CN106190887B - Bacillus subtilis T400 and preparation method of microbial inoculum thereof - Google Patents

Bacillus subtilis T400 and preparation method of microbial inoculum thereof Download PDF

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CN106190887B
CN106190887B CN201610336835.3A CN201610336835A CN106190887B CN 106190887 B CN106190887 B CN 106190887B CN 201610336835 A CN201610336835 A CN 201610336835A CN 106190887 B CN106190887 B CN 106190887B
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林敏�
杨国平
孙旭生
王亚君
尹坤
杨盼盼
张学贤
陈三凤
沈世华
谭志远
燕永亮
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Dongguan Baode Biological Engineering Co ltd
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Abstract

The invention relates to the technical field of microbial agents, in particular to bacillus subtilis T400 and a preparation method of the microbial agent, wherein the bacillus subtilis T400 is preserved in China center for type culture collection with the preservation number of CCTCCM2015755, and the bacillus subtilis T400 can supplement nitrogen required in the growth process of crops through self-generated nitrogen fixation and can also secrete auxin to promote the growth of plants. The preparation method of the bacillus subtilis T400 microbial inoculum comprises the following steps: (1) separating and screening; (2) purifying and storing; (3) culture in a culture medium: fermenting and culturing the single bacterial colony by using a fermentation culture medium to obtain a microbial liquid; (4) recovering thalli; (5) and (4) preparing a microbial inoculum. The process is simple and mature, large-scale production can be realized, and the prepared bacillus subtilis T400 has high nitrogenase activity, can secrete auxin, is wide in application range and high in economic benefit.

Description

Bacillus subtilis T400 and preparation method of microbial inoculum thereof
Technical Field
The invention relates to the technical field of microbial agents, and particularly relates to bacillus subtilis T400 and a preparation method of the microbial agent.
Background
With the increasing world population, the demand for chemical nitrogen fertilizers in global agriculture has also increased year by year. The use of the chemical nitrogen fertilizer greatly improves the yield of agricultural grains, but also brings serious negative effects such as environmental pollution, soil fertility reduction and the like. In nature, some prokaryotic microorganisms convert molecular nitrogen (N) in the atmosphere at normal temperature and pressure by nitrogenase2) Conversion to ammonia (NH)3) This process is called biological nitrogen fixation and this type of microorganism is called nitrogen-fixing microorganism. According to the statistics of grain and agriculture organization of the united nations, 70 percent of the total amount of combined nitrogen on the earth comes from biological nitrogen fixation, the amount of nitrogen fixed by global microorganisms can reach 2 hundred million tons every year, which accounts for about 3/4 times of the nitrogen demand of global crops and is equivalent to more than 3 times of that of industrial production nitrogen fertilizer. The method provides nitrogen sources for crops through biological nitrogen fixation, improves the yield, reduces the fertilizer dosage, reduces the production cost, promotes the sustainable development of agriculture, and is the most energy-saving, environment-friendly and eco-friendly nitrogen supply mode. Therefore, biological nitrogen fixation by various means is currently a major global concern.
According to the binding relationship between nitrogen-fixing microorganisms and hosts, nitrogen-fixing action can be classified into autogenous nitrogen-fixing such as azotobacter vinelandii, Klebsiella pneumoniae, Bacillus megaterium and the like, and symbiotic nitrogen-fixing such as Rhizobium and Actinomycetes. Among them, the rhizobia research has been over 100 years old, is the most clear biological nitrogen fixation system studied so far, and is widely used in the united states, brazil, argentina, australia, and the like. However, rhizobia has very harsh specific infectivity, is only limited to be used in leguminous crops, and cannot realize symbiotic nitrogen fixation with gramineous crops and other crops. Therefore, the self-growing nitrogen-fixing microorganisms which are planted in rhizosphere, root surface or enter cortex tissues of roots have very important application prospect in nitrogen fixation with gramineous crops such as rice, corn, sugarcane and the like.
The free-living nitrogen-fixing microorganisms are microorganisms which generally live independently in natural environments or culture media and can fix molecular nitrogen in the atmosphere as ammonia. The free-living nitrogen-fixing microorganisms can fix nitrogen without being combined with other organisms, the conditions are wide, the adaptability is wide, plant hormones can be secreted, the growth and development of roots and plants are stimulated, the free-living nitrogen-fixing bacteria can improve the micro-ecological community of plant rhizosphere, the plant growth is promoted, and the stress resistance of the plant is improved. But the research on the free-living nitrogen-fixing bacteria at home and abroad, particularly at home, is late, so that the application of the microbial agent in agriculture is greatly limited.
Disclosure of Invention
The invention aims to provide the bacillus subtilis T400 aiming at the defects in the prior art, and the bacillus subtilis T400 can supplement nitrogen required in the growth process of crops by self-generated nitrogen fixation and can secrete auxin to promote the growth of plants.
The invention also aims to overcome the defects in the prior art, and provides a preparation method of the bacillus subtilis T400 microbial inoculum, the process is simple and mature, the large-scale production can be realized, the prepared bacillus subtilis T400 has higher nitrogenase activity, can secrete auxin, and has wide application range and high economic benefit.
The purpose of the invention is realized by the following technical scheme.
A bacillus subtilis T400, classified under the name: bacillus subtilis T400, deposited in the China Center for type Collection, address: the preservation date of Wuhan university in Wuhan, China, 2015, 12 months and 15 days, the preservation number is as follows: CCTCC NO: m2015755.
The azotase activity of the bacillus subtilis T400 is 600.000-800.000 nmol/(mL-h), the indoleacetic acid IAA can be produced in the growth and metabolism process, and the indoleacetic acid concentration is 95-103 mg/L. Preferably, the bacillus subtilis T400 is a gram-positive bacterium containing spores.
