CN109456915B

CN109456915B - Bacillus safensis strain X3 and application thereof

Info

Publication number: CN109456915B
Application number: CN201811381569.1A
Authority: CN
Inventors: 姜瑛; 李培培; 王亮; 赵玮莉; 马月红; 刘晓丹; 王祎
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2018-11-20
Filing date: 2018-11-20
Publication date: 2020-08-21
Anticipated expiration: 2038-11-20
Also published as: CN109456915A

Abstract

The invention provides a bacillus safensis strain X3 and application thereof, belonging to the technical field of new application of microorganisms. The strain X3 has the phosphorus-dissolving capacity, the concentration of the degraded phosphorus reaches more than 476.6mg/L, the phosphorus which is difficult to utilize is converted into the available phosphorus, the content of the available phosphorus in the soil is increased, the utilization rate of the fertilizer is improved, and the growth and development of plants and the absorption of the fertilizer are promoted; meanwhile, the concentration of the indole acetic acid generated by the strain X3 reaches more than 34.78 mu g/mL. The application of the bacillus safensis strain X3 in plant planting can obviously improve the yield and quality of grain crop wheat and corn, oil crop peanut and economic crop tobacco.

Description

Bacillus safensis strain X3 and application thereof

Technical Field

The invention belongs to the technical field of new application of microorganisms, and particularly relates to a bacillus safensis strain X3 and application thereof.

Background

The sandy moisture soil has strong permeability, good shaking culture and dominant aerobic microorganism activity, can promote the decomposition of organic matters and accelerate the mineralization of the organic matters. And the soil is loose and easy to cultivate. The soil capillary has strong effect, fast water running and night tide phenomenon. The proper cultivation period is long, and seedlings are easy to stand; but the nutrient content is low, the fertilizer retention performance is poor, and the crops are easy to lose fertilizer and senilism in the later period. At present, most of the soil is used as cultivated land, the utilization rate reaches 92.3 percent, the soil is ripe twice a year, and the grain yield per mu is about 600kg in general. The future targeted improvement measures are as follows: due to soil planting, chemical fertilizer is reasonably applied, and formulated fertilization is carried out; applying organic fertilizer in multiple ways; the method is applied to the networking of the farmland and the forest, and develops the economic crops such as Chinese yam, watermelon and the like on the premise of ensuring the stable rise of grain production.

The growth-promoting rhizobacteria (PGPB) is defined as the Bacteria that live freely in soil, rhizosphere, root surface and phyllosphere and is favorable for Plant growth under certain conditions. These bacteria are capable of fixing nitrogen, solubilizing phosphorus, solubilizing iron, and producing plant hormones such as auxins, gibberellins, cytokinins, and ethylene. In addition, they can also improve plant stress resistance, including drought, high salt, heavy metal poisoning, and pesticides. A large number of phosphate solubilizing microorganisms exist in soil, which play an important role in the circulation of phosphorus and the growth and development of plants, and can improve the content of soluble phosphorus in the soil, so that the yield of crops is increased. The phosphate solubilizing bacteria can also increase the absorption of some trace nutrient elements (such as zinc, copper and the like) by the root system of the plant and enhance the disease resistance of the plant. Phosphate solubilizing bacteria are widely used as inoculants to increase the uptake of phosphorus and crop yield, based on their ability to solubilize poorly soluble inorganic phosphorus in the soil.

Plant hormones are active substances which are generated by plant cells under the induction of specific environmental signals and can regulate plant physiological responses at low concentration. The growth, development and differentiation of plants are regulated and controlled respectively or in mutual coordination in the aspects of cell division and elongation, tissue and organ differentiation, flowering and fructification, maturation and aging, dormancy and germination, isolated tissue culture and the like. There are many kinds of phytohormones, auxin (auxin), Gibberellin (GA), Cytokinin (CTK), abscisic acid (ABA), Ethylene (ETH), and Brassinosteroids (BR). In order to promote plant growth, some plant hormones are dependent on plant self-secretion, and some plant hormones can be artificially synthesized, such as indoleacetic acid. Although the sources of the synthetic indoleacetic acid are enlarged, the spraying mode, the content and the like need to be strictly controlled, the application is more complicated, and the application range of the indoleacetic acid is limited. Although rhizosphere bacteria symbiotically acting with plants can solve the problem, there is no report on rhizosphere bacteria having both a phosphate solubilizing function and a plant hormone production.

