CN110408567A - A saline-alkali-tolerant methylotrophic bacillus and its live bacterial preparation and application - Google Patents

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

A saline-alkali-tolerant methylotrophic bacillus and its live bacterial preparation and application

technical field

The invention relates to a saline-alkali-resistant methylotrophic bacillus and its live bacterial preparation and application, belonging to the field of agricultural biotechnology.

Background technique

Capsicum, alias: ox horn pepper, long pepper, vegetable pepper, bell pepper, is an annual or limited perennial herb of Magnolia, Solanaceae, Capsicum, and is one of the most widely planted vegetables in my country. Continuous cropping obstacle is one of the main restrictive factors affecting the yield and quality of pepper, and with the increase of continuous planting years, the problem of continuous cropping obstacle becomes more and more serious. The causes of continuous cropping obstacles are complex, mainly including: soil compaction and secondary salinization, unbalanced distribution of soil nutrients, deterioration of soil micro-ecological system, aggravation of soil-borne diseases and insect pests, and plant autotoxicity. Among them, plant autotoxicity has attracted more and more attention as one of the main factors causing continuous cropping obstacles.

The so-called autotoxicity is a chemical substance produced by plants that can be toxic to plants of the same species or family. These chemicals can be released into the environment through leaching of aboveground parts of plants, root exudates and plant residues, and then affect the growth of next crops. Phenolic acids are the main autotoxic substances produced by peppers, including benzoic acid, p-hydroxybenzoic acid, cinnamic acid, ferulic acid, etc., which have a significant inhibitory effect on the growth of peppers and can cause continuous cropping obstacles.

The main pathway for the decomposition of phenolic acids in soil is biodegradation, in which microorganisms are the main biological groups that cause biodegradation. It is a promising biological control measure to screen the microorganisms with strong degrading effect on phenolic acid substances from the soil and make live bacterial preparations to degrade the phenolic acid self-toxic substances in the soil to reduce continuous cropping obstacles. Many microorganisms have been reported to have the ability to degrade phenolic acids, including Phanerochaete chrysosporium, Serratia marcescens, Azospirillum adipogenetica, Bacillus amyloliquefaciens, Paenibacillus polymyxa, and yellow ochre chain There are more than 20 genera such as molds, but there are few reports on the degradation of phenolic acids by saline-alkali-tolerant methylotrophic bacillus.

Contents of the invention

The object of the present invention is to provide a methylotrophic bacillus (Bacillus methylotrophicus) YJJK-5 and its live bacterial preparation and application. Methylotrophic Bacillus YJJK-5 is isolated from pepper rhizosphere soil in saline-alkali land. It has many excellent characteristics such as saline-alkali tolerance, degradation of benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid, and promotion of plant growth. It can be used in production microbial fertilizer.

The purpose of the present invention adopts following technical scheme to realize:

A methylotrophic Bacillus (Bacillus methylotrophicus) YJJK-5 isolated from pepper rhizosphere soil in saline-alkaline land, the preservation number of this strain in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee is CGMCC NO.17952; the preservation address It is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is June 18, 2019.

The colony and cell characteristics of the methylotrophic Bacillus YJJK-5 strain: after being cultured in NA medium at 37° C. for 3 days, the colony is round, protruding, opaque, smooth, and milky white. The bacterium is rod-shaped, produces spores, the spores are round, not enlarged, without paraspore crystals, and the Gram stain is positive.

Physiological and biochemical characteristics of the methylotrophic Bacillus YJJK-5 strain: positive Gram staining test, positive motility test, positive catalase test, negative oxidase test, negative nitrate reduction test, casein hydrolysis Test positive, gelatin hydrolysis test positive, starch hydrolysis test negative, mannitol test positive, fructose test positive, galactose test positive, maltose test positive, trehalose test positive.

The invention also discloses a living bacteria preparation with methylotrophic bacillus YJJK-5 as the main active ingredient.

The preparation method of the live bacterial preparation of the methylotrophic bacillus YJJK-5 is characterized in that, after the methylotrophic bacillus YJJK-5 is expanded and cultivated (using NB medium), it is inoculated in the fermentation medium ( The inoculum size is 2-5%), cultured at 35-37° C. for 24-28 hours to obtain the fermentation liquid of methylotrophic bacillus YJJK-5. Add 0.01-0.02% (w/w) of propyl gallate, 1-2% (w/w) of β-cyclodextrin, 5- 7% (w/w), stir evenly, and spray dry to obtain the raw powder of live bacteria preparation; the raw powder of live bacteria preparation and soluble starch are mixed according to the mass ratio of 1: (5-10), which is methylotrophic spores Bacillus YJJK-5 live bacteria preparation.