Specifically, the method for measuring the azotase activity of the bacillus subtilis T400 comprises the following steps:
the test method comprises the following steps:
the azotase activity of each strain was measured by acetylene reduction. Activating each preserved strain by using VM-Ethanol solid culture medium, picking 1-ring thallus by using an inoculating loop into a 1.5mL sterile centrifuge tube, diluting the thallus by using sterile water, inoculating the thallus into a 10 mL test tube filled with 5mL semi-solid culture medium according to the same inoculation amount, and sealing the test tube by using a reverse rubber plug. After culturing at 37 ℃ for 24 hours, 1/10 vol of 10% acetylene gas was injected, the culture was continued for 24 hours, and 0.5mL of the gas was extracted from the test tube and injected into a gas chromatograph (SP-2100, Beijing Tianpu Analyzer) to measure the acetylene and ethylene contents. The activity of the azotase was calculated according to the following formula (edited by the microorganism specialties of Beijing university of agriculture, 1986):
C=(h x×c×V)/(24.9×h s×t) (2.1)
wherein the content of the first and second substances,h xis the peak area value of the sample;h sis standard C2H4Peak area value;cis standard C2H4Concentration (nmol/mL);
Vculture vessel volume (mL);tincubation time (h) for the sample;Cto produce C2H4Concentration [ nmol/(mL. h)]。
And (3) test results:
as a result, as shown in FIG. 4, the nitrogenase activity was found to be 600.000 to 800.000 nmol/(mL. h) by calculation according to the formula (2.1). Therefore, the bacillus subtilis T400 can generate specific nitrogen-fixing enzyme in the metabolic process and can spontaneously fix nitrogen in the atmosphere.
Specifically, the method for determining the qualitative content of indoleacetic acid in bacillus subtilis T400 comprises the following steps:
qualitative content determination of auxin
(1) Selecting strains and inoculating respectively (OD 600=1.0, 0.5mL of bacterial suspension) into a 250mL Erlenmeyer flask containing 50mL of King liquid medium, with 3 replicates per group.
(2) After inoculation, the triangular flask is placed on a shaker, cultured for 3d at 28 ℃ and 125rpm and is to be tested.
(3) And (3) placing 50 muL of the bacterial suspension growing on the King liquid culture medium for 3d in a transparent centrifuge tube, and adding 50 muL of colorimetric solution.
(4) Setting a positive control and a negative control, adding 50 muL of plant growth hormone (IAA) with the concentration of 10mg/L into the positive control, and simultaneously adding 50 muL of colorimetric solution. And adding 50 mu LKING liquid culture medium in the negative control, and simultaneously adding 50 mu L colorimetric solution.
(5) Placing the positive control solution, the negative control solution and the determination solution on a white ceramic plate, placing the white ceramic plate at room temperature for 15min, observing the color change, wherein the color change is positive when the color change is red, which indicates that the IAA can be secreted, and the deeper the color is, the stronger the ability of secreting the IAA is; if the strain is not discolored, the strain is negative, which indicates that the strain cannot secrete IAA, and the strain is used as a judgment basis for judgment and analysis. The culture medium is King culture medium (1L); the reagent formula is peptone 20g, K2HPO41.725g，MgSO4·7H2O1.5 g, glycerol 15mL, tryptophan 0.1g, and distilled water 1000 mL.
Quantitative determination of auxin
With minor modifications with reference to Riberio et al. The strain T400 is inoculated into LB liquid culture medium containing 1 g/L tryptophan, and is cultured for 48 hours at 30 ℃ and 180 r/min with shaking. The culture broth was centrifuged at 10000 r/min for 5min, 100mL of the supernatant was added to a 96-well plate, and mixed with 100mL of Salkowski's reagent (1 mL of 0.5 mol/L FeCl)3And 49 mL of 35% perchloric acid), standing at room temperature for 30 min, and measuring the absorbance at a wavelength of 530 nm by using a microplate reader. As shown in FIG. 5, a calibration curve was prepared using IAA standards at different concentrations, and the results are shown in Table 1.
TABLE 1 measurement results of Strain T400 IAA
Bacterial strains T400 T665 T808 T188
IAA concentration (mg/L) 98.36 90.12 86.86 21.57
As can be seen from FIG. 5 and Table 1, the auxin concentration of Bacillus subtilis T400 is 98.36mg/L, therefore, the Bacillus subtilis T400 can be judged to generate IAA in the growth and metabolism process and has the function of promoting the growth of crops.
The bacillus subtilis T400 has a DNA sequence of a sequence table NO. 1.
A preparation method of a bacillus subtilis T400 microbial inoculum comprises the following steps:
(1) separating and screening
Selecting a soil sample containing the azotobacter colony of the bacillus subtilis T400, separating, screening and culturing to obtain the azotobacter colony.
(2) Purifying and storing
And (3) carrying out streak purification on the azotobacter colony obtained by separation and screening on a purification preservation culture medium, culturing and separating to obtain a bacillus subtilis T400 single colony, and preserving the single colony for later use. FIG. 1 is a diagram showing a colony obtained by amplifying a single colony of Bacillus subtilis T400 under a microscope by 1000 times.
(3) Culture in a culture medium: fermenting and culturing a bacillus subtilis T400 single colony by using a fermentation culture medium to obtain a microbial liquid;
the culture conditions were: the environmental factors influencing the growth and the propagation of the microorganisms are many, and the liquid loading amount, the inoculation amount, the culture temperature, the rotating speed, the ventilation quantity, the pH value and the fermentation time have great influence on the fermentation.
(4) Recovery of bacterial cells
And (3) inoculating the microbial liquid produced by the fermentation tank into a sterile disc separator, centrifugally collecting the bacillus subtilis T400, quickly removing water, preparing dry pure bacterial powder, and measuring the number of spores to be 500 plus 800 hundred million/g.
