CN107586743B - A highly efficient Bacillus megaterium in the rhizosphere of forest trees and its application - Google Patents

Abstract

The invention discloses a strain of Bacillus megaterium with high efficiency for dissolving phosphorus in the rhizosphere of trees, the preservation number is CCTCC NO.M 2017454, and the classification name is Bacillus megaterium L205. Experiments show that the amount of dissolved phosphorus of Bacillus megaterium reaches 203.04 mg/L, and its liquid bacterial solution can effectively dissolve insoluble inorganic phosphorus, increase the content of available phosphorus in red-yellow soil, and promote the growth of tree seedlings. The invention enriches the population bank of Bacillus megaterium in the strain collection center, and provides convenience for better development and utilization of the microbial population resources of Bacillus megaterium. At the same time, Bacillus megaterium strain L205 can secrete organic acids, dissolve inorganic phosphorus, and improve the absorption and utilization of phosphorus by plants. It is especially suitable for the production of tree biological fertilizers and forest growth-promoting agents in the red-yellow soil area of South China. It can further develop microorganisms. Compound fertilizers and inoculants.

Description

A highly efficient Bacillus megaterium in the rhizosphere of forest trees and its application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a high-efficiency phosphorus-dissolving Bacillus megaterium in the rhizosphere of forest trees and its application.

Background technique

The rhizosphere is the place where soil, root system and microorganisms are closely combined and interacted, and it is also an important part of the soil micro-ecosystem. Sperber et al. found that most of the phosphorus-dissolving microorganisms appeared in the rhizosphere soil of plants, and the rhizosphere phosphorus-solubilizing bacteria and the root system had a mutual promotion effect. Most of the soil in China is in a state of phosphorus deficiency. More than 95% of the phosphorus in the soil in southern China is insoluble phosphorus and cannot be directly absorbed by plants. Phosphorus has become the main factor affecting plant growth. At the same time, most of the fast-growing and high-yielding forests in the south are pure artificial forests with simple biodiversity and weak ecosystems. The application of a large number of unreasonable chemical fertilizers has resulted in a decline in the quality of the ecological environment, and the problems of soil and water pollution and soil compaction have become increasingly serious.

The forest soil is rich in litter and organic matter, and soil microorganisms are more abundant than agricultural soils. Yin Ruiling's research found that among the phosphorus-dissolving microorganisms in China's dryland soils, bacteria account for the largest proportion. The main types of phosphorus-dissolving bacteria reported at present are: Bacillus, Pseudomonas, Salmonella, Alcaligenes, etc. Among them, Bacillus megaterium is the earliest and Phosphorus is the strain with the best effect and the most widely used area in agriculture.

The rhizosphere conditions of different plants are also different in different soil environment and climate characteristics, so the special soil environment may lead to the disadvantages of poor colonization ability of non-indigenous strains and high culling rate of strains. The soil in Guangxi is mainly red-yellow soil, which is also a typical soil type in southern my country. There are not many reports on the rhizosphere phosphorus-dissolving bacteria in the red-yellow soil area of my country. Due to its special soil-forming process, the clay, iron oxide and alumina in the soil are high, and the available phosphorus is deficient, which restricts the increase of the forest stock in this area. Dai Shenyan et al. isolated a high-efficiency phosphorus-dissolving bacteria Y5 from the paddy soil of Yingtan, Jiangxi, and its phosphorus-dissolving amount to calcium phosphate was 159.1 mg/L. At present, there are relatively few studies on the rhizosphere phosphorus-solubilizing bacteria in the red-yellow soil forests in China, and the selected ones are all derived from the crop soil of cultivated land, and their application effect in forest production is limited.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a high-efficiency phosphorus-dissolving Bacillus megaterium in the rhizosphere of trees with good phosphorus-dissolving effect, strong colonization ability, wide application range and low production cost, and its application. Production of forest bio-bacterial fertilizers and forest growth-promoting agents.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:

A strain of Bacillus megaterium with high efficiency for dissolving phosphorus in the rhizosphere of trees, the preservation number is CCTCC No.M 2017454, and the classification name is Bacillus megaterium L205.