Disclosure of Invention

In view of the above, the present invention aims to provide a bacillus safensis strain X3 and an application thereof, wherein the strain X3 not only has a strong phosphate solubilizing function, but also can produce indole acetic acid. When the strain X3 is applied to crop planting, the yield of crops can be greatly improved, and the stress resistance of plants such as disease resistance, drought resistance and the like can be improved.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a bacillus safensis strain X3, Bacillus safensis, wherein the preservation number of the strain X3 is CGMCC No. 12313.

The invention provides application of the bacillus safensis strain X3 in plant cultivation.

Preferably, the method for growing the plant comprises the following steps:

(1) carrying out amplification culture on the bacillus safensis strain X3 to obtain an amplification-cultured strain X3;

(2) and (3) planting the strain X3 subjected to the expanded culture into soil on which plants are planted, and keeping the water content of the soil to be 50% -60% of the maximum field water capacity.

Preferably, the culture medium for the expanded culture in the step (1) comprises the following components in percentage by mass per 1000ml of the PKO culture medium: 1% of carbon source and 0.05% -0.1% of nitrogen source; the pH value of the culture medium for the amplification culture is 5-8.

Preferably, the carbon source in step (1) comprises one or more of glucose, sucrose, mannitol, maltose and xylose;

the nitrogen source comprises one or more of ammonium nitrate, potassium nitrate, peptone, glutamic acid, yeast powder and ammonium sulfate;

the inorganic salt comprises one or more of sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, magnesium sulfate heptahydrate, manganese sulfate and ferrous sulfate heptahydrate.

Preferably, the time of the amplification culture in the step (1) is 20-60 h.

Preferably, the shaking culture is performed during the expanding culture in the step (1); the rotation speed of the shaking culture is 180-220 rpm;

the volume of the culture medium in the shaking culture is 25-75 ml/250ml conical flask.

Preferably, the microbial inoculum type of the strain X3 which is subjected to the enlarged culture in the step (2) comprises a microbial inoculum or a microbial inoculum;

the inoculation amount of the bacteria water agent is 1-9 × 10⁸CFU/g soil.

Preferably, the concentration of the strain X3 in the microbial agent through expanded culture is 1-9 × 10¹¹CFU/g；

The application amount of the microbial agent is 35-45 kg/hm²。

Preferably, the plant comprises a crop; the crops comprise food crops, economic crops or oil crops.

The invention provides a bacillus safensis strain X3, Bacillus safensis, wherein the preservation number of the strain X3 is CGMCC No. 12313. The strain X3 has the phosphorus-dissolving capacity, the concentration of the degraded phosphorus reaches more than 476.6mg/L, the phosphorus which is difficult to utilize is converted into the available phosphorus, the content of the available phosphorus in the soil is increased, the utilization rate of the fertilizer is improved, and the growth and development of plants and the absorption of the fertilizer are promoted; meanwhile, the concentration of the indoleacetic acid generated by the strain X3 reaches more than 34.78 mu g/mL, so that the elongation of the root system of the plant is directly promoted, and the opportunity of contacting with nutrient substances in soil is increased; the content of endogenous IAA in plants can be increased; inducing the expression of plant defense gene and raising the resistance of plant to diseases, drought, etc.

The invention provides application of the bacillus safensis strain X3 in plant cultivation. The strain X3 is applied to the field plant planting, and the yield and the quality of grain crop wheat and corn, oil crop peanut and economic crop tobacco are improved better. Experiments prove that: the strain X3 is applied to the wheat potted plant, so that the root system of the wheat is thicker, longer and has larger surface area, the absorption of the root system of the wheat to water and nutrients is facilitated, and the plant height of the upper part of the wheat field and the contents of nitrogen, phosphorus and potassium are higher than those of the control treatment. In the crop yield experiment, compared with a control, the strain X3 treatment increases the wheat yield by 88.18%, the corn yield by 16.8% and the peanut yield by 11.18%. The first-class smoke proportion in the tobacco reaches 68.8 percent, and is improved by 17.87 percent compared with the control. The potassium-chlorine ratio of the indexes for characterizing the tobacco quality is lower than that of a control, and the sugar-base ratio are both higher than that of the control.