(5) Preparation of microbial inoculum
And (3) completely mixing dry pure bacteria powder with a matrix material of talcum powder and grass peat, and detecting the number of viable bacteria, wherein the detection result shows that the range of the number of viable bacteria is 4-20 hundred million/g, and the number of viable bacteria of the product completely meets the requirement of more than 2 hundred million/g of national standard microbial inoculum GB20287-2006, so that the bacillus subtilis T400 microbial inoculum is obtained.
Preferably, in the step (1), the specific method for separating and screening is as follows: adding sterile water into a soil sample, shaking, standing, taking supernatant for centrifugal treatment, discarding supernatant, retaining precipitate, adding sterile water for suspension, centrifuging, removing precipitate, taking supernatant for re-centrifugation, discarding supernatant, retaining precipitate, adding phosphate buffer solution for suspension, and obtaining a sample solution; adding a sample solution into a phosphate buffer solution for suspension mixing to obtain a diluted bacterial suspension; and heating the diluted bacterial suspension in a water bath, naturally cooling, absorbing the bacterial suspension, coating the bacterial suspension on a nitrogen-free culture medium, and culturing to obtain a nitrogen-fixing bacterial colony.
More specifically, the separation and screening method comprises the following steps: 500 g of soil sample was collected from Chang Zhen Huang mud pond farm in Dongguan city, Guangdong province, 3L of sterile water containing 0.01% Tween 80 was added, shaking was carried out for 10min, standing was carried out for half an hour, and the supernatant was centrifuged at 8000rpm and 20 ℃ for 20 min. Discarding the supernatant, retaining the precipitate, adding 30-50 mL of sterile water containing 0.01% Tween 80 for suspension, centrifuging the liquid at 20 ℃ and 5000 rpm for 5 seconds, removing the precipitate, pouring the supernatant into a dry sterile centrifuge tube, centrifuging at 20 ℃ and 6000 rpm for 10min, discarding the supernatant, retaining the precipitate, suspending the precipitate with 10 mL of phosphate buffer solution with pH7.0, and suspending and mixing 1 mL of the sample solution with 9 mL of phosphate buffer solution to obtain 10-fold diluted bacterial suspension. And (3) placing the diluted bacterial suspension in a water bath at 75 ℃ for heating for 15min, naturally cooling, sucking 100 mu L of the diluted bacterial suspension, coating the diluted bacterial suspension on a nitrogen-free culture medium, and culturing at 30 ℃ for 36-48 hours to obtain a nitrogen-fixing bacteria colony containing the bacillus subtilis T400.
Preferably, the specific method for purifying and storing in step (2) is as follows: culturing the azotobacteria colonies obtained by the step (1) at the constant temperature of 28-33 ℃ for 36-48 hours, separating to obtain bacillus subtilis T400 single colonies, storing the single colonies appearing on the plate in a test tube, culturing at the constant temperature of 28-33 ℃ for 36-48 hours, and storing in a refrigerator at the temperature of 2-5 ℃.
Preferably, the culture medium of step (3) further defines the liquid loading amount, the inoculation amount, the culture temperature, the rotation speed, the aeration amount, the pH value and the fermentation time of the fermentation tank, and specifically comprises:
(3.1) liquid loading: the liquid filling amount of the fermentation tank is 70-75% of the volume of the tank body; the fermentation cylinder liquid loading volume will be regulated and control according to the actual size of fermentation cylinder, and the liquid loading is too many can lead to the pollution, and the liquid loading volume also can cause the extravagant condition of resource too little.
(3.2) inoculation amount: the inoculation amount of the bacillus subtilis T400 is 5-10%;
the inoculation amount is the ratio of the volume of the seed liquid to the volume of the fermentation liquid. The proper inoculation amount can adjust the viscosity and the dissolved oxygen content of the culture solution, shorten the time of reaching the peak of growth and lead the product to be synthesized in advance. The relationship between the fermentation time and the growth and the propagation of the thalli can be obtained through a growth curve, the optimum fermentation time can maximize the metabolic amount of the thalli, the phenomenon of thalli autolysis can not occur, and the accuracy of a fermentation result is ensured.
(3.3) culture temperature: the shake flask culture temperature of the bacillus subtilis T400 is 30-36 ℃;
both the growth of the microorganism and the synthesis of the product need to be carried out at their respective most suitable temperatures. The temperature is an important condition for ensuring the enzyme activity, so that the optimal temperature environment must be ensured in the fermentation process.
(3.4) rotational speed: the shaking table rotating speed of the bacillus subtilis T400 in the fermentation process is 150-300 rpm;
the rotating speed of the shaking table in the fermentation process enables the culture to be uniformly mixed, each liquid space is ensured to keep the same growth period, meanwhile, the rotating speed is increased to increase the contact area of air, and the concentration of dissolved oxygen in the culture medium is increased.
(3.5) ventilation: in the research aspect of fermentation process, aerobic fermentation is mainly used, and oxygen is mainly used as a terminal electron acceptor of a respiratory chain. Aerobic microorganisms convert organic matter into CO through aerobic respiration2、H2And O, and acquiring energy. The ventilation of Bacillus subtilis T400 is set according to different culture time, and is controlled at 0.9-1.2vvm, preferably 1.04vvm, and 1.15-1.6vvm, preferably 1.35vvm, and 43-48 hr, 1.4-1 after inoculation, 0-12 hr after inoculation8vvm, preferably, the ventilation is set to 1.63 vvm.
(3.6) pH: pH is a very important influencing parameter for microbial growth and product synthesis and is a comprehensive measure of metabolic activity. Changes in pH can affect various enzyme activities, the rate of substrate utilization by the bacteria, and the architecture of the cells, thereby affecting the growth of the bacteria and the synthesis of the product. In the fermentation process of the bacillus subtilis T400, the pH is adjusted to be 7.1-7.4 by an acid or alkali supplementing method.
(3.7) fermentation time: according to the growth condition of the bacillus subtilis T400 in the shake flask, 42-48 hours are set as the fermentation end point.
