CN110408567A - One plant of saline-alkali tolerant Methylotrophic bacillus and its active bacteria formulation and application - Google Patents
One plant of saline-alkali tolerant Methylotrophic bacillus and its active bacteria formulation and application Download PDFInfo
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Abstract
The invention discloses one plant of saline-alkali tolerant Methylotrophic bacillus and its active bacteria formulation and applications.The present invention is separated to one plant of Methylotrophic bacillus (Bacillus methylotrophicus) YJJK-5 from salt-soda soil capsicum rhizosphere soil, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.17952.The bacterial strain salt tolerant alkali ability is strong, and field planting rate is high in salt affected soil, and para Toluic Acid, P-hydroxybenzoic acid, cinnamic acid and ferulic acid etc. have preferable degradation effect, can be relieved continuous cropping obstacle caused by phenolic acid class suppression harmful bacteria.Propylgallate, beta-cyclodextrin and biochemical fulvic acid powder is added to fermentation liquid after the strain fermentation, spray drying obtains active bacteria formulation.Preparation method science, fermentation liquid bacteria concentration is high, and viable bacteria loss is few in spray-drying process, and stability of active bacteria formulation during preservation is high.
Description
Technical field
The present invention relates to one plant of saline-alkali tolerant Methylotrophic bacillus and its active bacteria formulation and applications, belong to agro-ecology
Technical field.
Background technique
Capsicum, alias: long red pepper, long capsicum, green pepper, bell pepper are Magnoliatae, Solanaceae, Capsicum 1 year or limited more
Year raw herbaceous plant, is one of the vegetables that China is most widely planted.Continuous cropping obstacle is to influence yield of hot pepper mainly to restrict with quality
One of factor, and with the increase of continuous Planting Years, Soil-sickness Problem is got worse.The reason of causing continuous cropping obstacle is multiple
It is miscellaneous, specifically include that soil hardening and secondary salinization, Distribution of Nutrient be unbalanced, soil micro-ecosystem system degradation, soil-borne disease
Insect pest exacerbation, plant Auto toxicity etc..Wherein, plant Auto toxicity is as causing one of principal element of continuous cropping obstacle increasingly
It attracts attention.
So-called Auto toxicity be generated by plant, can to of the same race or root of Roundfruit Licorice have toxic action chemical substance.
These chemical substances can be can be discharged into environment by approach such as overground part leaching, root exudates and the plant stubbles of plant,
And then influence the growth of second stubble crop.Phenolic acid is the main suppression harmful bacteria that capsicum generates, including benzoic acid, para hydroxybenzene
Formic acid, cinnamic acid, ferulic acid etc. have significant inhibiting effect to chili growth, can cause continuous cropping obstacle.
The main path that phenolic acid decomposes in soil is biodegrade, and wherein microorganism is to cause biodegradation
Primary biological monoid.The microorganism strong to phenolic acid degradation is screened from soil, active bacteria formulation is made, for dropping
It is a kind of up-and-coming bio-control method that the phenolic acid class suppression harmful bacteria in soil, which is solved, to mitigate continuous cropping obstacle.It is reported that
Many microorganisms all have the ability of degradation phenolic acid, including Phanerochaete chrysosporium, serratia marcesens, raw rouge fixed nitrogen spiral shell
Bacterium, bacillus amyloliquefaciens, Paenibacillus polymyxa and yellow ocher streptomycete etc. more than 20 belong to, but the methyl battalion of rare saline-alkali tolerant
Support the report of type bacillus degradation phenolic acid.
Summary of the invention
The object of the present invention is to provide one plant of Methylotrophic bacillus (Bacillus methylotrophicus)
YJJK-5 and its active bacteria formulation and application.Methylotrophic bacillus YJJK-5 is isolated from salt-soda soil capsicum rhizosphere soil, tool
There are saline-alkali tolerant, degradation benzoic acid, P-hydroxybenzoic acid, cinnamic acid and ferulic acid, promotes a variety of good characteristics such as plant growth, it can
For producing microbial manure.
The purpose of the present invention adopts the following technical scheme that realization:
One plant of Methylotrophic bacillus (Bacillus for being isolated from salt-soda soil capsicum rhizosphere soil
Methylotrophicus) YJJK-5, the bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC) deposit number is CGMCC NO.17952；Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China
Institute of microbiology, the academy of sciences, the deposit date is on June 18th, 2019.