The application of the above-mentioned high-efficiency phosphate-solubilizing Bacillus megaterium in the rhizosphere of forest trees in dissolving insoluble inorganic phosphorus.

Insoluble inorganic phosphorus comes from red-yellow soil.

The insoluble inorganic phosphorus is calcium phosphate.

In the above application, the propagation conditions of Bacillus megaterium are temperature 15-35° C., pH 6.0-9.0, dissolved oxygen 0.2-2.0 mg/L, and carbon-nitrogen mass ratio 2:1-5:1.

The application of the above-mentioned high-efficiency Bacillus megaterium in the rhizosphere of forest trees in promoting the growth of forest tree seedlings.

Tree seedlings grow in red-yellow soil.

In the above application, the propagation conditions of Bacillus megaterium are temperature 15-35° C., pH 6.0-9.0, dissolved oxygen 0.2-2.0 mg/L, and carbon-nitrogen mass ratio 2:1-5:1.

The above-mentioned high-efficiency Phosphorus megaterium in the rhizosphere of forest trees is used for producing forest tree biological bacterial fertilizer or forest tree growth-promoting bacterial agent.

The total number of Bacillus megaterium L205 or mixed bacteria is not less than 0.2*10 ⁸ cfu/mL (g).

The inventor isolated and bred an effective phosphorus-dissolving strain from the rhizosphere soil of trees in the red-yellow soil area of Guangxi, and identified it as Bacillus megaterium by Blast alignment of 16SrDNA with the sequences in GenBank. The forest tree rhizosphere highly efficient Bacillus megaterium has a deposit number of CCTCC No.M 2017454, and is classified and named as Bacillus megaterium (Bacillus megaterium) L205. In the research on the application of the strain, the inventor obtained the technology for the propagation of the strain through the expansion of the pure breed and the optimization of the liquid fermentation process. Experiments show that the amount of dissolved phosphorus of Bacillus megaterium reaches 203.04 mg/L, and its liquid bacterial solution can effectively dissolve insoluble inorganic phosphorus, increase the content of available phosphorus in red-yellow soil, and promote the growth of tree seedlings. The phosphorus-dissolving strain of the invention enriches the population library of Bacillus megaterium in the strain collection center, and provides convenience for better development and utilization of the microbial population resources of Bacillus megaterium. At the same time, Bacillus megaterium strain L205 can secrete organic acids, dissolve inorganic phosphorus, and improve the absorption and utilization of phosphorus by plants. It is especially suitable for the production of tree biological fertilizers and forest growth-promoting agents in the red-yellow soil area of South China. It can further develop microorganisms. Compound fertilizers and inoculants. In a word, the rhizosphere high-efficiency phosphate-solubilizing Bacillus megaterium of the present invention has good phosphate-dissolving effect, strong colonization ability, wide application range, low production cost, and has great market application potential.

Description of drawings

Figure 1 is a phylogenetic tree of Bacillus megaterium L205 of the present invention.

Fig. 2 is a graph showing the results of dissolving phosphorus of Bacillus megaterium L205 of the present invention, among the figures: the front view of the A plate, the back view of the B plate.

Description of preservation information

Bacillus megaterium L205, preservation number CCTCC NO.M 2017454, preservation date: August 28, 2017, preservation address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, zip code 430072, preservation unit : China Type Culture Collection.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments and the accompanying drawings.

1. Strain origin, breeding and gene sequencing of Bacillus megaterium

Source of strain: Bacillus megaterium L205 of the present invention is isolated and bred from the rhizosphere soil of trees in the red-yellow soil area of Guangxi, and the preservation information is the same as above.

The morphological characteristics of Bacillus megaterium L205 are as follows: the colony is white in color, the cell is rod-shaped and has spores.

The physiological and biochemical characteristics of Bacillus megaterium L205 are: Gram stain positive, contact enzyme positive, oxidase positive.