Preferably, in the step (3), the culture medium is composed of the following raw materials by mass: CaCO31.0-1.4g，MgSO4·7H2O 0.6-1.2g，K2HPO41.0-2.0g，NaCl 0.1-0.4g，FeSO4·7H2O 0.001-0.005g，NaMO4·2H20.05-0.1g of O, 5-10g of sucrose, 18-20g of agar and 1000mL of distilled water, wherein the pH value of the culture medium is 7.1-7.4. Of course, mass ratios according to the above masses also fall within the scope of the present invention.
Preferably, the following steps are further included between the step (2) and the step (3):
(S1) gram stain: gram staining is carried out on the purified single colony, and positive bacteria are obtained through screening;
(S2) spore staining: and (3) carrying out spore staining on the positive bacteria, and screening to obtain gram-positive bacteria single colonies containing spores.
Gram staining and spore staining are two common methods of bacterial identification, and staining may narrow the scope of identification. The undyed bacteria have small refractive index difference with the surrounding environment and are extremely difficult to observe under a microscope. The gram-stained bacteria are in sharp contrast to the environment, and the morphology and arrangement of the bacteria and the gram-positive nature of certain species can be clearly observed (G)+) Or gram-negative bacteria (G)-) For classification and identification. Gram-negative bacteria generally have potential safety hazards, and the structure is abandoned after direct high-temperature sterilization in the application process of agricultural microorganismsCharacteristically, there is a potential risk of possible pathogenicity. The spore dyeing dyes the spores in the fungus body, so that the size, the position, the shape and other characteristics of the spores can be visually observed, and the identification range of the spores is further narrowed. The bacillus has the characteristics of long shelf life and easy storage, and has wide application basis in agricultural microbial products.
As can be seen from the gram stain result chart of FIG. 2 and the spore stain result chart of FIG. 3, Bacillus subtilis T400 is a gram-positive bacterium and contains spores.
Preferably, the specific method of gram staining of step (S1) is:
(S1.1) smearing, namely taking a glass slide in a sterile operating platform, slightly baking the glass slide above a flame lamp, and removing impurities on the glass slide; dropping a drop of sterile water in the center of the glass slide, picking a single bacterial colony in the water drop, and uniformly smearing the bacterial colony by using a burned inoculating loop; the sample slide was passed back and forth over the flame for 3 times to fix the cells;
(S1.2) primary dyeing: dripping 2-5 drops of ammonium oxalate crystal violet dye solution, dyeing for 1min, pouring off the dye solution, and washing with running water until no purple color exists;
(S1.3) mordanting: washing with newly-prepared iodine solution (iodine 1.0g, potassium iodide 2.0g, and distilled water 300.0 mL), removing residual water, covering the coated surface with iodine solution for 1min, and washing with water;
(S1.4) decoloring: after removing residual water, dripping 95% alcohol for decoloring for about 15-20 seconds, and immediately washing with running water;
(S1.5) counterstaining: dripping 1 drop of safranin staining solution, staining for 3-5min, washing with water, and drying with absorbent paper;
(S1.6) microscopic examination: the slide glass was placed under an optical microscope to observe the staining results.
Preferably, the specific method for spore staining in the step (S2) is as follows: in a sterile operating table, a glass slide is taken and slightly baked above a flame lamp to remove impurities on the glass slide. And (3) dropping a drop of sterile water in the center of the glass slide, picking up a single colony water drop, and uniformly smearing the single colony water drop by using a burned inoculating ring. The sample slide was passed back and forth 3 times over the fire lamp to immobilize the cells. Dripping 1-2 drops of carbonate basic red dye solution into the zone coated with thallus, and dyeing for 3 min. The staining solution was rinsed off with distilled water, air dried, and the slides were placed under an optical microscope for observation.
The invention has the beneficial effects that:
(1) nitrogen fixation: the bacillus subtilis widely exists in soil, plant rhizosphere and other environments, has the advantages of safe strains, various functions, convenient production, long quality guarantee period and the like, is one of the strains widely applied to the production of microbial fertilizers in China at present, and has considerable economic value in the application field of the microbial fertilizers. The azotase activity of the bacillus subtilis T400 is 600.000-800.000 nmol/(mL-h), and the concentration of the indoleacetic acid generated in the growth and metabolism process is 95-103 mg/L.
(2) Reducing nitrogen input: in the corn field demonstration test, on the basis of basically consistent fertilization cost, the nitrogen saving rate of a test group reaches 17.2%, the yield increase rate reaches 29.24%, the yield of corn is remarkably improved, and the application of the T400 microbial inoculum can bring greater economic benefit while reducing the nitrogen input.
(3) Promoting the growth of crops: in the demonstration test of the corn field, the plant height, the stem thickness and the area of the stick leaves of a test group are all higher than those of a control group, the relative ear height and the lodging rate are all lower than those of the control group, and the fact that the T400 microbial inoculum has a remarkable promoting effect on the growth of corn is fully demonstrated.
Drawings
FIG. 1 is a diagram showing a colony obtained by amplifying a single colony of Bacillus subtilis T400 under a microscope by 1000 times.
FIG. 2 is a graph showing the gram staining results of Bacillus subtilis T400.
FIG. 3 is a graph showing the staining results of Bacillus subtilis T400 spores.
FIG. 4 is a graph showing the results of the determination of the nitrogenase activity of Bacillus subtilis T400.
FIG. 5 is a colorimetric effect graph of Bacillus subtilis T400 IAA.
FIG. 6 is a diagram showing the effect of a corn potting test using Bacillus subtilis T400.
Detailed Description
The invention will be further described with reference to the following examples, which are shown in FIGS. 1-6.