Biological material preservation information

Bacillus safensis (Bacillus safensis) is preserved in China general microbiological culture Collection center (CGMCC) at 28 days 3 and 28 years 2016, and the CGMCC is adopted as a unit, and is addressed to No. 3 of the West Lu No.1 of Beijing Korean district, the institute of microbiology of the Chinese academy of sciences, with the preservation number of CGMCC No.12313 and the strain number of X3.

Drawings

FIG. 1 is a colony map of strain X3 of the present invention;

FIG. 2 is a phylogenetic tree based on the 16SrDNA sequence of X3 and related strains, established by the adjacency method;

FIG. 3 shows the effect of different cultivation times on the phosphate-solubilizing ability of strain X3;

FIG. 4 shows the effect of different pH on the phosphate solubilizing ability of X3 strain;

FIG. 5 shows the effect of different aeration rates on the phosphate solubilizing ability of the X3 strain;

FIG. 6 shows the effect of different carbon sources on the phosphate solubilizing ability of the X3 strain;

FIG. 7 shows the effect of different nitrogen sources on the phosphate solubilizing ability of the X3 strain.

Detailed Description

In the present invention, the colony morphology of the strain X3 is as follows: the bacterial colony is white, the middle part is raised, the edge is complete, the surface is smooth but transparent, and the thallus is in a long rod shape. The physiological and biochemical characteristics of the strain X3 are as follows: gram-positive bacteria, starch hydrolysis positive, facultative anaerobic bacteria, catalase test, V-P test, gelatin liquefaction and nitrate reduction positive, and methyl red reaction and citrate utilization test negative.

In the invention, the strain X3 is subjected to morphological and 16S rDNA molecular identification, and the result shows that the strain X3 is Bacillus safensis.

In the invention, the phosphate solubilizing bacteria X3 have strong capability of decomposing inorganic phosphorus, and the capability reaches more than 476.6 mg/L. Meanwhile, the phosphate solubilizing bacteria X3 have the capability of secreting indoleacetic acid (IAA) which is more than 34.78 mu g/mL.

In the present invention, the method for growing the plant preferably comprises the following steps:

(2) and inoculating the expanded strain X3 into soil on which plants are planted, and keeping the water content of the soil to be 50-60% of the maximum field water capacity.

In the present invention, the culture medium for the expanded culture, preferably per 1000ml of PKO culture medium, comprises the following components by mass: 1% of carbon source and 0.05% -0.1% of nitrogen source; the pH value of the culture medium for the amplification culture is preferably 5-8, and more preferably 6.

In the present invention, the carbon source in the step (1) preferably includes one or more of glucose, sucrose, mannitol, maltose and xylose, more preferably glucose, sucrose or maltose; the nitrogen source preferably comprises one or more of ammonium nitrate, potassium nitrate, peptone, glutamic acid, yeast powder and ammonium sulfate, and more preferably peptone or yeast powder. The inorganic salt preferably comprises one or more of sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, magnesium sulfate heptahydrate, manganese sulfate, and ferrous sulfate heptahydrate. The sources of the carbon source, the nitrogen source and the inorganic salt are not particularly limited in the present invention, and those known to those skilled in the art can be used.

In the present invention, the time for the amplification culture is preferably 20 to 60 hours, and more preferably 50 to 56 hours. The temperature of the amplification culture is preferably 28-33 ℃, and more preferably 30 ℃. The period of the expanded culture is preferably shaking culture; the rotation speed of the shaking culture is 180-220 rpm; more preferably 200 rpm. The containing volume of the culture medium during the shaking culture is preferably 25-75 ml/250ml conical flask, and more preferably 50ml/250ml conical flask.