The bacterium colony and thallus feature of the Methylotrophic bacillus YJJK-5 bacterial strain: 3 are cultivated at 37 DEG C of NA culture medium
After it, bacterium colony is rounded, protrusion, opaque, smooth, milky.Thallus is in the shape of a rod, produces gemma, and gemma is round, does not expand, nothing
Parasporal crystal, Gram-positive.
The physiological and biochemical property of the Methylotrophic bacillus YJJK-5 bacterial strain: Gram stain test is positive, transports
Dynamic property test is positive, catalase test is positive, oxidase test is negative, nitrate reduction test is negative, casein hydrolysis
Test is positive, the gelatin hydrolysis test positive, Starch Hydrolysis negative, mannitol test are positive, fructose test is positive, galactolipin
Test is positive, maltose test is positive, trehalose test is positive.
The invention also discloses a kind of using Methylotrophic bacillus YJJK-5 as the active bacteria formulation of principle active component.
The preparation method of the Methylotrophic bacillus YJJK-5 bacterial strain active bacteria formulation, characterized in that seek methyl
Type bacillus YJJK-5 is supported after expansion culture (using NB culture medium), is inoculated in fermentation medium (inoculum concentration 2-5%),
35-37 DEG C of culture 24-28h obtains Methylotrophic bacillus YJJK-5 fermentation liquid.To Methylotrophic bacillus
Propylgallate 0.01-0.02% (w/w), beta-cyclodextrin 1-2% (w/w), biochemical fulvic acid are added in YJJK-5 fermentation liquid
Powder 5-7% (w/w), stirs evenly, and spray drying obtains active bacteria formulation original powder；Active bacteria formulation original powder and soluble starch press 1:
The mass ratio of (5~10) mixes, as Methylotrophic bacillus YJJK-5 active bacteria formulation.
The formula of the fermentation medium are as follows: bean cake powder 20-25g, Dried Corn Steep Liquor Powder 20-25g, corn flour 15-20g, phosphorus
Acid dihydride potassium 1.0g, magnesium sulfate 1.5g, manganese sulfate 0.1g, complex enzyme formulation 0.2g, water 1000mL, initial pH8.5.It is described compound
The composition of enzyme preparation are as follows: by weight, alkali protease 50%, neutral proteinase 20%, cellulase 20%, zytase
10%.
The application method of Methylotrophic bacillus YJJK-5 active bacteria formulation: Methylotrophic bacillus YJJK-5 is living
Bacteria preparation is diluted with water 100~200 times, when transplanting seedlings root dipping or watering when with water punching apply.
Beneficial effects of the present invention:
Methylotrophic bacillus YJJK-5 of the present invention can degrade benzoic acid, P-hydroxybenzoic acid, cinnamic acid simultaneously
With the plants suppression harmful bacteria such as ferulic acid, after cultivating 7d, the degradation rate to above-mentioned four kinds of phenolic acids is respectively 81.9%,
77.2%, 68.6% and 91.4%.The bacterial strain salt tolerant alkali ability is strong, can survive in salt affected soil, active bacteria formulation is suitable for
The suppression harmful bacteria that capsicum of degrading generates, mitigates capsicum continuous cropping obstacle.
The production method science of Methylotrophic bacillus YJJK-5 active bacteria formulation of the present invention, zymotic fluid viable count is high,
Production cost is low.Complex enzyme formulation is added in fermentation medium, to dregs of beans, Dried Corn Steep Liquor Powder, jade in temperature-rise period when sterilizing
The raw materials such as rice flour are hydrolyzed, and can shorten fermentation period of delay, improve raw material availability, improve viable bacteria concentration and bud in fermentation liquid
Spore production rate.Antioxidant propylgallate, embedding medium cyclodextrin are added when spray drying and there is protective effect to viable bacteria
Biochemical fulvic acid powder, it is possible to reduce the loss of number of viable in spray-drying process, and active bacteria formulation can be improved and saving
Stability in journey.