Example 1
The preparation method of the microbial inoculum of bacillus subtilis T400 comprises the following steps:
(1) separating and screening
Selecting a soil sample with a bacillus subtilis T400 azotobacter colony, separating, screening and culturing to obtain an azotobacter colony;
(2) purifying and storing
Carrying out streak purification on the azotobacter colony obtained by separation and screening on a purification preservation culture medium, culturing and separating to obtain a bacillus subtilis T400 single colony, and preserving the single colony for later use;
(3) culture in a culture medium: fermenting and culturing the single bacterial colony by using a fermentation culture medium to obtain a microbial liquid;
(4) recovery of bacterial cells
Inoculating the microbial liquid into a sterile disc separator, centrifugally collecting bacillus subtilis T400, quickly removing water, preparing dry pure bacterial powder, and measuring the number of spores to be 500 hundred million/g;
(5) preparation of microbial inoculum
And mixing the dry pure bacteria powder with a matrix material, and detecting the viable count, wherein the detection result shows that the viable count range is 5 hundred million/g, so that the bacillus subtilis T400 microbial inoculum is obtained.
The bacillus subtilis T400 is preserved in China center for type culture Collection with a preservation number of CCTCCM 2015755. The azotase activity of the bacillus subtilis T400 is 680.604 nmol/(mL & h), indole acetic acid can be produced in the growth and metabolism process, and the indole acetic acid concentration is 98.36 mg/L. The bacillus subtilis T400 has a DNA sequence of a sequence table NO. 1.
In the step (1), the specific method for separating and screening comprises the following steps: adding sterile water into a soil sample, shaking, standing, taking supernatant for centrifugal treatment, discarding supernatant, retaining precipitate, adding sterile water for suspension, centrifuging, removing precipitate, taking supernatant for re-centrifugation, discarding supernatant, retaining precipitate, adding phosphate buffer solution for suspension, and obtaining a sample solution; adding a sample solution into a phosphate buffer solution for suspension mixing to obtain a diluted bacterial suspension; and heating the diluted bacterial suspension in a water bath, naturally cooling, absorbing the bacterial suspension, coating the bacterial suspension on a nitrogen-free culture medium, and culturing to obtain a nitrogen-fixing bacterial colony.
The specific method for purifying and storing in the step (2) comprises the following steps: culturing the azotobacteria colonies obtained by the culture in the step (1) at a constant temperature of 32 ℃ for 38 hours, separating to obtain bacillus subtilis T400 single colonies, storing the single colonies appearing on the plate in a test tube, culturing at a constant temperature of 31 ℃ for 42 hours, and storing in a refrigerator at 4 ℃.
The culture medium culture of the step (3) further limits the liquid loading amount, the inoculation amount, the culture temperature, the rotating speed, the ventilation capacity, the pH value and the fermentation time of a fermentation tank, and specifically comprises the following steps:
(3.1) liquid loading: the liquid filling amount of the fermentation tank is 75% of the volume of the tank body;
(3.2) inoculation amount: the inoculation amount of the bacillus subtilis T400 is 5 percent;
(3.3) culture temperature: the shake flask culture temperature of the bacillus subtilis T400 is 32 ℃;
(3.4) rotational speed: the rotating speed of a shaking table of the bacillus subtilis T400 in the fermentation process is 200 rpm;
(3.5) ventilation: setting the ventilation of the bacillus subtilis T400 according to different culture times, wherein the ventilation is controlled to be 1.04vvm 0-12 hours after inoculation, the ventilation is controlled to be 1.35vvm 13-42 hours after inoculation, and the ventilation is set to be 1.63vvm 43-48 hours after inoculation;
(3.6) pH: in the fermentation process of the bacillus subtilis T400, the pH is adjusted to be 7.2 by an acid or alkali supplementing method;
(3.7) fermentation time: according to the growth of Bacillus subtilis T400 in the shake flask, 46 hours are set as the fermentation end point.
In the culture of the culture medium in the step (3), the culture medium is composed of the following raw materials by mass: CaCO31.0g，MgSO4·7H2O 0.6g，K2HPO41.0g，NaCl 0.1g，FeSO4·7H2O 0.0015g，NaMO4·2H20.05g of O, 5g of cane sugar, 18g of agar and 1000mL of distilled water, wherein the pH value of the culture medium is7.1。
Example 2
The present embodiment is different from embodiment 1 in that the following steps are further included between step (2) and step (3):
(S1) gram stain: gram staining is carried out on the purified single colony, and positive bacteria are obtained through screening;
(S2) spore staining: and (3) carrying out spore staining on the positive bacteria, and screening to obtain gram-positive bacteria single colonies containing spores.
The specific method of gram staining in the step (S1) is:
(S1.1) smearing, namely taking a glass slide in a sterile operating platform, slightly baking the glass slide above a flame lamp, and removing impurities on the glass slide; dropping a drop of sterile water in the center of the glass slide, picking a single bacterial colony in the water drop, and uniformly smearing the bacterial colony by using a burned inoculating loop; the sample slide was passed back and forth over the flame for 3 times to fix the cells;
(S1.2) primary dyeing: 4 drops of ammonium oxalate crystal violet dye solution are dripped for dyeing for 1min, the dye solution is poured out and washed by running water until no purple color exists;
(S1.3) mordanting: washing away residual water with newly-prepared iodine solution, covering the coated surface with iodine solution for 1min, and washing with water;
(S1.4) decoloring: after removing residual water, dripping 95% alcohol for decoloring for about 18 seconds, and immediately washing with running water;
(S1.5) counterstaining: dripping 1 drop of safranin staining solution, staining for 4min, washing with water, and blotting with absorbent paper;
(S1.6) microscopic examination: the slide glass was placed under an optical microscope to observe the staining results.
The specific method for spore staining in the step (S2) is as follows: in a sterile operating table, a glass slide is taken and slightly baked above a flame lamp to remove impurities on the glass slide. And (3) dropping a drop of sterile water in the center of the glass slide, picking up a single colony water drop, and uniformly smearing the single colony water drop by using a burned inoculating ring. The sample slide was passed back and forth 3 times over the fire lamp to immobilize the cells. 2 drops of carbonate basic red dye solution are dripped into the zone coated with the thalli for dyeing for 3 min.