In the present invention, the microbial inoculum of the expanded strain X3The type of the strain comprises a strain water agent or a microbial agent, wherein the inoculation amount of the strain water agent is preferably 1-9 × 10⁸CFU/g soil, more preferably 5 × 10⁸The microbial agent comprises an expanded strain X3 thallus and a carrier, and the concentration of the expanded strain X3 in the microbial agent is preferably 1-9 × 10¹¹CFU/g, more preferably 5 × 10¹¹CFU/g. The present invention is not particularly limited in the kind of the carrier, and a carrier of a microbial agent well known in the art may be used. In the present example, the carrier is bone meal. The method for preparing the microbial agent is not particularly limited, and a method for preparing a microbial agent known in the art may be used. The application amount of the microbial agent is preferably 35-45 kg/hm²More preferably 40kg/hm²。

In the present invention, the plant preferably includes a crop plant. The crop preferably comprises a food crop, a cash crop or an oil crop. The grain crops comprise wheat, millet, corn or sorghum and the like. The economic crops comprise tobacco, cotton, vegetables or fruit trees and the like. Oil crops include peanuts or rape and the like.

The present invention provides a bacillus safensis strain X3 and its application, which are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Preparation of culture Medium

Preparing the following culture media:

(1) PKO liquid medium (inorganic phosphorus bacteria culture medium)

0.3g of sodium chloride, 10g of glucose, 0.3g of potassium chloride, 5g of tricalcium phosphate, 0.5g of ammonium sulfate, 0.3g of magnesium sulfate heptahydrate, 0.03g of manganese sulfate, 0.03g of ferrous sulfate heptahydrate and 1000mL of distilled water, adjusting the pH value to 7.0-7.2, and sterilizing at 115 ℃ for 30 min. And adding 15g of agar powder into the culture solution to obtain the PKO solid culture medium.

(2) LB liquid culture Medium

10g of tryptone, 5g of yeast powder, 10g of sodium chloride and 1000mL of water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20 min. 15g of agar powder is added into the culture solution to obtain the LB solid culture medium.

Then, the plant growth promoting bacteria capable of secreting indoleacetic acid are screened out by qualitative determination and quantitative determination.

TABLE 1 basic soil Properties tested

2. Qualitative determination

The bacteria isolated and purified from the sandy soil (Table 1) were inoculated in LB liquid medium containing L-tryptophan (100mg/L) at 30 ℃ for 180 r.min^-1Shake culturing for 1 d. Dripping 50 μ L of the bacterial liquid on a white ceramic plate, and adding 50 μ L of Salkowski colorimetric solution (50mL 35% HClO)₄+1mL 0.5M FeCl₃). A colorimetric solution containing 50. mu.L of 50mg/L indole acetic acid was used as a positive control. The white ceramic plate is placed at room temperature in the dark for 30min and observed, and the condition that the color turns red shows that the indole acetic acid can be secreted.

3. Quantitative determination

The bacteria secreting IAA obtained by primary screening are quantitatively determined, and the culture conditions are the same as the above. Firstly, the OD of the bacteria aqua is measured by spectrophotometry₆₀₀Then the bacteria liquid agent is added at 10000 r.min^-1Centrifuging for 10min, collecting supernatant, adding equivalent Salkowski colorimetric solution, standing in dark place for 30min, and measuring OD₅₃₀The value is obtained. Calculating the bacterial concentration OD₆₀₀At a value of 1, the content of indoleacetic acid per unit volume of fermentation broth. The standard curve was prepared by gradient dilution of analytically pure indoleacetic acid. The result shows that the separated strain has the capability of secreting IAA, and reaches 34.78 mu g.mL^-1。

The obtained IAA-producing strain is subjected to screening and determination of phosphate solubilizing conditions, and the test strain is inoculated into a 250mL triangular flask containing 50mL of slightly soluble inorganic phosphate liquid culture medium at 30 ℃ for 200 r.min^-1After culturing for 72h, taking 10mL of culture solution cultured for 72h, centrifuging at 6000r/min for 20min, taking supernatant, and measuring the content of phosphorus in the supernatant by using an ultraviolet spectrophotometer. As a result, the strain has strong inorganic phosphorus decomposing capacity of 476.6 mg/L.

The strain screened and separated by the method is named as X3, 16SrDNA molecular identification is carried out according to reports of tens of thousands of soldiers (2016. screening identification and effect research of a corn rhizosphere multifunctional growth-promoting bacterium), the strain is sequenced by Beijing Meiyi bioengineering GmbH, http:// www.ncbi.nlm.nih.gov on-line query analysis is carried out according to the sequencing result of 16S rDNA, Blast software is utilized to carry out homology comparison with other 16S rDNA sequences in GenBank, and a similar sequence and a sequence of X3 are selected to construct a 16S rDNA phylogenetic tree of X3 by MEGAversion 3 software (figure 2).