Detailed description of the invention
Fig. 1 is the colonial morphology of Methylotrophic bacillus YJJK-5 bacterial strain；
Fig. 2 is the thalli morphology of Methylotrophic bacillus YJJK-5；
Fig. 3 is the phylogenetic tree constructed based on 16S rDNA partial sequence.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
Embodiment 1: the screening of Methylotrophic bacillus YJJK-5
(1) screening of saline-alkali tolerant bacterial strain
Methylotrophic bacillus YJJK-5 is isolated from capsicum rhizosphere soil.Soil sample is in Shandong Province Dongying City Kenli
Area is salt affected soil.Specific separation method are as follows: weigh 10g pedotheque, be added to equipped with 0.9% physiological saline of 90mL and lack
In the triangular flask for measuring bead, 30min is vibrated in 37 DEG C, 180rpm.Soil supension 1mL is taken to carry out 10-1-10-7Series of concentrations ladder
Degree dilution, then takes 10-5、10-6、10-7Three dilutions are applied on the plate containing Selective agar medium, are inverted in 35~37 DEG C
Cultivate 2d.It picks them separately single colonie to cross to the plate containing Selective agar medium, cultivates 2d in 37 DEG C of inversions.Picking single colonie turns
It is connected on storage medium test tube slant, in 37 DEG C of culture 2d, after covering with lawn, is placed in 4 DEG C of refrigerators and saves backup.Screening altogether
To 194 plants of saline-alkali tolerant bacterial strain.
The formula of the Selective agar medium are as follows: peptone 10g, yeast extract 5g, complex inorganic salt catalyst (NaCl: KCl: MgCl2=
10: 1: 1) 20g, agar 20g, distilled water 1000mL, pH9.0,121 DEG C of sterilizing 20min.
The storage medium uses NA culture medium, i.e. nutrient agar, formula are as follows: beef extract 3g, peptone
10g, NaCl 5g, agar 20g, water 1000mL, pH 7.0~8.0.
(2) screening of the plants such as benzoic acid, P-hydroxybenzoic acid, cinnamic acid, ferulic acid suppression harmful bacteria degradation bacteria strains
The single colonie for picking them separately the saline-alkali tolerant bacterium that step (1) filters out is crossed to the plate containing Selective agar medium, in
3d is cultivated in 35 DEG C of inversions.Picking single colonie is forwarded on storage medium test tube slant, in 35 DEG C of culture 3d, after covering with lawn,
It is placed in 4 DEG C of refrigerators and saves backup.Isolate the bacterial strain that 7 plants of colony characteristics and thalli morphology have differences.
The formula of the selective medium are as follows: phthalic acid 1000mg, ammonium sulfate 0.5g, complex inorganic salt catalyst (NaCl:
KCl∶MgCl2=10: 1: 1) 20g, dipotassium hydrogen phosphate 1.5g, distilled water 1000mL, agar 20g, pH7.0.The preservation culture
The formula of base are as follows: P-hydroxybenzoic acid 1000mg, beef extract 3g, peptone 10g, NaCl 5g, agar 20g, water 1000ml, pH
7.0。
7 plants of isolates are transferred respectively into addition 1000mg/L P-hydroxybenzoic acid, 1000mg/L benzoic acid, 1000mg/L
In the basal medium of cinnamic acid or 1000mg/L ferulic acid, after 35 DEG C, 180rpm shaken cultivation 3d, using high-efficient liquid phase color
Spectrometry measures the degradation rate of benzoic acid, P-hydroxybenzoic acid, cinnamic acid or ferulic acid, filter out 1 plant it is equal to four kinds of suppression harmful bacterias
Bacterial strain with preferable degradation effect, i.e. YJJK-5.
The formula of the basal medium are as follows: peptone 5g, ammonium sulfate 0.5g, magnesium sulfate 0.1g, potassium chloride 0.2g, chlorination
Sodium 0.5g, water 1000ml, pH 7.0.
The item used when high effective liquid chromatography for measuring benzoic acid, P-hydroxybenzoic acid, cinnamic acid and ferulic acid degradation rate
Part are as follows: with acetonitrile and water (adjusting pH2.8 with acetic acid) for mobile phase, flow velocity 1.0mL/min, 30 DEG C of column temperature, Detection wavelength
280nm.Using gradient elution, 0-30min, acetonitrile is increased to 40%, 30-40min from 5%, and acetonitrile keeps 40%, 40-
45min, acetonitrile drop to 5% from 40%.