The rest of this embodiment is the same as embodiment 1, and is not described herein again.
Example 3
This example is different from example 1 or 2 in that in the production method of this example, the bacterial cells in step (4) were collected and the number of spores was measured to be 600 hundred million/g; and (5) preparing the microbial inoculum, wherein the detection result shows that the viable count range is 8 hundred million/g. The azotase activity of the bacillus subtilis T400 is 692 nmol/(mL-h), indole acetic acid can be produced in the growth and metabolism process, and the indole acetic acid concentration is 98.2 mg/L.
The specific method for purifying and storing in the step (2) comprises the following steps: culturing the azotobacteria colonies obtained by the step (1) at the constant temperature of 28 ℃ for 48 hours, separating to obtain bacillus subtilis T400 single colonies, storing the single colonies appearing on the plate in a test tube, culturing at the constant temperature of 28 ℃ for 48 hours, and storing in a refrigerator at 2 ℃.
The culture medium culture of the step (3) further limits the liquid loading amount, the inoculation amount, the culture temperature, the rotating speed, the ventilation capacity, the pH value and the fermentation time of a fermentation tank, and specifically comprises the following steps:
(3.1) liquid loading: the liquid filling amount of the fermentation tank is 71 percent of the volume of the tank body;
(3.2) inoculation amount: the inoculation amount of the bacillus subtilis T400 is 6 percent;
(3.3) culture temperature: the shake flask culture temperature of the bacillus subtilis T400 is 31 ℃;
(3.4) rotational speed: the rotating speed of a shaking table of the bacillus subtilis T400 in the fermentation process is 180 rpm;
(3.5) ventilation: setting the ventilation of the bacillus subtilis T400 according to different culture times, wherein the ventilation is controlled to be 0.9vvm 0-12 hours after inoculation, 1.15 hours after inoculation and 1.4vvm after inoculation in 43-48 hours after inoculation;
(3.6) pH: in the fermentation process of the bacillus subtilis T400, the pH is adjusted to be 7.2 by an acid or alkali supplementing method;
(3.7) fermentation time: according to the growth condition of the bacillus subtilis T400 in the shake flask, 48 hours are set as the fermentation end point.
In the culture of the culture medium in the step (3), the culture medium is composed of the following raw materials by mass: CaCO31.2g，MgSO4·7H2O 0.8g，K2HPO41.3g，NaCl 0.2g，FeSO4·7H2O 0.002g，NaMO4·2H20.06g of O, 6g of sucrose, 18g of agar and 1000mL of distilled water, wherein the pH value of the culture medium is 7.2.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
Example 4
This example is different from example 1 or 2 in that in the production method of this example, the bacterial cells in step (4) were collected and the number of spores was measured to be 700 hundred million/g; and (5) preparing the microbial inoculum, wherein the detection result shows that the viable count range is 10 hundred million/g. The azotase activity of the bacillus subtilis T400 is 700 nmol/(mL-h), indole acetic acid can be produced in the growth and metabolism process, and the indole acetic acid concentration is 101.45 mg/L.
The specific method for purifying and storing in the step (2) comprises the following steps: culturing the azotobacteria colonies obtained by the culture in the step (1) at the constant temperature of 30 ℃ for 36 hours, separating to obtain bacillus subtilis T400 single colonies, storing the single colonies appearing on the plate in a test tube, culturing at the constant temperature of 30 ℃ for 36 hours, and storing in a refrigerator at 3 ℃.
The culture medium culture of the step (3) further limits the liquid loading amount, the inoculation amount, the culture temperature, the rotating speed, the ventilation capacity, the pH value and the fermentation time of a fermentation tank, and specifically comprises the following steps:
(3.1) liquid loading: the liquid filling amount of the fermentation tank is 73 percent of the volume of the tank body;
(3.2) inoculation amount: the inoculation amount of the bacillus subtilis T400 is 8 percent;
(3.3) culture temperature: the shake flask culture temperature of the bacillus subtilis T400 is 34 ℃;
(3.4) rotational speed: the rotating speed of a shaking table of the bacillus subtilis T400 in the fermentation process is 250 rpm;
(3.5) ventilation: setting the ventilation of the bacillus subtilis T400 according to different culture times, wherein the ventilation is controlled to be 1.0vvm 0-12 hours after inoculation, 1.21vvm 13-42 hours after inoculation, and 1.6vvm 43-48 hours after inoculation;
(3.6) pH: in the fermentation process of the bacillus subtilis T400, the pH is adjusted to be 7.3 by an acid or alkali supplementing method;
(3.7) fermentation time: according to the growth of the Bacillus subtilis T400 in the shake flask, 42 hours are set as the fermentation end point.
In the culture of the culture medium in the step (3), the culture medium is composed of the following raw materials by mass: CaCO31.3g，MgSO4·7H2O 1.0g，K2HPO41.6g，NaCl 0.3g，FeSO4·7H2O 0.003g，NaMO4·2H20.08g of O, 8g of sucrose, 19g of agar and 1000mL of distilled water, and the pH value of the culture medium is 7.3.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
Example 5
This example is different from example 1 or 2 in that in the production method of this example, the bacterial cells in step (4) were collected and the number of spores was measured to be 750 hundred million/g; and (5) preparing the microbial inoculum, wherein the detection result shows that the viable count range is 15 hundred million/g. The azotase activity of the bacillus subtilis T400 is 750.346 nmol/(mL & h), indole acetic acid can be produced in the growth and metabolism process, and the indole acetic acid concentration is 95.9 mg/L.
The specific method for purifying and storing in the step (2) comprises the following steps: culturing the azotobacteria colonies obtained by the culture in the step (1) at a constant temperature of 32 ℃ for 40 hours, separating to obtain bacillus subtilis T400 single colonies, storing the single colonies appearing on the plate in a test tube, culturing at the constant temperature of 32 ℃ for 40 hours, and storing in a refrigerator at 3 ℃.