Example 2

Aerobic test

The sterilized LB medium was poured into 3 sterilized test tubes, approximately at 2/3, and the slant-grown strain X3 was picked up with an inoculating needle on a sterile operating table and inoculated into the medium (which had to penetrate the bottom of the tube). The culture was carried out at 30 ℃ and the results were observed for 3 to 7 days, respectively. Aerobic bacteria grow on the surface of the agar column, and anaerobic bacteria or facultative anaerobic bacteria grow along the puncture line.

The test results showed that the colonies of strain X3 grew along the surface of the agar column, and colonies also grew in the puncture line, and they were facultative anaerobic (see Table 2).

Example 3

Determination of Catalase

Dropping 1 drop of 3% H on a clean glass slide₂O₂Taking a strain X3LB slant culture 1 ring cultured for 18-24H, and culturing in a culture medium H₂O₂And (4) smearing, wherein if bubbles are generated, the smearing is positive, and otherwise, the smearing is negative.

The test results showed that strain X3 was positive for catalase (see table 2).

Example 4

Methyl Red test (M.R test)

a. Culture medium and reagents: 5g of peptone, 5g of glucose, 5g of sodium chloride and 1000mL of distilled water, adjusting the pH value to 7.0-7.2, subpackaging the tubes with 4-5 mL each, and sterilizing at 121 ℃ for 20 min. Reagent: methyl Red 0.1g, 95% alcohol 300mL, distilled water 200 mL.

b. Strain culture and results observation the inoculated strain X3 was cultured in the above culture solution at 30 ℃ for l-2 days. A few drops of methyl red reagent are added into the culture solution, if the culture solution is red, the methyl red is positive, and the yellow is negative (the methyl red changes the color range from 4.4 red to 6.0 yellow).

The test results showed that strain X3 was M.R negative (see table 2).

Example 5

Ethanephthalein methyl carbinol test (VP test)

a. Medium culture was tested with methyl Red.

b. Culture of strains and result observation inoculation and culture are performed in the same methyl red test. When the VP test is carried out, the culture solution (about 2mL) is taken to be mixed with an equal amount of 40% NaOH solution, a small amount of creatine is added, and after sufficient shaking for 2-5 min, if the culture solution is red, the VP is positive.

The test results showed that strain X3 was VP positive (see Table 2).

Example 6

Starch hydrolysis test

a. Adding 0.2% soluble starch into broth peptone agar, packaging into triangular flask, and sterilizing at 121 deg.C for 20 min. Lugol's iodine: 1g of iodine tablet and 2g of potassium iodide, dissolving the potassium iodide by using a small amount of (3-5mL) distilled water, adding the iodine tablet at present, and adding water to dilute to 300mL after the iodine is completely dissolved.

b. And (3) strain culture and result observation, namely, inoculating an X3 strain on a flat plate, culturing for 2-4 days at 30 ℃ to form a strain, and after the strain is formed, dropwise adding a Rogowski iodine solution on the flat plate to cover the periphery of the strain, wherein the flat plate is blue, and if a colorless transparent ring appears around the strain, the starch is hydrolyzed. The size of the transparent circle generally indicates the amount of starch hydrolyzing ability.

The test results showed that strain X3 was positive for starch hydrolysis (see table 2).

Example 7

Hydrolysis test of gelatin

a. 5g of culture medium and reagent peptone, 120g of gelatin and 1000mL of distilled water. Adjusting the pH value to 7.2-7.4, subpackaging test tubes, enabling the height of a culture medium to be 4-5 cm, and sterilizing at 121 ℃ for 20 min.

b. Culture of strains and observation of results the strain X3 was inoculated in the center of the tube by puncture. Culturing in 30 deg.C incubator for one month, and observing whether gelatin is liquefied.

The test results showed that strain X3 was positive for gelatin hydrolysis (see Table 2).

