-NiF
施肥与优化处理一致，用荧光假单胞菌拌种，在播种时、入冬前、返青期、开春随水冲施荧光假单胞菌液，追肥时冲施菌液；
处理5：微生物菌剂-荧光假单胞菌CHA0-ΔretS-NiF
氮肥施用量是优化施肥的2/3，磷钾一致，其他施肥与优化处理一致， 用荧光假单胞菌拌种，在播种时、入冬前、返青期、开春随水冲施荧光假单胞菌液，追肥时冲施菌液；
微生物菌剂的剂型为液体，有效活菌数≥10亿/ml，用量：2公斤/亩。
2018年1月21日，蒜苗越冬前对大蒜的叶长，叶宽和茎粗，以及根系酶活力进行了测定(表1)。
表1 试验处理生物学性状统计表(1月21日)
Figure PCTCN2018103224-appb-000004
表2 产品示范试验处理生物学性状统计表(3月29日)
Figure PCTCN2018103224-appb-000005
上述两个表格的数据分别是在苗期，和冬后返青期测量的大蒜的生长指标，测得的数据同田间试验观察的结果吻合，荧光假单胞菌处理的3个处理在前期(苗期)对大蒜的叶宽和根系酶活力有明显的促进作用，其中促进最明显的为处理4，达到了38.5％。那是因为荧光假单胞菌剂替代了多宁拌种，多宁抑制了病菌生长也对蒜体周围的有益菌产生了伤害，间接阻碍了蒜生长初期的长势，而荧光假单胞菌剂处 理蒜苗期长势旺盛。
在大蒜的生长初期，叶面明显地长和宽，处理4和5的叶长比对照处理1的分别提高了23.6％和21.6％，叶宽分别提高了15.2％和14.8％。这与田间观测到的处理4和5的实际情况相吻合，这两个处理的叶面比对照处理1的叶面更伸展长势更旺。
大蒜的生长后期(冬后返青期)对叶宽的促进作用依然保持，而且荧光菌的两个处理大蒜的茎粗也明显的高于对照，处理4和5的叶长比对照处理1的分别提高了15.1％和11.7％，叶宽分别提高了10.2％和8.1％(图16)。
从10月大蒜定植到次年5月收获的过程进行计产，并换算成大蒜的亩产量，并对结果进行统计分析。
实验处理1-5的大蒜产量结果见表4和5。
表4 不同处理大蒜蒜头产量的差异
Figure PCTCN2018103224-appb-000006
蒜头产量较高的为试验组为添加了荧光假单胞菌菌剂的三个处理，增产率较对照的处理1农民习惯施肥方式组分别达到9.21％，19.25％和15.52％。蒜头处理产量最高的是荧光假单胞菌CHA0-ΔretS-NiF的处理，为2050.54公斤/亩：产量其次是荧光假单胞菌减氮处理，在荧光假单胞菌CHA0-ΔretS-NiF处理的基础上从减少施用氮肥30％，仍获得了1986.41公斤/亩产量。另一个市场上购买的荧光假单胞菌-云的处理产量1877.16公斤/亩，增产率为9.21％。荧光假单胞菌-云对蒜头产量的促进作用表现不如荧光假单胞菌CHA0-ΔretS-NiF工程菌。
表5 不同处理大蒜蒜薹产量的差异
Figure PCTCN2018103224-appb-000007
五个处理中，添加了荧光假单胞菌菌剂的三个处理的蒜薹产量较高，分别为347.73公斤/亩、375.74公斤/亩和365.89公斤/亩，比处理1的农民习惯方式处理组分别提高了9.06％，17.85％和14.76％。从蒜薹的产量来看，使用荧光假单胞菌菌剂处理后，均对大蒜的蒜薹产量有显著促进作用。其中促进作用最大的是荧光假单胞菌CHA0-ΔretS-NiF的处理，蒜薹产量较对照提高了17.85％，处理5荧光假单胞菌CHA0-ΔretS-NiF工程菌的减氮处理组的蒜薹产量比处理4略微有些减少，但总体保持在相对平均的水平。
由此可见，CHA0菌剂的施加对于大蒜品质的影响非常巨大，可以为广大客户带来巨大的经济实惠，而减少了1/3氮肥的使用，则可以在不影响产品品质的前提下，减少客户的生产成本，作用效果立竿见影。
不同试验处理大蒜病情指数的分析
大蒜主要的病害就是叶枯病和根腐病。大蒜叶枯病是大蒜上常见的病害之一，各菜区均有不同程度发生，主要危害露地栽培的大蒜。大蒜生长期间降水次数多、雨量大的年份发病重。发病严重时常造成病叶枯死、植株早衰、蒜头减产、蒜苔霉烂，直接影响产量。
中期的叶枯病情只能观察，最后收获时以通过计数小区的蒜头病害数，计算病情指数。
表6 不同处理对大蒜病害的影响
Figure PCTCN2018103224-appb-000008
播种前，施用荧光假单胞菌的处理4和处理5没有用多宁拌种，后期农户一共打了四次农药，处理4和处理5均没有喷施，从最后大蒜收获数据来看，处理4病情指数最低，处理5的病情指数也比农民习惯对照和优化施肥的指数略低。说明荧光假单胞菌对大蒜的根腐病有显著的防治作用。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Figure PCTCN2018103224-appb-000009
Figure PCTCN2018103224-appb-000010
Figure PCTCN2018103224-appb-000011
Figure PCTCN2018103224-appb-000012