The identification of 2 Methylotrophic bacillus YJJK-5 of embodiment
(1) form and Physiology and biochemistry spy feature
The bacterium colony and thallus feature of the Methylotrophic bacillus YJJK-5 bacterial strain: 3 are cultivated at 37 DEG C of NA culture medium
After it, bacterium colony is rounded, protrusion, opaque, smooth, milky, as shown in Figure 1.Thallus is in the shape of a rod, produces gemma, and gemma is round,
It does not expand, no parasporal crystal, Gram-positive, as shown in Figure 2.NA culture medium, that is, the nutrient agar, matches
Side is beef extract 3g, peptone 10g, NaCl 5g, agar 20g, water 1000mL, pH 7.0~8.0.
The physiological and biochemical property of the Methylotrophic bacillus YJJK-5 bacterial strain: Gram stain test is positive, transports
Dynamic property test is positive, catalase test is positive, oxidase test is negative, nitrate reduction test is negative, casein hydrolysis
Test is positive, the gelatin hydrolysis test positive, Starch Hydrolysis negative, mannitol test are positive, fructose test is positive, galactolipin
Test is positive, maltose test is positive, trehalose test is positive.
(2) 16S rDNA sequence is analyzed
By YJJK-5 strain inoculated into NB culture medium, in 37 DEG C, 180r/min shaking table culture is for 24 hours.The NB culture medium,
It is formulated are as follows: beef extract 3g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0.Collect thallus, extract total DNA, then with
It is template, in the universal primer F27:5 '-AGA GTT TGA TCA TGG CTC AG-3 ' of prokaryotes 16S rRNA gene
The PCR amplification of 16S rDNA gene is carried out under guidance with F27:5 '-AGA GTT TGA TCA TGG CTC AG-3 '.Amplification
Product is recycled after the separation of 1% agarose gel electrophoresis using plastic recovery kit, and the raw limited public affairs of work biotechnology in Shanghai are transferred to
Department's sequencing, gained sequence is as shown in sequence table SEQ No.1.By the sequence in surveyed 16S rDNA sequence and GenBank database
It compares, and carries out multisequencing homology analysis, and phylogenetic tree construction with 7.0 software of MEGA, as shown in Figure 3.
By the analysis of form, physiological and biochemical property and 16S rDNA sequence it is found that the bacterial strain is Methylotrophic gemma bar
Bacterium is named as Methylotrophic bacillus (Bacillus methylotrophicus) YJJK-5.The bacterial strain is in 2019
June 18 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is
CGMCC NO.17952。
The salt tolerant alkali ability of 3 Methylotrophic bacillus YJJK-5 bacterial strain of embodiment
Using the saline-alkali tolerant energy of the selecting property culture medium verifying Methylotrophic bacillus YJJK-5 bacterial strain of different saline and alkalines
Power.The formula of the Selective agar medium are as follows: peptone 10g, yeast extract 5g, complex inorganic salt catalyst (NaCl: KCl: MgCl2=10: 1:
1) 20~100g, agar 20g, distilled water 1000mL, pH8~10,121 DEG C of sterilizing 20min.By Methylotrophic bacillus
YJJK-5 bacterial strain, on the selective medium plate of different saline and alkalines, is cultivated from slant medium streak inoculation in 35~37 DEG C
2~3d judges the whether resistance to corresponding saline and alkaline of the bacterial strain so that whether Methylotrophic bacillus YJJK-5 is longer.Methyl type
Growing state of the bacillus YJJK-5 on high salt, high-alkali culture medium is as shown in table 1 below, Methylotrophic bacillus
YJJK-5 have stronger salt tolerant alkali ability, can salt content 10%, pH10 high salinity culture medium on grow.
Growing state of the 1 methyl type bacillus YJJK-5 of table on high salt, high-alkali culture medium
The preparation of 4 Methylotrophic bacillus YJJK-5 microbial inoculum of embodiment
The preparation method of the Methylotrophic bacillus YJJK-5 bacterial strain active bacteria formulation the following steps are included:
1) actication of culture: Methylotrophic bacillus YJJK-5 is transferred into NA culture medium test tube slant, is trained in 35 DEG C
It supports and is activated for 24 hours.The formula of the NA culture medium are as follows: beef extract 3g, peptone 10g, NaCl 5g, agar 20g, water
1000ml, pH 7.0.
2) prepared by triangular flask seed: with the Methylotrophic bacillus YJJK-5 lawn after oese scraping activation, connecing
Kind is in NB culture medium, and 35 DEG C of cultures are for 24 hours.NB culture medium, that is, the nutrient broth medium, formula are as follows: beef extract 3g, egg
White peptone 10g, NaCl 5g, water 1000ml, pH 7.0.