The formula of the fermentation medium is: soybean meal powder 20-25g, corn steep liquor dry powder 20-25g, corn flour 15-20g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 1.5g, manganese sulfate 0.1g, compound enzyme preparation 0.2g 1. Water 1000mL, initial pH 8.5. The composition of the compound enzyme preparation is: by weight, 50% of alkaline protease, 20% of neutral protease, 20% of cellulase and 10% of xylanase.

How to use the live methylotrophic bacillus YJJK-5 preparation: dilute the methylotrophic bacillus YJJK-5 live preparation with water 100 to 200 times, dip the roots when transplanting seedlings or rinse with water when watering.

Beneficial effects of the present invention:

The methylotrophic Bacillus YJJK-5 of the present invention can simultaneously degrade plant autotoxic substances such as benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid. After culturing for 7 days, the degradation rates of the above four phenolic acids were respectively 81.9%, 77.2%, 68.6% and 91.4%. The strain has strong salt-alkali tolerance and can survive in saline-alkali soil, and its live bacterial preparation is suitable for degrading autotoxic substances produced by peppers and reducing obstacles to continuous cropping of peppers.

The production method of the methylotrophic bacillus YJJK-5 living bacteria preparation of the invention is scientific, the number of viable bacteria in the fermentation liquid is high, and the production cost is low. The compound enzyme preparation is added to the fermentation medium, and the raw materials such as soybean meal, corn steep liquor dry powder, and corn flour are hydrolyzed during the heating process during sterilization, which can shorten the fermentation delay period, improve the utilization rate of raw materials, and increase the concentration and concentration of live bacteria in the fermentation liquid. Sporulation rate. The addition of antioxidant propyl gallate, embedding agent cyclodextrin and biochemical fulvic acid powder which has a protective effect on live bacteria can reduce the loss of live bacteria in the spray drying process and improve the live bacteria preparation in the spray drying process. Stability during storage.

Description of drawings

Fig. 1 is the colony form of methylotrophic bacillus YJJK-5 bacterial strain;

Fig. 2 is the thalline morphology of methylotrophic bacillus YJJK-5;

Figure 3 is a phylogenetic tree constructed based on the partial sequence of 16S rDNA.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1: Screening of methylotrophic bacillus YJJK-5

(1) Screening of saline-alkali-tolerant strains

Methylotrophic Bacillus YJJK-5 was isolated from capsicum rhizosphere soil. The soil was sampled in Kenli District, Dongying City, Shandong Province, which is saline-alkali soil. The specific separation method is as follows: weigh 10 g of soil sample, add it into a conical flask filled with 90 mL of 0.9% physiological saline and a small amount of glass beads, shake at 37° C. and 180 rpm for 30 min. Take 1mL of the soil suspension and make a serial concentration gradient dilution of 10 ^-1 -10 ^-7 , then take three dilutions of 10 ^-5 , 10 ^-6 , and 10 ^-7 and spread them on the plate containing the selection medium. Cultivate upside down for 2 days. Single colonies were picked and streaked onto plates containing selection medium, and cultured upside down at 37°C for 2 days. Pick a single colony and transfer it to the slant of the preservation medium test tube, culture it at 37°C for 2 days, and store it in a refrigerator at 4°C after it is covered with a lawn. A total of 194 saline-alkali-tolerant strains were screened.

The formula of the selection medium is: 10g of peptone, 5g of yeast extract, 20g of compound inorganic salt (NaCl:KCl:MgCl ₂ =10:1:1), 20g of agar, 1000mL of distilled water, pH9.0, sterilized at 121°C for 20min .

The preservation medium adopts NA medium, that is, nutrient agar medium, and its formula is: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water, and pH 7.0-8.0.

(2) Screening of strains degrading plant autotoxic substances such as benzoic acid, p-hydroxybenzoic acid, cinnamic acid, and ferulic acid

Single colonies of saline-alkali-tolerant bacteria screened in step (1) were picked and streaked onto a plate containing a selection medium, and cultured upside down at 35°C for 3 days. Pick a single colony and transfer it to the slant of the preservation medium test tube, culture it at 35°C for 3 days, and store it in a refrigerator at 4°C after it is covered with a lawn. A total of 7 strains with differences in colony characteristics and cell morphology were isolated.