Example 8

Nitrate reduction test

a. Culture medium and reagent nitrate liquid culture medium: peptone 10g, KNO₃1g, 1000mL of distilled water, and adjusting the pH value to 7.0-7.4. Gries reagent: solution A: 0.5g of sulfanilic acid and 150mL of dilute acetic acid (about 10 percent); and B, liquid B: 0.1g of alpha-naphthol, 20mL of distilled water and 150mL of dilute acetic acid (about 10%). Diphenylamine reagent 0.5g was dissolved in l00mL concentrated sulfuric acid and diluted with 20mL of distilled water.

b. Strain culture and result observation Strain X3 was inoculated in a nitrate liquid medium and cultured at 30 ℃ for 1, 3, 5 days. A little culture solution is poured into a small hole of a white porcelain plate, and then 1 drop of reagent A and reagent B is respectively dropped into the small hole, when the culture solution turns pink, rose red, orange or brown and the like, the existence of nitrite is indicated, and the reduction is positive for nitrate, otherwise, the reduction is negative.

Test results the strain showed positive for nitrate reduction X3 (see table 2).

Example 9

Utilization of citrate

a. Culture medium and reagent sodium citrate 2g, NaCl 5g, MgSO₄·7H₂O 0.2g，(NH₄)₂·HPO₄1g, 10mL of 1% bromothymol blue aqueous solution, 20g of agar and 1000mL of distilled water, adjusting the pH value to 6.8-7.0, and sterilizing at 121 ℃ for 20 min.

b. And (4) strain culture and result observation, inoculating young (cultured for 18-24 h) X3 strain on a slant, and culturing at 30 ℃ for 3-7 days, wherein the strain is positive when the culture medium is alkaline (blue) and negative when the culture medium is unchanged.

The results of the citrate utilization test showed that strain X3 was negative (see table 2).

TABLE 2 physio-biochemical characteristics of the X3 Strain

As can be seen from the above, the isolated strain X3 was gram-positive, white, raised, with intact edges, smooth surfaces and opaque colonies. Aerobic or facultative anaerobic. The optimum pH value is 6, and the nitrate is reduced positively. According to the physiological and biochemical characteristics of the strain, the strain is identified as bacillus safensis (Bacillus safensis). The strain is preserved in the China general microbiological culture Collection center at 2016, 3, 28 days, with the preservation number of CGMCCNo.12313.

Example 10

The phosphate solubilizing bacteria with phosphate solubilizing function can be obtained by qualitatively screening and quantitatively determining the strain X3

(1) And (3) qualitative determination: after the test strain X3 is diluted with sterile water in a gradient manner, a proper gradient is coated on a PKO plate, the PKO plate is placed in an incubator at the temperature of 30 ℃ for culture, the phosphorus-solubilizing bacteria are obtained when a transparent ring colony appears, and the phosphorus-solubilizing bacteria are purified and then stored. The bacterial strain X3 shows a transparent ring colony, which indicates that the bacterial strain X3 provided by the invention is phosphate solubilizing bacteria.

(2) And quantitatively determining, namely inoculating the strain X3 into a triangular flask containing 50mL of PKO liquid culture medium, placing the triangular flask in a 30 ℃ shaking table, culturing for 96h at 180r/min, transferring the culture solution into a centrifuge tube, centrifuging for 15min at 4 ℃ at 10000r/min, collecting the supernatant, and determining the content of available phosphorus in the fermentation liquor by a molybdenum blue colorimetric method. Can convert inorganic phosphorus which is difficult to utilize into available phosphorus, and has the capability of dissolving inorganic phosphorus and organic phosphorus. As a result, the strain has strong inorganic phosphorus decomposing capacity of 476.6 mg/L.

According to the Chinese patent with the application number of 201410761484.1, the tobacco growth-promoting rhizobacteria YC8 is disclosed and classified as Bacillus megaterium. The strain X3 provided by the invention is classified and identified as Bacillus safensis (Bacillus safensis), and belongs to the Bacillus with the strain YC8, but the phosphorus-dissolving capacity of the strain X3 is more than 476.6mg/L, and the strain has obvious advantages compared with the phosphorus-dissolving capacity of the strain YC8 (the conversion amount of YC8 to tricalcium phosphate is 163.16 mg/L).

Example 11

In order to further verify the phosphate solubilizing ability and the optimum conditions of the phosphate solubilizing bacteria X3 obtained in example 10, the effect on the phosphate solubilizing ability was investigated for different carbon sources, different nitrogen sources, different amounts of shaking culture, different pH, and different culture times.