3) prepared by seeding tank strain: triangular flask seed is transferred with 2% inoculum concentration into the 10L equipped with 6L NB culture medium
In seeding tank, in 35 DEG C of culture 20h, whole speed of agitator 200rpm, 0-6h ventilation quantity is 3L/min, and 6-20h ventilating ratio is 6L/
min。
4) fermented and cultured: seeding tank strain is transferred with 2% inoculum concentration into the fermentor of 500L.The built-in fermentation of fermentor
300L in culture medium, 35 DEG C of culture 28h, the as fermentation liquid of Methylotrophic bacillus YJJK-5.Whole speed of agitator is
180rpm, 0-6h ventilation quantity are 200L/min, and 6-28h ventilation quantity is 300L/min.After fermentation, methylotrophy in fermentation liquid
The viable count of type bacillus YJJK-5 is (1~2) × 1010Cfu/ml, gemma production rate are 90%~95%.
The formula of the step 4) fermentation medium are as follows: bean cake powder 20g, Dried Corn Steep Liquor Powder 20g, corn flour 15g, di(2-ethylhexyl)phosphate
Hydrogen potassium 1.0g, magnesium sulfate 1.5g, manganese sulfate 0.1g, complex enzyme formulation 0.2g, water 1000mL, initial pH8.5.The complex enzyme system
The composition of agent are as follows: by weight, alkali protease 50%, neutral proteinase 20%, cellulase 20%, zytase 10%.
Neutral proteinase and alkali protease are produced by Tai'an letter Biotechnology Co., Ltd that gets profit, product specification be respectively 50,000 U/g with
100000 U/g, enzyme activity unit definition execute national standard GB/T 23527-2009 " protease preparation " with detection method.It is fine
Plain enzyme and zytase to be tieed up to be produced by Shandong Long great Biotechnology Co., Ltd, product specification is respectively 100,000 U/g and 50,000 U/g,
Enzyme activity unit definition and the detection method of cellulase execute People's Republic of China's light industry standard QB 2583-2003
" cellulase preparation ", enzyme activity unit definition and the detection method of zytase execute People's Republic of China's light industry mark
Quasi- QB/T 4483-2013 " xylanase preparation ".
Step 4) the fermentation medium the preparation method comprises the following steps: weigh raw material according to formula, it is soluble in water, be heated to 52
DEG C, 1.5h is kept the temperature, then heats to 121 DEG C, heat preservation 25min sterilizes.
5) it the preparation of active bacteria formulation: is added into Methylotrophic bacillus YJJK-5 fermentation liquid described in step 4) and does not eat
Sub- propyl propionate (PG) 0.01% (w/w), beta-cyclodextrin 1% (w/w), biochemical fulvic acid powder 5% (w/w), stir evenly, then in
It is spray-dried under conditions of 200 DEG C of inlet temperature, 80 DEG C of outlet temperature, obtains active bacteria formulation original powder.It is described biochemical yellow rotten
Sour powder has the production of fertilizer Co., Ltd by Shandong Quan Linjia, and fulvic acid content is 40%.
Methylotrophic bacillus YJJK-5 active bacteria formulation original powder and the soluble starch ratio of 1:7.5 in mass ratio are mixed
Even, as Methylotrophic bacillus YJJK-5 active bacteria formulation, viable bacteria content are preferably 1.5 × 1010cfu/g。
5 Methylotrophic bacillus YJJK-5 para Toluic Acid of embodiment, P-hydroxybenzoic acid, cinnamic acid and ferulic acid
Degrading experiment
Benzoated culture medium is prepared, is formulated are as follows: benzoic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, compound nothing
Machine salt (NaCl: KCl: MgCl2=10: 1: 1) 20g, water 1000ml, pH 7.0.
Prepare the culture medium containing P-hydroxybenzoic acid, formula are as follows: P-hydroxybenzoic acid 1000mg, peptone 5g, sulfuric acid
Ammonium 0.5g, complex inorganic salt catalyst (NaCl: KCl: MgCl2=10: 1: 1) 20g, water 1000ml, pH 7.0.
Prepare the culture medium containing cinnamic acid, formula are as follows: cinnamic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, compound nothing
Machine salt (NaCl: KCl: MgCl2=10: 1: 1) 20g, water 1000ml, pH 7.0.