The formula of the selective medium is: phthalic acid 1000mg, ammonium sulfate 0.5g, compound inorganic salt (NaCl:KCl:MgCl ₂ =10:1:1) 20g, dipotassium hydrogen phosphate 1.5g, distilled water 1000mL, Agar 20g, pH7.0. The formula of the preservation medium is: p-hydroxybenzoic acid 1000mg, beef extract 3g, peptone 10g, NaCl 5g, agar 20g, water 1000ml, pH7.0.

The seven isolates were respectively transferred to the basal medium supplemented with 1000mg/L p-hydroxybenzoic acid, 1000mg/L benzoic acid, 1000mg/L cinnamic acid or 1000mg/L ferulic acid, and cultured at 35°C and 180rpm for 3 days Finally, the degradation rate of benzoic acid, p-hydroxybenzoic acid, cinnamic acid or ferulic acid was measured by high performance liquid chromatography, and a strain with good degradation effect on the four kinds of autotoxic substances was screened out, namely YJJK-5 .

The formula of the basal medium is: peptone 5g, ammonium sulfate 0.5g, magnesium sulfate 0.1g, potassium chloride 0.2g, sodium chloride 0.5g, water 1000ml, pH 7.0.

The conditions adopted when high-performance liquid chromatography measures the degradation rate of benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid are: with acetonitrile and water (regulating pH2.8 with acetic acid) as mobile phase, flow rate 1.0mL/min, The column temperature is 30°C, and the detection wavelength is 280nm. Using gradient elution, 0-30min, acetonitrile increased from 5% to 40%, 30-40min, acetonitrile maintained at 40%, 40-45min, acetonitrile decreased from 40% to 5%.

The identification of embodiment 2 methylotrophic bacillus YJJK-5

(1) Morphological and physiological and biochemical characteristics

The colony and cell characteristics of the methylotrophic Bacillus YJJK-5 strain: after being cultured in NA medium at 37° C. for 3 days, the colony is round, protruding, opaque, smooth, and milky white, as shown in FIG. 1 . The bacteria are rod-shaped, spore-forming, and the spores are round, not enlarged, without paraspore crystals, and Gram staining is positive, as shown in Figure 2. The NA medium is nutrient agar medium, and its formula is 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water, and pH 7.0-8.0.

Physiological and biochemical characteristics of the methylotrophic Bacillus YJJK-5 strain: positive Gram staining test, positive motility test, positive catalase test, negative oxidase test, negative nitrate reduction test, casein hydrolysis Test positive, gelatin hydrolysis test positive, starch hydrolysis test negative, mannitol test positive, fructose test positive, galactose test positive, maltose test positive, trehalose test positive.

(2) 16S rDNA sequence analysis

The YJJK-5 strain was inoculated into NB medium, and cultured on a shaker at 180 r/min for 24 hours at 37°C. The formula of the NB medium is: beef extract 3g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0. Collect the bacteria, extract the total DNA, and then use it as a template to apply universal primers F27:5′-AGA GTT TGA TCA TGG CTC AG-3′ and F27:5′-AGA GTT TGA TCA TGG CTC in the prokaryotic 16S rRNA gene The PCR amplification of 16S rDNA gene was carried out under the guidance of AG-3′. After the amplified product was separated by 1% agarose gel electrophoresis, it was recovered with a gel recovery kit and submitted to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The obtained sequence is shown in the sequence table SEQ No.1. The measured 16S rDNA sequence was compared with the sequence in the GenBank database, and MEGA 7.0 software was used for multiple sequence homology analysis, and a phylogenetic tree was constructed, as shown in Figure 3.

According to the morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain was a methylotrophic Bacillus, named Bacillus methylotrophicus (Bacillus methylotrophicus) YJJK-5. The strain was deposited in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms on June 18, 2019, with the preservation number CGMCC NO.17952.

The saline-alkali tolerance of embodiment 3 methylotrophic bacillus YJJK-5 bacterial strains

The selective medium with different salinity was used to test the saline-alkali tolerance of methylotrophic Bacillus YJJK-5 strain. The formula of the selection medium is: peptone 10g, yeast extract 5g, compound inorganic salt (NaCl:KCl:MgCl ₂ =10:1:1) 20-100g, agar 20g, distilled water 1000mL, pH 8-10, extinguished at 121°C Bacteria 20min. Methylotrophic Bacillus YJJK-5 strain was streaked from the slant culture medium on selective medium plates with different salinity, cultured at 35-37°C for 2-3 days, and methylotrophic Bacillus YJJK- 5 Whether it grows out to judge whether the strain is resistant to the corresponding salinity. The growth of Methylotrophic Bacillus YJJK-5 on high-salt and high-alkaline medium is shown in Table 1 below. Methylotrophic Bacillus YJJK-5 has strong salt-alkali tolerance and can 10%, pH 10 high-salt medium growth.