Bacillus megaterium L205 was obtained by the following specific isolation steps:

A Soil collection: Take the rhizosphere soil of trees with good growth in Wuzhou, Ningming, Luzhai, Tianlin and other places in the red-yellow soil area of Guangxi, take 0-20cm rhizosphere non-rhizosphere soil and root samples, and put them in unopened fresh-keeping bags , labelled, sealed, placed in an ice pack and brought back to the laboratory immediately for isolation of phosphate-solubilizing strains.

The measurement results of various indicators of the soil samples are shown in Table 1. It can be seen from Table 1 that the organic matter content of the soil samples collected from Wuzhou, Ningming, Luzhai, and Tianlin in Guangxi were all greater than 36.75 mg/kg, and the organic matter content of the four soil samples was between 36.75 and 56.51 mg/kg, and the soil with the lowest organic matter content. Samples were taken from Luzhai. The total phosphorus content of the soil collected from Wuzhou was the highest at 1.48g/kg, and the soil available content of the samples collected from the field forest was the highest at 16.53mg/kg. The pH value of each soil sample was slightly acidic between 4.15 and 5.02.

Table 1 Analysis results of soil samples collected from four different areas in Guangxi

B strain isolation: Weigh 5g samples of eucalyptus rhizosphere soil from different regions, put them in a sterilized 150mL conical flask containing 45mL of 0.85% NaCl solution, and shake for 30min to make a suspension. Use a 1mL micropipette to draw 1mL of suspension and inject it into a test tube containing 9mL of 0.85% sterile NaCl solution, mix well, and then draw 1mL from this test tube and inject it into another test tube containing 9mL of 0.85% sterile NaCl solution , and so on, to prepare three kinds of soil suspensions with dilution gradients of 10 ^-3 , 10 ^-4 , and 10 ^-5 in the ratio of content (g) to NaCl solution (mL). Pipette 50 μL of spread, 3 replicates per gradient, and label. The constant temperature is 28°C, cultured for 2-5 days, and the strains with the phosphorus-dissolving circle on the medium of the phosphorus-dissolving bacteria are screened. The strains with stronger phosphorus ability were inoculated onto LB plates for purification, and then stored on LB slant medium and stored in a refrigerator at 4°C for future use.

Determination of C phosphorus-dissolving ability: put 100 mL of liquid PKO medium into a 250 mL conical flask, sterilize at 121°C for 20 min, and after cooling, inoculate 1 mL of the bacterial suspension of each strain to be tested (10 ⁸ cfu/mL) into the conical flask. Three replicates were set for each strain, and the control was not inoculated with 1 ml of sterile water instead. The flasks were placed at 28°C and incubated at 160 r/min on a shaker for 3 d, and the pH value of the culture solution was measured with a pH meter. And centrifuge at 100 00r/min at 4℃ for 10min, take 5mL of supernatant and use molybdenum antimony scandium colorimetry to measure the available phosphorus content at a wavelength of 700nm.

D Liquid culture: Pick a ring of Bacillus megaterium from the slant of a single colony test tube, inoculate it into a liquid medium, and cultivate it on a shaker at 150-220 rpm at 32°C for 24 hours, and the number of viable bacteria reaches about 6×10 ⁸ cfu/mL .

E strain preservation: the above-mentioned liquid fermentation is obtained to obtain Bacillus megaterium bacterial liquid and add the glycerol through high temperature sterilization, so that the final concentration of glycerol in the seed liquid is 20% (V/V), make Bacillus megaterium glycerol freezing tube, store Store in a -80°C refrigerator, or store in a -196°C liquid nitrogen tank for ultra-low temperature preservation.

Determination of F gene sequence: The test tube of Bacillus megaterium was sent to Shanghai Meiji Biotechnology Company for 16sRNA gene sequencing, and the 16s rRNA gene sequence was as shown in SEQ.ID.No.1 in the sequence table. Its phylogenetic tree is shown in Figure 1.