(1) Influence of different culture time on growth and phosphate-solubilizing capability of strain X3

The strain X3 was inoculated into PKO medium and cultured with shaking at 30 ℃ and the strain was sampled at each time point to determine the phosphorus decomposing ability.

As can be seen from FIG. 3, the strain X3 is in the growth stage within 0-10 h, so the phosphate solubilizing ability of the strain is not strong, the phosphorus content is low and the growth is slow. And (3) entering a logarithmic phase within 15-20 h, gradually enhancing the decomposition capability of phosphorus, entering a plateau phase within a time period of 20-36 h, reducing the unit increment, and continuously increasing the phosphorus content after 36 h.

(2) Effect of different pH on growth and phosphate-solubilizing ability of Strain X3

Adjusting the PKO culture medium to different pH values (4, 5, 6, 7, 8, 9 and 10), respectively, taking 50mL of the PKO culture medium, placing the 50mL of the PKO culture medium in a 250mL triangular flask, inoculating X3 in logarithmic growth phase according to the inoculation amount of 1% (v/v), placing the culture medium at 30 ℃ for 180 r.min^-1Shaking culture for 24h, and determining available phosphorus content by quantitative determination method, wherein the optimum pH of the strain with the strongest phosphorus-solubilizing ability is about 6 in a slightly acidic environment as shown in FIG. 4.

(3) Effect of different aeration amounts on growth and phosphate-solubilizing ability of Strain X3

Placing PKO liquid culture medium in 25mL, 50mL, 75mL, 100mL and 150mL in 250mL triangular flask, inoculating X3 in logarithmic growth phase according to 1% (v/v) inoculation quantity, placing at 30 deg.C and 180 r.min^-1Shake culturing for 24h, and measuring the content of available phosphorus by quantitative determination method. As shown in FIG. 5, since the strain X3 is facultative anaerobic, the aeration rate affects the phosphate-solubilizing ability of the strain, and when 50mL of the liquid is filled, the phosphate-solubilizing ability of the strain is strongest, and then the content of available phosphorus decreases with the decrease of the liquid filled, indicating that the phosphate-solubilizing ability gradually decreases。

(4) Influence of different carbon sources on growth and phosphate-solubilizing ability of strain X3

Adding 1% (w/v) carbon source (glucose, xylose, sucrose, fructose, mannitol, lactose, maltose) into PKO culture medium (containing no glucose), respectively, taking 50ml, placing in 250ml triangular flask, inoculating X3 in logarithmic phase according to 1% (v/v) inoculation amount, placing at 30 deg.C for 180 r.min^-1Shaking culturing for 24h, and determining available phosphorus content by quantitative determination method. As a result, as shown in FIG. 6, this strain exhibited the strongest phosphate solubilizing ability when supplied with maltose, followed by sucrose.

(5) Influence of different nitrogen sources on growth and phosphate-solubilizing capability of strain X3

Respectively adding 0.1% (w/v) nitrogen source (ammonium nitrate, ammonium sulfate, potassium nitrate, peptone, yeast powder, alanine, urea, etc.) into PKO culture medium (excluding ammonium sulfate), inoculating 50ml of X3 in 250ml triangular flask according to 1% (v/v) inoculation amount, placing at 30 deg.C for 180 r.min^-1Shaking culturing for 24h, and determining available phosphorus content by quantitative determination method. The results are shown in FIG. 7, which illustrates that the most available phosphorus content is obtained when yeast powder is used as the nitrogen source, followed by peptone.

Example 12

The strain X3 of the invention has obvious growth promoting effect on wheat, and is explained by a pot experiment.

And (3) filling about 200g of soil to be tested into a pot, wherein the wheat variety used in the test is Zheng wheat 9023, disinfecting the surface of the wheat, repeatedly washing the surface of the wheat with sterile water for multiple times, accelerating germination for 2 days, selecting seeds with consistent germination in the pot according to 4 wheat seeds per pot, and performing final singling to 3 seeds after one week. Culturing test strain X3, and making into liquid preparation according to the formula 10⁸The inoculation amount of CFU/g was inoculated into the soil, 4 replicates of each treatment were used as control against the non-inoculated strain, the water content was kept to 60% of the maximum field capacity, and the pots were placed in a light culture room. Sampling after 30 days, obtaining images of wheat root systems by using a root system scanner (LA1600+ scanner, Canada), analyzing related root system indexes by using root system analysis software (Winrizo 2003b, Canada), and measuring soilBasic physical and chemical properties and plant nitrogen, phosphorus and potassium content. The results show that the application of the strain X3 in the wheat potted plant can make the root system of the wheat thicker and longer and has larger surface area (Table 3), is beneficial to the absorption of the root system of the wheat on water and nutrients, and makes the plant height of the upper part of the wheat and the contents of nitrogen, phosphorus and potassium higher than those of the control treatment (Table 4).