The growth situation of table 1 bacillus methyl group YJJK-5 on the medium of high salt and high alkalinity

The preparation of embodiment 4 methylotrophic bacillus YJJK-5 inoculum

The preparation method of described methylotrophic bacillus YJJK-5 bacterial strain live bacteria preparation comprises the following steps:

1) Strain activation: Methylotrophic Bacillus YJJK-5 was transferred to the slant of NA medium test tube, and cultured at 35° C. for 24 hours for activation. The formula of the NA medium is: beef extract 3g, peptone 10g, NaCl 5g, agar 20g, water 1000ml, pH 7.0.

2) Triangular flask seed preparation: Scrape the activated lawn of methylotrophic Bacillus YJJK-5 with an inoculation loop, inoculate it in NB medium, and incubate at 35°C for 24 hours. The NB medium is a nutrient broth medium, and its formula is: 3g of beef extract, 10g of peptone, 5g of NaCl, 1000ml of water, and pH 7.0.

3) Preparation of seed tank strains: Transfer the seeds of the triangular flask to a 10L seed tank equipped with 6L NB medium with an inoculation amount of 2%, and cultivate them at 35°C for 20h, with a stirring speed of 200rpm throughout the whole process, and a ventilation volume of 0-6h. 3L/min, 6-20h ventilation ratio is 6L/min.

4) Fermentation culture: Transfer the seed tank bacteria into a 500L fermenter with an inoculum amount of 2%. Put 300L of fermentation medium in the fermenter and incubate at 35°C for 28h, which is the fermentation broth of methylotrophic Bacillus YJJK-5. The whole stirring speed is 180rpm, the ventilation rate is 200L/min for 0-6h, and 300L/min for 6-28h. After the fermentation, the number of viable bacteria of the methylotrophic bacillus YJJK-5 in the fermentation broth is (1-2)×10 ¹⁰ cfu/ml, and the sporulation rate is 90%-95%.

Step 4) The formula of the fermentation medium is: soybean meal powder 20g, corn steep liquor dry powder 20g, corn flour 15g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 1.5g, manganese sulfate 0.1g, compound enzyme preparation 0.2g, water 1000mL , initial pH 8.5. The composition of the compound enzyme preparation is: by weight, 50% of alkaline protease, 20% of neutral protease, 20% of cellulase and 10% of xylanase. Neutral protease and alkaline protease are produced by Taian Xindeli Biotechnology Co., Ltd. The product specifications are 50,000 U/g and 100,000 U/g respectively. The definition of the enzyme activity unit and the detection method follow the national standard GB/T 23527- 2009 "Protease Preparation". Cellulase and xylanase are produced by Shandong Longda Biotechnology Co., Ltd. The product specifications are 100,000 U/g and 50,000 U/g respectively. The industry standard QB 2583-2003 "Cellulase Preparation", the definition and detection method of the enzyme activity unit of xylanase follow the People's Republic of China light industry standard QB/T 4483-2013 "Xylanase Preparation".

Step 4) The preparation method of the fermentation medium is as follows: Weigh the raw materials according to the formula, dissolve them in water, heat to 52°C, keep warm for 1.5h, then raise the temperature to 121°C, keep warm for 25min to sterilize.

5) Preparation of live bacteria preparation: add propyl gallate (PG) 0.01% (w/w), β-cyclodextrin 1% ( w/w), biochemical fulvic acid powder 5% (w/w), stirred evenly, and then spray-dried under the conditions of an inlet temperature of 200°C and an outlet temperature of 80°C to obtain the raw powder of live bacteria preparation. The biochemical fulvic acid powder is produced by Shandong Quanlin Jiayou Fertilizer Co., Ltd., and its fulvic acid content is 40%.

Methylotrophic Bacillus YJJK-5 live bacteria preparation powder and soluble starch are mixed in a mass ratio of 1:7.5, which is the methylotrophic Bacillus YJJK-5 live bacteria preparation, and the live bacteria content is preferably 1.5 ×10 ¹⁰ cfu/g.

Degradation test of embodiment 5 methylotrophic bacillus YJJK-5 p-benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid

The medium containing benzoic acid was prepared, and its formula was: 1000 mg of benzoic acid, 5 g of peptone, 0.5 g of ammonium sulfate, 20 g of compound inorganic salt (NaCl:KCl:MgCl ₂ =10:1:1), 1000 ml of water, pH 7.0.