2. Phosphorus dissolution test

PKO (Pikovskaya's Medium) medium: glucose 10g, calcium phosphate 3g, ammonium sulfate 0.5g, sodium chloride 0.1g, magnesium sulfate heptahydrate 0.1g, potassium chloride 0.2g, manganese sulfate 0.004g, ferrous sulfate 0.002g, Yeast paste 0.5g, water 1000ml, pH 7.2.

Bacillus megaterium CCTCC M 2017454 was inoculated on PK0 medium, cultured at 30°C for 5 d, and the ratio of the diameter (D) of the transparent circle (D)/colony size (d) was determined to be 2.67. It shows that the Bacillus megaterium has a good phosphorus-dissolving effect. 3. Determination of dissolved phosphorus

PKO (Pikovskaya's Medium) medium: glucose 10g, calcium phosphate 3g, ammonium sulfate 0.5g, sodium chloride 0.1g, magnesium sulfate heptahydrate 0.1g, potassium chloride 0.2g, manganese sulfate 0.004g, ferrous sulfate 0.002g, Yeast paste 0.5g, water 1000ml, pH 7.2.

Put 100 mL of liquid PK0 medium into a 250 mL conical flask, sterilize at 121° C. for 20 min, and after cooling, inoculate 1 mL of Bacillus megaterium CCTCC M 2017454 (10 ⁸ cfu/mL) into the conical flask. Three replicates were set for each strain, and the control was not inoculated with 1 ml of sterile water instead. The flasks were placed at 28°C and incubated at 160 r/min on a shaker for 3 d, and the pH value of the culture solution was measured with a pH meter. And centrifuge at 100 00r/min at 4℃ for 10min, take 5mL of supernatant and use molybdenum antimony scandium colorimetry to measure the available phosphorus content at a wavelength of 700nm.

According to the phosphorus-dissolving ability of L205 under liquid shaking flask culture, the results showed that the effective phosphorus increment of this strain was 203.04 mg/L.

4. Preparation of Phosphorus Dissolving Microbial Liquid from Forest Trees

(1) Seed preparation

Seed medium: tryptone 10g, yeast extract powder 5g, NaCl 10g, pH 7.0, distilled water 1000mL. Inoculate one loop of Bacillus megaterium CCTCC M 2017454 in a 250 mL conical flask containing 100 mL of the above seed medium, and cultivate it at 30°C for 24 h on a rotary shaker at 180 rpm to obtain seeds suitable for inoculation.

(2) Liquid fermentation culture

Fermentation medium: tryptone 10g, yeast extract powder 5g, NaCl 10g, pH 7.0, distilled water 1000mL. The Bacillus megaterium seeds obtained in (1) were inoculated into a liquid fermentation medium according to 5% of the volume of the fermentation medium, and cultured at 30° C. for 48 hours under a rotating shaker at 180 rpm.

(3) After the fermentation, the pH value of the fermentation broth is adjusted to be between 5.0 and 6.0, and the liquid bacterial agent can be obtained.

5. Preparation of Phosphorus Resolving Microorganisms for Forest Trees

(1) Seed preparation

Seed medium: tryptone 10g, yeast extract powder 5g, NaCl 10g, pH 7.0, distilled water 1000mL. Inoculate one loop of Bacillus megaterium CCTCC M 2017454 in a 250 mL conical flask containing 100 mL of the above seed medium, and cultivate it at 30°C for 24 h on a rotary shaker at 180 rpm to obtain seeds suitable for inoculation.

(2) Liquid fermentation culture

Fermentation medium: tryptone 10g, yeast extract powder 5g, NaCl 10g, pH 7.0, distilled water 1000mL. The Bacillus megaterium seeds obtained in (1) were inoculated into a liquid fermentation medium according to 5% of the volume of the fermentation medium, and cultured at 30° C. for 48 hours under a rotating shaker at 180 rpm.

(3) After the fermentation, the liquid fermentation medium was taken and placed in a high-speed centrifuge for centrifugation for 5 minutes, the supernatant was removed, 1% Tween 80 was added, and freeze-dried to obtain a forest tree phosphorus-resolving microbial inoculum.