TABLE 3 Effect of Strain X3 on wheat potted root systems

TABLE 4 Effect of Strain X3 on wheat potted plants

Example 13

Preparing bacterial strain X3 into bacterial agent with bone meal as carrier, and effective viable count of 10¹¹CFU/g, performing field test to verify the effect of X3, planting grain crop wheat and corn, oil crop peanut and economic crop tobacco at suitable planting time of various crops, setting 3 times of repetition and random block arrangement for the test, and setting the area of a cell to be 6 × 3m². All treatments are carried out by applying 15-15-15 compound fertilizer as base fertilizer with the application amount of 600 kg.hm^-2Simultaneously applying 40kg hm of X3 microbial agent^-2The control treatment applied equal amounts of bone meal and the yield and corresponding quality index were determined during harvest.

The results show that strain X3 treatment produced 87.85% increase in wheat (table 5), 16.55% increase in corn (table 6), and 10.89% increase in peanut (table 7) compared to the control. The first-class smoke proportion in tobacco reached 65.1%, which was increased by 11.86% compared to the control (table 8). The indexes characterizing tobacco quality were lower in potassium to chloride ratio than the control, and higher in both sugar to base ratio and sugar to sugar ratio than the control (Table 9).

TABLE 5 influence of X3 on wheat yield traits

TABLE 6 influence of X3 on maize yield and its constitutive factors

TABLE 7 influence of X3 on the amount of flower production and its constitutive factors

TABLE 8 influence of X3 treatment on the yield traits of flue-cured tobacco

TABLE 9 influence of X3 on the general chemical composition of tobacco Central leaf

The embodiment shows that the application of the strain X3 in plant cultivation can obviously improve the growth speed of plants and the crop yield and the product quality.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Bacillus safensisBacillus safensis) The strain X3 is characterized in that the preservation number of the strain X3 is CGMCC No. 12313.

2. The use of the bacillus safensis strain X3 as claimed in claim 1 for plant cultivation, wherein the method of plant cultivation comprises the steps of:

(2) and inoculating the expanded strain X3 into soil on which plants are planted, and keeping the water content of the soil to be 50% -60% of the maximum field water capacity.

3. The use of claim 2, wherein the medium for the expanded culture in the step (1) comprises the following components in percentage by mass per 1000ml of PKO medium: 1% of carbon source and 0.05% -0.1% of nitrogen source; the pH value of the culture medium for the amplification culture is 5-8.

4. The use of claim 3, wherein the carbon source in step (1) comprises one or more of glucose, sucrose, mannitol, maltose and xylose;

the nitrogen source comprises one or more of ammonium nitrate, potassium nitrate, peptone, glutamic acid, yeast powder and ammonium sulfate.

5. The use according to claim 2, wherein the time for the scale-up culture in step (1) is 20-60 h.

6. The use according to any one of claims 2 to 5, wherein the shaking culture is performed during the expansion culture in the step (1); the rotation speed of the shaking culture is 180-220 rpm;

7. The use as claimed in claim 2, wherein the microbial inoculum type of the strain X3 which is expanded and cultured in the step (2) is a microbial inoculum agent;

the inoculum size of the bacteria aqua is 1 × 10⁸~9×10⁸CFU/g soil.

8. The use of claim 2, wherein the microbial inoculum of the strain X3 in the step (2) is a microbial inoculum, and the concentration of the strain X3 in the microbial inoculum is 1 × 10¹¹~9×10¹¹CFU/g；

The application amount of the microbial agent is 35-45 kg/hm²。

9. Use according to any one of claims 2 to 5, 7 and 8, wherein the plant is a crop; the crops are grain crops, economic crops or oil crops.