Prepare the medium containing p-hydroxybenzoic acid, its formula is: p-hydroxybenzoic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, compound inorganic salt (NaCl: KCl: MgCl ₂ =10: 1: 1) 20g, water 1000ml, pH 7.0.

A medium containing cinnamic acid was prepared, and its formula was: 1000 mg cinnamic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g compound inorganic salt (NaCl:KCl:MgCl ₂ =10:1:1), 1000 ml water, pH 7.0.

Prepare the culture medium containing ferulic acid, its formula is: ferulic acid 1000mg, peptone 5g, ammonium sulfate 0.5g, compound inorganic salt (NaCl:KCl:MgCl ₂ =10:1:1) 20g, water 1000ml, pH 7.0 .

Methylotrophic Bacillus YJJK-5 was transferred into the above four mediums, and cultured at 35°C and 180rpm with shaking for 7 days. Take 10 mL of the culture solution, centrifuge at 3500 r/min for 15 min, discard the precipitate, and extract the supernatant with 10 mL of dichloromethane. The extract phase was evaporated to dryness with a vacuum rotary evaporator, and the residue was dissolved in 1 mL of methanol for later use.

The residues of benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid in the samples were detected by high performance liquid chromatography, and the degradation rate was calculated. The parameter conditions are: acetonitrile and water (adjust pH 2.8 with acetic acid) as mobile phase, flow rate 1.0mL/min, column temperature 30°C, detection wavelength 280nm. Using gradient elution, 0-30min, acetonitrile increased from 5% to 40%, 30-40min, acetonitrile maintained at 40%, 40-45min, acetonitrile decreased from 40% to 5%.

The results of high-performance liquid chromatography showed that the methylotrophic Bacillus YJJK-5 was cultured for 7 days, and the degradation rates of benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid were 81.9% and 77.2% respectively. , 68.6% and 91.4%.

SEQUENCE LISTING

<110> Shandong Satian Biotechnology Co., Ltd.

<120> A saline-alkali-tolerant methylotrophic Bacillus and its live bacterial preparation and application

<130> 0

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1414

<212>DNA

<213> 16S rDNA gene of 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 ccacactgggg actgagacac ggcccagact cctacggggag 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. A salt-alkali-tolerant methylotrophic Bacillus (Bacillus methylotrophicus) YJJK-5, the preservation number of which is CGMCC NO.17952. 2. the application of the methylotrophic bacillus YJJK-5 described in claim 1 in degrading benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid. 3. A live bacteria preparation with the methylotrophic bacillus YJJK-5 described in claim 1 as the main active ingredient. 4. The preparation method of the live bacteria preparation described in claim 3 is characterized in that, after expanding the cultivation of methylotrophic bacillus YJJK-5, it is inoculated in the fermentation medium, cultivated at 35-37°C for 24-28h, and obtains Methylotrophic Bacillus YJJK-5 fermentation broth, and then further made into live bacteria preparations. 5. The preparation method according to claim 4, wherein the fermentation medium is: 20-25g soybean meal powder, 20-25g corn steep liquor dry powder, 15-20g corn flour, 1.0g potassium dihydrogen phosphate, sulfuric acid Magnesium 1.5g, manganese sulfate 0.1g, compound enzyme preparation 0.2g, water 1000mL, initial pH8.5; The composition of described compound enzyme preparation is, by weight: alkaline protease 50%, neutral protease 20%, cellulose Enzyme 20%, xylanase 10%. 6. the preparation method of live bacteria preparation as claimed in claim 4 or 5 is characterized in that, in methylotrophic bacillus YJJK-5 fermented liquid, add propyl gallate 0.01-0.02%, β-cyclodextrin 1-2%, 5-7% of biochemical fulvic acid powder, stir evenly, spray dry to obtain the original powder of live bacteria preparation; the original powder of live bacteria preparation and soluble starch are mixed according to the mass ratio of 1: (5 ~ 10), It is the live bacterial preparation of methylotrophic Bacillus YJJK-5. 7. the application of live bacteria preparation described in claim 3 in degrading benzoic acid, p-hydroxybenzoic acid, cinnamic acid and ferulic acid. 8. The method for using the live bacterial preparation as claimed in claim 3, characterized in that, the methylotrophic bacillus YJJK-5 live bacterial preparation is diluted 100 to 200 times with water, and when transplanting seedlings, dip the roots or water with water. Chong Shi.