The culture medium culture of the step (3) further limits the liquid loading amount, the inoculation amount, the culture temperature, the rotating speed, the ventilation capacity, the pH value and the fermentation time of a fermentation tank, and specifically comprises the following steps:
(3.1) liquid loading: the liquid filling amount of the fermentation tank is 74% of the volume of the tank body;
(3.2) inoculation amount: the inoculation amount of the bacillus subtilis T400 is 9 percent;
(3.3) culture temperature: the shake flask culture temperature of the bacillus subtilis T400 is 34 ℃;
(3.4) rotational speed: the rotating speed of a shaking table of the bacillus subtilis T400 in the fermentation process is 285 rpm;
(3.5) ventilation: setting the ventilation of the bacillus subtilis T400 according to different culture times, wherein the ventilation is controlled to be 1.1vvm 0-12 hours after inoculation, 1.45vvm 13-42 hours after inoculation, and 1.72vvm 43-48 hours after inoculation;
(3.6) pH: in the fermentation process of the bacillus subtilis T400, the pH is adjusted to be 7.4 by an acid or alkali supplementing method;
(3.7) fermentation time: according to the growth condition of the bacillus subtilis T400 in the shake flask, 48 hours are set as the fermentation end point.
In the culture of the culture medium in the step (3), the culture medium is composed of the following raw materials by mass: CaCO31.3g，MgSO4·7H2O 1.1g，K2HPO41.8g，NaCl 0.3g，FeSO4·7H2O 0.004g，NaMO4·2H20.09g of O, 9g of sucrose, 20g of agar and 1000mL of distilled water, wherein the pH value of the culture medium is 7.4.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
Example 6
This example is different from example 1 or 2 in that in the production method of this example, the bacterial cells in step (4) were collected and the number of spores was measured to be 800 hundred million/g; and (5) preparing the microbial inoculum, wherein the detection result shows that the viable count range is 20 hundred million/g. The azotase activity of the bacillus subtilis T400 is 786.375 nmol/(mL & h), indole acetic acid can be produced in the growth and metabolism process, and the indole acetic acid concentration is 99.2 mg/L.
The specific method for purifying and storing in the step (2) comprises the following steps: culturing the azotobacteria colonies obtained by the culture in the step (1) at the constant temperature of 33 ℃ for 36 hours, separating to obtain bacillus subtilis T400 single colonies, storing the single colonies appearing on the plate in a test tube, culturing at the constant temperature of 33 ℃ for 36 hours, and storing in a refrigerator at 5 ℃.
The culture medium culture of the step (3) further limits the liquid loading amount, the inoculation amount, the culture temperature, the rotating speed, the ventilation capacity, the pH value and the fermentation time of a fermentation tank, and specifically comprises the following steps:
(3.1) liquid loading: the liquid filling amount of the fermentation tank is 75% of the volume of the tank body;
(3.2) inoculation amount: the inoculation amount of the bacillus subtilis T400 is 10 percent;
(3.3) culture temperature: the shake flask culture temperature of the bacillus subtilis T400 is 36 ℃;
(3.4) rotational speed: the rotating speed of a shaking table of the bacillus subtilis T400 in the fermentation process is 300 rpm;
(3.5) ventilation: setting the ventilation of the bacillus subtilis T400 according to different culture times, wherein the ventilation is controlled to be 1.2vvm 0-12 hours after inoculation, 1.6vvm 13-42 hours after inoculation, and 1.8vvm 43-48 hours after inoculation;
(3.6) pH: in the fermentation process of the bacillus subtilis T400, the pH is adjusted to be 7.4 by an acid or alkali supplementing method;
(3.7) fermentation time: according to the growth of the Bacillus subtilis T400 in the shake flask, 42 hours are set as the fermentation end point.
In the culture of the culture medium in the step (3), the culture medium is composed of the following raw materials by mass: CaCO31.4g，MgSO4·7H2O 1.2g，K2HPO42.0g，NaCl0.4g，FeSO4·7H2O 0.005g，NaMO4·2H20.1g of O, 10g of sucrose, 20g of agar and 1000mL of distilled water, wherein the pH value of the culture medium is 7.4.
The rest of this embodiment is the same as embodiment 1 or 2, and is not described again here.
The identification of 16S rDNA gene sequence strain of Bacillus subtilis T400
The bacteria are tiny in individuals and simple in shape, and the traditional method for identifying the bacteria is often used as a main basis for classification and identification according to different physiological and biochemical reactions of the bacteria. Since the late 70 s in the 20 th century, the international general "official" or "official" classification of bacteria was based on Bergey's Manual of bacteriology of identification. In physiological and biochemical identification, one or more physiological indexes do not conform to the unique properties of the strain, and the strain is difficult to be clearly identified. Currently, methods for identifying bacteria usually combine physiological and biochemical indicators of strains with molecular biological characteristics to draw more reliable conclusions. Wherein the 16S rRNA gene evolutionary development system of DNA sequence analysis has become a common technical means for the heterogeneous classification and identification of bacteria in the international (Kim et al, 2004; Prap et al, 1997).
The ribosome 16S rDNA gene sequence has a total length of about 1550bp and consists of alternative conserved region and variable region. The 16S rDNA fragments of all bacteria can be amplified by using a universal primer designed by a conserved region. The 16S rDNA sequence analysis technology is based on the basic principle that 16S rDNA fragments are extracted from a microorganism sample, 16S rDNA sequence information is obtained through cloning, sequencing or enzyme digestion and probe hybridization, and then the sequence information is compared with sequence data or other data of a 16S rDNA database to determine the position of the sequence information in an evolutionary tree, so that the possible microorganism species in the sample can be identified. The universal primer designed by using the conserved region of the 16S rDNA fragment can not be complementary to non-bacterial DNA, and the difference of the 16S rDNA variable region of bacteria can be used for distinguishing different bacteria. It is therefore generally accepted that the final identification is obtained by sequencing the 16S rDNA of a strain.