Prepare the culture medium containing ferulic acid, formula are as follows: ferulic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, compound nothing
Machine salt (NaCl: KCl: MgCl2=10: 1: 1) 20g, water 1000ml, pH 7.0.
Methylotrophic bacillus YJJK-5 is transferred in above-mentioned four kinds of culture mediums, in 35 DEG C, 180rpm oscillation training
Support 7d.It takes culture solution 10mL, 3500r/min to be centrifuged 15min, abandons precipitating, supernatant is extracted using 10mL methylene chloride.Extraction phase
It is evaporated using vacuum rotary evaporator, residue 1mL methanol dissolves, spare.
Using the residual of benzoic acid, P-hydroxybenzoic acid, cinnamic acid and ferulic acid in high performance liquid chromatography test sample
Amount, and calculate degradation rate.Parameter Conditions are as follows: with acetonitrile and water (adjusting pH2.8 with acetic acid) for mobile phase, flow velocity 1.0mL/min,
30 DEG C of column temperature, Detection wavelength 280nm.Using gradient elution, 0-30min, acetonitrile is increased to 40%, 30-40min, acetonitrile from 5%
40%, 40-45min is kept, acetonitrile drops to 5% from 40%.
High performance liquid chromatography testing result shows that Methylotrophic bacillus YJJK-5 is cultivated 7 days, to suppression harmful bacteria
Benzoic acid, P-hydroxybenzoic acid, cinnamic acid and ferulic acid degradation rate be respectively 81.9%, 77.2%, 68.6% and 91.4%.
SEQUENCE LISTING
<110>Tian Shi Biotechnology Co., Ltd is helped in Shandong
<120>one plants of saline-alkali tolerant Methylotrophic bacillus and its active bacteria formulation and applications
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1414
<212> DNA
<213>the 16S rDNA gene of Methylotrophic bacillus (Bacillus methylotrophicus) YJJK-5
<400> 1
tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tggttgtttg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180
gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360
atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420
ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480
gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540
ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600
acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660
ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720
cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780
gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960
cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080
cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200
aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260
cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380
ccgaagtcgg tgaggtaacc ttttaggagc cagc 1414
Claims (8)
1. one plant of saline-alkali tolerant Methylotrophic bacillus (Bacillus methylotrophicus) YJJK-5, the bacterial strain
Deposit number be CGMCC NO.17952.
2. Methylotrophic bacillus YJJK-5 described in claim 1 is in degradation benzoic acid, P-hydroxybenzoic acid, cinnamic acid
With the application in ferulic acid.
3. a kind of using Methylotrophic bacillus YJJK-5 described in claim 1 as the active bacteria formulation of principle active component.
4. the preparation method of active bacteria formulation as claimed in claim 3, characterized in that pass through Methylotrophic bacillus YJJK-5
After expanding culture, it is inoculated in fermentation medium, 35-37 DEG C of culture 24-28h obtains Methylotrophic bacillus YJJK-5 hair
Then active bacteria formulation is further made in zymotic fluid.
5. preparation method as claimed in claim 4, characterized in that the fermentation medium are as follows: bean cake powder 20-25g, corn pulp
Dry powder 20-25g, corn flour 15-20g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 1.5g, manganese sulfate 0.1g, complex enzyme formulation 0.2g,
Water 1000mL, initial pH8.5；The group of the complex enzyme formulation becomes, by weight: alkali protease 50%, neutral proteinase
20%, cellulase 20%, zytase 10%.
6. the preparation method of active bacteria formulation as described in claim 4 or 5, characterized in that Methylotrophic bacillus
Propylgallate 0.01-0.02%, beta-cyclodextrin 1-2%, biochemical fulvic acid powder 5-7% are added in YJJK-5 fermentation liquid, stirs
It mixes uniformly, spray drying obtains active bacteria formulation original powder；Active bacteria formulation original powder and soluble starch press 1:(5~10) mass ratio
It mixes, as Methylotrophic bacillus YJJK-5 active bacteria formulation.
7. active bacteria formulation as claimed in claim 3 answering in degradation benzoic acid, P-hydroxybenzoic acid, cinnamic acid and ferulic acid
With.
8. the application method of active bacteria formulation as claimed in claim 3, characterized in that Methylotrophic bacillus YJJK-5 is living
Bacteria preparation is diluted with water 100~200 times, when transplanting seedlings root dipping or watering when with water punching apply.
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