6. The application of forest tree phosphorus-resolving microbial bacterial solution in the cultivation of tree cup seedlings

Using the root irrigation method, the bacterial solution obtained in step 4 was diluted 10 times with sterile water. Take the pine and fir seedlings with the same growth potential, water 10mL of the diluted bacterial solution, water 50mL of sterile water every 2d, and apply phosphorus-deficient Hoagland nutrient solution every 10d. 25 strains were treated at each concentration, with clear water as the control, and the test temperature was 15-30 °C. After 30 days of inoculation, the survival number of seedlings, average seedling height and ground diameter of each treatment were calculated.

The measurement results showed that: the pine cup seedlings added with phosphorus-dissolving microbial inoculants increased the height by 12.3% and the ground diameter by 10.6% compared with the control group; . It shows that the microbial preparation of the present invention has a relatively good growth-promoting effect on forest trees.

sequence listing

<110> Guangxi Zhuang Autonomous Region Forestry Research Institute

<120> A high-efficiency phosphate-solubilizing Bacillus megaterium in the rhizosphere of forest trees and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1398

<212> DNA

<213> Bacillus megaterium L205 (Bacillus megaterium L205)

<400> 1

gcagtcgagc gaactgatta gaagcttgct tctatgacgt tagcggcgga cgggtgagta 60

acacgtgggc aacctgcctg taagactggg ataacttcgg gaaaccgaag ctaataccgg 120

ataggatctt ctccttcatg ggagatgatt gaaagatggt ttcggctatc acttacagat 180

gggcccgcgg tgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcatag 240

ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300

ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360

gatgaaggct ttcgggtcgt aaaactctgt tgttagggaa gaacaagtac gagagtaact 420

gcttgtacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480

gtaatacgta ggtggcaagc gttatccgga attattgggc gtaaagcgcg cgcaggcggt 540

ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga gggtcattgg aaactgggga 600

acttgagtgc agaagagaaa agcggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660

ggaggaacac cagtggcgaa ggcggctttt tggtctgtaa ctgacgctga ggcgcgaaag 720

cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780

gtgttagagg gtttccgccc tttagtgctg cagctaacgc attaagcact ccgcctgggg 840

agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900

atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaac 960

tctagagata gagcgttccc cttcggggga cagagtgaca ggtggtgcat ggttgtcgtc 1020

agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt 1080

gccagcattt agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg 1140

atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt 1200

acaaagggct gcaagaccgc gaggtcaagc caatcccata aaaccattct cagttcggat 1260

tgtaggctgc aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc 1320

gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac 1380

acccgaagtc ggtggagt 1444

Claims (8)

1. A high-efficiency phosphorus-solubilizing Bacillus megaterium in the rhizosphere of trees is characterized in that the deposit number is CCTCC NO.M2017454, and the classification is named as Bacillus megaterium (Bacillus megaterium) L205. 2. the application of high-efficiency phosphorus-solubilizing Bacillus megaterium in the rhizosphere of claim 1 in dissolving insoluble inorganic phosphorus. 3. The application according to claim 2, wherein the insoluble inorganic phosphorus comes from red-yellow soil. 4. The application according to claim 2, wherein the insoluble inorganic phosphorus is calcium phosphate. 5. the application of high-efficiency Phosphorus megaterium in the rhizosphere of claim 1 in promoting the growth of forest tree seedlings. 6. application according to claim 5 is characterized in that: described forest tree seedling grows in red-yellow soil soil. 7. the application of high-efficiency phosphorus-solubilizing Bacillus megaterium in the rhizosphere of claim 1 in the production of forest biological bacterial fertilizer or forest growth-promoting bacterial agent. 8. application according to claim 7, is characterized in that: in described forest tree biological bacterial fertilizer or forest tree growth promoting microbial inoculant, Bacillus megaterium L205 total number is not less than 0.2*10 ⁸ cfu/mL or 0.2*10 ⁸ cfu/ g.

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