And a method of identification
(1) PCR reaction (25. mu.L):
10×PCR Buffer 2.5μL
dNTP(2.5mM) m 2.0μL
primer 27F (10. mu.M) 0.5. mu.L
Primer 1492R (10. mu.M) 0.5. mu.L
DNA template 100ng
Taq enzyme (5U/. mu.L) 0.5. mu.L
ddH2O 19μL
(2) And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 72 ℃ for 80 s, 35 cycles, and extension at 72 ℃ for 10 min. DNA sequencing was performed using an ABI 3730 xl DNA Analyzer (applied biosystems).
Sequencing results
tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg
atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaca
3. Homology analysis
Identifying the bacterium asBacillus subtilisBacillus subtilis.
Application example: the bacillus subtilis T400 microbial inoculum of the invention is used for carrying out corn and rice pot experiment
1. Seedling raising
Soaking corn and rice seeds in warm water overnight, sowing in a plastic cup filled with soil, raising seedlings for 12-14 days under natural conditions, and transplanting the corn and rice with uniform growth into a flowerpot when the plant is about 10 cm.
2. Soil pretreatment
After the soil is air-dried, the soil is sieved by a 1 mm sieve. Each process set 3 replicates. Each flowerpot was filled with 4.0 kg of soil, 407 mg of urea, 162 mg of calcium superphosphate and 418 mg of potassium sulfate. .
3. Test set-up
A bacillus subtilis T400 microbial inoculum with the functions of self-generation nitrogen fixation and crop growth promotion is selected for carrying out a pot effect comparison test, and 1g of microbial inoculum is added in each pot. The bentonite addition was set as a control experiment, and 1g of bentonite was added per pot. Proper amount of water is watered every day, the water amount of each pot is the same, the water is uniformly spread, and the water is not allowed to flow out of the pot bottom so as to avoid the error caused by fertilizer loss.
4. Test results
After 45 days of seedling transplantation, the tillering number, the plant height and the chlorophyll content of the rice are measured. Data were statistically analyzed using SPSS 17.0. After 64 days, the height of the corn plants, the number of leaves and the fresh weight of each plant were measured. It can be shown that the inoculation of the T400Bacillus subtilis preparation can obviously promote the growth of corn and rice, and the results are shown in Table 2, Table 3 and FIG. 6.
TABLE 2 microbial inoculum T400 rice pot experiment results
Figure DEST_PATH_IMAGE001
Note: the same column with different lower case letters indicates significant difference (P<0.05).
TABLE 3 bacterial agent T400 corn pot experiment results
Bacterial strains Plant height (cm) Number of blades (sheet) Fresh weight of single plant (g)
T400 106.0 10.5 42.2
CK 77.6 8.8 29.2
As can be seen from tables 2, 3 and 6, when the bacillus subtilis T400 microbial inoculum of the invention is used for rice and corn pot culture tests, the microbial inoculum T400 remarkably improves the tillering number, the plant height and the chlorophyll content of rice, the increase rate of the tillering number is 70.67%, the increase rate of the plant height is 19.34% and the increase rate of the chlorophyll content is 49.34%. The plant height of the corn plant is 1.36 times of that of the control-treated plant, the leaf number is 1.19 times of that of the control-treated plant, and the fresh weight of the single plant is 1.44 times of that of the control-treated plant.
The bacillus subtilis T400 and the microbial inoculum thereof prepared by the method are subsidized and researched by introducing an innovative entrepreneurial team project in Guangdong province, have wide market prospect and high economic benefit.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
<0001>
SEQUENCE LISTING
<110> Dongguan City, Purcha bioengineering Co., Ltd
<120> preparation methods of bacillus subtilis T400 and microbial inoculum thereof
<130>0
<160>1
<170>PatentIn version 3.3
<210>1
<211>959
<212>DNA
<213> 16s rDNA gene sequence of Bacillus subtilis T400
<400>1
tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaca 959

Claims (3)

1. A Bacillus subtilis T400, which is characterized in that: the bacillus subtilis T400 is preserved in China center for type culture Collection with the preservation number of CCTCCM 2015755.
2. The Bacillus subtilis T400 of claim 1, wherein: the azotase activity of the bacillus subtilis T400 is 600.000-800.000 nmol/(mL-h), and the concentration of the indoleacetic acid generated in the growth and metabolism process is 95-103 mg/L.
3. The Bacillus subtilis T400 of claim 1, wherein: the bacillus subtilis T400 has a DNA sequence of a sequence table NO. 1.
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CN103275895A (en) * 2013-05-24 2013-09-04 南京农业大学 Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof
CN104560789A (en) * 2014-12-13 2015-04-29 郑州市污水净化有限公司 Peanut growth promoting rhizobacteria HS2 and application thereof
CN104611252A (en) * 2014-12-13 2015-05-13 苏新宏 Tobacco plant growth promoting bacterium TC6 and application thereof
CN104630090A (en) * 2014-12-13 2015-05-20 河南农业大学 Corn rhizosphere growth promoting bacteria YM3 and application thereof

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Publication number Priority date Publication date Assignee Title
CN103275895A (en) * 2013-05-24 2013-09-04 南京农业大学 Saline-alkali-tolerant heteroauxin-producing Bacillus subtilis and application thereof
CN104560789A (en) * 2014-12-13 2015-04-29 郑州市污水净化有限公司 Peanut growth promoting rhizobacteria HS2 and application thereof
CN104611252A (en) * 2014-12-13 2015-05-13 苏新宏 Tobacco plant growth promoting bacterium TC6 and application thereof
CN104630090A (en) * 2014-12-13 2015-05-20 河南农业大学 Corn rhizosphere growth promoting bacteria YM3 and application thereof

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