Claims (10)

  1. 荧光假单胞菌(Pseudomonas protegens CHA0)突变菌株CHA0-ΔretS-NiF，其保藏编号为：CGMCC No.14476。
  2. 一种组合物，例如微生物菌剂，其特征在于，其活性成分为权利要求1所述的荧光假单胞菌突变菌株CHA0-ΔretS-NiF。
  3. 权利要求1所述荧光假单胞菌突变菌株CHA0-ΔretS-NiF在对植物杀菌、固氮、促进植物生长、增加植物产量和/或防治植物病害方面的应用。
  4. 生产荧光假单胞菌突变菌株CHA0-ΔretS-NiF9的方法，其包括以下步骤：
    a)敲除荧光假单胞菌CHA0基因组中的retS基因；和
    b)将固氮斯氏假单胞菌DSM4166基因组中的NiF固氮基因岛整体克隆到步骤a)得到的菌株中，使之异源表达。
  5. 对植物杀菌、固氮、促进植物生长、增加植物产量和/或防治植物病害的方法，包括给植物或其种子施用权利要求1所述的荧光假单胞菌突变菌株CHA0-ΔretS-NiF或权利要求2的组合物。
  6. 权利要求1所述的突变菌株CHA0-ΔretS-NiF的发酵培养方法，其特征在于，包括以下步骤：
    (1)种子活化：从-80℃超低温冷冻存储箱中取出含有CHA0-ΔretS-NiF菌种的甘油管，待解冻后取少量菌液划线于LB+genta20平板上，于30℃恒温生化培养箱中倒置培养20h，从平板上随机挑选5个单菌落进行菌落PCR验证以确保获得正确的目的菌株；
    (2)摇瓶种子培养：将活化好的CHA0-ΔretS-NiF菌种接入KB培养基中，放置在全温振荡培养箱中培养20h，得到种子液；
    (3)发酵罐培养：将种子液接种到装有KB培养基发酵罐中，接种量为5～10％，即每100mlKB培养基加入5～10ml种子液，接种完毕后进行通气量，溶氧，温度，转速，pH设置，每隔6h取菌液测定细胞密度，发酵周期为96h。
  7. 根据权利要求6所述的发酵培养方法，其特征在于，步骤(2)与步骤(3)所述KB培养基的配方为：每1000mL水中含甘油10mL，蛋白胨20g，K 2HPO 41.5g，MgSO 4·7H 2O 1.5g。
  8. 根据权利要求6所述的发酵培养方法，其特征在于，步骤(2)中培养条件为30℃，200rpm。
  9. 根据权利要求6所述的发酵培养方法，其特征在于，步骤(3)所述发酵罐培养条件为：温度为26～32℃，pH为6～7.5，转速为300～600rpm，通气量为0.8～4.0L/min，溶氧0.8～1.0L/min，溶氧不与转速串联，培养12～24小时后流加25～100mL的质量分数为50％葡萄糖水溶液，此后每间隔2～6小时流加一次25～100mL的50％葡萄糖水溶液，直到发酵结束，期间用体积比为20％磷酸，氨水维持pH的稳定，体积比50％消泡剂进行消泡。
  10. 根据权利要求9所述的发酵培养方法，其特征在于，步骤(3)所述发酵培养条件为：温度为28℃，pH为7，转速为600rpm。
PCT/CN2018/103224 2017-10-27 2018-08-30 一株杀菌固氮荧光假单胞菌及其发酵方法与应用 WO2019080638A1 (zh)

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